| Diversity of vascular smooth muscle cell (VSMC) function may be important in physiology and pathophysiology, allowing responses to vasodilators and vasoconstrictors to vary within or between vascular beds. Heterogeneity of VSMC is related to many kinds of recepts and ion channels distributed in their membrane. The normal of ion channels in VSMC plays pivotal role not only in the regulation of vascular smooth muscle contraction, but also in the maintenance of cellular excitation and membrane stability. Potassium channel, a family of widely expressed in VSMC, is required for maintenance of vascular tone by providing a repolarizing current to counteract and balance vasoconstrictive in?uences. Relatively a few potassium current may vary membrane potential severely. Potassium efflux from VSMC results in membrane repolarization and, by promoting the close for voltaged-dependent calcium channel, reduces intracellular free calcium concentration. As an important therapeutic target, some potassium channel regulators have been used clinically. However, as the most complicated classification (including functional and structural classification) among the membrane ion channels, various subfamilies of potassium channels result in the low selectivity and satisfaction of potassium channel regulators which had been used in clinical therapy at present.Four main classes of K+ channels have been described in VSMC. Two types of K+ channels that are known to be prevalent and contribute significantly to the repolarization of VSMC membranes are the high-conductance Ca2+-activated K+ (BK) channels and the voltage-gated K+ (Kv) channels. While most VSMC identified to date contain both BK and Kv channels, the expression of individual channel occurs in a tissue-specific manner, thereby providing functional specificity. The heterogeneity of channel subunits in VSMC ensures the appropriate responses of VSMC to diverse stimuli. Although BK channel and Kv channel had been studied in some arteries, the function and expression of them in the VSMC of different circulatory beds remain largely unknown. Vascular tone of coronary and cerebral artery is thought to regulate the blood flow of heart and brain. It is essential to establish the precise diversity of VSMC Kv channel and BK channel in coronary artery and middle cerebral artery in physiology, pathophysiology and pharmacology.In the last few years, several laboratories have focused their attentions on the investigation of the cellular mechanisms underlying the effects of taurine, a well known sulfonic amino acid distributed widely in different tissues and abundantly in cardiovascular tissues. These concerns come from the multiple and surprising effects exerted by taurine involved in many physiological processes and the positive effects induced by supplement of taurine in some cardiovascular pathologic state.The targets of taurine are complicated. The cellular mechanism of action of taurine is under investigation and appears to involve the interaction with several ion channels including potassium channels. Previous findings from our laboratory have demonstrated that taurine changes vasomotion by interaction with VSMC potassium channels.In this study, VSMCs were freshly isolated from rat coronary arteries and middle cerebral arteries. Whole cell patch clamp and multicell RT-PCR techniques were used in the experiments to investigate the function and expression diversities of Kv channel and BK channel between coronary artery and middle cerebral artery. The changes of Kv currents and BK currents after taurine perfusion were recorded as well.The results were as follows:1. VSMC Kv channel of rat coronary artery and middle cerebral arteryThe VSMC resting membrane potential of coronary artery and middle cerebral artery was -28.91±3.93 mV (n = 15) and -30.82±2.79 mV (n = 19), respectively. The capacitance was 17.66±0.95pF (n = 46) and 14.28±1.01pF (n = 42), respectively. Both of them had insignificant difference between coronary artery and middle cerebral artery (P> 0.05).In rat coronary artery and middle cerebral artery, the VSMC Kv currents were voltage dependent, delayed rectifying and outward rectifying. The currents were activated about -40 mV and slowly inactivated during the 500ms recording period. 4-aminopyridine (4-AP; 3 mM), the classic Kv channel blocker, inhibited Kv currents significantly. Fitted to a conventional Boltzmann distribution equation, membrane potential producing half-maximal activation and the slope were -4.39±1.44 mV and 12.42±1.19 in coronary artery SMC, whereas -9.89±1.72 mV and 13.03±1.62 in middle cerebral artery SMC. Membrane potential producing half-maximal inactivation and the slope were -11.78±1.47 mV and 14.73±1.54 in coronary artery SMC, whereas -25.77±2.29 mV and 16.37±2.33 in middle cerebral artery SMC. The test potentials at which VSMC outward current was half-activated or half-inactivated were significantly shifted toward more positive membrane potentials in coronary artery compared with middle cerebral artery (P< 0.05, n=11), whereas slope was not different.To identify the tetramer composition of Kv channels in rat VSMCs, multicell RT-PCR was used to screen for mRNAs encoding each of the five Kv subfamilies (Kv1.1, Kv1.2, Kv1.5, Kv1.6 and Kv2.1) which have delayed rectifier properties. Kv1.2 and Kv1.5 subfamilies were detected outstandingly both in rat coronary artery and middle cerebral artery SMC. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a housekeeping gene. The expression of VSMC Kv1.2 relative to GAPDH was 94.4±7.59% and 98.1±5.08% (P>0.05, n=7) in coronary artery and middle cerebral artery, respectively. The amounts of VSMC Kv1.5 transcripts relative to GAPDH were 87.3±4.20% and 80.2±6.06% (P>0.05, n=7) in coronary artery and middle cerebral artery, respectively. VSMC Kv1.1 subfamily was detected moderately in rat middle cerebral artery and the amounts of Kv1.1 transcripts relative to GAPDH were 54.7±3.05%. However, in rat middle cerebral artery, a faint band of VSMC Kv1.1 expression was detected in one group and its amount of transcripts relative to GAPDH was 37.6%. The positive expression rate of VSMC Kv1.1 was 100% in middle cerebral artery and was 14.3% in coronary artery. VSMC Kv1.6 and Kv2.1 were silent both in rat coronary artery and middle cerebral artery.2. VSMC BK channel of rat coronary artery and middle cerebral arteryThe BK current recorded was voltage dependent, outward rectifying and intracellular calcium sensitive. The rectifying component of the outward current was inhibited by tetraethylammonium (TEA, 1 mM), a BK channel blocker.The expression of VSMC BKαsubunit in rat coronary artery was less than which in middle cerebral artery. The amounts of VSMC BKαsubunit transcripts relative to GAPDH were 48±8.80% in coronary artery whereas it was 80.9±8.37% in middle cerebral artery (P<0.01, n=7). The level of VSMC BKβ1 transcripts in coronary artery was comparable to that in middle cerebral artery. The amounts of VSMC BKβ1 transcripts relative to GAPDH were 23.5±5.33% and 22.7±5.27% (P>0.05, n=7) in coronary artery and middle cerebral artery, respectively.3. Effect of taurine on VSMC Kv current and BK current of rat coronary artery and middle cerebral artery1) The effect of taurine on VSMC Kv current of rat coronary arteryKv current was attenuated after perfusing 1mM, 3mM or 10mM taurine whereas it was increased by 40mM or 60mM taurine.Kv current was decreased significantly by 1mM (n=11) taurine at +20 mV test potential (5.28±0.23 pA/pF) (P<0.05, compared with absence of taurine, 6.29±0.2 pA/pF). Kv current was decreased significantly by 3mM (n=12), 10mM (n=11) taurine at +20 mV, +10 mV and 0 mV test potential (P<0.05, compared with absence of taurine). At +20 mV test potential, Kv current was 6.29±0.2pA/pF, 4.47±0.26 pA/pF and 4.19±0.28 pA/pF in absence of taurine or perfused with 3mM and 10mM taurine, respectively. At +10 mV test potential, Kv current was 5.18±0.28 pA/pF, 3.71±0.29 pA/pF and 3.72±0.14 pA/pF in absence of taurine or perfused with 3mM and 10mM taurine. At 0 mV test potential, Kv current was 6.29±0.2 pA/pF, 4.47±0.26 pA/pF and 4.19±0.28 pA/pF in absence of taurine or perfused with 3mM and 10mM taurine, respectively.Kv current was not altered by 20mM taurine (P>0.05).Kv current was increased by 40mM (n=11) and 60mM taurine (n=11). In the presence of 60mM taurine, Kv current was 6.34±0.54pA/pF and 7.27±0.35 pA/pF at +10 mV and +20 mV test potential that was augmented significantly compared with absence of taurine (5.18±0.28 pA/pF and 6.29±0.2 pA/pF, P<0.05).2) The effect of taurine on VSMC Kv current of rat middle cerebral arteryKv current was decreased by 1mM and 3mM taurine (n=11). At +10 mV and +20 mV test potential, Kv current was attenuated significantly by 1mM taurine (4.18±0.21 pA/pF and 4.29±0.18 pA/pF) (P<0.05, compared with absence of taurine, 4.99±0.31 pA/pF and 5.94±0.26 pA/pF).Kv current was not altered by 10mM but was significantly increased by 20mM (n=11), 40mM (n=11) and 60mM (n=11) taurine concentration-dependently.At +20 mV test potential, Kv current was 5.94±0.26 pA/pF, 6.87±0.25 pA/pF and 7.74±0.28 pA/pF in absence of taurine or perfusing 40mM and 60mM taurine, respectively. At +10 mV test potential, Kv current was 4.99±0.31 pA/pF, 5.91±0.25pA/pF and 6.89±0.43pA/pF in absence of taurine or perfused with 40mM and 60mM taurine, respectively. Campared with absence of taurine, the increase of Kv current in presence of taurine was significant (P<0.05). Campared with presence of 40mM taurine, the increase of Kv current in presence of 60mM taurine was significant at +20 mV and +10 mV test potential (P<0.05).3) Time-dependent and reversible effect of taurine (1mM) on VSMC Kv currentBoth in rat coronary artery and middle cerebral artery SMC, Kv current decreased after 1min taurine perfusion and attenuated significantly at 5min perfusion. The current recovered to about 90% level of pretaurine after washout taurine for 5min.4) The effect of taurine (60mM) on VSMC BK current of rat coronary artery and middle cerebral arteryIn rat coronary artery SMC, BK current was increased at negative membrane potential whereas it was attenuated at positive membrane potential after perfusion of taurine (60mM, n=12). Compared with absence of taurine (3.29±0.31pA/pf, 3.82±0.35pA/pF and 4.42±0.38pA/pF), BK current was augmented significantly to 4.9±0.46pA/pF, 5.35±0.49pA/pF and 6.47±0.6pA/pF at -20 mV, -10 mV and 0 mV test potential, respectively (P< 0.05). Compared with absence of taurine (24.26±1.55 pA/pF, 29.31±1.86 pA/pF and 32.89±2.02 pA/pF), BK current was attenuated significantly to 16.26±1.51pA/pF,19.81±1.84pA/pF and 24.04±2.24 pA/pF at +60 mV, +70 mV and +80 mV test potential, respectively (P< 0.05).In rat middle cerebral artery SMC, BK current was increased significantly by taurine (60mM, n=9) from -40 mV to +10 mV test potential (P< 0.05, compared with absence of taurine) whereas was not altered above +40 mV test potential. From -40 mV to +10 mV test potential, BK current was 4.65±1.35 pA/pF, 5.18±1.32 pA/pF, 5.98±1.4pA/pF, 6.48±1.51 pA/pF, 8.2±1.5 pA/pF and 10.05±1.43 pA/pF in the presence of taurine. Absence of taurine, BK current was 1.33±0.25 pA/pF, 1.74±0.37 pA/pF, 1.94±0.46 pA/pF, 2.34±0.5 pA/pF, 2.84±0.59 pA/pF and 3.54±0.52 pA/pF from -40 mV to +10 mV test potential, respectively.In summary, the function and expression of both VSMC Kv channel and BK channel are disparity between rat coronary artery and middle cerebral artery. The VSMC Kv channel thresholds of both activation and inactivation potential were significantly higher in coronary artery than in middle cerebral artery. Among the detected five Kv channel subfamilies, the member was Kv 1.2 and Kv1.5 subfamily in rat coronary artery SMC and the member was Kv 1.2, Kv1.5 and Kv 1.1 subfamily in rat middle cerebral artery SMC. The expression level of VSMC BKαsubunit was lower in coronary artery than in middle cerebral artery. The precise functions of these members under physiological and pathological conditions should be determined by further studies.In the presence of taurine, Kv currents of rat coronary artery and middle cerebral artery SMC were augmented in higher concentration whereas inhibited in low concentration time-dependently and reversibly. As for the effects of taurine on BK channel, it was different between rat coronary artery and middle cerebral artery SMC. In coronary artery, VSMC BK currents were increased at negative membrane potential and decreased at positive membrane potential after taurine perfusion. In middle cerebral artery, taurine inhibited VSMC BK currents when the membrane potential was lower than +40 mV test potential and had no effect above +40 mV test potential. The effect of taurine on VSMC Kv current and BK current seems to be different and complicated in rat coronary artery and middle cerebral artery. |
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