Coexpression of voltage-dependent calcium channels Cav1.2, 2.1a, and 2.1b in vascular myocytes

Voltage-dependent Ca2+ channels Cav1.2 (L type) and Cav2.1 (P/Q type) are expressed in vascular smooth muscle cells (VSMCs) and are important for the contraction of renal resistance vessels. In the present study we examined whether native renal VSMCs coexpress L-, P-, and Q-type Ca2+ currents. The expression of both Cav2.1a (P-type) and Cav2.1b (Q-type) mRNA was demonstrated by RT-PCR in renal preglomerular vessels from rats and mice. Immunolabeling was performed on A7r5 cells, renal cryosections, and freshly isolated renal VSMCs with anti-Cav1.2 and anti-Cav2.1 antibodies. Conventional and confocal microscopy revealed expression of both channels in all of the smooth muscle cells. Whole-cell patch clamp on single preglomerular VSMCs from mice showed L-, P-, and Q-type currents. Blockade of the L-type currents by calciseptine (20 nmol/L) inhibited 35.6+/-3.9% of the voltage-dependent Ca2+ current, and blocking P-type currents (omega-agatoxin IVA 10 nmol/L) led to 20.2+/-3.0% inhibition, whereas 300 nmol/L of omega agatoxin IVA (blocking P/Q-type) inhibited 45.0+/-7.3%. In rat aortic smooth muscle cells (A7r5), blockade of L-type channels resulted in 28.5+/-6.1% inhibition, simultaneous blockade of L-type and P-type channels inhibited 58.0+/-11.8%, and simultaneous inhibition of L-, P-, and Q-type channels led to blockade (88.7+/-5.6%) of the Ca2+ current. We conclude that aortic and renal preglomerular smooth muscle cells express L-, P-, and Q-type voltage-dependent Ca2+ channels in the rat and mouse.     

TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels

Vasodilatory factors produced by the endothelium are critical for the maintenance of normal blood pressure and flow. We hypothesized that endothelial signals are transduced to underlying vascular smooth muscle by vanilloid transient receptor potential (TRPV) channels. TRPV4 message was detected in RNA from cerebral artery smooth muscle cells. In patch-clamp experiments using freshly isolated cerebral myocytes, outwardly rectifying whole-cell currents with properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) (5 micromol/L) and the endothelium-derived arachidonic acid metabolite 11,12 epoxyeicosatrienoic acid (11,12 EET) (300 nmol/L). Using high-speed laser-scanning confocal microscopy, we found that 11,12 EET increased the frequency of unitary Ca2+ release events (Ca2+ sparks) via ryanodine receptors located on the sarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca2+ sparks activated nearby sarcolemmal large-conductance Ca2+-activated K+ (BKCa) channels, measured as an increase in the frequency of transient K+ currents (referred to as "spontaneous transient outward currents" [STOCs]). 11,12 EET-induced increases in Ca2+ spark and STOC frequency were inhibited by lowering external Ca2+ from 2 mmol/L to 10 micromol/L but not by voltage-dependent Ca2+ channel inhibitors, suggesting that these responses require extracellular Ca2+ influx via channels other than voltage-dependent Ca2+ channels. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteries prevented 11,12 EET-induced smooth muscle hyperpolarization and vasodilation. Thus, we conclude that TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release in response to an endothelial-derived factor.     

Voltage-dependent Ca2+ channels in resistance arteries from Dahl salt-sensitive rats.

To determine whether chronic salt-loading would alter voltage-dependent Ca2+ channels in resistance arteries of Dahl salt-sensitive rats, whole-cell voltage-clamp experiments were performed on single cells that were isolated from small mesenteric arteries. Dahl salt-sensitive rats were fed either an 8% NaCl diet (high-NaCl group) or a 0.3% NaCl diet (low-NaCl group) from the age of 6 or 7 weeks. After 4 to 5 weeks, systolic blood pressure was significantly higher in the high-NaCl group than in the low-NaCl group. In the high-NaCl group, the threshold potential for Ca2+ channel current was more negative and the current amplitude that was normalized by cell capacitance was higher at negative command potentials (-40 mV to -20 mV), as compared with the low-NaCl group. When the current was separated into fast transient current and slow sustained (L-type) current, the alteration in the high-NaCl group was attributable to the change in L-type current. The steady-state inactivation curve was not different between the high-NaCl and low-NaCl groups. In conclusion, L-type Ca2+ channels in resistance arteries of Dahl salt-sensitive rats became more available for opening near the resting potential after dietary salt-loading.     

Characterization of a G-protein-regulated outward K+ current in mesophyll cells of vicia faba L.

Whole-cell voltage-dependent currents in isolated mesophyll protoplasts of Vicia faba were investigated by patch-clamp techniques. With 104 mM K+ in the cytosol and 13 mM K+ in the external solution, depolarization of the plasma membrane from -47 mV to potentials between -15 and +85 mV activated a voltage- and time-dependent outward current (Iout). The average magnitude of Iout at +85 mV was 28.5 +/- 3.3 pA.pF-1. No inward voltage-dependent current was observed upon hyperpolarization of the plasma membrane from -55 mV to potentials as negative as -175 mV. Time-activated outward current was blocked by Ba2+ (1 mM BaCl2) and was not observed when K+ was eliminated from the external and internal solutions, indicating that this outward current was carried primarily by K+ ions. The voltage dependency of outward K+ current revealed a possible mechanism for K+ efflux from mesophyll cells. A GDP analogue guanosine 5'-[beta-thio]diphosphate (500 microM) significantly enhanced outward K+ current. The outward K+ current was inhibited by the GTP analogue guanosine 5'-[gamma-thio]triphosphate (500 microM) and by an increase in cytoplasmic free Ca2+ concentrations. Cholera toxin, which ADP-ribosylates guanine nucleotide-binding regulatory proteins, also inhibited outward K+ current. These findings illustrate the presence in mesophyll cells of outward-rectifying K+ channels that are regulated by GTP-binding proteins and calcium.     

Oxygen-sensitive calcium channels in vascular smooth muscle and their possible role in hypoxic arterial relaxation.

We have investigated the modifications of cytosolic [Ca2+] and the activity of Ca2+ channels in freshly dispersed arterial myocytes to test whether lowering O2 tension (PO2) directly influences Ca2+ homeostasis in these cells. Unclamped cells loaded with fura-2 AM exhibit oscillations of cytosolic Ca2+ whose frequency depends on extracellular Ca2+ influx. Switching from a PO2 of 150 to 20 mmHg leads to a reversible attenuation of the Ca2+ oscillations. In voltage-clamped cells, hypoxia reversibly reduces the influx of Ca2+ through voltage-dependent channels, which can account for the inhibition of the Ca2+ oscillations. Low PO2 selectively inhibits L-type Ca2+ channel activity, whereas the current mediated by T-type channels is unaltered by hypoxia. The effect of low PO2 on the L-type channels is markedly voltage dependent, being more apparent with moderate depolarizations. These findings demonstrate the existence of O2-sensitive, voltage-dependent, Ca2+ channels in vascular smooth muscle that may critically contribute to the local regulation of circulation.     

L- and T-type Ca2+ channels in canine cardiac Purkinje cells. Single-channel demonstration of L-type Ca2+ window current.

Canine cardiac Purkinje cells contain both L- and T-type calcium currents, yet the single Ca2+ channels have not been characterized from these cells. Additionally, previous studies have shown an overlap between the steady-state inactivation and activations curves for L-type Ca2+ currents, suggesting the presence of L-type Ca2+ "window" current. We used the on-cell, patch-clamp technique to study Ca2+ channels from isolated cardiac Purkinje cells. Patches contained one or more Ca2+ channels 75% of the time. L-type channels were seen in 69% and T-type channels in 73% of these patches. With 110 mM Ba2+ as the charge carrier, the conductances of the L- and T-type Ca2+ channels were 24.2 +/- 0.8 pS (n = 9) and 9.0 +/- 0.5 pS (n = 8), respectively (mean +/- SEM). With 110 mM Ca2+ as the charge carrier, the conductance of the L-type Ca2+ channel decreased to 9.7 +/- 1.2 pS (n = 4), whereas the T-type Ca2+ channel conductance was unchanged. Voltage-dependent inactivation was shown for both L- and T-type Ca2+ channels, although for L-type Ca2+ channel with Ba2+ as the charge carrier, inactivation took at least 30 seconds at a potential of +40 mV. After channel inactivation was complete, L-type Ca2+ channel reopenings were observed following repolarizing steps into the window voltage range. Thus, our data identify both L- and T-type Ca2+ channels in cardiac Purkinje cells and demonstrate, at the single-channel level, L-type channel transitions expected for a window current. Window current may play an important role in shaping the action potential and in arrhythmogenesis.     

Calcium currents in GH3 cultured pituitary cells under whole-cell voltage-clamp: inhibition by voltage-dependent potassium currents.

To isolate inward Ca2+ currents in GH3 rat pituitary cells, an inward Na+ current as well as two outward K+ currents, a transient voltage-dependent current (IKV) and a slowly rising Ca2+-activated current (IKCa), must be suppressed. Blockage of these outward currents, usually achieved by replacement of intracellular K+ with Cs+, reveals sustained inward currents. Selective blockage of either K+ current can be accomplished in the presence of intracellular K+ by use of quaternary ammonium ions. When IKCa and Na+ currents are blocked, the net current elicited by stepping the membrane potential (Vm) from -60 to 0 mV is inward first, becomes outward and peaks in 10-30 msec, and finally becomes inward again. Under this condition, in which both IKV and Ca2+ currents should be present throughout the duration of the voltage step, the Ca2+ current was not detected at the time of peak outward current. That is, plots of peak outward current vs. Vm are monotonic and are not modified by nisoldipine or low external Ca2+ as would be expected if Ca2+ currents were present. However, similar plots at times other than at peak current are not monotonic and are altered by nisoldipine or low Ca2+ (i.e., inward currents decrease and plots become monotonic). When K+ channels are first inactivated by holding Vm at -30 mV, a sustained Ca2+ current is always observed upon stepping Vm to 0 mV. Furthermore, substitution of Ba2+ for Ca2+ causes blockage of IKV and inhibition of this current results in inward Ba2+ currents with square wave kinetics. These data indicate that the Ca2+ current is completely inhibited at peak outward IKV and that Ca2+ conductance is progressively disinhibited as the transient K+ current declines due to channel inactivation. This suggests that in GH3 cells Ca2+ channels are regulated by IKV.     

Calciseptine, a peptide isolated from black mamba venom, is a specific blocker of the L-type calcium channel.

The venom of the black mamba contains a 60-amino acid peptide called calciseptine. The peptide has been fully sequenced. It is a smooth muscle relaxant and an inhibitor of cardiac contractions. Its physiological action resembles that of drugs, such as the 1,4-dihydropyridines, which are important in the treatment of cardiovascular diseases. Calciseptine, like the 1,4-dihydropyridines, selectively blocks L-type Ca2+ channels and is totally inactive on other voltage-dependent Ca2+ channels such as N-type and T-type channels. To our knowledge, it is the only natural polypeptide that has been shown to be a specific inhibitor of L-type Ca2+ channels.     

Crosstalk between voltage-independent Ca2+ channels and L-type Ca2+ channels in A7r5 vascular smooth muscle cells at elevated intracellular pH: evidence for functional coupling between L-type Ca2+ channels and a 2-APB-sensitive cation channel

This study was designed to investigate the role of voltage-independent and voltage-dependent Ca2+ channels in the Ca2+ signaling associated with intracellular alkalinization in A7r5 vascular smooth muscle cells. Extracellular administration of ammonium chloride (20 mmol/L) resulted in elevation of intracellular pH and activation of a sustained Ca2+ entry that was inhibited by 2-amino-ethoxydiphenyl borate (2-APB, 200 micromol/L) but not by verapamil (10 micro;mol/L). Alkalosis-induced Ca2+ entry was mediated by a voltage-independent cation conductance that allowed permeation of Ca2+ (PCa/PNa approximately 6), and was associated with inhibition of L-type Ca2+ currents. Alkalosis-induced inhibition of L-type Ca2+ currents was dependent on the presence of extracellular Ca2+ and was prevented by expression of a dominant-negative mutant of calmodulin. In the absence of extracellular Ca2+, with Ba2+ or Na+ as charge carrier, intracellular alkalosis failed to inhibit but potentiated L-type Ca2+ channel currents. Inhibition of Ca2+ currents through voltage-independent cation channels by 2-APB prevented alkalosis-induced inhibition of L-type Ca2+ currents. Similarly, 2-APB prevented vasopressin-induced activation of nonselective cation channels and inhibition of L-type Ca2+ currents. We suggest the existence of a pH-controlled Ca2+ entry pathway that governs the activity of smooth muscle L-type Ca2+ channels due to control of Ca2+/calmodulin-dependent negative feedback regulation. This Ca2+ entry pathway exhibits striking similarity with the pathway activated by stimulation of phospholipase-C-coupled receptors, and may involve a similar type of cation channel. We demonstrate for the first time the tight functional coupling between these voltage-independent Ca2+ channels and classical voltage-gated L-type Ca2+ channels.     

Effects of external pH on ionic currents in smooth muscle cells from the basilar artery of the guinea pig.

pHo is an important determinant of vascular tone in cerebral blood vessels. We investigated the effects of changes in pHo on isolated smooth muscle cells from the basilar artery of the guinea pig. Single cells contracted rapidly in response to an elevation in pHo (constant CO2), and contraction was blocked by nifedipine, suggesting a role for dihydropyridine-sensitive Ca2+ channels. In whole-cell patch-clamp experiments, changes in pHo (pHo 5.7-8.1, pHi 7.2 with 10 mM HEPES) strongly affected the amplitude of the peak Ca2+ channel current (10 mM Ba2+, +15 mV, holding potential of -55 mV), with an apparent pK of 6.9. The current-voltage curves were minimally shifted, indicating no important effect of surface charge. To separate the slowly inactivating L-type Ca2+ channel current from the more rapidly inactivating B-type current, the decaying portions of inward currents from cells studied with repetitive 1-second pulses (+15 mV, holding potential of -55 mV) were fit to a two-component model. Titration curves for the L-type and B-type currents indicated maximum increases by factors of 3.65 and 1.28 at alkaline pHo and gave apparent pK values of 7.71 and 6.47 (Hill coefficient unity). The time constant of inactivation for the B-type current at +15 mV was little affected by pHo, whereas that for the L-type current increased somewhat with increasing pHo. Additional experiments showed no significant effect of pHo on holding current or on voltage-activated outward currents (pCai 7 with 11 mM EGTA). Our results provide additional evidence for participation of Ca2+ channels in regulating basal tone in cerebral smooth muscle and indicate that pHo regulates current through slowly inactivating, dihydropyridine-sensitive L-type Ca2+ channels.     

L- and T-type voltage-gated Ca2+ channels in human granulosa cells: functional characterization and cholinergic regulation

Using the whole-cell configuration of the patch-clamp technique, we have characterized two types of ionic currents through voltage-dependent Ca2+ channels in human granulosa cells. One is long-lasting, activates at approximately -20 mV, reaches the peak at approximately +20 mV, has an inactivation time constant of 132.5 +/- 5.6 msec at 20 mV, and is sensitive to dihydropyridines. The other is transient, activates at approximately -40 mV, peaks at approximately -10 mV, has an inactivation time constant of 38.8 +/- 1.8 msec at -10 mV, displays a voltage-dependent inactivation, and is sensitive to 100 microm Ni2+, but not to dihydropyridines. Biophysical and pharmacological properties of these currents indicate that they are gated through L- and T-type calcium channels, respectively. The cholinergic receptor agonist carbachol (50 microm) reduces the amplitude of the currents through both L-type (-34.7 +/- 6.4%; n = 10) and T-type (-52.6 +/- 7.4%; n = 8) channels, suggesting a possible role of these channels in the cholinergic regulation of human ovarian functions.     

Two types of calcium channels in human ovarian endocrine cells: involvement in steroidogenesis

Nature, regulation, and functional role of ion channels of human ovarian endocrine cells are not well known. In our present study, we show two types of voltage-activated Ca(2+) currents (I(Ca)) in cultured human luteinized granulosa cells (GCs), as assessed by whole-cell patch-clamp experiments. Electrophysiological properties, namely low threshold of activation, pronounced time-dependent inactivation, slow and voltage-dependent deactivation kinetics, insensitivity to SNX-482, and high sensitivity to Ni(2+), defined the predominant I(Ca) as a T-type Ca(2+) current (I(Ca.T)). In 4% of cells a Ni(2+)-insensitive I(Ca) was measured alone or together with I(Ca.T). This Ca(2+) current was high voltage activated and highly sensitive to dihydropyridine, indicative of an L-type Ca(2+) current. RT-PCR analysis demonstrated the presence of mRNA coding for alpha(1)-subunits of two different Ca(2+) channels (T-type Ca(v)3.2 and L-type Ca(v)1.2) in GCs. In addition, these two types were detected in the human corpus luteum by RT-PCR (Ca(v)3.2) and immunohistochemistry (Ca(v)1.2). Although stimulation of cultured GCs with human chorionic gonadotropin did not change the characteristics of recorded I(Ca.T), it markedly increased the percentage of cells displaying I(Ca) from 29 to 63% and significantly increased (2.2-fold) the density of I(Ca.T). Furthermore, the stimulatory effect of human chorionic gonadotropin on progesterone production was diminished by pharmacological blockage of I(Ca.T) by Ni(2+) or flunarizine. Thus, our study provides evidence that human GCs in vivo and in vitro express T- and L-type Ca(2+) channels and that the Ca(v)3.2 (also called alpha(1H)) isoform is involved in a fundamental endocrine function of these cells.     

The dietary flavonoid quercetin activates BKCa currents in coronary arteries via production of H2O2. Role in vasodilatation

OBJECTIVE: Large conductance Ca(2+)-activated K(+) channels (BKCa) regulate coronary artery tone in vivo, play a key role in blood pressure regulation, and have been suggested as novel potential drug targets in hypertension. Quercetin exerts systemic and coronary vasodilator effects in vitro and reduces blood pressure in several rat models of hypertension, and its consumption is associated with a lower mortality rate from coronary heart disease in epidemiological studies. We hypothesized that quercetin might activate BKCa channel in isolated myocytes from rat coronary arteries and that this mechanism might be involved in its coronary artery relaxant effects. METHODS: Membrane currents were measured using the whole-cell configuration of the patch-clamp technique. Contractile tension was recorded in rat coronary artery rings mounted in a myograph. RESULTS: Quercetin (>0.1 muM) increased the outward currents in the whole range of test potentials, hyperpolarized cell membranes, and increased the frequency of spontaneous transient outward currents (STOCs) carried by BKCa channels. These effects were abolished by the selective BKCa blocker iberiotoxin and by catalase. Quercetin increased dichlorofluorescein fluorescence in coronary arteries in a polyethylenglycol-catalase-sensitive manner, indicating that it increased cytosolic H(2)O(2). The membrane-permeable analogue of H(2)O(2)t-butylhydroperoxide mimicked the effects of quercetin on outward currents. The vasodilator effect of quercetin in isolated rat coronary arteries was partially inhibited by iberiotoxin. CONCLUSION: Quercetin increased BKCa currents via production of intracellular H(2)O(2). This effect is involved, at least partly, in the coronary vasodilator effects of quercetin.     

Evidence for the stimulatory effect of resveratrol on Ca(2+)-activated K+ current in vascular endothelial cells

OBJECTIVE: Resveratrol, a natural phytoalexin compound, is present in grapes and wine, and it can produce vasorelaxation. However, little is known of its mechanisms of action on ionic currents in endothelial cells. METHODS: The effect of resveratrol on Ca(2+)-activated K+ currents in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein was investigated with the aid of the patch-clamp technique. RESULTS: In the whole-cell configuration, resveratrol reversibly increased the amplitude of K+ outward currents. The increase in outward current caused by resveratrol was greatly inhibited by iberiotoxin (200 nM) or paxilline (1 microM), but not by glibenclamide (10 microM), tamoxifen (10 microM), or beta-bungarotoxin (200 nM). Thus, this outward current is believed to be Ca(2+)-activated K+ current (I K(Ca)). In the inside-out configuration, bath application of resveratrol (30 microM) caused no change in the single-channel conductance, but increased the activity of large-conductance Ca(2+)-activated K+ (BKCa) channels. Resveratrol enhanced the channel activity in a concentration-dependent manner. The EC50 value for resveratrol-induced channel activity was 20 microM. The resveratrol-stimulated increase in the channel activity was independent of internal Ca2+. Resveratrol (30 microM) also shifted the activation curve of BKCa channels to less positive membrane potentials. The change in the kinetic behavior of BKCa channels caused by resveratrol in these cells in due to an increase in mean open time and a decrease in mean closed time. In a pancreatic islet endothelial cell line (MS1), resveratrol (30 microM) also increased the activity of intermediate-conductance KCa channels. CONCLUSIONS: These results provide evidence that in addition to the presence of antioxidative activity, resveratrol can also stimulate KCa channels in endothelial cells. The direct stimulation of these KCa channels by resveratrol may be responsible for its effect on the functional activities of endothelial cells.     

Inhibitory effects of carvedilol on calcium channels in vascular smooth muscle cells

Carvedilol has hypotensive effects and inhibits agonist-induced cell proliferation of vascular smooth muscle and then prevents vascular remodeling. However, the basic mechanisms have not been clarified. We examined the effects of carvedilol on [Ca2+]i mobilization and voltage-dependent L-type Ca2+ current (ICa.L) in vascular smooth muscle cells, and compared them with metoprolol. [Ca2+]i was measured using fura-2 AM and patch clamp techniques in rat embryonic aortic smooth muscle cells (A7r5). In the presence of extracellular Ca2+, vasopressin and endothelin-1 increased [Ca2+]i due first to the Ca2+ release from store sites, and subsequently Ca2+ entry. Carvedilol did not inhibit the Ca2+ release, but significantly suppressed the sustained rise due to Ca2+ entry concentration-dependently. Nilfedipine and nicardipine (10 microM) partly inhibited the sustained rise, but carvedilol inhibited it more effectively than the Ca2+ channel blockers. Under voltage clamp conditions, carvedilol (0.2-10 microM) reversibly inhibited the ICa.L concentration-dependently without any changes in the current-voltage relationships of ICa.L. Carvedilol shifted the steady-state inactivation for ICa.L to more negative potentials and inhibited ICa.L in a voltage-dependent manner. In addition, carvedilol did not inhibit Ca2+ release from store sites induced by thapsigargin, but significantly inhibited the sustained rise due to capacitative Ca2+ entry unrelated to ICa.L. In contrast, metoprolol did not mimic these effects of carvedilol. These results provide evidence that carvedilol inhibits ICa.L and may also inhibit the channels for agonist (vasopressin and endothelin-1)-induced Ca2+ entry in vascular smooth muscle cells, which might contribute to the vasorelaxing and antiproliferative effects of carvedilol.     

Role of voltage-dependent and Ca(2+)-activated K(+) channels on the regulation of isometric force in porcine coronary artery

We investigated the role of K(+) channels in the regulation of vascular tone in de-endothelialized porcine coronary artery. Isometric force and intracellular Ca(2+) ([Ca(2+)](i)) under resting conditions were increased by treatment with 4-aminopyridine (4-AP, 1 mM), an inhibitor of voltage-dependent K(+) (K(v)) channels, but not by tetraethylammonium chloride (TEA, 1 mM) or charybdotoxin (100 nM), both inhibitors of Ca(2+)-activated K(+) (K(Ca)) channels, or glibenclamide (10 microM), an inhibitor of ATP-sensitive K(+) channels. Under stimulated conditions with 9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F(2alpha) (U46619), 4-AP as well as TEA or charybdotoxin increased isometric force and [Ca(2+)](i), but not glibenclamide. 4-AP was the most potent in terms of depolarization of membrane potential compared with TEA or glibenclamide in the presence or absence of EGTA. In the presence of U46619, a high concentration of 4-AP (10 mM) caused a further contraction with oscillations. The force oscillations induced by 4-AP were inhibited by diltiazem (10 microM), an inhibitor of voltage-dependent Ca(2+) channels, or TEA (1 mM), but not by glibenclamide (10 microM). These force oscillations may be associated with the periodic activation of K(Ca) channels. These findings suggested that 4-AP-sensitive K(v) channels play an important role in the control of vascular tone in both resting and stimulated conditions. Moreover, under stimulated conditions, K(Ca) channels also have an important role in the regulation of vascular tone. Dysfunction of these channels induces abnormal vasoconstriction and may be implicated in vascular diseases such as hypertension and vasospasm.     

Dopamine inhibits two characterized voltage-dependent calcium currents in identified rat lactotroph cells

The effects of dopamine (DA) on voltage-dependent Ca2+ currents were investigated in cultured rat lactotroph cells using the patch clamp recording technique. Each recorded cell was identified by the reverse hemolytic plaque assay. In the whole-cell configuration, two types of Ca2+ currents, L and T, were characterized on the basis of their kinetics, voltage sensitivity, and pharmacology. The L component had a threshold of -25 mV, showed little inactivation during a 150-msec voltage step, and was maximal at +10 mV. Cadmium ions (100 microM) significantly reduced its amplitude (75%). The T component was activated at a membrane potential close to -50 mV, was maximal at -10 mV, and showed a voltage-dependent inactivation between -90 and -30 mV. It was quickly inactivated during a maintained depolarization (time constant, 27 ms at -30 mV) and was strongly reduced (80%) by nickel ions (100 microM). Bath application of DA (10 nM) caused a markedly general depression of inward Ca2+ currents, acting differently on the T- and L-type currents. DA application shifted the voltage-dependence of the L-type current activation toward depolarization values (8 mV) without modifying its time- and voltage-dependent inactivation. In contrast, DA enhanced the inactivation of the T-type current by accelerating its time-dependent inactivation (25% decrease in the time constant of inactivation) and by shifting the voltage-dependence of the T-type current inactivation toward hyperpolarizing values (-63 mV in control vs. -77 mV in the presence of DA). These effects of DA were dose-dependent and involved the activation of a D2 receptor type. They were mimicked by bromocriptine application (10 nM), whereas sulpiride (100 nM) blocked the DA-evoked response. The D1 antagonist SCH 23390 was ineffective up to 100 microM. All of these DA-induced modifications in Ca2+ currents were abolished using a GTP-free pipette solution or after pretreatment of cells with pertussis toxin, suggesting that DA can regulate the function of Ca2+ channels through GTP-binding proteins (G-proteins). Our results show that DA acts simultaneously by reducing both voltage-dependent Ca2+ currents on lactotroph cells. Thus, DA reduces the entry of Ca2+ ions across the surface membrane and thereby influences electrical activity and the cytosolic free Ca2+ concentration involved in both basal and evoked PRL release.     

Properties and Modulation of Ca channels in adult human atrial cells.

Ca-channel currents have been investigated in single cells isolated from adult human atrium using the whole-cell patch clamp technique. Ca-channel currents are activated at voltage positive to -40 mV, peak between -10 and 0 mV and inactivate with a slow decay when Ba2+ ions (5 mM) are used as charges carrier. These properties correspond to those of the high voltage activated, DHP-sensitive, (L-type) Ca channel. No low voltage activated (T-type) currents have been evidenced. The present work also provides the first report about the modulation of Ca channels in adult human atrial cells by beta-adrenergic agonists and dihydropyridines (agonists and antagonists). Electrophysiological and pharmacological properties of these Ca channels are qualitatively similar to those of the L-type Ca currents recorded from cardiac animal cells. However, at a physiological calcium concentration (2 mM), basal Ca currents are often very small or even absent but are revealed following the addition of the dihydropyridine (DHP) agonist Bay K 8644. Whether the decrease of the basal Ca current amplitude may be related to the chronic pretreatment of the patients by Ca channel blockers or to the pathology is discussed.     

Activation thresholds of K(V), BK andCl(Ca) channels in smooth muscle cells in pial precapillary arterioles

We have previously shown expression of voltage-gated K+ channels (K(V)) in smooth muscle of cerebral arterioles and suggested the channels function to oppose voltage-dependent Ca2+ entry. However, other studies indicate that large conductance Ca2+-activated K+ (BK) channels serve this function and chloride (Cl-) channels may have the opposite effect. In this study we compared the activation thresholds and absolute current amplitudes for K(V) channels, BK channels and Cl- channels at physiological membrane potentials in intact precapillary arterioles from the rabbit cerebral circulation. Patch-clamp recordings were made to measure current and membrane potential, and a video scan line was used to detect external diameter. Two strategies to determine the basal current-voltage relationship of BK channels showed the channels contributed current only at voltages positive of -35 mV, even though voltage-dependent Ca2+-entry occurred. Ca2+-activated and niflumic acid-sensitive Cl- current was detected but, between -50 and -10 mV, both BK and Cl- channel currents were much smaller and contributed less to the membrane potential compared with K(V) channel current. Furthermore, in the absence of an exogenous vasoconstrictor agent, block of K(V) channels but not BK or Cl- channels caused constriction, although in the presence of endothelin-1 block of BK or K(V) channels caused constriction. The data indicate K(V) channels are the first inhibitory mechanism to activate when there is depolarisation in precapillary arteriolar smooth muscle cells of the cerebral circulation.