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¹ Department of Anaesthesiology, Division of Molecular Medicine
² Department of Molecular and Medical Pharmacology
³ Department of Physiology
⁴ Brain Research Institute, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA 90095-1778, USA

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Large-conductance, voltage- and Ca²⁺-activated K⁺ channels (MaxiK, BK) are key regulators of vascular tone. Vascular MaxiK are formed by the pore-forming subunit and the modulatory ß1 subunit, which imprints unique kinetics, Ca²⁺/voltage sensitivities and pharmacology to the channel. As age progresses, subunit functional expression and protein levels diminish in coronary myocytes. However, whether ageing modifies ß1 subunit expression or the mechanism of subunit reduction is unknown. Thus, we examined functional and pharmacological characteristics of MaxiK, as well as and ß1 transcript levels in coronary myocytes from young and old F344 rats. The mechanism of age-dependent subunit protein reduction involves its transcript downregulation. A corresponding loss of ß1 transcripts was also detected in old myocytes, suggesting a proportional age-dependent decrease of ß1 to subunit protein. Indeed, MaxiK channel properties, defined by coassembly of ß1 and subunits, were equivalent in young versus old, for example in terms of (i) activation kinetics, (ii) sensitivity to Ca²⁺ levels > 1 µM (iii) dehydrosoyasaponin-I-induced activation, and (iv) iberiotoxin blockade. Consistent with less MaxiK expression/function in older myocytes, the ability of iberiotoxin to contract coronary rings was reduced 50% with ageing confirming our previous findings. 5-Hydroxytryptamine (5-HT) contractile efficacy was reduced by iberiotoxin pretreatment in young > old coronary arteries (explained by larger iberiotoxin-induced contraction and decreased dynamic range for 5-HT contraction in young versus old) with no apparent differences in nitroglycerine-induced relaxation. We propose that the age-related MaxiK reduction involves a parallel decrease of and ß1 functional expression via a transcript downregulatory mechanism; a major impact on basal and possibly stimulated coronary contraction may contribute to altered coronary flow regulation and coronary morbidity in the elderly.

(Received 24 May 2004; accepted after revision 16 July 2004; first published online 22 July 2004)
Corresponding author L. Toro: Division of Molecular Medicine, Department of Anaesthesiology, David Geffen School of Medicine at UCLA, BH-509A CHS, Box 957115, Los Angeles, CA 90095-7115, USA. Email: ltoro{at}ucla.edu

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Advancing age causes changes in the structure and function of blood vessels which lead to an increase in the incidence of cardiovascular diseases. In the elderly, coronary arteries are hyperreactive and this hyperreactivity may cause sudden and intense coronary spasm (Toro et al. 2002). One factor that influences the coronary contractile state is the activity of ion channels on the plasmalemmal surface of smooth muscle cells. In particular, large-conductance, voltage- and Ca²⁺-activated K⁺ (MaxiK, BK) channels play a critical role in maintaining arterial tone by regulating both electrical and chemical excitability since they are sensors of voltage, intracellular Ca²⁺ and metabolic activity (e.g. P_O2, phosphorylation). In this regard, MaxiK channels participate in both vasorelaxation (e.g. NO pathway via protein kinase G (PKG)-induced channel activation; Alioua et al. 1998; Marijic & Toro, 2001; Schubert & Nelson, 2001) and vasoconstriction (e.g. 5-HT, angiotensin II pathways via c-Src tyrosine kinase-induced channel inhibition; Alioua et al. 2002). In coronary smooth muscle, MaxiK channels are assembled by at least two non-covalently associated subunits; the pore-forming subunit and a regulatory ß1 subunit (Tanaka et al. 1997; Brenner et al. 2000). Although the subunit is responsible for the basic ion flux function of MaxiK channels, the regulatory ß1 subunit can dramatically affect channel conduction by changing channel kinetics, voltage/Ca²⁺ sensitivities and pharmacology (Toro et al. 1998; Meera et al. 2001). Thus, MaxiK channel molecular components represent excellent candidates for ageing-associated changes that can contribute to altered vascular reactivity in the elderly. In line with this view, we have recently reported that ageing can affect the expression of the pore-forming subunit in the coronary arteries (Marijic et al. 2001). Both the number of functional channels and the expression of subunit protein are diminished in old coronary arteries from human and rats. A diminished K⁺ channel activity due to a reduced number of active conductive pores would favour a more depolarized smooth muscle, and consequently increased reactivity, and increased vascular tension in the elderly. These ageing-induced coronary derangements could be even more pronounced if the channel has altered Ca²⁺ sensitivity or if ß1 subunit functional expression is altered as well. In line with this view, ß1 subunit gene ablation is known to induce mouse hypertension (Brenner et al. 2000; Pluger et al. 2000). However, it is still unknown whether ageing alters ß1 subunit expression/function or if it changes the functional characteristics of MaxiK currents in coronary myocytes. Thus, the main purpose of this work was to determine whether ß1 subunit expression and functional coupling with subunit changes as a function of age. We reasoned that if ageing induced a larger suppression of subunits with respect to ß1 subunits or an uncoupling of +ß1 subunit complexes, then the macroscopic current properties should be altered. This is expected since, as mentioned above, studies in heterologous systems have shown that the ß1 subunit imparts to the subunit fingerprint characteristics such as: (a) slower kinetics (Meera et al. 1996; Dworetzky et al. 1996), (b) higher Ca²⁺ sensitivity (McManus et al. 1995; Meera et al. 1996), (c) a slower on-rate of iberiotoxin (IbTx) association with the channel pore (Meera et al. 2000), and (d) sensitivity to nanomolar concentrations of dehydrosoyasaponin I (DHS-I) (McManus et al. 1995; Wallner et al. 1999). Therefore, we first investigated age-related changes in these MaxiK current properties. We also examined if diminished and/or ß1 transcription could be a mechanism in MaxiK channel reduction during ageing, and the functional consequences on active tension experiments.

We now show that the main effect of ageing is a decreased expression of MaxiK channels without major changes in their biophysical and pharmacological properties typically modulated by the ß1 subunit, suggesting a parallel decrease in and ß1 subunits. The process of MaxiK current and subunit protein loss by ageing entails a parallel reduction of its ß1 subunit functional expression via transcript downregulatory mechanisms of both and ß1 subunits. The decreased expression of MaxiK channel and ß1 subunits have a major functional impact on basal tone and, probably, stimulated contraction, that may lead to vascular morbidity in the elderly.

	Methods

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F344 male rats (young, aged 3–4 months; old, aged 28–30 months) were used. Animals were killed with an overdose of anaesthetic (inhaled isoflurane, 2 ml (100 g)^–1). Protocols were approved by the UCLA Institutional Animal Care and Use Committee (Chancellor's Animal Research Committee) in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and the USDA Animal Welfare Act Regulations.

Cell isolation

The main branches of rat coronary arteries were used to dissociate single smooth muscle cells as previously described (Marijic et al. 2001).

Patch clamp recording

Membrane currents were recorded at room temperature using whole-cell and inside-out configurations. To inactivate other voltage-activated K⁺ currents, the holding potential (V_h) was set to 0 mV. Pipette resistances were 2–3 M. Data were filtered at 1/5th the sampling frequency which was 10 kHz. In whole-cell recording, the external solution was (mM): sodium methanesulphonate (Na-Mes) 135, K-Mes 5, CaCl₂ 0.1, MgCl₂ 2, Hepes 10, glucose 5, pH 7.4. The internal solution was (mM): K-Mes 140, CaCl₂ 0.1, MgCl₂ 2, Na₂-ATP 0.1, Na₂-GTP 0.1, EGTA 0.145, Hepes 10, pH 7.3, pCa 6.35 (450 nM free Ca²⁺). Cell capacitance (C_m) was obtained by a 10 mV pulse and calculated from C_m=dQ/dV. In inside-out recordings, the internal and external solutions were the same (mM): K-Mes 140, Hepes 10, HEDTA 5, pH 7.4. Variable amounts of CaCl₂ and MgCl₂, were added to the solution to give the desired free Ca²⁺ concentrations and 2 mM free Mg²⁺ (Fabiato, 1988). Free Ca²⁺ was measured with a Ca²⁺ electrode.

RNA isolation and cDNA preparation

We used right and left coronary arteries and their branches or isolated myocytes from young and old male rats. For isolated myocytes, only spindle-shaped coronary myocytes (300 from each enzymatic isolation) were collected individually by suction into glass pipettes. Total RNA was isolated from rat coronary arteries using the Trizol method (Invitrogen) and from isolated myocytes using the RNeasy minikit (QIAGEN). To avoid DNA contamination, DNase (RNase-free DNase set, QIAGEN) was combined with the RNeasy kit. First strand cDNA was synthesized from total RNA preparations using the Sensiscript Reverse Transcriptase kit (QIAGEN) with specific reverse primers for MaxiK and ß1 subunits, and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Mock reverse transcription was performed using H₂O instead of reverse transcriptase.

Real-time PCR

Real-time PCR was performed using the iCycler iQ Real Time PCR (BioRad) and cDNAs obtained by reverse transcription of experimental RNA samples (see above) or with cloned cDNAs (for standard curves), SYBR Green I (Molecular Probes) as the fluorescent probe, and Platinum Quantitative PCR Supermix-UDG (Gibco BRL). Specific primers were designed to amplify segments of 200 base pairs of MaxiK , MaxiK ß1 and GAPDH (Table 1). To check for genomic DNA contamination, ß1 primers were designed to flank intron 3 (4.5 kb) (Jiang et al. 1999) or mock product of reverse transcription (without reverse transcriptase) was used for amplification. GAPDH was used as negative control since it showed no major changes as a function of age. Another control used H₂O instead of cDNA. Amplification conditions were: 5 min at 95°C, followed by 45 cycles at 95°C for 45 s, 65°C for 45 s and 72°C for 45 s. MaxiK , MaxiK ß1 and GAPDH standard curves were obtained using known concentrations of cloned cDNAs (amplicons) and the same primers used to amplify the experimental samples. Amplicons used were: (a) full length human MaxiK subunit (primers share 100% identity with human and rat cDNAs), (b) full length rat MaxiK ß1 subunit, and (c) 200 bp fragment of rat GAPDH isolated from heart using the primers in Table 1. To obtain the standard curves, a threshold was assigned in the linear range of the fluorescence versus cycle number graphs and plotted as a function of [cDNA]. Data were fitted to a straight line, where a slope of –3.3 represents 100% efficiency. [cDNA] in experimental samples was obtained by extrapolation from the corresponding standard curve and expressed as attomoles of RNA per 100 cells (absolute quantification), which assumes a 1: 1 conversion of RNA to cDNA in the reverse transcription reaction performed prior to real-time PCR. Relative quantification to GAPDH was performed by the 2^–CT method (Livak & Schmittgen, 2001). As required for accurate measurements when using SYBR Green I as a fluorescent probe, melting curves showed the presence of a single product. To corroborate the amplification of a single product, we directly visualized the final products of all experiments using agarose gel electrophoresis and ethidium bromide fluorescence. Consistent with the melting curves, single bands of the expected size were always detected with no product at 4.5 kb for ß1. In experiments using coronary artery preparations, the real-time PCR was stopped at cycle 33 instead of cycle 45 to visualize by agarose gel electrophoresis the relative abundance of MaxiK and ß1 subunits in young and old rats.

The three main branches of coronary arteries (left, right and septal) were quickly isolated, cut into 3 mm-long rings and mounted on small wire hooks in 2 ml organ baths. Baths were filled with modified Krebs buffer solution at 37°C continuously gassed with a 95% O₂–5% CO₂ mixture. The composition of the Krebs buffer was (mM): NaCl 119, KCl 4.7, CaCl₂ 1.6, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 22, Hepes 8, creatine 5, taurine 20, pyruvate 5 and glucose 5, pH 7.4. Coronary artery rings were equilibrated for 60 min under optimal resting tension (120 mg for young and 200 mg for old). Nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME, 100 µM) was present during the whole experiment to avoid the contribution of endothelium-derived NO on basal tone. L-NAME caused a variable increase in tension most likely reflecting different degrees of endothelium loss during mounting of the rings. Arterial rings were incubated for 20 min with or without IbTx (100 nM) prior to cumulative stimulation with 5-HT (3 x 10^–8 to 1 x 10^–5M). Nitroglycerine (NTG) 1 x 10^–9 to 1 x 10^–4M was added at the time of maximum contraction induced by 5-HT. Tension was recorded with WINDAQ/200 (Dataq Instruments, USA). Responses were expressed as a percentage of the maximum (max) contraction or as a percentage of the maximum relaxation. Maximum contraction = limiting contraction at 1 x 10^–5M 5-HT – initial tension. 5-HT-induced contractions were measured as increments of tension from zero [5-HT] tension level; in the case of vessels partially precontracted by the addition of IbTx, zero [5-HT] level was set at the IbTx-induced tension level. NTG-induced relaxations were measured as decrements of tension from maximal contraction. One hundred per cent relaxation was the full recovery to the initial tension. The percentage IbTx contraction = 100 x (tension increment induced by 100 nM IbTx)/maximum contraction.

Statistical analysis

Data are expressed as mean ±S.E.M. Repeated measures ANOVA and Student's t test were used to determine P values. P < 0.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Activation kinetics in young versus old coronary myocytes

The decrease in MaxiK current activation kinetics caused by the presence of ß1 subunits is better observed at nanomolar Ca²⁺ concentrations (Meera et al. 1996). Therefore, this parameter was investigated by measuring the activation kinetics of whole-cell currents with 450 nM free Ca²⁺ in the pipette solution.

Figure 1A shows typical recordings of MaxiK currents in young versus old coronary artery myocytes. Consistent with our previous data, at potentials over +40 mV, MaxiK current density was significantly diminished in old coronary myocytes (inset). Activation time constants () were determined at +70 to +90 mV using a single exponential fitting. Current traces under +60 mV were not analysed because the small current amplitudes in old coronary myocytes prevented a good quality fit. As illustrated in Fig. 1B, the activation kinetics of whole-cell MaxiK currents at +90 mV were practically identical in coronary myocytes from old (activation rate constant, 1/= 0.019 ± 0.001 ms^–1, n= 38) and young (1/= 0.017 ± 0.0008 ms^–1, n= 53) rats (P= 0.24). Similarly there were no substantial changes found for the other potentials analysed (Fig. 1C). This first set of experiments demonstrated that MaxiK currents in old and in young coronary arteries activate with similar kinetics upon depolarization. A simple explanation for this result is that in old coronary arteries, the ß1 subunit protein decreases concomitantly with a decrease in subunit protein expression, and in a similar ratio, maintaining a 1: 1 stoichiometry (Garcia-Calvo et al. 1994). Alternatively, the amount of ß1 subunit protein may not change with ageing leading to an excess of ß1 subunits in old coronary arteries; however, if this were the case, the coupling ratio of ß1/ subunits would have to remain constant in old and young coronary arteries to achieve similar activation kinetics.

View larger version (19K): [in this window] [in a new window]   Figure 1.  MaxiK currents in young versus old coronary myocytes A, examples of whole-cell currents in young and old coronary myocytes. Vh= 0 mV. Test pulses of 250 ms duration were from –80 mV to +90 mV in 10 mV increments (template at the bottom). Inset: mean current density–voltage relationships. *P < 0.05. Measurement of cell capacitance showed a small but significant difference in size between young (15.2 ± 0.5 pF, n= 50) and old (22.4 ± 1.6 pF, n= 34) myocytes (P < 0.001). B, typical records of whole-cell K+ currents elicited with depolarizing steps from 0 mV to +90 mV in young and old myocytes. C, mean values of activation rate constants (1/) were, in young versus old myocytes, respectively: 0.015 ± 0.0009 ms–1 (n= 52) versus 0.016 ± 0.001 ms–1 (n= 38) at +70 mV; 0.015 ± 0.0007 ms–1 (n= 53) versus 0.018 ± 0.001 ms–1 (n= 38) at +80 mV; and 0.017 ± 0.0008 ms–1 (n= 53) versus 0.019 ± 0.001 ms–1 (n= 38) at +90 mV.

We have reported that MaxiK channels formed exclusively by subunits respond to rises in intracellular Ca²⁺ ([Ca²⁺]_i) beyond 100 nM Ca²⁺ by increasing their open probability (P_o). The same is true if channels are assembled by and ß1 subunits. However, the presence of the modulatory ß1 subunit elicits a remarkable increase in Ca²⁺ efficacy to open the channel when intracellular Ca²⁺ rises above 1 µM Ca²⁺. This increased Ca²⁺ efficacy induced by the ß1 subunit is reflected in the fractional open probability versus voltage curves (voltage activation curves) that show a leftward shift of the half-activation potential (V_1/2) of near 90 mV (Meera et al. 1996; Toro et al. 1998). Therefore, a change in Ca²⁺/voltage sensitivities was expected (at [Ca²⁺]_i > 1 µM) if the coupling of ß1 and subunits is diminished during ageing.

The analysis of voltage activation curves at different Ca²⁺ concentrations revealed that ageing produced channels that were significantly less responsive to Ca²⁺ changes from 100 to 700 nM than those from young myocytes. In contrast, at [Ca²⁺]_i near and beyond 1 µM, currents from young and old myocytes responded equally well to Ca²⁺ changes. Examples of inside-out macroscopic MaxiK currents in a young and in an old coronary myocyte are shown in Fig. 2A. Note that in the old myocyte (lower traces), current amplitudes were practically the same at 100 nM[Ca²⁺]_i when compared to 700 nM Ca²⁺. This is also reflected in the mean activation curves (fractional P_oversus voltage) depicted in Fig. 2B (right panel: , 100 nM; •, 700 nM) where V_1/2 values were close to each other (+84 ± 15 mV, n= 5, versus +71 ± 7 mV, n= 6, P= 0.2). This is in sharp contrast to the channel behaviour in young coronary myocytes where an increase in Ca²⁺ from 100 to 700 nM Ca²⁺ caused a significant increase in current (Fig. 2A, upper traces) and a leftward shift in the V_1/2 value from +107 ± 5 mV to +67 ± 4 mV (n= 7, P < 0.0001) (Fig. 2B, left panel, versus •). As shown in Fig. 2C, the reduced change in old myocytes when changing solution from 100 to 700 nM Ca²⁺ is caused by a less positive V_1/2 value at 100 nM[Ca²⁺]_i in old versus young myocytes (V_1/2,old=+84 ± 9 mV, n= 5, versus V_1/2,young=+107 ± 5 mV, n= 7; P < 0.05). This result indicates an increased P_o in old myocytes at 100 nM[Ca²⁺]_i. In a two-state model, an increased P_o results from a higher probability of occupancy of the open state that is due to a larger on-rate () to the open state or a smaller off-rate (ß) from the open state. Given that P_o=/(+ß) and 1/=+ß, a higher P_o derives from a higher activation rate constant, 1/ (due to an increase in and/or reduction of ß), that is, a faster activation time constant, . In fact, current traces in Fig. 2D show that at a high potential of 140 mV that should mostly reflect the on-rate, , 1/ was larger in old (0.039 ms^–1, continuous grey line) than in young (0.018 ms^–1, continuous black line). Mean values were: 1/_old= 0.065 ± 0.04 (n= 5) versus 1/_young= 0.02 ± 0.005 (n= 7), P < 0.05, at 140 mV and 100 nM Ca²⁺. Furthermore, plotting activation rate constants (1/) as a function of membrane potential 1/–V curves in Fig. 2D showed that the 1/–V curves at 100 nM[Ca²⁺]_i had a higher voltage dependence (slope) in old (grey graph) when compared to young (black graph) myocytes. The mean slope values were: for young, 2.3 x 10^–4± 7 x 10^–5 ms^–1 mV^–1; and for old, 8.05 x 10^–4± 1.2 x 10^–4 ms^–1 mV^–1 (±S.D., P < 0.001). In contrast, at 700 nM[Ca²⁺]_i there was no significant difference. The mean slope values were: for young, 6.7 x 10^–4± 9 x 10^–5 ms^–1 mV^–1, and for old, 8 x 10^–4± 1 x 10^–4 ms^–1 mV^–1 (P > 0.05). Further, the difference between the regression coefficient (slope of the best fit) of young versus old was statistically significant at 100 nM Ca²⁺ (25.7 x 10^–5± 3.7 x 10^–5 ms^–1 mV^–1, P < 0.05) but not at 700 nM Ca²⁺ (12 x 10^–5± 15 x 10^–5 ms^–1 mV^–1, P > 0.05). Thus, the change in activation kinetics (slopes in 1/–V curves) at 100 nM Ca²⁺ or lack thereof at 700 nM Ca²⁺ between young and old agree with the positions in the voltage axis of the voltage activation curves or V_1/2 values in young versus old coronary myocytes at these Ca²⁺ concentrations.

View larger version (41K): [in this window] [in a new window]   Figure 2.  Voltage and Ca2+ dependency of MaxiK currents in young and old myocytes A, examples of macroscopic inside-out MaxiK currents at various internal Ca2+ concentrations (numbers at top of traces) in a young and an old coronary myocyte; symbols correspond to graphs in B. Each sweep is the ensemble average of 15 recordings. Vh= 0 mV. Pulses of 100 ms duration were applied every 10 mV from –100 to +190 mV (100 and 700 nM Ca2+), –150 to +150 mV (3 µM Ca2+) and –190 to +100 mV (25 µM Ca2+). Tail currents were elicited to –50 mV (100 nM Ca2+), –100 mV (700 nM Ca2+), –150 mV (3 µM Ca2+), and –180 mV (25 µM Ca2+) for 50 ms before returning to 0 mV. B, voltage–activation relationships at various internal Ca2+ concentrations in young versus old coronary myocytes. Data were obtained from tail current measurements to avoid current blockade by divalent cations. C, mean V1/2 values at various internal Ca2+ concentrations. V1/2 values were calculated by fitting data to a Boltzmann distribution: FPo =G/Gmax= 1/{1 + exp[(V1/2–V)zF/RT]}, where FPo represents fractional open probability, G conductance, Gmax limiting maximum conductance, V1/2 potential of 50% limiting open probability or half-activation potential, z effective valency, and F, R and T have their usual thermodynamic meanings. Mean V1/2 values were, for young versus old (mV): +107 ± 5 (n= 7) versus +84 ± 9 (n= 5) at 100 nM Ca2+, +68 ± 4 (n= 7) versus +71 ± 7 (n= 6) at 700 nM Ca2+, –27 ± 4 (n= 7) versus –31 ± 6 at 3 µM Ca2+, and –86 ± 4 (n= 6) versus –87 ± 6 (n= 6) at 25 µM Ca2+. *Significantly different (P < 0.05). Dashed boxes in B and C mark differences between young and old myocytes. D, left panel: current traces at 100 nM[Ca2+]i, elicited from Vh= 0 mV to a test pulse of 140 mV, were fitted to a single exponential function (continuous lines) to obtain activation time constants, . Young, traces in black; old, traces in grey. Numbers in parentheses are rate constants, 1/. Dashed line, voltage step. Right panel: 1/–V relationships for young (grey symbols) and old (black symbols) at 100 nM[Ca2+]i. Fitted curves gave significantly different voltage dependencies. Values are given in the text.

Pharmacological evidence for an age-dependent parallel decrease of and ß1 subunits

Further evidence supporting the view of a parallel decrease in expression/coupling of +ß1 subunits during ageing was provided by experiments using iberiotoxin (IbTx) and dehydrosoyasaponin I (DHS-I) as pharmacological probes.

Iberiotoxin.  Smooth muscle MaxiK currents are typically blocked by IbTx. In heterologous systems, IbTx is able to differentiate between channels formed by the subunit and channels assembled by +ß1 subunits. The on-rate of toxin block is made slower by at least one order of magnitude by the ß1 subunit from 2 x 10⁶M^–1 s^–1 to 1 x 10⁵M^–1 s^–1 (Meera et al. 2000). Thus, we took advantage of this differential property and examined the on-rate of IbTx block in young versus old coronary myocytes. Figure 3A shows typical blockade of MaxiK whole-cell currents by extracellular application of 100 nM IbTx. After IbTx perfusion to the recording chamber, the remaining K⁺ currents at +80 mV were 4.3 ± 1.9% (young, n= 6) and 4.9 ± 0.8% (old, n= 4) of the initial currents. These results confirmed the identity of MaxiK currents. Association rate constants (k_on) were determined by measuring current amplitudes to +80 mV every 5 s after application of 100 nM IbTx and assuming a bimolecular reaction. Consistent with the view that functional +ß1 subunit complexes are maintained in young versus old coronary arteries, k_on values were practically identical in young (3.4 x 10⁵± 0.3 x 10⁵M^–1 s^–1, n= 6) and old coronary myocytes (3.0 x 10⁵± 1.4 x 10⁵M^–1 s^–1, n= 6). Furthermore, these experiments confirmed the expression of MaxiK channels as predominant +ß1 complexes in rat coronary myocytes, since k_on values were close to those reported for heterologously expressed +ß1 subunits (1.1 x 10⁵± 0.5 x 10⁵M^–1 s^–1) (Meera et al. 2000). For comparison, Fig. 3B also shows the theoretical curve (dashed line) expected for blockade of channels formed by the subunit alone.

View larger version (16K): [in this window] [in a new window]   Figure 3.  Blockade of MaxiK currents by IbTx in young versus old coronary myocytes A, IbTx, a specific MaxiK channel blocker, inhibits whole-cell outward K+ currents. Recordings are from a young coronary myocyte; test pulses of 250 ms were from –80 to +90 mV in 10 mV increments. Vh= 0 mV. B, time course of K+ current inhibition by IbTx (100 nM). Constant pulses of +80 mV were applied at 0.2 Hz. An arrow marks the time (time zero) when IbTx reached the recording chamber. Currents were normalized to the current value at time zero. kon values for young and old myocytes are given in the text and correspond to +ß1 complexes. Dashed line is the blocking curve expected for the subunit alone assuming kon= 2 x 106M–1 s–1 and koff= 2 x 10–3 s–1 (Meera et al. 2000).

View larger version (22K): [in this window] [in a new window]   Figure 4.  Dehydrosoyasaponin I (DHS-I) activates MaxiK currents from old and young coronary myocytes A, typical records of macroscopic MaxiK currents in an old myocyte in the absence (control) and presence of DHS-I (250 nM). Test pulses are to –100, –50, 0, +50 and +100 mV from Vh= 0 mV. Potential after the test pulse was –60 mV before returning to 0 mV. B and C, mean voltage–activation relationships in the absence and presence of DHS-I. FPo represents fractional open probability. The small decrease in current near 100 mV could be due to a Ca2+ or Mg2+ block phenomenon. D, mean DHS-I-induced shift of V1/2 values. V1/2 values were calculated as in Fig. 2.

Tetraethylammonium.  We also examined if MaxiK channels from old and young coronary arteries could have subtle differences in their pore properties. To this end, we used tetraethylammonium (TEA) which is able to block MaxiK channels from the external surface at micromolar concentrations but loses this property by a single point mutation in the pore region (Adelman et al. 1992). We found that coronary myocytes from both young and old animals responded equally well to TEA. Extracellularly applied TEA inhibited the K⁺ current in a concentration-dependent manner with IC₅₀ values of 329 ± 45 µM (young, n= 5) and 456 ± 147 µM (old, n= 5) at +80 mV. These values are in good agreement with those obtained in reconstituted MaxiK channels (Toro et al. 1991).

Transcriptional downregulation of both and ß1 subunits in ageing coronary arteries and isolated myocytes

Consistent with the view that ageing may induce a parallel decrease in and ß1 subunit proteins, real-time PCR experiments demonstrated that both and ß1 subunit transcripts were significantly diminished in old versus young coronary arteries (Fig. 5) and isolated coronary myocytes (Fig. 6). Figure 5A and B shows real-time PCR experiments halted during the linear range of amplification (cycle 33) of reverse-transcribed and ß1 transcripts using coronary arteries from young and old animals as a source of RNA (coronary arteries from 3 young and 3 old rats). As control (Ctr), when mock (without reverse transcriptase) reverse transcription product from young (or old) coronary arteries was used, the detected fluorescence was null with zero amplification (squares in Fig. 5B) discarding the possibility of genomic DNA amplification. Figure 5C shows the results of parallel amplification of GAPDH cDNA using the same RNA preparations as for and ß1 subunits. After halting the reactions, products were visualized with ethidium bromide on agarose gel electrophoresis (Fig. 5D). It is clear, from both the real-time fluorescence curves and in-gel ethidium bromide fluorescence, that both and ß1 transcripts (as shown by amplified cDNA) were less abundant in ageing coronary arteries, whereas GAPDH was fairly constant. Similar results were obtained if isolated myocytes were used to perform quantitative real-time PCR reactions as described below.

View larger version (37K): [in this window] [in a new window]   Figure 5.  Age-related RNA changes of and ß1 subunits in coronary arteries A, B and C, real-time PCR fluorescence plots as a function of cycle number for the MaxiK and ß1 subunits, and GAPDH. Coronary artery RNA was isolated from 3 young () and 3 old (•) rats and reverse transcribed to obtain gene-specific cDNAs. Real-time PCR amplifications of gene-specific cDNAs were done in triplicate; reactions were halted at cycle 33. In B, represent a real-time PCR reaction from young coronary arteries using a mock product of reverse transcription with no reverse transcriptase (control, Ctr) and using ß1 primers for amplification; data points show zero fluorescence demonstrating no genomic DNA contamination. Similar results were obtained using a sample from old coronary arteries. D, products (at cycle 33) were separated using agarose gel electrophoresis, and visualized with ethidium bromide fluorescence. Note a single product for , ß and GAPDH, and no product for control (Ctr). A clear decrease in signal was observed for and ß1 subunits reflecting decreased transcript expression of both subunits in samples from old (O) when compared to young (Y) coronary arteries. First lane, DNA ladder.

View larger version (26K): [in this window] [in a new window]   Figure 6.  Transcript downregulation of and ß1 MaxiK subunits in isolated myocytes during ageing A, B and C, mean real-time PCR plots for the MaxiK and ß1 subunits, and GAPDH. Gene-specific cDNAs were from young () and old (•) coronary myocytes. Reactions for each gene (independent cell and cDNA preparations of 4 old and 4 young rats) including the corresponding standard curve and controls were performed in triplicate in a single 96-well plate and using a master mix of reagents (without cDNA) to boost amplification fidelity. Each data point was done in triplicate and averaged prior to averaging the 4 experiments. Insets: melting curves (dF/dTversus temperature) of PCR products showing a single peak. D, E and F, calculated mean absolute transcript levels of , ß1 and GAPDH in young versus old coronary myocytes of samples in A–C. Values for the subunit were: young= 3 x 10–3± 0.2 x 10–3 amol RNA (100 coronary myocytes)–1 (n= 4); old= 1.7 x 10–3± 0.4 x 10–3 amol RNA (100 coronary myocytes)–1 (n= 4). Values for the ß1 subunit were: ß1young= 14 x 10–3± 2 x 10–3 amol RNA (100 coronary myocytes)–1 (n= 3); ß1old= 7 x 10–3± 1 x 10–3 amol RNA (100 coronary myocytes)–1 (n= 4). In one preparation, levels of ß1young were higher, reaching 50 x 10–3 amol RNA (100 coronary myocytes)–1. Insets: calibration curves amplifying known concentrations of corresponding cDNAs (amplicon); slope of –3.3 reflects a 100% efficiency, R2 > 0.99 in all cases. Similar diminished transcript expression in old coronary arteries was obtained if the 2–CT method (Livak & Schmittgen, 2001) was used to evaluate relative expression normalized to GAPDH. Normalized values relative to young (1) were: old= 0.7 ± 0.005 and ßold= 0.49 ± 0.012.

Figure 6B and E show that ß1 subunit transcript expression is also diminished during ageing. Ageing had a similar effect on ß1 as on the subunit, which was to increase its threshold cycle (Fig. 6B). Moreover, when expression values were evaluated, ß1 transcripts were significantly diminished with age by about 50% (ß1_young= 14 x 10^–3± 2 x 10^–3 amol RNA (100 coronary myocytes)^–1, n= 3, versus ß1_old= 7 x 10^–3± 1 x 10^–3 amol RNA (100 coronary myocytes)^–1, n= 4). Possible changes in GAPDH transcript levels were more difficult to resolve as shown by real-time fluorescence curves (Fig. 6C), and quantities were not significantly different, with values for young of 249 x 10^–3± 17 x 10^–3 amol RNA (100 coronary myocytes)^–1 and for old of 186 x 10^–3± 40 x 10^–3 amol RNA (100 coronary myocytes)^–1 (Fig. 6C and F). Note that transcript abundance followed the sequence GAPDH >>>ß1 > subunit.

Functional impact of age-related changes in MaxiK channel expression

To investigate the functional impact of our findings at the cell and molecular levels, we performed tension experiments in old versus young coronary rings, and used iberiotoxin as a tool to examine the MaxiK role (Fig. 7). Three parameters known to involve MaxiK channel activity were investigated: (a) basal muscle tone, (b) 5-hydroxytryptamine (5-HT)-stimulated contraction, and (c) relaxation of 5-HT-precontracted vessels induced by nitroglycerine (NTG). The role of MaxiK on basal muscle tone was examined by measuring the tension increment induced by IbTx, as it indicates blockade of functional MaxiK channels contributing to arterial tone. Stimulated contraction and relaxation were examined by comparing 5-HT-induced contraction and NTG-induced relaxation in control conditions and after IbTx-induced precontraction (see Methods).

View larger version (35K): [in this window] [in a new window]   Figure 7.  Ageing and MaxiK role in coronary artery contraction A, B and C, examples of isometric tension recordings as a function of time for young (A and B) and old (C) coronary artery rings. The arrows mark the addition of 100 µML-NAME to the bath solution. Asterisks marks the time of addition of 100 nM IbTx. Cumulative dose–response experiments were performed for 5-HT (0.03–10 µM) followed by cumulative dose–response experiments with NTG (0.001–100 µM); filled circles mark the time of addition of each dose. Bars at the bottom of traces indicate the duration of 5-HT and NTG application. Drugs were washed (w) at the end of the experiment (arrowheads, grey bar). D, mean dose–response curves for 5-HT expressed as percentage maximum contraction as a function of [5-HT] in young (Y) and old (O) coronary artery rings; in the absence of IbTx (control, squares) or after contraction with IbTx (circles). 5-HT-induced contractions in partially precontracted vessels by IbTx were measured taking as reference IbTx-induced tension level (see Methods). Mean values were fitted to a Hill function. Fitted values ±S.E.M. were for: Controlyoung (), Maxcontraction= 85 ± 1.5% and EC50= 134 ± 9 nM (n= 6–7) (R2= 0.997); for Controlold (), Maxcontraction= 82 ± 0.4% and EC50= 124 ± 3 nM (n= 7–8) (R2= 0.999); for IbTx-treatedyoung (), Maxcontraction= 19 ± 0.6% and EC50= 153 ± 20 nM (n= 6–7) (R2= 0.987); for IbTx-treatedold (•), Maxcontraction= 47 ± 0.2% and EC50= 621 ± 8 nM (n= 6–7) (R2= 1). Similar results were obtained when control experiments were normalized to the contraction elicited by 80 mM KCl (not shown). Inset:%IbTx-induced contraction in young (58 ± 6%; n= 7) and old (31 ± 6%; n= 8); * statistically significant. E, dose–response curves for NTG-induced relaxation of 5-HT precontracted rings expressed as percentage maximum relaxation as a function of [NTG] in young (Y) and old (O) coronary artery rings; in the absence (control, squares) or presence of IbTx (circles). No significant differences were observed for NTG action in young versus old. Fitted values were for: Controlyoung (), Maxrelaxation= 106 ± 3% and EC50= 1.7 ± 0.4 µM (n= 6–7) (R2= 0.994); for Controlold (), Maxrelaxation= 104 ± 2% and EC50= 0.5 ± 0.07 µM (n= 7–8) (R2= 0.997); for IbTx-treatedyoung (), Maxrelaxation= 81 ± 4% and EC50= 2 ± 0.7 µM (n= 6–7) (R2= 0.984); for IbTx-treatedold (•), Maxrelaxation= 78 ± 4% and EC50= 2 ± 1 µM (n= 7–8) (R2= 0.988).

5-HT was 84 ± 5% (n= 7 rings, 6 rats) in the absence and 21 ± 5% (n= 7 rings, 6 rats) in the presence of IbTx (P < 0.001). Figure 7D shows the fitting of all data points to: Percentage maximum contraction = maximum contraction/(1 + (EC₅₀/[drug])^N) where N is the Hill coefficient and EC₅₀ is the concentration needed to cause 50% of the effect. Fits (continuous curves) gave maximum contraction of 85 ± 1.5% (n= 6–7 rings) in control (, Y) versus only 19 ± 0.6% (n= 6–7 rings) when young rings were preincubated with IbTx (, Y). EC₅₀ values for 5-HT were similar without (134 ± 9 nM) and with IbTx (153 ± 20 nM). As described for many other vessels, stimulated contractions were readily reversed by NTG and its relaxatory capacity was hampered when IbTx was present (compare Fig. 7A and B). For instance, the percentage of maximum relaxation achieved by 10 µM NTG was 68 ± 10% (n= 7) without IbTx versus 42 ± 9% (n= 7) when IbTx was present (P < 0.05). Figure 7E shows the fits of all data points with maximum relaxation values of 106 ± 3% in the absence and 81 ± 4% in the presence of iberiotoxin, and EC₅₀ values of 1 ± 0.2 µMversus 2 ± 0.7 µM, respectively.

Ageing produced a significant decrease of nearly 50% in the ability of IbTx to induce basal contraction; IbTx-induced contraction was reduced from 58 ± 6% in young (n= 7) to 31 ± 6% in old (n= 8) coronary rings (compare Fig. 7B and C; Fig. 7D, inset). These results are consistent with a decreased MaxiK expression/function during ageing and confirm our published findings (Marijic et al. 2001). The decreased IbTx-induced contraction with ageing resulted in an increased capacity of 5-HT to induce contraction in old myocytes pretreated with IbTx (compare Fig. 7C and B, dashed lines; Fig. 7D, • (O) versus (Y)). At 10 µM 5-HT, contraction was 16 ± 8% (n= 6–7) in young and increased to 42 ± 13% in old (n= 6–8) coronary arteries pretreated with IbTx. In the presence of IbTx, the fitted maximum 5-HT contraction was 19 ± 0.6% (n= 6–7) in young rings, whereas in old rings it was increased to 47 ± 0.2% (n= 6–7). Interestingly, EC₅₀ values were apparently increased with age when IbTx was present from 153 ± 20 nM in young to 621 ± 8 nM in old coronary rings. In contrast, ageing did not affect the response to 5-HT alone (Fig. 7D, control, squares) or that of NTG either in control (squares, Fig. 7E) or when iberiotoxin was present (circles, Fig. 7E).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Ageing is the main risk factor for cardiovascular disease which remains the number one killer in industrialized societies (Wenger, 1997; Shepherd, 2001; Wu & Von Eckardstein, 2003). Because MaxiK channels regulate vascular tone by fine tuning the level of membrane potential and intracellular Ca²⁺ (Brayden & Nelson, 1992), any change in their function will influence the health status of the coronary arteries. Previously, we demonstrated that coronary arteries suffer a substantial decrease in MaxiK channel subunit protein during ageing (Marijic et al. 2001) that may translate into a diminished hyperpolarizing force and an increased risk for coronary vasospasm. Thus, knowing the mechanisms of MaxiK protein decrease in the elderly should provide tools for molecular medicine for the well-being of the growing ageing population (Shepherd, 2001). In this work, we show that the mechanism of MaxiK subunit reduction involves transcript downregulation. Further, the reduction in MaxiK channel subunit protein is accompanied by a parallel reduction in ß1 subunit functional expression via downregulation of its mRNA.

An interesting finding is the altered response of MaxiK channels to nanomolar Ca²⁺ in ageing coronary myocytes. This altered Ca²⁺ response in old myocytes, given by less positive V_1/2 values at 100 nM[Ca²⁺]_i, predicts MaxiK channels with higher open probability at 'resting' Ca²⁺ in old coronary arteries. In agreement, we found that MaxiK channels from old coronary arteries have higher activation rate constants at 100 nM[Ca²⁺]_i than those from young coronary arteries (Fig. 2). Mechanistically, the higher P_o and faster activation kinetics in old versus young can be explained either by changes in the MaxiK voltage sensor leading to an increased voltage sensitivity or changes in the Ca²⁺ sensor(s) leading to an increase in Ca²⁺ sensitivity. MaxiK channels are characterized by being insensitive to Ca²⁺ changes in the low nanomolar range below 100 nM Ca²⁺ (‘Ca²⁺-independent mode’) where they are intrinsically activated by voltage (Meera et al. 1996; Cui et al. 1997; Stefani et al. 1997), and start to respond to Ca²⁺ changes around this [Ca²⁺]_i concentration. The fact that at 700 nM Ca²⁺, where the channel is already in the ‘Ca²⁺-modulated mode’, there were no differences in old versus young activation rates causes us to speculate that the change observed at 100 nM Ca²⁺ may result from changes in the way the channel senses voltage, since at 100 nM Ca²⁺ MaxiK channels mainly operate in the Ca²⁺-independent mode. Regardless of the sensor(s) (voltage and/or Ca²⁺) being modified, this specific change in open probability may be a compensatory mechanism that maintains the coronary muscle cell with a healthy membrane potential and counterbalances the loss of MaxiK protein during ageing. We recently reported that in contrast to coronary myocytes, rat cerebral myocytes do not show a significant loss of MaxiK current density and surface expression of its subunits during ageing (Nishimaru et al. 2004) which may reflect different mechanisms involved in the ageing of coronary and cerebral myocytes. However, and although not significantly different, cerebral myocytes show a similar trend to coronary myocytes in having a more positive V_1/2 value at 100 nM Ca²⁺ in old myocytes. Thus, this tendency may be a common ageing feature of these vascular beds. The limited response of MaxiK to a Ca²⁺ rise from 100 to 700 nM in the old coronary and cerebral arterial myocytes may cause other cell functions to fail, including, but not restricted to, the activation of signalling enzymes and gene transcription.

The MaxiK ß1 subunit dramatically modifies the functional properties (kinetics, pharmacology, Ca²⁺/voltage sensitivities) of the pore-forming subunit (Toro et al. 1998); thus, a balanced coupling of both subunits is critical to maintain the coronary myocytes in a healthy state. Therefore, it was important to determine whether ß1 subunit functional expression is altered during coronary ageing. We predicted that if the ß1/ subunit ratio changes with age, we would observe altered functional properties when comparing young versus old coronary arteries. All functional experiments performed in isolated cells, including kinetics, and sensitivities to high Ca²⁺ (micromolar levels), DHS-I and iberiotoxin, conveyed the same result (Figs 1–4), that is, equivalent properties in old and young coronary myocytes. Since subunit protein is known to diminish with age (Marijic et al. 2001), the equivalent properties in young versus old can be explained by a parallel protein reduction of both subunits or by an unaltered /ß1 ratio during ageing. At present, we do not have an alternative way of measuring ß1 protein expression in coronary arteries other than function and pharmacology as the antibodies tested, both commercial and custom-made, failed to pass specificity tests in native tissues (rat aorta, rat coronary arteries and mouse myometrium) (not shown). Antibodies tested in most cases labelled a band of the expected size of ß1 in transfected cells where the protein is overexpressed but were unable to give specific labelling in native tissue samples, i.e. labelling a band(s) with molecular size that can be attributable to native ß1 in its glycosylated (29–31 kDa) or non-glycosylated (22 kDa) forms (Knaus et al. 1994; Jiang et al. 1999), and signals that could be eliminated by preincubation of the antibody (Ab) with the antigen. For example, using a polyclonal Ab from Affinity BioReagents (lot no. 484-104), we could observe a weak immunoreactive band in cell lysates of HEK cells expressing c-Myc-tagged ß1 (c-Myc epitope was used to confirm ß1 expression with anti-c-Myc Ab) of the appropriate size (29 kDa) but one of larger size (37 kDa) in native tissues that is absent when the Ab is preincubated with the antigenic peptide. Although blocked by the antigenic peptide, the nature of the 37 kDa signal is uncertain because (i) the immunolabelled protein has a higher molecular size than that expected for ß1, and (ii) a shift to lower mass after deglycosylation with N-glycosidase F could not be observed (author's unpublished observations). In addition, the epitope YHTEDTRDQNQQC used to raise the polyclonal antibody has the potential to cross-react with at least two other proteins of 37 kDa (NCBI, Blast program) one of them being the MaxiK ß2 subunit (39 kDa when glycosylated; YHTEETMKINQKC, 62% identical) (Wallner et al. 1999). Although a measurement of ß1 protein by immunoblot was not possible at this time, the parallel reduction in and ß1 transcripts (Figs 5 and 6) favours a parallel protein reduction of both subunits. Furthermore, since subunit protein levels and functional expression diminish to about one-half in old coronary myocytes (Marijic et al. 2001) (Fig. 1), and transcript levels of both subunits are also reduced by 50% (Fig. 6) it is safe to postulate that transcription is the main mechanism that governs the expression of both and ß1 subunits in coronary myocytes during ageing.

The single-cell type real-time PCR experiments demonstrate that both and ß1 subunit genes are under age-dependent transcript control leading to a proportional diminution of both transcripts in ageing coronary myocytes. At least two possibilities can account for decreased transcript levels: (a) a general increased rate of mRNA degradation in ageing tissues, or (b) specific transcript downregulation. We favour the second possibility since GAPDH transcript levels remained fairly constant with age (Fig. 6F). Age-dependent changes in transcription factor activities (e.g. c-fos, NF-B) that upregulate the expression of senescent vascular smooth muscle cell proteins (e.g. cell cycle regulatory proteins, iNOS) have been reported (Yan et al. 1999; Rivard et al. 2000). Recently, we have shown that the MaxiK subunit transcript and protein expression may also be under hormonal control since it is diminished during pregnancy (Song et al. 1999). It would be interesting to determine if the age-dependent loss of MaxiK channel and ß subunits could be recovered upon hormonal treatment as is the case of the vitamin D receptor in ageing bone cells (Duque et al. 2002).

At the organ level, the impact of age-related changes in MaxiK channel expression was pronounced in basal tension and stimulated contraction as assessed by blocking iberiotoxin-sensitive MaxiK channels (Fig. 7). The efficacy of 5-HT to produce contraction was substantially decreased after IbTx-induced precontraction in young > old coronary rings, probably due to the fact that MaxiK blockade and subsequent effectors lead to the use of most of the contractile capacity of the coronary rings in young myocytes. In other words, there is a larger IbTx-induced contraction and decreased dynamic range for 5-HT contraction in young versus old. In fact, in some young coronary rings, IbTx-induced contraction was so potent that 5-HT barely had an effect (not shown). This IbTx-induced contraction indicates that MaxiK channels are functionally active under basal conditions, contributing to the maintenance of arterial tone. Interestingly, the EC₅₀ of 5-HT was increased with age when IbTx was present which may support a tight functional coupling between MaxiK and 5-HT receptors (Alioua et al. 2002). The functional role of the ß1 subunit in vascular reactivity has been attributed to its ability to increase the channel open probability near Ca²⁺ spark sites favouring vasorelaxation and/or regulating tone (Toro et al. 1998; Brenner et al. 2000). The importance of this subunit in coronary function is highlighted in our studies by the ß1 RNA surplus in coronary myocytes when compared to subunit transcript levels in both young and old animals (Fig. 6), which would favour a full /ß subunit coupling even in ageing myocytes as demonstrated in cell functional studies (Figs 1–4). Consistent with a reduced expression of MaxiK channels ( and most probably ß1) with ageing, iberiotoxin induced a weaker contraction (Marijic et al. 2001) and made more effective 5-HT-stimulated contraction in old coronary arteries. In contrast, ageing did not have an apparent effect on NTG-induced relaxation including its MaxiK-related component (assessed by IbTx). In summary, tension experiments demonstrate that the reduction of MaxiK channel protein/function in coronary vessels may lead mainly to altered tone contributing to ailing coronary artery behaviours.

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