Last two issues

Human cutaneous microvascular ageing: potential insights into underlying physiological mechanisms of endothelial function and dysfunction

Holowatz, L. A. — 2008-07-14

TRPA1: irritant detector of the airways

Gerhold, K. A., Bautista, D. M. — 2008-07-14

In the flicker of an eye

Pedersen, N. P., Fuller, P. M., Lu, J., Saper, C. B. — 2008-07-14

Unresolved issues and controversies in purinergic signalling

Burnstock, G. — 2008-07-14

Some areas of current interest in the rapidly expanding purinergic signalling field that are controversial or are unresolved are highlighted in this review. These include the mechanisms underlying: ATP transport across cell and vesicle membranes; the interaction of multiple receptors for purines and pyrimidines on single cells; the blocking effect of antagonists to P2X₄ and P2X₇ receptors expressed by microglial cells in neuropathic and inflammatory pain; and the complex actions mediated by P2X₇ receptors. Some desirable areas for further research are also discussed including: comparative studies of the evolution of purinergic signalling; studies of purinergic signalling in development and regeneration, including the involvement of stem cells; behavioural studies; and therapeutic strategies.

Novel aspects of the molecular mechanisms controlling insulin secretion

2008-07-14

Pancreatic β-cells secrete insulin by Ca²⁺-dependent exocytosis of secretory granules. β-cell exocytosis involves SNARE (soluble NSF-attachment protein receptor) proteins similar to those controlling neurotransmitter release and depends on the close association of L-type Ca²⁺ channels and granules. In most cases, the secretory granules fuse individually but there is ultrastructural and biophysical evidence of multivesicular exocytosis. Estimates of the secretory rate in β-cells in intact islets indicate a release rate of ~15 granules per β-cell per second, 100-fold higher than that observed in biochemical assays. Single-vesicle capacitance measurements reveal that the diameter of the fusion pore connecting the granule lumen with the exterior is ~1.4 nm. This is considerably smaller than the size of insulin and membrane fusion is therefore not obligatorily associated with release of the cargo, a feature that may contribute to the different rates of secretion detected by the biochemical and biophysical measurements. However, small molecules like ATP and GABA, which are stored together with insulin in the granules, are small enough to be released via the narrow fusion pore, which accordingly functions as a molecular sieve. We finally consider the possibility that defective fusion pore expansion accounts for the decrease in insulin secretion observed in pathophysiological states including long-term exposure to lipids.

Differential dissociation of G protein heterotrimers

Digby, G. J., Sethi, P. R., Lambert, N. A. — 2008-07-14

Signalling by heterotrimeric G proteins is often isoform-specific, meaning certain effectors are regulated exclusively by one family of heterotrimers. For example, in excitable cells inwardly rectifying potassium (GIRK) channels are activated by Gβ dimers derived specifically from G_i/o heterotrimers. Since all active heterotrimers are thought to dissociate and release free Gβ dimers, it is unclear why these channels respond primarily to dimers released by G_i/o heterotrimers. We reconstituted GIRK channel activation in cells where we could quantify heterotrimer expression at the plasma membrane, GIRK channel activation, and heterotrimer dissociation. We find that G_oA heterotrimers are more effective activators of GIRK channels than G_s heterotrimers when comparable amounts of each are available. We also find that active G_oA heterotrimers dissociate more readily than active G_s heterotrimers. Differential dissociation may thus provide a simple explanation for G-specific activation of GIRK channels and other Gβ-sensitive effectors.

Preferential motor unit loss in the SOD1G93A transgenic mouse model of amyotrophic lateral sclerosis

Hegedus, J., Putman, C. T., Tyreman, N., Gordon, T. — 2008-07-14

The present study investigated motor unit (MU) loss in a murine model of familial amyotrophic lateral sclerosis (ALS). The fast-twitch tibialis anterior (TA) and medial gastrocnemius (MG) muscles of transgenic SOD1^G93A and SOD1^WT mice were studied during the presymptomatic phase of disease progression at 60 days of age. Whole muscle maximum isometric twitch and tetanic forces were 80% lower (P < 0.01) in the TA muscles of SOD1^G93A compared to SOD1^WT mice. Enumeration of total MU numbers within TA muscles showed a 60% reduction (P < 0.01) within SOD1^G93A mice (38 ± 7) compared with SOD1^WT controls (95 ± 12); this was attributed to a lower proportion of the most forceful fast-fatigable (FF) MU in SOD1^G93A mice, as seen by a significant (P < 0.01) leftward shift in the cumulative frequency histogram of single MU forces. Similar patterns of MU loss and corresponding decreases in isometric twitch force were observed in the MG. Immunocytochemical analyses of the entire cross-sectional area (CSA) of serial sections of TA muscles stained with anti-neural cell adhesion molecule (NCAM) and various monoclonal antibodies for myosin heavy chain (MHC) isoforms showed respective 65% (P < 0.01) and 28% (P < 0.05) decreases in the number of innervated IIB and IID/X muscle fibres in SOD1^G93A, which paralleled the 60% decrease (P < 0.01) in the force generating capacity of individual fibres. The loss of fast MUs was partially compensated by activity-dependent fast-to-slower fibre type transitions, as determined by increases (P < 0.04) in the CSA and proportion of IIA fibres (from 4% to 14%) and IID/X fibres (from 31% to 39%), and decreases (P < 0.001) in the CSA and proportion of type IIB fibres (from 65% to 44%). We conclude that preferential loss of IIB fibres is incomplete at 60 days of age, and is consistent with a selective albeit gradual loss of FF MUs that is not fully compensated by sprouting of the remaining motoneurons that innervate type IIA or IID/X muscle fibres. Our findings indicate that disease progression in fast-twitch muscles of SOD1^G93A mice involves parallel processes: (1) gradual selective motor axon die-back of the FF motor units that contain large type IIB muscle fibres, and of fatigue-intermediate motor units that innervate type IID/X muscle fibres, and (2) activity-dependent conversion of motor units to those innervated by smaller motor axons innervating type IIA fatigue-resistant muscle fibres.

High frequency action potential bursts (>= 100 Hz) in L2/3 and L5B thick tufted neurons in anaesthetized and awake rat primary somatosensory cortex

de Kock, C. P. J., Sakmann, B. — 2008-07-14

High frequency (≥ 100 Hz) bursts of action potentials (APs) generated by neocortical neurons are thought to increase information content and, through back-propagation, to influence synaptic integration and efficacy in distal dendritic compartments. It was recently shown in acute slice experiments that intrinsic bursting properties differ between neocortical L2/3 and L5B (thick tufted) neurons. In L2/3 neurons for instance, dendritic APs were brief and generated only one additional AP after the initial somatic AP. In L5B neurons, dendritic plateau potentials facilitated the generation of trains of three or more APs. We recently showed in vivo that spiking frequencies are very different for L2/3 and L5B thick tufted neurons under anaesthesia. Here, we addressed the question whether in vivo the bursting properties are different for these two cell types. We recorded from L2/3 and L5B thick tufted neurons of rat primary somatosensory (barrel) cortex under anaesthetized and awake conditions and found that AP activity is dominated by single APs. In addition, we found that in the anaesthetized animal also bursts of two APs were observed in L2/3 neurons but the relative occurrence of these bursts was low. In L5B thick tufted neurons, bursts consisting of up to six APs were recorded and their relative occurrence was significantly higher. Frequencies within bursts were also significantly higher in L5B thick tufted neurons than in L2/3 neurons. In awake (head-restrained) animals, average spike frequencies of L2/3 and L5B thick tufted neurons were surprisingly similar to spike rates under anaesthesia. However, bursting behaviour in L2/3 neurons was comparable to L5B thick tufted neurons. Thus, the distribution of interspike intervals was changed in L2/3 neurons without affecting the average spiking rate. We observed bursts consisting of up to five APs in both cell types and both probability of bursts and AP frequency within bursts were similar for L2/3 and L5B thick tufted neurons. Our analysis shows that most cortical APs occur as single APs, although a minor fraction of APs in L2/3 and L5B thick tufted neurons are part of high frequency bursts (15%). This AP bursting is dependent on the behavioural state of the animal in a cell-type dependent manner.

Ca2+-dependent inactivation of Ca2+-induced Ca2+ release in bullfrog sympathetic neurons

Akita, T., Kuba, K. — 2008-07-14

We studied inactivation of Ca²⁺-induced Ca²⁺ release (CICR) via ryanodine receptors (RyRs) in bullfrog sympathetic neurons. The rate of rise in [Ca²⁺]_i due to CICR evoked by a depolarizing pulse decreased markedly within 10–20 ms to a much slower rate despite persistent Ca²⁺ entry and little depletion of Ca²⁺ stores. The Ca²⁺ entry elicited by the subsequent pulse within 50 ms, during which the [Ca²⁺]_i level remained unchanged, did not generate a distinct [Ca²⁺]_i rise. This mode of [Ca²⁺]_i rise was unaffected by a mitochondrial uncoupler, carbonyl cyanide p-trifluromethoxy-phenylhydrazone (FCCP, 1 µm). Paired pulses of varying interval and duration revealed that recovery from inactivation became distinct ≥ 50 ms after depolarization and depended on [Ca²⁺]_i. The inactivation was prevented by BAPTA (≥ 100 µm) but not by EGTA (≤ 10 mm), whereas the activation was less affected by BAPTA. When CICR was partially activated, some of the non-activated RyRs were also inactivated directly. Thus, the inactivation in these neurons is induced by Ca²⁺ binding to the high-affinity regulatory sites residing very close to Ca²⁺ channels and/or RyRs, although the sites for activation are located much closer to those Ca²⁺ sources. The rate of [Ca²⁺]_i decay after the pulse decreased with increasing pulse duration longer than 10 ms, and this was abolished by BAPTA. Thus, some mechanism counteracting Ca²⁺ clearance is induced after full inactivation and potentiated during the pulse. Possible models for RyR inactivation were proposed and the roles of inactivation in Ca²⁺ signalling were discussed.

Intracortical modulation of cortical-bulbar responses for the masseter muscle

Ortu, E., Deriu, F., Suppa, A., Giaconi, E., Tolu, E., Rothwell, J. C. — 2008-07-14

Short interval intracortical inhibition (SICI) and intracortical facilitation (ICF) were evaluated in the masseter muscles of 12 subjects and the cortical silent period (SP) in nine subjects. Motor evoked potentials (MEPs) were recorded from contralateral (cMM) and ipsilateral (iMM) masseters, activated at 10% of maximal voluntary contraction (MVC). Interstimulus intervals (ISIs) were 2 and 3 ms for SICI, 10 and 15 ms for ICF. TMS of the left masseteric cortex induced MEPs that were larger in the cMM than the iMM; stimulation of right masseteric cortex produced a similar asymmetry in response amplitude. SICI was only observed using a CS intensity of 70% AMT and was equal in both cMM and iMM. SICI was stronger at higher TS intensities, was abolished by muscle activation greater than 10% MVC, and was unaffected by coil orientation changes. Control experiments confirmed that SICI was not contaminated by any inhibitory peripheral reflexes. However, ICF could not be obtained because it was masked by bilateral reflex depression of masseter EMG caused by auditory input from the coil discharge. The SP was bilateral and symmetric; its duration ranged from 35 to 70 ms depending on TS intensity and coil orientation. We conclude that SICI is present in the cortical representation of masseter muscles. The similarity of SICI in cMM and iMM suggests either that a single pool of inhibitory interneurons controls ipsi- and contralateral corticotrigeminal projections or that inhibition is directed to bilaterally projecting corticotrigeminal fibres. Finally, the corticotrigeminal projection seems to be weakly influenced by inhibitory interneurons mediating the cortical SP.

Mouse models of human KCNQ2 and KCNQ3 mutations for benign familial neonatal convulsions show seizures and neuronal plasticity without synaptic reorganization

2008-07-14

The childhood epilepsy syndrome of benign familial neonatal convulsions (BFNC) exhibits the remarkable feature of clinical remission within a few weeks of onset and a favourable prognosis, sparing cognitive abilities despite persistent expression of the mutant KCNQ2 or KCNQ3 potassium channels throughout adulthood. To better understand such dynamic neuroprotective plasticity within the developing brain, we introduced missense mutations that underlie human BFNC into the orthologous murine Kcnq2 (Kv7.2) and Kcnq3 (Kv7.3) genes. Mutant mice were examined for altered thresholds to induced seizures, spontaneous seizure characteristics, hippocampal histology, and M-current properties of CA1 hippocampal pyramidal neurons. Adult Kcnq2^A306T/+ and Kcnq3^G311V/+ heterozygous knock-in mice exhibited reduced thresholds to electrically induced seizures compared to wild-type littermate mice. Both Kcnq2^A306T/A306T and Kcnq3^G311V/G311V homozygous mutant mice exhibited early onset spontaneous generalized tonic-clonic seizures concurrent with a significant reduction in amplitude and increased deactivation kinetics of the neuronal M-current. Mice had recurrent seizures into adulthood that triggered molecular plasticity including ectopic neuropeptide Y (NPY) expression in granule cells, but without hippocampal mossy fibre sprouting or neuronal loss. These novel knockin mice recapitulate proconvulsant features of the human disorder yet show that inherited M-current defects spare granule cells from reactive changes in adult hippocampal networks. The absence of seizure-induced pathology found in these epileptic mouse models parallels the benign neurodevelopmental cognitive profile exhibited by the majority of BFNC patients.

Glutamatergic calcium dynamics and deregulation of rat retinal ganglion cells

Hartwick, A. T. E., Hamilton, C. M., Baldridge, W. H. — 2008-07-14

A rise in intracellular calcium levels ([Ca²⁺]_i) is a key trigger for the lethal effects of the excitatory neurotransmitter glutamate in various central neurons, but a consensus has not been reached on the pathways that mediate glutamate-dependent increases of [Ca²⁺]_i in retinal ganglion cells (RGCs). Using Ca²⁺ imaging techniques we demonstrated that, in the absence of external Mg²⁺, the Ca²⁺ signal evoked by glutamate was predominantly mediated by NMDA-type glutamate receptors (NMDA-Rs) in immunopanned RGCs isolated from neonatal or adult rats. Voltage-gated Ca²⁺ channels and AMPA/kainate-Rs contributed a smaller portion of the Ca²⁺ response at saturating concentrations of glutamate. Consistent with NMDA-R involvement, extracellular Mg²⁺ inhibited RGC glutamate responses, while glycine had a potentiating effect. With Mg²⁺ present externally, the effect of AMPA/kainate-R antagonists was enhanced and both NMDA- and AMPA/kainate-R antagonists greatly reduced the glutamate-induced increases of RGC [Ca²⁺]_i. This finding indicates that the primary contribution of AMPA/kainate-Rs to RGC glutamatergic Ca²⁺ dynamics is through the depolarization-dependent relief of the Mg²⁺ block of NMDA-R channels. The effect of glutamate receptor antagonists on glutamatergic Ca²⁺ signals from RGCs in adult rat retinal wholemounts yielded results similar to those obtained using immunopanned RGCs. Additional experiments on isolated RGCs revealed that during a 1 h glutamate (10–1000 µm) exposure, 18–28% of RGCs exhibited delayed Ca²⁺ deregulation (DCD) and the RGCs that underwent DCD were positive for the death marker annexin V. RGCs with larger glutamate-evoked Ca²⁺ signals were more likely to undergo DCD, and NMDA-R blockade significantly reduced the occurrence of DCD. Identifying the mechanisms underlying RGC excitotoxicity aids in our understanding of the pathophysiology of retinal ischaemia, and this work establishes a major role for NMDA-R-mediated increases in [Ca²⁺]_i in glutamate-related RGC death.

Relative contributions of TRPA1 and TRPV1 channels in the activation of vagal bronchopulmonary C-fibres by the endogenous autacoid 4-oxononenal

2008-07-14

Transient receptor potential (TRP) A1 channels are cation channels found preferentially on nociceptive sensory neurones, including capsaicin-sensitive TRPV1-expressing vagal bronchopulmonary C-fibres, and are activated by electrophilic compounds such as mustard oil and cinnamaldehyde. Oxidative stress, a pathological feature of many respiratory diseases, causes the endogenous formation of a number of reactive electrophilic alkenals via lipid peroxidation. One such alkenal, 4-hydroxynonenal (4HNE), activates TRPA1 in cultured sensory neurones. However, our data demonstrate that 100 µm 4HNE was unable to evoke significant action potential discharge or tachykinin release from bronchopulmonary C-fibre terminals. Instead, another endogenously produced alkenal, 4-oxononenal (4ONE, 10 µm), which is far more electrophilic than 4HNE, caused substantial action potential discharge and tachykinin release from bronchopulmonary C-fibre terminals. The activation of mouse bronchopulmonary C-fibre terminals by 4ONE (10–100 µm) was mediated entirely by TRPA1 channels, based on the absence of responses in C-fibre terminals from TRPA1 knockout mice. Interestingly, although the robust increases in calcium caused by 4ONE (0.1–10 µm) in dissociated vagal neurones were essentially abolished in TRPA1 knockout mice, at 100 µm 4ONE caused a large TRPV1-dependent response. Furthermore, 4ONE (100 µm) was shown to activate TRPV1 channel-expressing HEK cells. In conclusion, the data support the hypothesis that 4-ONE is a relevant endogenous activator of vagal C-fibres via an interaction with TRPA1, and at less relevant concentrations, it may activate nerves via TRPV1.

Tonic and phasic phenomena underlying eye movements during sleep in the cat

Marquez-Ruiz, J., Escudero, M. — 2008-07-14

Mammalian sleep is not a homogenous state, and different variables have traditionally been used to distinguish different periods during sleep. Of these variables, eye movement is one of the most paradigmatic, and has been used to differentiate between the so-called rapid eye movement (REM) and non-REM (NREM) sleep periods. Despite this, eye movements during sleep are poorly understood, and the behaviour of the oculomotor system remains almost unknown. In the present work, we recorded binocular eye movements during the sleep–wake cycle of adult cats by the scleral search-coil technique. During alertness, eye movements consisted of conjugated saccades and eye fixations. During NREM sleep, eye movements were slow and mostly unconjugated. The two eyes moved upwardly and in the abducting direction, producing a tonic divergence and elevation of the visual axis. During the transition period between NREM and REM sleep, rapid monocular eye movements of low amplitude in the abducting direction occurred in coincidence with ponto-geniculo-occipital waves. Along REM sleep, the eyes tended to maintain a tonic convergence and depression, broken by high-frequency bursts of complex rapid eye movements. In the horizontal plane, each eye movement in the burst comprised two consecutive movements in opposite directions, which were more evident in the eye that performed the abducting movements. In the vertical plane, rapid eye movements were always upward. Comparisons of the characteristics of eye movements during the sleep–wake cycle reveal the uniqueness of eye movements during sleep, and the noteworthy existence of tonic and phasic phenomena in the oculomotor system, not observed until now.

Tonic inhibition and ponto-geniculo-occipital-related activities shape abducens motoneuron discharge during REM sleep

Escudero, M., Marquez-Ruiz, J. — 2008-07-14

Eye movements, ponto-geniculo-occipital (PGO) waves, muscular atonia and desynchronized cortical activity are the main characteristics of rapid eye movement (REM) sleep. Although eye movements designate this phase, little is known about the activity of the oculomotor system during REM sleep. In this work, we recorded binocular eye movements by the scleral search-coil technique and the activity of identified abducens (ABD) motoneurons along the sleep–wake cycle in behaving cats. The activity of ABD motoneurons during REM sleep was characterized by a tonic decrease of their mean firing rate throughout this period, and short bursts and pauses coinciding with the occurrence of PGO waves. We demonstrate that the decrease in the mean firing discharge was due to an active inhibition of ABD motoneurons, and that the occurrence of primary and secondary PGO waves induced a pattern of simultaneous but opposed phasic activation and inhibition on each ABD nucleus. With regard to eye movements, during REM sleep ABD motoneurons failed to codify eye position as during alertness, but continued to codify eye velocity. The pattern of tonic inhibition and the phasic activations and inhibitions shown by ABD motoneurons coincide with those reported in other non-oculomotor motoneurons, indicating that the oculomotor system – contrary to what has been accepted until now – is not different from other motor systems during REM sleep, and that all motor systems are receiving similar command signals during this period.

Initial segment Kv2.2 channels mediate a slow delayed rectifier and maintain high frequency action potential firing in medial nucleus of the trapezoid body neurons

2008-07-14

The medial nucleus of the trapezoid body (MNTB) is specialized for high frequency firing by expression of Kv3 channels, which minimize action potential (AP) duration, and Kv1 channels, which suppress multiple AP firing, during each calyceal giant EPSC. However, the outward K⁺ current in MNTB neurons is dominated by another unidentified delayed rectifier. It has slow kinetics and a peak conductance of ~37 nS; it is half-activated at –9.2 ± 2.1 mV and half-inactivated at –35.9 ± 1.5 mV. It is blocked by several non-specific potassium channel antagonists including quinine (100 µm) and high concentrations of extracellular tetraethylammonium (TEA; IC₅₀ = 11.8 mm), but no specific antagonists were found. These characteristics are similar to recombinant Kv2-mediated currents. Quantitative RT-PCR showed that Kv2.2 mRNA was much more prevalent than Kv2.1 in the MNTB. A Kv2.2 antibody showed specific staining and Western blots confirmed that it recognized a protein ~110 kDa which was absent in brainstem tissue from a Kv2.2 knockout mouse. Confocal imaging showed that Kv2.2 was highly expressed in axon initial segments of MNTB neurons. In the absence of a specific antagonist, Hodgkin–Huxley modelling of voltage-gated conductances showed that Kv2.2 has a minor role during single APs (due to its slow activation) but assists recovery of voltage-gated sodium channels (Nav) from inactivation by hyperpolarizing interspike potentials during repetitive AP firing. Current-clamp recordings during high frequency firing and characterization of Nav inactivation confirmed this hypothesis. We conclude that Kv2.2-containing channels have a distinctive initial segment location and crucial function in maintaining AP amplitude by regulating the interspike potential during high frequency firing.

Exercise prevents age-related decline in nitric-oxide-mediated vasodilator function in cutaneous microvessels

Black, M. A., Green, D. J., Cable, N. T. — 2008-07-14

Ageing is associated with impaired endothelium-derived nitric oxide (NO) function in human microvessels. We investigated the impact of cardiorespiratory fitness and exercise training on physiological and pharmacological NO-mediated microvascular responses in older subjects. NO-mediated vasodilatation was examined in young, older sedentary and older fit subjects who had two microdialysis fibres embedded into the skin on the ventral aspect of the forearm and laser Doppler probes placed over these sites. Both sites were then heated to 42°C, with Ringer solution infused in one probe and N-nitro-l-arginine methyl ester (l-NAME) through the second. In another study, three doses of ACh were infused in the presence or absence of l-NAME in similar subjects. The older sedentary subjects then undertook exercise training, with repeat studies at 12 and 24 weeks. The NO component of the heat-induced rise in cutaneous vascular conductance (CVC) was diminished in the older sedentary subjects after 30 min of prolonged heating at 42°C (26.9 ± 3.9%CVC_max), compared to older fit (46.2 ± 7.0%CVC_max, P < 0.05) and young subjects (41.2 ± 5.2%CVC_max, P < 0.05), whereas exercise training in the older sedentary group enhanced NO-vasodilator function in response to incremental heating (P < 0.05). Similarly, the NO contribution to ACh responses was impaired in the older sedentary versus older fit subjects (low dose 3.2 ± 1.3 versus 6.6 ± 1.3%CVC_max; mid dose 11.4 ± 2.4 versus 21.6 ± 4.5%CVC_max; high dose 35.2 ± 6.0 versus 52.6 ± 7.9%CVC_max, P < 0.05) and training reversed this (12 weeks: 13.7 ± 3.6, 28.9 ± 5.3, 56.1 ± 3.9%CVC_max, P < 0.05). These findings indicate that maintaining a high level of fitness, or undertaking exercise training, prevents age-related decline in indices of physiological and pharmacological microvascular NO-mediated vasodilator function. Since higher levels of NO confer anti-atherogenic benefit, this study has potential implications for the prevention of microvascular dysfunction in humans.

Acute and chronic effects of vitamin C on endothelial fibrinolytic function in overweight and obese adult humans

2008-07-14

We determined the effects of acute intra-arterial vitamin C administration and chronic oral vitamin C supplementation on the capacity of the endothelium to release t-PA in overweight and obese adults. Net endothelial t-PA release was determined in vivo in response to intrabrachial infusions of bradykinin and sodium nitroprusside in 33 sedentary adults: 10 normal-weight (BMI: 23.4 ± 0.5 kg m^–2; 7M/3F); and 23 overweight/obese (BMI: 31.2 ± 0.8 kg m^–2; 15M/8F). In 10 normal weight and eight overweight/obese adults the dose–response curves to bradykinin and sodium nitroprusside were repeated with a coinfusion of the antioxidant vitamin C (24 mg min^–1). Seventeen of the 23 overweight/obese adults completed a 3 month chronic oral vitamin C (500 mg day^–1) supplementation intervention. Intra-arterial administration of vitamin C significantly potentiated t-PA release in overweight/obese adults. Net release of t-PA was ~95% higher (P < 0.01) after (from –0.9 ± 1.1 to 94.6 ± 16.2 ng (100 ml tissue)^–1 min^–1) compared with before (from –0.8 ± 0.8 to 49.9 ± 7.7 ng (100 ml tissue)^–1 min^–1) vitamin C administration. Daily vitamin C supplementation significantly increased t-PA release in overweight/obese adults (from 0.2 ± 0.9 to 48.2 ± 6.5 ng (100 ml tissue)^–1 min^–1) before supplementation versus (0.3 ± 0.5 to 66.3 ± 8.7 ng (100 ml tissue)^–1 min^–1) after supplementation. These results indicate that the antioxidant vitamin C favourably affects the capacity of the endothelium to release t-PA in overweight/obese adults. Daily vitamin C supplementation represents an effective lifestyle intervention strategy for improving endothelial fibrinolytic regulation in this at-risk population.

Restricted N-terminal truncation of cardiac troponin T: a novel mechanism for functional adaptation to energetic crisis

Feng, H.-Z., Biesiadecki, B. J., Yu, Z.-B., Hossain, M. M., Jin, J.-P. — 2008-07-14

The N-terminal variable region of cardiac troponin T (TnT) is a regulatory structure that can be selectively removed during myocardial ischaemia reperfusion by µ-calpain proteolysis. Here we investigated the pathophysiological significance of this post-translational modification that removes amino acids 1–71 of cardiac TnT. Working heart preparations were employed to study rat acute myocardial infarction and transgenic mouse hearts over-expressing the N-terminal truncated cardiac TnT (cTnT-ND). Ex vivo myocardial infarction by ligation of the left anterior descending coronary artery induced heart failure and produced cTnT-ND not only in the infarct but also in remote zones, including the right ventricular free wall, indicating a whole organ response in the absence of systemic neurohumoral mechanisms. Left ventricular pressure overload in mouse working hearts produced increased cTnT-ND in both ventricles, suggesting a role of haemodynamic stress in triggering an acute whole organ proteolytic regulation. Transgenic mouse hearts in which the endogenous intact cardiac TnT was partially replaced by cTnT-ND showed lowered contractile velocity. When afterload increased from 55 mmHg to 90 mmHg, stroke volume decreased in the wild type but not in the transgenic mouse hearts. Correspondingly, the left ventricular rapid-ejection time of the transgenic mouse hearts was significantly longer than that of wild type hearts, especially at high afterload. The restricted deletion of the N-terminal variable region of cardiac troponin T demonstrates a novel mechanism by which the thin filament regulation adapts to sustain cardiac function under stress conditions.

Calprotectin is released from human skeletal muscle tissue during exercise

2008-07-14

Skeletal muscle has been identified as a secretory organ. We hypothesized that IL-6, a cytokine secreted from skeletal muscle during exercise, could induce production of other secreted factors in skeletal muscle. IL-6 was infused for 3 h into healthy young males (n = 7) and muscle biopsies obtained at time points 0, 3 and 6 h in these individuals and in resting controls. Affymetrix microarray analysis of gene expression changes in skeletal muscle biopsies identified a small set of genes changed by IL-6 infusion. RT-PCR validation confirmed that S100A8 and S100A9 mRNA were up-regulated 3-fold in skeletal muscle following IL-6 infusion compared to controls. Furthermore, S100A8 and S100A9 mRNA levels were up-regulated 5-fold in human skeletal muscle following cycle ergometer exercise for 3 h at ~60% of in young healthy males (n = 8). S100A8 and S100A9 form calprotectin, which is known as an acute phase reactant. Plasma calprotectin increased 5-fold following acute cycle ergometer exercise in humans, but not following IL-6 infusion. To identify the source of calprotectin, healthy males (n = 7) performed two-legged dynamic knee extensor exercise for 3 h with a work load of ~50% of peak power output and arterial–femoral venous differences were obtained. Arterial plasma concentrations for calprotectin increased 2-fold compared to rest and there was a net release of calprotectin from the working muscle. In conclusion, IL-6 infusion and muscle contractions induce expression of S100A8 and S100A9 in skeletal muscle. However, IL-6 alone is not a sufficient stimulus to facilitate release of calprotectin from skeletal muscle.

Do nerve terminal sprouts contribute to functional recovery from botulinum neurotoxin A?

Ko, C.-P. — 2008-07-01

Anterior pituitary cells excited by GABA

Kreft, M., Zorec, R. — 2008-07-01

Unravelling the mysteries of the exercise pressor reflex at the cellular level

Mitchell, J. H., Smith, S. A. — 2008-07-01

Muscle [phosphocreatine] dynamics during exercise: implication for understanding the regulation of muscle oxidative metabolism

Menuet, C., Arsac, L. M. — 2008-07-01

Changes in motoneuron properties following spinal cord transection: does afferent input play a role?

Laird, A. S., Wu, A., Lauschke, J. L. — 2008-07-01

Store-operated channels: mechanisms and function

Parekh, A. B. — 2008-07-01

Orai, STIM1 and iPLA2{beta}: a view from a different perspective

Bolotina, V. M. — 2008-07-01

The mechanism of store-operated Ca²⁺ entry (SOCE) remains one of the intriguing mysteries in the field of Ca²⁺ signalling. Recent discoveries have resulted in the molecular identification of STIM1 as a Ca²⁺ sensor in endoplasmic reticulum, Orai1 (CRACM1) as a plasma membrane channel that is activated by the store-operated pathway, and iPLA₂β as an essential component of signal transduction from the stores to the plasma membrane channels. Numerous studies have confirmed that molecular knock-down of any one of these three molecules impair SOCE in a wide variety of cell types, but their mutual relations are far from being understood. This report will focus on the functional roles of Orai1, STIM1 and iPLA₂β, and will address some specific questions about Orai1 and TRPC1, and their relation to SOC channels in excitable and non-excitable cells. Also, it will analyse the novel role of STIM1 as a trigger for CIF production, and the complex relationship between STIM1 and Orai1 expression, puncta formation and SOCE activation. It will highlight some of the most recent findings that may challenge simple conformational coupling models of SOCE, and will offer some new perspectives on the complex relationships between Orai1, STIM1 and iPLA₂β in the SOCE pathway.

Ca2+ microdomains near plasma membrane Ca2+ channels: impact on cell function

Parekh, A. B. — 2008-07-01

In eukaryotic cells, a rise in cytoplasmic Ca²⁺ can activate a plethora of responses that operate on time scales ranging from milliseconds to days. Inherent to the use of a promiscuous signal like Ca²⁺ is the problem of specificity: how can Ca²⁺ activate some responses but not others? We now know that the spatial profile of the Ca²⁺ signal is important Ca²⁺ does not simply rise uniformly throughout the cytoplasm upon stimulation but can reach very high levels locally, creating spatial gradients. The most fundamental local Ca²⁺ signal is the Ca²⁺ microdomain that develops rapidly near open plasmalemmal Ca²⁺ channels like voltage-gated L-type (Cav1.2) and store-operated CRAC channels. Recent work has revealed that Ca²⁺ microdomains arising from these channels are remarkably versatile in triggering a range of responses that differ enormously in both temporal and spatial profile. Here, I delineate basic features of Ca²⁺ microdomains and then describe how these highly local signals are used by Ca²⁺-permeable channels to drive cellular responses.

Cytoplasmic calcium oscillations and store-operated calcium influx

Putney, J. W., Bird, G. S. — 2008-07-01

Intracellular calcium oscillations have fascinated scientists for decades. They provide an important cellular signal which, unlike most signalling mechanisms, is digitally encoded. While it is generally agreed that oscillations most frequently arise from cyclical release and re-uptake of intracellularly stored calcium, it is becoming increasingly clear that influx of calcium across the plasma membrane also plays a critical role in their maintenance and even in delivering their signal to the correct cellular locus. In this review we will discuss the role played by Ca²⁺ entry mechanisms in Ca²⁺ oscillations, and approaches to understanding the molecular nature of this Ca²⁺ entry pathway.

2-Aminoethoxydiphenyl borate directly facilitates and indirectly inhibits STIM1-dependent gating of CRAC channels

Peinelt, C., Lis, A., Beck, A., Fleig, A., Penner, R. — 2008-07-01

2-Aminoethoxydiphenyl borate (2-APB) has emerged as a useful pharmacological tool in the study of store-operated Ca²⁺ entry (SOCE). It has been shown to potentiate store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) currents at low micromolar concentrations and to inhibit them at higher concentrations. Initial experiments with the three CRAC channel subtypes CRACM1, CRACM2 and CRACM3 have indicated that they might be differentially affected by 2-APB. We now present a thorough pharmacological profile of 2-APB and report that it can activate CRACM3 channels in a store-independent manner without the requirement of STIM1, whereas CRACM2 by itself is completely unresponsive to 2-APB and CRACM1 is only very weakly activated. However, when coexpressed with STIM1 and activated via store depletion, CRACM1 and CRACM2 are facilitated at low 2-APB concentrations and inhibited at higher concentrations, while CRACM3 only exhibits potentiated currents. Consistently, the 2-APB-induced CRAC currents exhibit altered selectivities that are characterized by a leftward shift in reversal potential and the emergence of large outward currents that are carried by normally impermeant monovalent cations such as Cs⁺ or K⁺. These results suggest that 2-APB has agonistic and antagonistic modes of action on CRAC channels, acting at the channel level as a store-independent and direct gating agonist for CRACM3 and a potentiating agonist for CRACM1 and CRACM2 following store-operated and STIM1-dependent activation. The inhibition of CRACM1 channels by high concentrations of 2-APB appears to involve a direct block at the channel level and an additional uncoupling of STIM1 and CRACM1, since the compound reversed the store-dependent multimerization of STIM1. Finally, we demonstrate that single-point mutations of critical amino acids in the selectivity filter of the CRACM1 pore (E106D and E190A) enable 2-APB to gate CRACM1 in a STIM1-independent manner, suggesting that 2-APB facilitates CRAC channels by altering the pore architecture.

Three C-terminal residues from the sulphonylurea receptor contribute to the functional coupling between the KATP channel subunits SUR2A and Kir6.2

Dupuis, J. P., Revilloud, J., Moreau, C. J., Vivaudou, M. — 2008-07-01

Cardiac ATP-sensitive potassium (K_ATP) channels are metabolic sensors formed by the association of the inward rectifier potassium channel Kir6.2 and the sulphonylurea receptor SUR2A. SUR2A adjusts channel gating as a function of intracellular ATP and ADP and is the target of pharmaceutical openers and blockers which, respectively, up- and down-regulate Kir6.2. In an effort to understand how effector binding to SUR2A translates into Kir6.2 gating modulation, we examined the role of a 65-residue SUR2A fragment linking transmembrane domain TMD2 and nucleotide-binding domain NBD2 that has been shown to interact with Kir6.2. This fragment of SUR2A was replaced by the equivalent residues of its close homologue, the multidrug resistance protein MRP1. The chimeric construct was expressed in Xenopus oocytes and characterized using the patch-clamp technique. We found that activation by MgADP and synthetic openers was greatly attenuated although apparent affinities were unchanged. Further chimeragenetic and mutagenetic studies showed that mutation of three residues, E1305, I1310 and L1313 (rat numbering), was sufficient to confer this defective phenotype. The same mutations had no effects on channel block by the sulphonylurea glibenclamide or by ATP, suggesting a role for these residues in activatory – but not inhibitory – transduction processes. These results indicate that, within the K_ATP channel complex, the proximal C-terminal of SUR2A is a critical link between ligand binding to SUR2A and Kir6.2 up-regulation.

Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5-bisphosphate in mesenteric artery myocytes

Albert, A. P., Saleh, S. N., Large, W. A. — 2008-07-01

The present work investigates the effect of phosphatidylinositol-4,5-bisphosphate (PIP₂) on native TRPC6 channel activity in freshly dispersed rabbit mesenteric artery myocytes using patch clamp recording and co-immunoprecipitation methods. Inclusion of 100 µm diC8-PIP₂ in the patch pipette and bathing solutions, respectively, inhibited angiotensin II (Ang II)-evoked whole-cell cation currents and TRPC6 channel activity by over 90%. In inside-out patches diC8-PIP₂ also inhibited TRPC6 activity induced by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) with an IC₅₀ of 7.6 µm. Anti-PIP₂ antibodies potentiated Ang II- and OAG-evoked TRPC6 activity by about 2-fold. Depleters of tissue PIP₂ wortmannin and LY294002 stimulated TRPC6 activity, as did the polycation PIP₂ scavenger poly-l-lysine. Wortmannin reduced Ang II-evoked TRPC6 activity by over 75% but increased OAG-induced TRPC6 activity by over 50-fold. Co-immunoprecipitation studies demonstrated association between PIP₂ and TRPC6 proteins in tissue lysates. Pre-treatment with Ang II, OAG and wortmannin reduced TRPC6 association with PIP₂. These results provide for the first time compelling evidence that constitutively produced PIP₂ exerts a powerful inhibitory action on native TRPC6 channels.

Molecular, pharmacological and functional properties of GABAA receptors in anterior pituitary cells

Zemkova, H. W., Bjelobaba, I., Tomic, M., Zemkova, H., Stojilkovic, S. S. — 2008-07-01

Anterior pituitary cells express -aminobutyric acid (GABA)-A receptor-channels, but their structure, distribution within the secretory cell types, and nature of action have not been clarified. Here we addressed these questions using cultured anterior pituitary cells from postpubertal female rats and immortalized T3-1 and GH₃ cells. Our results show that mRNAs for all GABA_A receptor subunits are expressed in pituitary cells and that 1/β1 subunit proteins are present in all secretory cells. In voltage-clamped gramicidin-perforated cells, GABA induced dose-dependent increases in current amplitude that were inhibited by bicuculline and picrotoxin and facilitated by diazepam and zolpidem in a concentration-dependent manner. In intact cells, GABA and the GABA_A receptor agonist muscimol caused a rapid and transient increase in intracellular calcium, whereas the GABA_B receptor agonist baclofen was ineffective, suggesting that chloride-mediated depolarization activates voltage-gated calcium channels. Consistent with this finding, RT-PCR analysis indicated high expression of NKCC1, but not KCC2 cation/chloride transporter mRNAs in pituitary cells. Furthermore, the GABA_A channel reversal potential for chloride ions was positive to the baseline membrane potential in most cells and the activation of ion channels by GABA resulted in depolarization of cells and modulation of spontaneous electrical activity. These results indicate that secretory pituitary cells express functional GABA_A receptor-channels that are depolarizing.

Brain-derived neurotrophic factor enhances the excitability of rat sensory neurons through activation of the p75 neurotrophin receptor and the sphingomyelin pathway

Zhang, Y. H., Chi, X. X., Nicol, G. D. — 2008-07-01

Neurotrophin-mediated signalling cascades can be initiated by activation of either the p75 neurotrophin receptor (p75^NTR) or the more selective tyrosine kinase receptors. Previously, we demonstrated that nerve growth factor (NGF) increased the excitability of sensory neurons through activation of p75^NTR to liberate sphingosine 1-phosphate. If neurotrophins can modulate the excitability of small diameter sensory neurons through activation of p75^NTR, then brain-derived neurotrophic factor (BDNF) should produce the same sensitizing action as did NGF. In this report, we show that focally applied BDNF increases the number of action potentials (APs) evoked by a ramp of depolarizing current by reducing the rheobase without altering the firing threshold. This increased excitability results, in part, from the capacity of BDNF to enhance a tetrodotoxin-resistant sodium current (TTX-R I_Na) and to suppress a delayed rectifier-like potassium current (I_K). The idea that BDNF acts via p75^NTR is supported by the following observations. The sensitizing action of BDNF is prevented by pretreatment with a blocking antibody to p75^NTR or an inhibitor of sphingosine kinase (dimethylsphingosine), but not by inhibitors of tyrosine kinase receptors (K252a or AG879). Furthermore, using single-cell RT-PCR, neurons that were sensitized by BDNF expressed the mRNA for p75^NTR but not TrkB. These results demonstrate that neurotrophins can modulate the excitability of small diameter capsaicin-sensitive sensory neurons through the activation of p75^NTR and its downstream sphingomyelin signalling cascade. Neurotrophins released upon activation of a variety of immuno-competent cells may be important mediators that give rise to the enhanced neuronal sensitivity associated with the inflammatory response.

Interactions between multiple sources of short-term plasticity during evoked and spontaneous activity at the rat calyx of Held

Hennig, M. H., Postlethwaite, M., Forsythe, I. D., Graham, B. P. — 2008-07-01

Sustained activity at most central synapses is accompanied by a number of short-term changes in synaptic strength which act over a range of time scales. Here we examine experimental data and develop a model of synaptic depression at the calyx of Held synaptic terminal that combines many of these mechanisms (acting at differing sites and across a range of time scales). This new model incorporates vesicle recycling, facilitation, activity-dependent vesicle retrieval and multiple mechanisms affecting calcium channel activity and release probability. It can accurately reproduce the time course of experimentally measured short-term depression across different stimulus frequencies and exhibits a slow decay in EPSC amplitude during sustained stimulation. We show that the slow decay is a consequence of vesicle release inhibition by multiple mechanisms and is accompanied by a partial recovery of the releasable vesicle pool. This prediction is supported by patch-clamp data, using long duration repetitive EPSC stimulation at up to 400 Hz. The model also explains the recovery from depression in terms of interaction between these multiple processes, which together generate a stimulus-history-dependent recovery after repetitive stimulation. Given the high rates of spontaneous activity in the auditory pathway, the model also demonstrates how these multiple interactions cause chronic synaptic depression under in vivo conditions. While the magnitude of the depression converges to the same steady state for a given frequency, the time courses of onset and recovery are faster in the presence of spontaneous activity. We conclude that interactions between multiple sources of short-term plasticity can account for the complex kinetics during high frequency stimulation and cause stimulus-history-dependent recovery at this relay synapse.

Short-term potentiation of mEPSCs requires N-, P/Q- and L-type Ca2+ channels and mitochondria in the supraoptic nucleus

Quinlan, M. E., Alberto, C. O., Hirasawa, M. — 2008-07-01

The glutamatergic synapses of the supraoptic nucleus display a unique activity-dependent plasticity characterized by a barrage of tetrodotoxin-resistant miniature EPSCs (mEPSCs) persisting for 5–20 min, causing postsynaptic excitation. We investigated how this short-term synaptic potentiation (STP) induced by a brief high-frequency stimulation (HFS) of afferents was initiated and maintained without lingering presynaptic firing, using in vitro patch-clamp recording on rat brain slices. We found that following the immediate rise in mEPSC frequency, STP decayed with two-exponential functions indicative of two discrete phases. STP depends entirely on extracellular Ca²⁺ which enters the presynaptic terminals through voltage-gated Ca²⁺ channels but also, to a much lesser degree, through a pathway independent of these channels or reverse mode of the plasma membrane Na⁺–Ca²⁺ exchanger. Initiation of STP is largely mediated by any of the N-, P/Q- or L-type channels, and only a simultaneous application of specific blockers for all these channels attenuates STP. Furthermore, the second phase of STP is curtailed by the inhibition of mitochondrial Ca²⁺ uptake or mitochondrial Na⁺–Ca²⁺ exchanger. mEPSCs amplitude is also potentiated by HFS which requires extracellular Ca²⁺. In conclusion, induction of mEPSC-STP is redundantly mediated by presynaptic N-, P/Q- and L-type Ca²⁺ channels while the second phase depends on mitochondrial Ca²⁺ sequestration and release. Since glutamate influences unique firing patterns that optimize hormone release by supraoptic magnocellular neurons, a prolonged barrage of spontaneous excitatory transmission may aid in the induction of respective firing activities.

Recovery of mouse neuromuscular junctions from single and repeated injections of botulinum neurotoxin A

Rogozhin, A. A., Pang, K. K., Bukharaeva, E., Young, C., Slater, C. R. — 2008-07-01

Botulinum neurotoxin type A (BoNT/A) paralyses muscles by blocking acetylcholine (ACh) release from motor nerve terminals. Although highly toxic, it is used clinically to weaken muscles whose contraction is undesirable, as in dystonias. The effects of an injection of BoNT/A wear off after 3–4 months so repeated injections are often used. Recovery of neuromuscular transmission is accompanied by the formation of motor axon sprouts, some of which form new synaptic contacts. However, the functional importance of these new contacts is unknown. Using intracellular and focal extracellular recording we show that in the mouse epitrochleoanconeus (ETA), quantal release from the region of the original neuromuscular junction (NMJ) can be detected as soon as from new synaptic contacts, and generally accounts for > 80% of total release. During recovery the synaptic delay and the rise and decay times of endplate potentials (EPPs) become prolonged approximately 3-fold, but return to normal after 2–3 months. When studied after 3–4 months, the response to repetitive stimulation at frequencies up to 100 Hz is normal. When two or three injections of BoNT/A are given at intervals of 3–4 months, quantal release returns to normal values more slowly than after a single injection (11 and 15 weeks to reach 50% of control values versus 6 weeks after a single injection). In addition, branching of the intramuscular muscular motor axons, the distribution of the NMJs and the structure of many individual NMJs remain abnormal. These findings highlight the plasticity of the mammalian NMJ but also suggest important limits to it.

Local subcutaneous and muscle pain impairs detection of passive movements at the human thumb

Weerakkody, N. S., Blouin, J. S., Taylor, J. L., Gandevia, S. C. — 2008-07-01

Activity in both muscle spindle endings and cutaneous stretch receptors contributes to the sensation of joint movement. The present experiments assessed whether muscle pain and subcutaneous pain distort proprioception in humans. The ability to detect the direction of passive movements at the interphalangeal joint of the thumb was measured when pain was induced experimentally in four sites: the flexor pollicis longus (FPL), the subcutaneous tissue overlying this muscle, the flexor carpi radialis (FCR) muscle and the subcutaneous tissue distal to the metacarpophalangeal joint of thumb. Tests were conducted when pain was at a similar subjective intensity. There was no significant difference in the ability to detect flexion or extension under any painful or non-painful condition. The detection of movement was significantly impaired when pain was induced in the FPL muscle, but pain in the FCR, a nearby muscle that does not act on the thumb, had no effect. Subcutaneous pain also significantly impaired movement detection when initiated in skin overlying the thumb, but not in skin overlying the FPL muscle in the forearm. These findings suggest that while both muscle and skin pain can disturb the detection of the direction of movement, the impairment is site-specific and involves regions and tissues that have a proprioceptive role at the joint. Also, pain induced in FPL did not significantly increase the perceived size of the thumb. Proprioceptive mechanisms signalling perceived body size are less disturbed by a relevant muscle nociceptive input than those subserving movement detection. The results highlight the complex relationship between nociceptive inputs and their influence on proprioception and motor control.

Protein kinase CK2 modulates synaptic plasticity by modification of synaptic NMDA receptors in the hippocampus

Kimura, R., Matsuki, N. — 2008-07-01

Synaptic plasticity is the foundation of learning and memory. The protein kinase CK2 phosphorylates many proteins related to synaptic plasticity, but whether it is directly involved in it has not been clarified. Here, we examined the role of CK2 in synaptic plasticity in hippocampal slices using the CK2 selective inhibitors 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) and 4,5,6,7-tetrabromobenzotriazole (TBB). These significantly inhibited N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP). DRB also inhibited NMDA receptor-mediated synaptic transmission, while leaving NMDA receptor-independent LTP unaffected. NMDA receptors thus appear to be the primary targets of CK2. Although both long-term depression (LTD) and LTP are induced by the influx of Ca²⁺ through NMDA receptors, surprisingly, LTD was not affected by CK2 inhibitors. We postulated that the LTP-selective modulation by CK2 is due to selective modulation of NMDA receptors, and tested two hypotheses concerning the modulation of NMDA receptors: (i) CK2 selectively modulates NR2A subunits possibly related to LTP, but not NR2B subunits possibly related to LTD; and (ii) CK2 selectively affects synaptic but not extrasynaptic NMDA receptors whose activation is sufficient to induce LTD. DRB decreased NMDA receptor-mediated synaptic transmission in the presence of selective NR2A subunit antagonist. The former hypothesis thus appears unlikely to be correct. However, DRB decreased synaptic NMDA receptor responses in cultured hippocampal neurons without affecting extrasynaptic NMDA receptor current. These findings support the latter hypothesis, that CK2 selectively affects LTP by selective modification of synaptic NMDA receptors in a receptor-location-specific manner.

Bradykinin- and sodium nitroprusside-induced increases in capillary tube haematocrit in mouse cremaster muscle are associated with impaired glycocalyx barrier properties

2008-07-01

Previous studies have suggested that agonists may increase functionally perfused capillary volume by modulation of blood-excluding glycocalyx volume, but direct evidence for this association is lacking at the moment. Using intravital microscopic visualization of mouse cremaster muscle, we determined the effects of bradykinin (10^–5 m) and sodium nitroprusside (10^–6 m) on capillary tube haematocrit and glycocalyx barrier properties. In control C57Bl/6 mice (n = 10), tube haematocrit in capillaries (n = 71) increased (P < 0.05) from 8.7 ± 0.3% during baseline to 21.2 ± 1.2 and 22.2 ± 0.9% during superfusion with bradykinin and nitroprusside, respectively. In parallel, the exclusion zone of FITC-labelled 70 kDa dextrans decreased (P < 0.05) from 0.37 ± 0.01 µm during baseline to 0.17 ± 0.01 µm with bradykinin and 0.15 ± 0.01 µm with nitroprusside. Bradykinin and nitroprusside had no effect on dextran exclusion and tube haematocrit in capillaries (n = 55) of hyperlipidemic ApoE3-Leiden mice, which showed impaired exclusion of 70 kDa dextrans (0.05 ± 0.02 µm; P < 0.05 versus C57Bl/6) and increased capillary tube haematocrit (23 ± 0.8%; P < 0.05 versus C57Bl/6) under baseline conditions, indicating glycocalyx degradation. Our data show that vasodilator substances increase functionally perfused capillary volume and that this effect is associated with a reduction in glycocalyx exclusion of 70 kDa dextrans. Modulation of glycocalyx volume might represent a novel mechanism of perfusion control at the capillary level.

Offspring from mothers fed a 'junk food' diet in pregnancy and lactation exhibit exacerbated adiposity that is more pronounced in females

Bayol, S. A., Simbi, B. H., Bertrand, J. A., Stickland, N. C. — 2008-07-01

We have shown previously that a maternal junk food diet during pregnancy and lactation plays a role in predisposing offspring to obesity. Here we show that rat offspring born to mothers fed the same junk food diet rich in fat, sugar and salt develop exacerbated adiposity accompanied by raised circulating glucose, insulin, triglyceride and/or cholesterol by the end of adolescence (10 weeks postpartum) compared with offspring also given free access to junk food from weaning but whose mothers were exclusively fed a balanced chow diet in pregnancy and lactation. Results also showed that offspring from mothers fed the junk food diet in pregnancy and lactation, and which were then switched to a balanced chow diet from weaning, exhibited increased perirenal fat pad mass relative to body weight and adipocyte hypertrophy compared with offspring which were never exposed to the junk food diet. This study shows that the increased adiposity was more enhanced in female than male offspring and gene expression analyses showed raised insulin-like growth factor-1 (IGF-1), insulin receptor substrate (IRS)-1, vascular endothelial growth factor (VEGF)-A, peroxisome proliferator-activated receptor- (PPAR), leptin, adiponectin, adipsin, lipoprotein lipase (LPL), Glut 1, Glut 3, but not Glut 4 mRNA expression in females fed the junk food diet throughout the study compared with females never given access to junk food. Changes in gene expression were not as marked in male offspring with only IRS-1, VEGF-A, Glut 4 and LPL being up-regulated in those fed the junk food diet throughout the study compared with males never given access to junk food. This study therefore shows that a maternal junk food diet promotes adiposity in offspring and the earlier onset of hyperglycemia, hyperinsulinemia and/or hyperlipidemia. Male and female offspring also display a different metabolic, cellular and molecular response to junk-food-diet-induced adiposity.

Congenital tracheal malformation in cystic fibrosis transmembrane conductance regulator-deficient mice

2008-07-01

In cystic fibrosis (CF) patients, the major alteration in pulmonary function is due to peripheral airway obstruction. In the present study, we investigated the possibility that alterations in the extrathoracic airways, particularly in the trachea that expresses high levels of CFTR (CF transmembrane conductance regulator), may contribute to respiratory dysfunction. We performed morphological analyses of the trachea and airway functional studies in adult Cftr knockout (Cftr^–/–) and F508del-CFTR mice and their controls. Macroscopic and histological examination of the trachea showed the presence of one to seven disrupted or incomplete cartilage rings in Cftr^–/– mice (23/25) while only a few Cftr^+/+ mice (6/25) had one abnormal ring. Tracheal defects were mainly localized in the proximal trachea. In 14 Cftr^–/– mice, frontal disruption of the first three to six rings below the cricoid cartilage were associated with upper tracheal constriction. Similar tracheal abnormalities were detected in adult F508del-CFTR and in newborn Cftr^–/– and F508del-CFTR mice. Tracheal and ventilatory function analyses showed in Cftr^–/– mice a decreased contractile response of the proximal trachea and a reduced breathing rate due to an increase in the inspiratory and expiratory times. In F508del-CFTR mice, the expiratory time was longer than in controls. Therefore, these structural and functional abnormalities detected in adult and newborn CF mouse models may represent congenital malformations related to CFTR dysfunction. These results raise important questions concerning the mechanisms governing tracheal development within the context of CFTR protein dysfunction and the implication of such abnormalities in the pathogenesis of airway disease in CF.

Differential responses of sensory neurones innervating glycolytic and oxidative muscle to protons and capsaicin

Xing, J., Sinoway, L., Li, J. — 2008-07-01

Activation of thin fibre muscle afferent nerves by metabolic by-products plays a critical role in the initiation and maintenance of the autonomic response to exercise and the metabolic profile of active muscle can influence the response. The purpose of this report was to determine the responsiveness of sensory neurones innervating muscles comprising predominantly glycolytic and oxidative fibres to protons and capsaicin using whole-cell patch clamp methods. Dorsal root ganglion (DRG) neurones from 4- to 6-week-old rats were labelled by injecting the fluorescence tracer DiI into the muscle 3–5 days prior to the recording experiments. The percentage of the DRG neurones innervating glycolytic and oxidative muscle was similar in response to both protons and capsaicin. However, the neurones innervating glycolytic muscle had greater inward current amplitude responses to protons and capsaicin as compared with oxidative muscle. The peak current amplitudes to pH 6.0 were 0.84 ± 0.06 nA (oxidative muscle) versus 1.36 ± 0.07 nA (glycolytic muscle, P < 0.05). The capsaicin-induced current amplitudes were 2.3 ± 0.15 nA (oxidative muscle) versus 3.1 ± 0.21 nA (glycolytic muscle, P < 0.05). Of neurones that responded to pH 6.0 with a sustained current, 88% also responded to capsaicin. Capsaicin exposure enhanced the proton responsiveness in the neurones innervating the muscle; and protons also increased the capsaicin response. These data suggest that (1) receptors mediating protons and capsaicin responses coexist in the DRG neurones innervating muscle; (2) the responsiveness of acidosis and capsaicin can be sensitized by each other; and (3) DRG neurones with nerve endings in a glycolytic muscle developed greater inward current responses to protons and capsaicin than did those with nerve endings in an oxidative muscle.

Increased sympathetic outflow in juvenile rats submitted to chronic intermittent hypoxia correlates with enhanced expiratory activity

2008-07-01

Chronic intermittent hypoxia (CIH) in rats produces changes in the central regulation of cardiovascular and respiratory systems by unknown mechanisms. We hypothesized that CIH (6% O₂ for 40 s, every 9 min, 8 h day^–1) for 10 days alters the central respiratory modulation of sympathetic activity. After CIH, awake rats (n = 14) exhibited higher levels of mean arterial pressure than controls (101 ± 3 versus 89 ± 3 mmHg, n = 15, P < 0.01). Recordings of phrenic, thoracic sympathetic, cervical vagus and abdominal nerves were performed in the in situ working heart–brainstem preparations of control and CIH juvenile rats. The data obtained in CIH rats revealed that: (i) abdominal (Abd) nerves exhibited an additional burst discharge in late expiration; (ii) thoracic sympathetic nerve activity (tSNA) was greater during late expiration than in controls (52 ± 5 versus 40 ± 3%; n = 11, P < 0.05; values expressed according to the maximal activity observed during inspiration and the noise level recorded at the end of each experiment), which was not dependent on peripheral chemoreceptors; (iii) the additional late expiratory activity in the Abd nerve correlated with the increased tSNA; (iv) the enhanced late expiratory activity in the Abd nerve unique to CIH rats was accompanied by reduced post-inspiratory activity in cervical vagus nerve compared to controls. The data indicate that CIH rats present an altered pattern of central sympathetic–respiratory coupling, with increased tSNA that correlates with enhanced late expiratory discharge in the Abd nerve. Thus, CIH alters the coupling between the central respiratory generator and sympathetic networks that may contribute to the induced hypertension in this experimental model.

Analysis of cardiac mitochondrial Na+-Ca2+ exchanger kinetics with a biophysical model of mitochondrial Ca2+ handing suggests a 3: 1 stoichiometry

Dash, R. K., Beard, D. A. — 2008-07-01

Calcium is a key ion and is known to mediate signalling pathways between cytosol and mitochondria and modulate mitochondrial energy metabolism. To gain a quantitative, biophysical understanding of mitochondrial Ca²⁺ regulation, we developed a thermodynamically balanced model of mitochondrial Ca²⁺ handling and bioenergetics by integrating kinetic models of mitochondrial Ca²⁺ uniporter (CU), Na⁺–Ca²⁺ exchanger (NCE), and Na⁺–H⁺ exchanger (NHE) into an existing computational model of mitochondrial oxidative phosphorylation. Kinetic flux expressions for the CU, NCE and NHE were developed and individually parameterized based on independent data sets on flux rates measured in purified mitochondria. While available data support a wide range of possible values for the overall activity of the CU in cardiac and liver mitochondria, even at the highest estimated values, the Ca²⁺ current through the CU does not have a significant effect on mitochondrial membrane potential. This integrated model was then used to analyse additional data on the dynamics and steady-states of mitochondrial Ca²⁺ governed by mitochondrial CU and NCE. Our analysis of the data on the time course of matrix free [Ca²⁺] in respiring mitochondria purified from rabbit heart with addition of different levels of Na⁺ to the external buffer medium (with the CU blocked) with two separate models – one with a 2: 1 stoichiometry and the other with a 3: 1 stoichiometry for the NCE – supports the hypothesis that the NCE is electrogenic with a stoichiometry of 3: 1. This hypothesis was further tested by simulating an additional independent data set on the steady-state variations of matrix free [Ca²⁺] with respect to the variations in external free [Ca²⁺] in purified respiring mitochondria from rat heart to show that only the 3: 1 stoichiometry model predictions are consistent with the data. Based on these analyses, it is concluded that the mitochondrial NCE is electrogenic with a stoichiometry of 3: 1.

Undiscovered role of endogenous thromboxane A2 in activation of cardiac sympathetic afferents during ischaemia

Fu, L.-W., Guo, Z.-L., Longhurst, J. C. — 2008-07-01

Myocardial ischaemia activates blood platelets, which in turn stimulate cardiac sympathetic afferents, leading to chest pain and sympathoexcitatory reflex cardiovascular responses. Previous studies have shown that activated platelets stimulate ischaemically sensitive cardiac sympathetic afferents, and that thromboxane A₂ (TxA₂) is one of the mediators released from activated platelets during myocardial ischaemia. The present study tested the hypothesis that endogenous TxA₂ stimulates cardiac afferents during ischaemia through direct activation of TxA₂ (TP) receptors coupled with the phospholipase C–protein kinase C (PLC–PKC) cellular pathway. Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicantes (T₂–T₅) in anaesthetized cats. Single fields of 39 afferents (conduction velocity = 0.27–3.65 m s^–1) were identified in the left or right ventricle initially with mechanical stimulation and confirmed with a stimulating electrode. Five minutes of myocardial ischaemia stimulated all 39 cardiac afferents (8 A-, 31 C-fibres) and the responses of these 39 afferents to chemical stimuli were further studied in the following four protocols. In the first protocol, 2.5, 5 and 10 µg of the TxA₂ mimetic, U46619, injected into the left atrium (LA), stimulated seven ischaemically sensitive cardiac afferents in a dose-dependent manner. Second, BM13,177, a selective TxA₂ receptor antagonist, abolished the responses of six afferents to 5 µg of U46619 injected into the left atrium and attenuated the ischaemia-related increase in activity of seven other afferents by 44%. In contrast, cardiac afferents, in the absence of TP receptor blockade responded consistently to repeated administration of U46619 (n = 6) and to recurrent myocardial ischaemia (n = 7). In the fourth protocol, administration of PKC-(19–36), a selective PKC inhibitor, attenuated the responses of six other cardiac afferents to U46619 by 38%. Finally, using an immunohistochemical staining approach, we observed that TP receptors were expressed in cardiac sensory neurons in thoracic dorsal root ganglia. Taken together, these data indicate that endogenous TxA₂ contributes to the activation of cardiac afferents during myocardial ischaemia through direct stimulation of TP receptors probably located in the cardiac sensory nervous system and that the stimulating effect of TxA₂ on cardiac afferents is dependent, at least in part, upon the PLC–PKC cellular pathway.