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The involvement of inositol 1,4,5-trisphosphate (IP(3)) formation in the voltage-dependent regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) was examined in smooth muscle cells of the porcine coronary artery. Slow ramp depolarization from -90 to 0 mV induced progressive [Ca(2+)](i) increase. The slope was reduced or increased in the presence of Cd(2+) or (±)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]-phenyl)pyridine-3-carboxlic acid methyl ester (Bay K 8644), respectively. The decrease in [Ca(2+)](i) via the membrane hyperpolarization induced by K(+) channel openers (levcromakalim and Evans blue) under current clamp was identical to that under voltage clamp. The step hyperpolarization from -40 to -80 mV reduced [Ca(2+)](i) uniformly over the whole-cell area with a time constant of ∼10 s. The [Ca(2+)](i) at either potential was unaffected by heparin, an inhibitor of IP(3) receptors. Alternatively, [Ca(2+)](i) rapidly increased in the peripheral regions by depolarization from -80 to 0 mV and stayed at that level (∼400 nM) during a 60-s pulse. When the pipette solution contained IP(3) pathway blockers [heparin, 2-aminoethoxydiphenylborate, xestospongin C, or 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U73122)], the peak [Ca(2+)](i) was unchanged, but the sustained [Ca(2+)](i) was gradually reduced by ∼250 nM within 30 s. In the presence of Cd(2+), a long depolarization period slightly increased the [Ca(2+)](i), which was lower than that in the presence of heparin alone. In coronary arterial myocytes, the sustained increase in the [Ca(2+)](i) during depolarization was partly caused by the Ca(2+) release mediated by the enhanced formation of IP(3). The initial [Ca(2+)](i) elevation triggered by the Ca(2+) influx though voltage-dependent Ca(2+) channels may be predominantly responsible for the activation of phospholipase C for IP(3) formation.

In this study, we provide evidence of critical changes in the expression of non-selective cation currents (NSCC) during culture in rat aortic myocytes. A selective TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate (4alphaPDD), had little effect on membrane currents and intracellular Ca(2+) (Ca(2+)(i)) in freshly isolated cells from the aorta. In contrast, in cultured aortic myocytes with and without serum, 4alphaPDD at a concentration range between 0.3 and 3 muM effectively elevated Ca(2+)(i), which was abolished in the absence of external Ca(2+). Application of 4alphaPDD to cultured aortic myocytes also activated NSCC, which had a reversal potential of +3 mV. Both of these signals were blocked by ruthenium red (RuR), an effective blocker of TRPVs. Although the expression of TRPV4 mRNA transcript was found in cultured as well as non-cultured aortic myocytes, significant immunoreactivity to TRPV4 protein was only detected in cultured rat aortic myocytes. Moreover, cultured human pulmonary arterial smooth muscle cells (hPASM) had a substantial response to 4alphaPDD, which was susceptible to the removal of external Ca(2+) and application of RuR. These results provide a strong basis for our proposal that endogenous TRPV4 functions as an important regulator of Ca(2+)(i) in vascular myocytes under some physiological and pathophysiological conditions.

Changes in K(+) conductances and their contribution to membrane depolarization in the setting of an acidic pH environment have been studied in myocytes from aortic smooth muscle cells of spontaneously hypertensive rats (SHR) compared with those from Wistar-Kyoto (WKY) rats. The resting membrane potential (RMP) of aortic smooth muscle at extracellular pH (pH(o)) of 7.4 was significantly more depolarized in SHR than in WKY rats. Acidification to pH(o) 6.5 made this difference in RMP between SHR and WKY rats more significant by further depolarizing the SHR myocytes. Large-conductance Ca(2+)-activated K(+) (BK) currents, which were markedly suppressed by acidification, were larger in aortic myocytes of SHR than in those of WKY rats. In contrast, acid-sensitive, non-BK currents were smaller in SHR. Western blot analyses showed that expression of BK-alpha- and -beta(1) subunits in SHR aortas was upregulated and comparable with those in WKY rats, respectively. Additional electrophysiological and molecular studies showed that pH- and halothane-sensitive two-pore domain weakly inward rectifying K(+) channel (TWIK)-like acid-sensitive K(+) (TASK) channel subtypes were functionally expressed in aortas, and TASK1 expression was significantly higher in WKY than in SHR. Although the background current through TASK channels at normal pH(o) (7.4) was small and may not contribute significantly to the regulation of RMP, TASK channel activation by halothane or alkalization (pH(o) 8.0) induced significant hyperpolarization in WKY but not in SHR. In conclusion, the larger depolarization and subsequent abnormal contractions after acidification in aortic myocytes in the setting of SHR hypertension are mainly attributable to the larger contribution of BK current to the total membrane conductance than in WKY aortas.

In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1-phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5-TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation.

Effects of sphingosine-1-phosphate (S1P), a putative lipid mediator, was examined in vascular smooth muscle and endothelial cells by means of measuring intracellular Ca2+ concentration ([Ca2+]i), whole-cell membrane currents and tension. Application of S1P in a concentration range from 30 to 1000 nM to human umbilical vein endothelial cells (HUVECs) induced a slowly developing increase in [Ca2+]i. and concomitant non-selelctive cationic inward currents. Pretreatment of HUVECs with pertussis toxin (PTX) almost abolished the S1P-induced elevation of [Ca2+]i, suggesting that PTX sensitive edg1 receptors are involved in the S1P-induced response in HUVECs. Moreover, treatment of HUVECs with S1P for 24 hrs caused cell-proliferation to 150% of the control. On the other hand, application of S1P had no effect on [Ca2+]i in rabbit, rat and mouse aortic myocytes. When S1P applied to rabbit aorta with endothelium, the muscle ring had small relaxation which disappeared in endothelium-denuded aorta. In contrast, rat cerebral arterial myocytes had increase in [Ca2+]i in the presence of S1P. These results suggest that responses of vascular tissues to S1P vary from cell to cell. To clarify physiological and pharmacological significance of S1P in cardiovascular system, comprehensive analyses are required.

The mechanisms underlying the activation of large conductance Ca2+-activated K+ (BK) channel by nordihydroguaiaretic acid (NDGA) were examined in human embryonic kidney (HEK293) cells, where BK channel alpha (BKalpha) or a plus beta1 subunit (BKalphabeta1) was heterologously expressed, and also in freshly isolated porcine coronary arterial smooth muscle cells (PCASMCs). The activity of both BKalpha and BKalphabeta1 channels was increased by 10 microM NDGA in similar manners, indicating the selective action on the a subunit to increase Ca2+ sensitivity. The application of NDGA to PCASMCs induced outward current and hyperpolarization under voltage and current clamp, respectively, in a concentration-dependent manner (> or = 3 microM). These effects were blocked by 100 nM iberiotoxin. Electrical events induced by NDGA (> or = 10 microM) were, unexpectedly, associated with the increase in [Ca2+]i. After the treatment with caffeine and ryanodine, the [Ca2+]i increase by NDGA was markedly reduced and the hyperpolarization by NDGA was attenuated. The Ca2+ release by 10 microM NDGA was preceded by membrane depolarization of mitochondria. These results indicate that BK channel opening by NDGA in PCASMCs is due to the direct action on a subunit and also to Ca2+ release from sarcoplasmic reticulum, presumably via, at least in part, the inhibition of mitochondria respiration.

Simultaneous recording of Ca2+-images in one confocal plane from vascular smooth muscle cells (SMCs) and endothelial cells (ECs) of an intact rat femoral artery segment was performed using indo-1 and a confocal microscope. During application of 10 microM acetylcholine (ACh), elevation and oscillation of intracellular Ca2+ concentration ([Ca2+]i) were observed in ECs but not in SMCs. Sequential conduction of Ca2+ oscillation from an EC to the neighboring ECs in one longitudinal direction was often observed in the presence of ACh. On the other hand, the activation of voltage-dependent Ca2+ channels by external 30 mM K+ resulted in the elevation of [Ca2+]i only in SMCs. When 10 microM ACh was added in the presence of 30 mM K+, it was observed in one confocal plane that [Ca2+]i in ECs and SMCs was almost simultaneously increased and decreased, respectively. The simultaneous recording method in this intact preparation will provide a line of valuable information about the interactions between SMCs and ECs, based on spatio-temporal analyses of absolute values of [Ca2+]i in individual cells.

1. Effects of NS-1619, an opener of large conductance Ca2+-activated K+ (BK) channel, on intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were examined in single myocytes freshly isolated from porcine coronary artery. 2. Under current clamp mode, the application of 1-30 microM NS-1619 hyperpolarized the membrane in concentration-dependent manner. The NS-1619-induced hyperpolarization was abolished by the presence of 100 nM iberiotoxin. 3. Application of 1-10 microM NS-1619 hyperpolarized the membrane by approximately 6 mV or less but did not change significantly the [Ca2+]i. When membrane hyperpolarization of 12 mV or so was caused by 30 microM NS-1619, [Ca2+]i was unexpectedly increased by approximately 200 nM. This increase in [Ca2+]i and the concomitant outward current activation were also observed under voltage-clamp at holding potential of -40 mV. 4. The increase in [Ca2+]i by 30 microM NS-1619 occurred mainly in peripheral regions than in the centre of the myocytes. The removal of extracellular Ca2+ affected neither the membrane hyperpolarization nor the increase in [Ca2+]i. 5. In the presence of 10 mM caffeine and 10 microM ryanodine, the increase in [Ca2+]i by 30 microM NS-1619 was not observed and the membrane hyperpolarization was reduced to approximately 67% of the control. 6. These results indicate that the opening of BK channels by NS-1619 at 30 microM, which is the most frequently used concentration of this agent, is partly due to Ca2+ release from caffeine/ryanodine-sensitive intracellular storage sites but is mainly due to the direct activation of the channels.

The relationship between the density of ionic currents through major two channels, voltage-dependent L-type Ca2+ channels (L-type VDCC) and large-conductance, Ca2+-activated K+ channels (BKC), and the mRNA expression levels of alpha1C subunit of L-type VDCC (alpha1C) and alpha/beta subunits of BKC (alphaBK/betaBK) were compared in smooth muscle cells (SMC) of rabbit aorta and vas deferens using whole cell-voltage clamp and reverse transcription-polymerase chain reaction (RT-PCR) analyses. The density of the currents through VDCC (ICa) and BKC (IK,Ca) at +10 mV in aortic SMC was approximately one-seventh and one-sixth respectively of that in vas deferens. Whilst application of the Ca2+ channel agonist Bay K 8644 increased ICa by 75-90% in these SMC, the increase in IK,Ca was far greater in aorta than in vas deferens. The expression of the alpha1C transcript in vas deferens was approximately 3.5 times higher than that in aorta. In contrast, expression of alphaBK/betaBK was almost identical in both tissues, indicating the dissociation of IK,Ca density from the expression levels of BKC transcripts in aorta. The results were supported by Western blot and immunocytochemical analyses using subunit-specific antibodies. The lower Ca2+ influx through VDCC in aorta activates only a very limited fraction of BKC compared with that in vas deferens. The greater expression of BKC than of VDCC in aortic SMC contributes to a strong negative feed-back mechanism that minimizes membrane depolarization and acts as a safety margin to maintain low membrane excitability.

Voltage-dependent calcium channels are crucially important for calcium influx and the following smooth muscle contraction. Beta subunits of these channels are known to modify calcium currents through pore-forming alpha subunits. Among the four reported independent beta subunits, the beta3 subunit is expressed in smooth muscle cells and thought to compose L-type calcium channels in the tissue. To determine the role of the beta3 subunit in the cardiovascular system, we have analyzed beta3-null mice. Electrophysiological examinations proved the existence of dihydropyridine (DHP)-sensitive. L-type calcium channels in the smooth muscle cells. Beta3-null mice show no apparent changes in smooth muscle contraction and sensitivity to DHP, and normal blood pressure when they are raised on a normal diet, but the 13 subunit deficient mice show elevated blood pressure in response to a high-salt diet, with significant reductions in plasma catecholamine concentrations. Our finding strongly suggests a close relationship between voltage-dependent channels and high blood pressure.

1. Effects of K+ channel opener, levcromakalim, on vascular endothelial cells were examined. Under voltage- and current-clamp conditions, application of acetylcholine to dispersed endothelial cells isolated from rabbit superior mesenteric artery (dispersed RMAECs) produced hyperpolarization and outward currents. On the other hand, dispersed RMAECs did not respond to levcromakalim. 2. When membrane potential was recorded from endothelium in a mesenteric arterial segment, exposure to levcromakalim in a concentration range of 0.1 to 3 microM caused concentration-dependent hyperpolarization. The hyperpolarization was observed in the absence of external Ca2+ and was inhibited by 10 microM glibenclamide. 3. The presence of 1 mM heptanol did not affect the levcromakalin-induced hyperpolarization, whereas treatment of the mesenteric arterial segment with 20 microM 18 beta-glycyrrhetinic acid significantly reduced the hyperpolarization. The response to acetylcholine of RMAECs in an arterial segment with 18 beta-glycyrrhetinic acid was, however, similar to that without 18 beta-glycyrrhetinic acid. 4. These suggest that although RMAECs themselves are functionally insensitive to levcromakalim, those in an arterial segment are hyperpolarized by levcromakalim via myo-endothelial electrical communication.

The effects of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor and an antioxidant, on membrane currents were examined in single smooth muscle cells isolated from porcine coronary artery. Spontaneous transient outward currents (STOCs) recorded at -30 mV were markedly enhanced by NDGA (>/=10 microM). Pretreatment with caffeine and ryanodine abolished STOCs and reduced NDGA-induced increase in outward current at -30 mV by approximately 60%. NDGA showed dual action on an outward current elicited by step depolarization from -60 to 0 mV: inhibition and enhancement at concentrations of 3 and >/=10 microM, respectively. In the presence of Cd(2+), the inhibition of outward current by NDGA disappeared and the enhancement remained. NDGA inhibited both the voltage-dependent Ca(2+) channel current (IC(50) = 2.5 microM) and the delayed rectifier K(+) current (IC(50) = 9.8 microM). The NDGA-induced enhancement of STOCs and outward currents on depolarization was abolished by 100 nM iberiotoxin but was not affected by glibenclamide or apamin. Under current clamp mode, 30 microM NDGA significantly hyperpolarized myocytes. The application of lipoxygenase inhibitors (caffeic acid and esculetin), a cyclooxygenase inhibitor (indomethacin), antioxidants (ascorbic acid and erythorbic acid), and structural-related compounds of NDGA (catechol and dopamine) did not enhance K(+) currents. These results indicate that the opening of the large conductance Ca(2+)-dependent K(+) channel by NDGA, which is independent of its lipoxygenase inhibition or antioxidant effect, results in membrane hyperpolarization.