Current issue only

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.