Role of Ca 2+Activated K + Channels on Adrenergic Responses of Human Saphenous Vein (original) (raw)
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Autonomic and Autacoid Pharmacology, 2006
1 As shown in a parallel study the magnitude of depolarization induced in human saphenous vein by raising external potassium ([K + ] e ) falls markedly below the theoretical values predicted by the Goldman-Hodgkin-Katz equations. This anomaly prompted us to re-examine the relaxant actions of L-type (nifedipine) and T-type (mibefradil) Ca 2+ channel antagonists, and relaxant and electrophysiological effects of the K + channel opener, pinacidil, on saphenous veins contracted by the elevation of [K + ] e . 2 Nifedipine produced concentration-dependent relaxations in tissues contracted at various high [K + ] e . In tissues contracted with 20 mM [K + ] e , the pIC 50 for nifedipine was significantly (8.20 ± 0.05; n ¼ 6; mean ± SEM; P < 0.05) greater than in tissues contracted with ‡40 mM [K + ] e . 3 Tissues contracted with 20 mM [K + ] e also relaxed in response to mibefradil (pIC 50 ¼ 6.1 ± 0.14) and pinacidil (pIC 50 ¼ 6.45 ± 0.08), the latter being almost completely reversed (93.4 ± 9.9%) by addition of glibenclamide (10 lM). 4 The resting E m of smooth muscle cells of saphenous vein was )77.0 ± 0.7 mV (n ¼ 52), and 20 mM [K + ] e produced a modest but significant depolarization to )73.0 ± 0.7 mV (n ¼ 52). Incubation with pinacidil plus 20 mM [K + ] e resulted in a significant hyperpolarization of the E m to )82 ± 0.6 mV (n ¼ 52). 5 N x -nitro-L-arginine methyl ester did not impede the relaxant responses of nifedipine, mibefradil or pinacidil. 6 In conclusion, the relaxant effects of nifedipine and pinacidil (i) occurred at an E m distinctly below the presumed threshold for the opening of the classic (Ca V 1.3a 1 ) L-type Ca 2+ channels, and (ii) did not depend on generation of nitric oxide.
L-type Ca 2+ channels activation and contraction elicited by myricetin on vascular smooth muscles
Naunyn-Schmiedeberg's Archives of Pharmacology, 2003
The effects of myricetin (3,3',4',5,5',7-hesahydroxyflavone), a natural flavonoid found in edible plants, were studied on vascular smooth muscle L-type Ca 2+ channels by comparing its mechanical, radioligand binding, and electrophysiological properties to those of the Ca 2+ channel agonist (S)-(-)-Bay K 8644. In rat aorta rings, both myricetin and (S)-(-)-Bay K 8644 induced contractile responses, which were dependent upon prior exposure to K +. At 15 mM K + (K15) the pEC 50 values for myricetin and (S)-(-)-Bay K 8644 were 4.43±0.03 and 7.92±0.13, respectively. Furthermore, the maximum tension response to myricetin was not significantly different from that elicited by either (S)-(-)-Bay K 8644 or K60. The Ca 2+ channel blockers nifedipine, verapamil and diltiazem antagonised and fully reverted myricetin-, (S)-(-)-Bay K 8644-as well as K60-induced contractions. Both myricetin and (S)-(-)-Bay K 8644 potentiated rat aorta ring responses to K + , shifting the K + concentration-response curve to the left. (S)-(-)-Bay K 8644, but not myricetin, inhibited in a concentration-dependent manner (+)-[ 3 H]PN200-110 binding in porcine aortic membranes. Electrophysiological recordings from single rat tail artery myocytes, under amphotericin B-perforated as well as conventional methods, showed that both myricetin and (S)-(-)-Bay K 8644 increased L-type Ba 2+ current (I Ba(L)) and shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarising direction, without, however, modifying the threshold potential. Furthermore, (S)-(-)-Bay K 8644 accelerated both activation and inactivation kinetics of I Ba(L) while myricetin slowed down the activation kinetics. Finally, both (S)-(-)-Bay K 8644 and myricetin slowed down deactivation kinetics of I Ba(L). These results suggest that myricetin induces vasoconstriction by activating L-type Ca 2+ channel with similar efficacy but a site of action different to that of (S)-(-)-Bay K 8644. Keywords (S)-(-)-Bay K 8644 • Myricetin • Vascular smooth muscle • Whole-cell patch-clamp • Wine polyphenols Abbreviations I Ba(L) L-type Ba 2+ current • PSS Physiological salt solution • V h Holding potential
AJP: Cell Physiology, 2012
Hristov KL, Parajuli SP, Soder RP, Cheng Q, Rovner ES, Petkov GV. Suppression of human detrusor smooth muscle excitability and contractility via pharmacological activation of large conductance Ca 2ϩ -activated K ϩ channels. Overactive bladder syndrome is frequently associated with increased detrusor smooth muscle (DSM) contractility. We tested the hypothesis that pharmacological activation of the large-conductance voltage-and Ca 2ϩ -activated K ϩ (BK) channel with NS-1619, a selective BK channel opener, reduces the excitability and contractility of human DSM. We used the amphotericinperforated whole cell patch-clamp technique on freshly isolated human DSM cells, live-cell Ca 2ϩ imaging, and isometric DSM tension recordings of human DSM strips obtained from open bladder surgeries. NS-1619 (30 M) significantly increased the amplitude of the voltage step-induced whole cell BK currents, and this effect was abolished by pretreatment with 200 nM iberiotoxin (IBTX), a selective BK channel inhibitor. In current-clamp mode, NS-1619 (30 M) significantly hyperpolarized the resting membrane potential, and the hyperpolarization was reversed by IBTX (200 nM). NS-1619 (30 M) significantly decreased the intracellular Ca 2ϩ level in isolated human DSM cells. BK channel activation with NS-1619 (30 M) significantly inhibited the amplitude, muscle force, frequency, duration, and tone of the spontaneous phasic and pharmacologically induced DSM contractions from human DSM isolated strips. IBTX (200 nM) suppressed the inhibitory effects of NS-1619 on spontaneous contractions. The amplitude of electrical field stimulation (0.5-50 Hz)induced contractions was significantly reduced by NS-1619 (30 M). Our data suggest that pharmacological activation of BK channels could represent a novel treatment option to control bladder dysfunction in humans.
Acta Physiologica Scandinavica, 1998
Local calcium transients (`Ca 2+ sparks') are thought to be elementary Ca 2+ signals in heart, skeletal and smooth muscle cells. Ca 2+ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca 2+ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately A40 mV, an elevation of arterial wall [Ca 2+ ] i and contraction (`myogenic tone') of arteries. Ca 2+ sparks activate calcium-sensitive K + (K Ca ) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca 2+ sparks by ryanodine, or of K Ca channels by iberiotoxin, leads to membrane depolarization, activation of L-type voltage-gated Ca 2+ channels, and vasoconstriction. Conversely, activation of Ca 2+ sparks can lead to vasodilation through activation of K Ca channels. Our recent work is aimed at studying the properties and roles of Ca 2+ sparks in the regulation of arterial smooth muscle function. The modulation of Ca 2+ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca 2+ entry through voltagedependent Ca 2+ channels in the regulation of Ca 2+ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca 2+ channels, RyR channels, and K Ca channels function as a coupled unit, through Ca 2+ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.
Circulation Research, 2010
Rationale: Calcium channel blockers (CCBs) exert their antihypertensive effect by reducing cardiac afterload but not preload, suggesting that Ca 2؉ influx through L-type Ca 2؉ channels (LTCC) mediates arterial but not venous tone. Objective: The object of this study was to resolve the mechanism of venous resistance to CCBs. Methods and Results: We compared the sensitivity of depolarization (KCl)-induced constriction of rat small mesenteric arteries (MAs) and veins (MVs) to the dilator effect of CCBs. Initial findings confirmed that nifedipine progressively dilated depolarization-induced constrictions in MAs but not MVs. However, Western blots showed a similar expression of the ␣ 1C pore-forming subunit of the LTCC in both vessels. Patch-clamp studies revealed a similar density of whole-cell Ca 2؉ channel current between single smooth muscle cells (SMCs) of MAs and MVs. Based on these findings, we hypothesized that LTCCs are expressed but "silenced" by intracellular Ca 2؉ in venous SMCs. After depletion of intracellular Ca 2؉ stores by the SERCA pump inhibitor thapsigargin, depolarization-induced constrictions in MVs were blocked 80% by nifedipine suggesting restoration of Ca 2؉ influx through LTCCs. Similarly, KCl-induced constrictions were sensitive to block by nifedipine after depletion of intracellular Ca 2؉ stores by caffeine, ryanodine, or 2-aminoethoxydiphenyl borate. Cell-attached patch recordings of unitary LTCC currents confirmed rare channel openings during depolarization of venous compared to arterial SMCs, but chelating intracellular Ca 2؉ significantly increased the open-state probability of venous LTCCs. Conclusions: We report that intracellular Ca 2؉ inactivates LTCCs in venous SMCs to confer venous resistance to CCB-induced dilation, a fundamental drug property that was previously unexplained. (Circ Res. 2010;106:739-747.
Identification of the Ca2+ current activated by vasoconstrictors in vascular smooth muscle cells
Pflügers Archiv European Journal of Physiology, 1994
The noncontractile aortic cell line A7r5 was chosen to study the effect of the vasoconstrictor peptide vasopressin on transmembrane Ca 2+ movements, using conventional whole-cell patch recording techniques. Conditions in which previously characterised vasoconstrictor-modulated currents were suppressed revealed a tiny inward current component (-18+2 pA, n=50, at -61 mV in i10 mM CaC12). The vasopressin-activated inward current was absent when Ca 2+ was absent from the extracellular solution, and the current amplitude increased with [Ca 2+] (0.01-110 mM), with an apparent dissociation constant for Ca 2+ of 9.7 raM. It was highly selective for Ca 2+ over monovalent cations (permeability ratio Ca/Cs greater than 17). It was not voltage gated, except that the current/potential characteristic showed some inwards rectification. Amplitudes of the evoked inward currents had the same order of magnitude in Sr 2+ and Ca 2+, whereas they were much smaller in Mn 2 § suggesting that this pathway is highly permeable to Sr 2 § but poorly permeable to Mn 2 § Inward currents evoked in Ca 2+ were inhibited by other cations with the following order of potency: La3+>Cd2+>Co2+-Ni2+-Mn 2+. The channel producing this current corresponds most probably to the ionic pathway originally called the receptoroperated calcium channel, which produces a long-lasting, constrictor-induced plateau of increased intracellular free calcium concentration in smooth muscle.
British Journal of Pharmacology, 2001
1 The subcellular mechanisms regulating stimulus-contraction coupling in detrusor remain to be determined. We used Ca 2+ -free solutions, Ca 2+ channel blockers, cyclopiazonic acid (CPA), and RhoA kinase (ROK) inhibitors to test the hypothesis that Ca 2+ in¯ux and Ca 2+ sensitization play primary roles. 2 In rabbit detrusor, peak bethanechol (BE)-induced force was inhibited 90% by incubation for 3 min in a Ca 2+ -free solution. By comparison, a 20 min incubation of rabbit femoral artery in a Ca 2+ -free solution reduced receptor-induced force by only 5%. 3 In detrusor, inhibition of sarcoplasmic reticular (SR) Ca 2+ release by 2APB, or depletion of SR Ca 2+ by CPA, inhibited BE-induced force by only 27%. The CPA-insensitive force was abolished by LaCl 3 . By comparison, 2APB inhibited receptor-induced force in rabbit femoral artery by 71%. 4 In the presence of the non-selective cation channel (NSCC) inhibitor, LOE-908, BE did not produce an increase in [Ca 2+ ] i but did produce weak increases in myosin phosphorylation and force. 5 Inhibitors of ROK-induced Ca 2+ sensitization, HA-1077 and Y-27632, inhibited BE-induced force by *50%, and in combination with LOE-908, nearly abolished force. 6 These data suggest that two principal muscarinic receptor-stimulated detrusor contractile mechanisms include NSCC activation, that elevates [Ca 2+ ] i and ROK activation, that sensitizes cross bridges to Ca 2+ .