Activation of Ca2+-activated Cl- channels by store-operated Ca2+ entry in arterial smooth muscle cells does not require reverse-mode Na+/Ca2+ exchange

The main purpose of this study was to characterize the stimulation of Ca(2+)-activated Cl(-) (Cl(Ca)) by store-operated Ca(2+) entry (SOCE) channels in rabbit pulmonary arterial smooth muscle cells (PASMCs) and determine if this process requires reverse-mode Na(+)/Ca(2+) exchange (NCX). In whole-cell voltage clamped PASMCs incubated with 1 μmol/L nifedipine (Nif) to inhibit Ca(2+) channels, 30 μmol/L cyclopiazonic acid (CPA), a SERCA pump inhibitor, activated a nonselective cation conductance permeable to Na(+) (I(SOC)) during an initial 1-3 s step, ranging from-120 to +60 mV, and Ca(2+)-activated Cl(-) current (I(Cl(Ca))) during a second step to +90 mV that increased with the level of the preceding hyperpolarizing step. Niflumic acid (100 μmol/L), a Cl(Ca) channel blocker, abolished I(Cl(Ca)) but had no effect on I(SOC), whereas the I(SOC) blocker SKF-96365 (50 μmol/L) suppressed both currents. Dual patch clamp and Fluo-4 fluorescence measurements revealed the appearance of CPA-induced Ca(2+) transients of increasing magnitude with increasing hyperpolarizing steps, which correlated with I(Cl(Ca)) amplitude. The absence of Ca(2+) transients at positive potentials following a hyperpolarizing step combined with the observation that SOCE-stimulated I(Cl(Ca)) was unaffected by the NCX blocker KB-R7943 (1 μmol/L) suggest that the SOCE/Cl(Ca) interaction does not require reverse-mode NCX in our conditions.     

Calcineurin Aalpha but not Abeta augments ICl(Ca) in rabbit pulmonary artery smooth muscle cells

Activation of Ca(2+)-dependent Cl(-) currents (I(Cl(Ca))) increases membrane excitability in vascular smooth muscle cells. Previous studies showed that Ca(2+)-dependent phosphorylation suppresses I(Cl(Ca)) in pulmonary artery myocytes, and the aim of the present study was to determine the role of the Ca(2+)-dependent phosphatase calcineurin on chloride channel activity. Immunocytochemical and Western blot studies with isoform-specific antibodies revealed that the alpha and beta forms of the CaN catalytic subunit are expressed in PA cells but that only the alpha variant translocated to the cell periphery upon a rise in intracellular [Ca(2+)]. I(Cl(Ca)) evoked by pipette solutions containing a [Ca(2+)] set at 500 nm was considerably larger when the pipette solution included constitutively active CaN containing the alpha catalytic isoform. This stimulatory effect was lost by boiling the enzyme or by the inclusion of a specific CaN inhibitory peptide and was not shared by the inclusion of the beta form of the catalytic subunit. In the absence of constitutively active CaN, cyclosporin A, an inhibitor of CaN, suppressed I(Cl(Ca)) evoked by 500 nm Ca(2+) when the current amplitude was relatively large but was ineffective in cells with smaller currents. In perforated patch recordings, cyclosporin A consistently inhibited I(Cl(Ca)) evoked as a consequence of Ca(2+) influx through voltage-dependent calcium channels. These novel data show that in PA myocytes activation of I(Cl(Ca)) is enhanced by Ca(2+)-dependent dephosphorylation and that the regulation of this conductance is highly isoform-specific.     

Effects of (-)-epigallocatechin-3-gallate in Ca2+ -permeable non-selective cation channels and voltage-operated Ca2+ channels in vascular smooth muscle cells

The effects of (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of tea, on Ca(2+)-permeable non-selective cation currents (NSCC) and voltage-operated Ca(2+) channels (VOCC) have been investigated in cultured rat aortic smooth muscle cells using the whole-cell voltage-clamp technique. Under the Cs(+)/tetraethylammonium (TEA)-containing internal solution, and in the presence of nifedipine (1 microM), EGCG (30 microM) activated a long-lasting inward current, with a reversal potential (E(rev)) of approximately 0 mV. This current was not significantly altered by the replacement of [Cl(-)](i) or [Cl(-)](o), implying that the inward current was not a chloride channel, but a NSCC. SKF 96365 (30 microM) and Cd(2+) (500 microM) almost completely abolished the EGCG-induced NSCC. A higher dose of EGCG (100 microM) additionally activated a nifedipine-sensitive inward current in the absence of depolarization protocol. EGCG (100 microM) also potentiated a nifedipine-sensitive voltage-dependent Ba(2+)-current during the first 5 min of incubation. However, after > 10 min of incubation with EGCG, this current was significantly inhibited. Our results suggest that EGCG caused a Ca(2+) influx into smooth muscle cells via VOCC (probably L-type) and other SKF-96365- and Cd(2+)-sensitive Ca(2+)-permeable channels. The action described here may be responsible for the contraction induced by EGCG in rat aortic rings and for the rise of the intracellular concentration of Ca(2+) in rat aortic smooth muscle cells evoked by this catechin. On the other hand, the inhibition of VOCC after > 10 min of incubation may be, in part, responsible for the relaxation of rat aorta induced by EGCG.     

Low-intensity interval exercise training attenuates coronary vascular dysfunction and preserves Ca²⁺-sensitive K⁺ current in miniature swine with LV hypertrophy

Coronary vascular dysfunction has been observed in several models of heart failure (HF). Recent evidence indicates that exercise training is beneficial for patients with HF, but the precise intensity and underlying mechanisms are unknown. Left ventricular (LV) hypertrophy can play a significant role in the development of HF; therefore, the purpose of this study was to assess the effects of low-intensity interval exercise training on coronary vascular function in sedentary (HF) and exercise trained (HF-TR) aortic-banded miniature swine displaying LV hypertrophy. Six months postsurgery, in vivo coronary vascular responses to endothelin-1 (ET-1) and adenosine were measured in the left anterior descending coronary artery. Baseline and maximal coronary vascular conductance were similar between all groups. ET-1-induced reductions in coronary vascular conductance (P < 0.05) were greater in HF vs. sedentary control and HF-TR groups. Pretreatment with the ET type A (ET(A)) receptor blocker BQ-123 prevented ET-1 hypersensitivity in HF animals. Whole cell voltage clamp was used to characterize composite K(+) currents (I(K(+))) in coronary smooth muscle cells. Raising internal Ca(2+) from 200 to 500 nM increased Ca(2+)-sensitive K(+) current in HF-TR and control, but not HF animals. In conclusion, an ET(A)-receptor-mediated hypersensitivity to ET-1, elevated resting LV wall tension, and decreased coronary smooth muscle cell Ca(2+)-sensitive I(K(+)) was found in sedentary animals with LV hypertrophy. Low-intensity interval exercise training preserved normal coronary vascular function and smooth muscle cell Ca(2+)-sensitive I(K(+)), illustrating a potential mechanism underlying coronary vascular dysfunction in a large-animal model of LV hypertrophy. Our results demonstrate the potential clinical impact of exercise on coronary vascular function in HF patients displaying pathological LV hypertrophy.     

L, P-/Q- and T-type Ca2+ channels in smooth muscle cells from human umbilical artery.

The electrophysiological and pharmacological properties of Ca(2+) current (I(Ca)) were determined by the whole-cell configuration of the patch-clamp technique in smooth muscle cells from human umbilical artery. Using 5 mM extracellular Ca(2+), depolarizing step pulses from -60 to 50 mV from a holding membrane potential of -80 mV evoked an I(Ca) which activated at membrane potentials more positive than -50 mV and exhibited a maximum current density in a range of 10-20 mV. Steady-state inactivation protocols using a V(test) of 10 mV gave a voltage at one-half inactivation and a slope factor of -35.6 mV and 9.5 mV, respectively. Nifedipine (1 microM), an L-type Ca(2+) channels antagonist, completely inhibited I(Ca), while the L-type Ca(2+) channels agonist Bay-K 8644 (1 microM) significantly increased I(Ca) amplitude. Moreover, the selective blocker of P-/Q-type Ca(2+) channels omega-agatoxin IVA partially blocked I(Ca) (about 40 % inhibition at +20 mV by 20 nM). These pharmacological results suggest that L- and P-/Q-type Ca(2+) channels, both nifedipine-sensitive, underlie the I(Ca) registered using low extracellular Ca(2+). The presence of the P-/Q-type Ca(2+) channels was confirmed by immunoblot analysis. When I(Ca) was recorded in a high concentration (30 mM) of extracellular Ca(2+) or Ba(2+) as current carrier, it was evident the presence of a nifedipine-insensitive component which completely inactivated during the course of the voltage-step (75 ms) at all potentials tested, and was blocked by the T-type Ca(2+) channels blocker mibefradil (10 microM). Summarizing, this work shows for the first time the electrophysiological and pharmacological properties of voltage-activated Ca(2+) currents in human umbilical artery smooth muscle cells.     

Effect of NYD-SP27 down-regulation on ATP-induced Ca2+-dependent pancreatic duct anion secretion in cystic fibrosis cells

Our previous study demonstrated that NYD-SP27 is a novel inhibitory PLC isoform expressed endogenously in human pancreas and upregulated in CFPAC-1 cells. The present study investigated the effect of NYD-SP27 down-regulation on the ATP-stimulated and Ca(2+)-dependent pancreatic anion secretion by CFPAC-1 cell line using short-circuit current (I(SC)) recording. NYD-SP27 antisense-transfected CFPAC-1 (AT-CF) cells exhibited a significantly higher basal transmembrane potential difference and current than those of empty vector-transfected CFPAC-1 (VT-CF) cells. Cl(-) channel blocker, DPC or Glibenclamide (1mM), and inhibitor of Na(+)-K(+)-Cl(-) cotransporter, bumetanide (100 microM) significantly inhibited the basal current in AT-CF cells. The inhibitor of adenylate cyclase, MDL12330A (20 microM), and Ca(2+)-dependent Cl(-) channel (CaCC) blocker, DIDS (100 microM) also significantly reduced the basal current in AT-CF. Apical application of ATP (10 microM) stimulated a fast transient I(SC) increase in VT-CF cells, but a more sustained rise with slower decline in AT-CF cells. Pretreatment with BAPTA-AM (50 microM) reduced the ATP-induced I(SC) response in AT-CF cells by 77.9%. PMA (1 microM), a PKC activator, inhibited the ATP-stimulated current increase (the transient peak) in VT-CF cells, but had no effect on the AT-CF cells. However, PKC inhibitor, staurosporine (40 microM) could inhibit the ATP-induced I(SC) response in AT-CF cells. The present results confirm the previously proposed inhibitory role of NYD-SP27 in the PLC pathway and demonstrate that the suppression of its expression could result in an enhancement of ATP-stimulated Ca(2+) dependent pancreatic anion secretion.     

Intracellular Cl- fluxes play a novel role in Ca2+ handling in airway smooth muscle

Intracellular Ca(2+) is actively sequestered into the sarcoplasmic reticulum (SR), whereas the release of Ca(2+) from the SR can be triggered by activation of the inositol 1,4,5-trisphosphate and ryanodine receptors. Uptake and release of Ca(2+) across the SR membrane are electrogenic processes; accumulation of positive or negative charge across the SR membrane could electrostatically hinder the movement of Ca(2+) into or out of the SR, respectively. We hypothesized that the movement of intracellular Cl(-) (Cl(i)(-)) across the SR membrane neutralizes the accumulation of charge that accompanies uptake and release of Ca(2+). Thus inhibition of SR Cl(-) fluxes will reduce Ca(2+) sequestration and agonist-induced release. The Cl(-) channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10(-4) M), previously shown to inhibit SR Cl(-) channels, significantly reduced the magnitude of successive acetylcholine-induced contractions of airway smooth muscle (ASM), suggesting a "run down" of sequestered Ca(2+) within the SR. Niflumic acid (10(-4) M), a structurally different Cl(-) channel blocker, had no such effect. Furthermore, NPPB significantly reduced caffeine-induced contraction and increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Depletion of Cl(i)(-), accomplished by bathing ASM strips in Cl(-)-free buffer, significantly reduced the magnitude of successive acetylcholine-induced contractions. In addition, Cl(-) depletion significantly reduced caffeine-induced increases in [Ca(2+)](i). Together these data suggest a novel role for Cl(i)(-) fluxes in Ca(2+) handling in smooth muscle. Because the release of sequestered Ca(2+) is the predominate source of Ca(2+) for contraction of ASM, targeting Cl(i)(-) fluxes may prove useful in the control of ASM hyperresponsiveness associated with asthma.     

Characterization of stretch-activated cation current in coronary smooth muscle cells

Stretch-activated ion currents were recorded from vascular smooth muscle (VSM) after enzymatic isolation of single cells from porcine coronary arterioles. Patch pipettes were used to record whole cell current and control cell length. Under voltage clamp in physiological saline solution, an inward cation current (I(CAT)) was activated by 105--135% longitudinal stretch. I(CAT) coincided with an increase in intracellular Ca(2+) concentration. Under current clamp, membrane depolarization was induced by stretch. The magnitude of I(CAT) varied from -0.8 to -6.9 pA/pF at a holding potential of -60 mV. I(CAT) was graded with stretch, inactivated on release, and could be repeatedly induced. A potassium current (I(K)) activated in unstretched cells by depolarization was also enhanced by stretch. In Ca(2+)-free bath solution, stretch-induced enhancement of I(K) was blocked, but I(CAT) was still present. Hexamethyleneamiloride (50 microM), a reputed inhibitor of mechanosensitive channels, blocked I(CAT) and the stretch-induced increase in I(K) but not basal I(K). Grammostolla spatulata venom (1:100,000) blocked basal I(K), blocked stretch-induced increases in I(K), and blocked I(CAT). Iberiotoxin, a specific Ca(2+)-activated K(+) channel blocker, did not alter I(CAT) but blocked the stretch-induced increase in I(K) and increased the magnitude of stretch-induced depolarization. We concluded that longitudinal stretch directly activates a cation current and secondarily activates a Ca(2+)-activated K(+) current in isolated coronary myocytes. Although these two currents would partially counteract each other, the predominance of I(CAT) at physiological potentials is likely to explain the depolarization and contraction observed in intact coronary VSM during pressure elevation.     

Ca2+-activated Cl- channels in corpus cavernosum smooth muscle: a novel mechanism for control of penile erection.

Little is known of the excitatory mechanisms that contribute to the tonic contraction of the corpus cavernosum smooth muscle in the flaccid state. We used patch-clamp electrophysiology to investigate a previously unidentified inward current in freshly isolated rat and human corporal myocytes. Phenylephrine (PE) contracted cells and activated whole cell currents. Outward current was identified as large-conductance Ca(2+)-activated K(+) current. The inward current elicited by PE was dependent on the Cl(-) gradient and was inhibited by niflumic acid, indicative of a Ca(2+)-activated Cl(-) (Cl(Ca)) current. Furthermore, spontaneous transient outward and inward currents (STOCs and STICs, respectively) were identified in both rat and human corporal myocytes and derived from large-conductance Ca(2+)-activated K(+) and Cl(Ca) channel activity. STICs and STOCs were inhibited by PE and A-23187, and combined 8-bromoadenosine cAMP and 8-bromoadenosine cGMP decreased their frequency. When studied in vivo, chloride channel blockers transiently increased intracavernosal pressure and prolonged nerve-evoked erections. This report reveals for the first time Cl(Ca) current in rat and human corpus cavernosum smooth muscle cells and demonstrates its key functional role in the regulation of penile erection.     

Adenosine triphosphate induces inhibition of Na(+) absorption in mouse endometrial epithelium: a Ca(2+)-dependent mechanism

The present study investigated the inhibitory effect of extracellular ATP on Na(+) absorption and the possible underlying mechanism in cultured mouse endometrial epithelium using the short-circuit current (I(SC)) technique. The cultured epithelia exhibited a Na(+)-dependent basal current that could be predominately blocked by the epithelial Na(+) channel (ENaC) blocker, amiloride (10 microM). Apical addition of ATP (10 microM) induced a reduction in basal I(SC). However, in the presence of amiloride or when apical Na(+) was removed, the ATP-induced reduction was abolished and an increase in the I(SC) was observed with kinetic characteristics similar to those reported previously for the ATP-induced Cl(-) secretion, indicating that ATP could induce both Cl(-) secretion and inhibition of Na(+) absorption. Further reduction in I(SC) after ATP challenge could be obtained with forskolin (10 microM), which indicates that different inhibitory mechanisms are involved. The ATP-induced inhibition of Na(+) absorption, but not that induced by forskolin, could be abolished by the P(2) receptor antagonist, reactive blue (100 microM), indicating the involvement of a P(2) receptor in mediating the ATP response. ATP and uridine 5'-diphosphate (UDP; 100 microM), a relatively selective agonist for the pyrimidinoceptor, induced separate I(SC) reduction, and distinct I(SC) increases in the presence of amiloride, regardless of the order of drug administration, indicating the involvement of two receptor populations. The ATP-induced inhibition of Na(+) absorption was mimicked by the Ca(2+) ionophore, ionomycin (1 microM), whereas the Ca(2+) chelators, EGTA and BAPTA-AM, abolished the ATP-induced, but not the forskolin-induced, inhibition of Na(+) absorption, suggesting the involvement of a Ca(2+)-dependent pathway. In the presence of the Cl(-) channel blocker, DIDS (100 microM), both inhibitory and stimulatory responses to ATP were abolished, suggesting the involvement of a Ca(2+)-activated Cl(-) channels (CaCCs) in mediating both ATP responses. The ATP-induced as well as the forskolin-induced reduction in I(SC) was not observed when Cl(-) was removed from the bathing solution, indicating that Cl(-) permeation is important for the inhibition of Na(+) absorption. The results suggest the presence of a Ca(2+)-dependent ENaC-inhibiting mechanism involving CaCC in mouse endometrial epithelial cells. Thus, extracellular nucleotides may play an important role in the fine-tuning of the uterine fluid microenvironment by regulating both Cl(-) secretion and Na(+) absorption across the endometrium.     

A close association of RyRs with highly dense clusters of Ca2+-activated Cl- channels underlies the activation of STICs by Ca2+ sparks in mouse airway smooth muscle

Ca(2+) sparks are highly localized, transient releases of Ca(2+) from sarcoplasmic reticulum through ryanodine receptors (RyRs). In smooth muscle, Ca(2+) sparks trigger spontaneous transient outward currents (STOCs) by opening nearby clusters of large-conductance Ca(2+)-activated K(+) channels, and also gate Ca(2+)-activated Cl(-) (Cl((Ca))) channels to induce spontaneous transient inward currents (STICs). While the molecular mechanisms underlying the activation of STOCs by Ca(2+) sparks is well understood, little information is available on how Ca(2+) sparks activate STICs. In the present study, we investigated the spatial organization of RyRs and Cl((Ca)) channels in spark sites in airway myocytes from mouse. Ca(2+) sparks and STICs were simultaneously recorded, respectively, with high-speed, widefield digital microscopy and whole-cell patch-clamp. An image-based approach was applied to measure the Ca(2+) current underlying a Ca(2+) spark (I(Ca(spark))), with an appropriate correction for endogenous fixed Ca(2+) buffer, which was characterized by flash photolysis of NPEGTA. We found that I(Ca(spark)) rises to a peak in 9 ms and decays with a single exponential with a time constant of 12 ms, suggesting that Ca(2+) sparks result from the nonsimultaneous opening and closure of multiple RyRs. The onset of the STIC lags the onset of the I(Ca(spark)) by less than 3 ms, and its rising phase matches the duration of the I(Ca(spark)). We further determined that Cl((Ca)) channels on average are exposed to a [Ca(2+)] of 2.4 microM or greater during Ca(2+) sparks. The area of the plasma membrane reaching this level is <600 nm in radius, as revealed by the spatiotemporal profile of [Ca(2+)] produced by a reaction-diffusion simulation with measured I(Ca(spark)). Finally we estimated that the number of Cl((Ca)) channels localized in Ca(2+) spark sites could account for all the Cl((Ca)) channels in the entire cell. Taken together these results lead us to propose a model in which RyRs and Cl((Ca)) channels in Ca(2+) spark sites localize near to each other, and, moreover, Cl((Ca)) channels concentrate in an area with a radius of approximately 600 nm, where their density reaches as high as 300 channels/microm(2). This model reveals that Cl((Ca)) channels are tightly controlled by Ca(2+) sparks via local Ca(2+) signaling.     

Properties of a tonically active, sodium-permeable current in mouse urinary bladder smooth muscle

Urinary bladder smooth muscle (UBSM) elicits depolarizing action potentials, which underlie contractile events of the urinary bladder. The resting membrane potential of UBSM is approximately -40 mV and is critical for action potential generation, with hyperpolarization reducing action potential frequency. We hypothesized that a tonic, depolarizing conductance was present in UBSM, functioning to maintain the membrane potential significantly positive to the equilibrium potential for K(+) (E(K); -85 mV) and thereby facilitate action potentials. Under conditions eliminating the contribution of K(+) and voltage-dependent Ca(2+) channels, and with a clear separation of cation- and Cl(-)-selective conductances, we identified a novel background conductance (I(cat)) in mouse UBSM cells. I(cat) was mediated predominantly by the influx of Na(+), although a small inward Ca(2+) current was detectable with Ca(2+) as the sole cation in the bathing solution. Extracellular Ca(2+), Mg(2+), and Gd(3+) blocked I(cat) in a voltage-dependent manner, with K(i) values at -40 mV of 115, 133, and 1.3 microM, respectively. Although UBSM I(cat) is extensively blocked by physiological extracellular Ca(2+) and Mg(2+), a tonic, depolarizing I(cat) was detected at -40 mV. In addition, inhibition of I(cat) demonstrated a hyperpolarization of the UBSM membrane potential and decreased the amplitude of phasic contractions of isolated UBSM strips. We suggest that I(cat) contributes tonically to the depolarization of the UBSM resting membrane potential, facilitating action potential generation and thereby a maintenance of urinary bladder tone.     

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.     

Sphingosylphosphorylcholine induces Ca(2+)-sensitization of vascular smooth muscle contraction: possible involvement of rho-kinase

Sphingosylphosphorylcholine (SPC), a sphingolipid, concentration-dependently (1-50 microM) induced contraction and slight elevation of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in smooth muscle of the pig coronary artery, the result being a marked increase in the force/[Ca(2+)](i) ratio. In alpha-toxin- or beta-escin-permeabilized, but not Triton X-100-permeabilized, vascular strips, SPC induced contraction at constant [Ca(2+)](i) (pCa 6.3) in the absence of GTP, whereas a G-protein-coupled receptor agonist, histamine, required the presence of GTP to induce the contraction. The Rho-kinase blocker, Y-27632 (10 microM) abolished the SPC-induced Ca(2+)-sensitization, without affecting the Ca(2+)-induced contraction. These results suggest that SPC induces Ca(2+)-sensitization of force in vascular smooth muscle, presumably through the activation of Rho-kinase (or a related kinase).     

Essential role for calcium waves in migration of human vascular smooth muscle cells

Vascular smooth muscle cell (SMC) migration is characterized by extension of the lamellipodia at the leading edge, lamellipodial attachment to substrate, and release of the rear (uropod) of the cell, all of which enable forward movement. However, little is known regarding the role of intracellular cytosolic Ca(2+) concentration ([Ca(2+)](i)) in coordinating these distinct activities of migrating SMCs. The objective of our study was to determine whether regional changes of Ca(2+) orchestrate the migratory cycle in human vascular SMCs. We carried out Ca(2+) imaging using digital fluorescence microscopy of fura-2 loaded human smooth muscle cells. We found that motile SMCs exhibited Ca(2+) waves that characteristically swept from the rear of polarized cells toward the leading edge. Ca(2+) waves were less evident in nonpolarized, stationary cells, although acute stimulation of these SMCs with the agonists platelet-derived growth factor-BB or histamine could elicit transient rise of [Ca(2+)](i). To investigate a role for Ca(2+) waves in the migratory cycle, we loaded cells with the Ca(2+) chelator BAPTA, which abolished Ca(2+) waves and significantly reduced retraction, supporting a causal role for Ca(2+) in initiation of retraction. However, lamellipod motility was still evident in BAPTA-loaded cells. The incidence of Ca(2+) oscillations was reduced when Ca(2+) release from intracellular stores was disrupted with the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin or by treatment with the inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxy-diphenyl borate or xestospongin C, implicating Ca(2+) stores in generation of waves. We conclude that Ca(2+) waves are essential for migration of human vascular SMCs and can encode cell polarity.     

Ca(2+)- and volume-sensitive chloride currents are differentially regulated by agonists and store-operated Ca2+ entry

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca2+]i in a Ca(2+)-free medium followed by a secondary [Ca2+]i rise upon Ca2+ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca(2+)-activated (I(Cl(Ca))) and volume-regulated (I(Cl, swell)) chloride currents. NPPB and DIDS more efficiently inhibited I(Cl(Ca)) and I(Cl, swell), respectively. Cell swelling caused by hypotonic solution invariably activated I(Cl, swell) while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling-induced I(Cl, swell), while its inactive analogue U-73343 had no effect. I(Cl(Ca)) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca2+]i in the pipette solution at 500 nM. In contrast, I(Cl, swell) could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP3 infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced I(Cl, swell) in a nonadditive manner, suggesting their convergence on a common pathway. I(Cl, swell) and I(Cl(Ca)) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated I(Cl(Ca)), but abolished I(Cl, swell), thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that I(Cl, swell) can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a "selection" switch between closely localized channels.     

Protein kinase C-dependent phosphorylation of transient receptor potential canonical 6 (TRPC6) on serine 448 causes channel inhibition

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry following the stimulation of a G(q)-protein coupled or tyrosine kinase receptor. A dysregulation of TRPC6 activity causes abnormal proliferation of smooth muscle cells and glomerulosclerosis. In the present study, we investigated the regulation of TRPC6 activity by protein kinase C (PKC). We showed that inhibiting PKC with GF1 or activating it with phorbol 12-myristate 13-acetate potentiated and inhibited agonist-induced Ca(2+) entry, respectively, into cells expressing TRPC6. Similar results were obtained when TRPC6 was directly activated with 1-oleyl-2-acetyl-sn-glycerol. Activation of the cells with carbachol increased the phosphorylation of TRPC6, an effect that was prevented by the inhibition of PKC. The target residue of PKC was identified by an alanine screen of all canonical PKC sites on TRPC6. Unexpectedly, all the mutants, including TRPC6(S768A) (a residue previously proposed to be a target for PKC), displayed PKC-dependent inhibition of channel activity. Phosphorylation prediction software suggested that Ser(448), in a non-canonical PKC consensus sequence, was a potential target for PKCδ. Ba(2+) and Ca(2+) entry experiments revealed that GF1 did not potentiate TRPC6(S448A) activity. Moreover, activation of PKC did not enhance the phosphorylation state of TRPC6(S448A). Using A7r5 vascular smooth muscle cells, which endogenously express TRPC6, we observed that a novel PKC isoform is involved in the inhibition of the vasopressin-induced Ca(2+) entry. Furthermore, knocking down PKCδ in A7r5 cells potentiated vasopressin-induced Ca(2+) entry. In summary, we provide evidence that PKCδ exerts a negative feedback effect on TRPC6 through the phosphorylation of Ser(448).     

Mechanism of internal anal sphincter smooth muscle relaxation by phorbol 12,13-dibutyrate

We investigated the mechanism of the inhibitory action of phorbol 12,13-dibutyrate (PDBu), one of the typical protein kinase C (PKC) activators, in in vitro smooth muscle strips and in isolated smooth muscle cells of the opossum internal anal sphincter (IAS). The inhibitory action of PDBu on IAS smooth muscle (observed in the presence of guanethidine + atropine) was partly attenuated by tetrodotoxin, suggesting that a part of the inhibitory action of PDBu is via the nonadrenergic, noncholinergic neurons. A major part of the action of PDBu in IAS smooth muscle was, however, via its direct action at the smooth muscle cells, accompanied by a decrease in free intracellular Ca(2+) concentration ([Ca(2+)](i)) and inhibition of PKC translocation. PDBu-induced IAS smooth muscle relaxation was unaffected by agents that block Ca(2+) mobilization and Na+-K+-ATPase. The PDBu-induced fall in basal IAS smooth muscle tone and [Ca(2+)](i) resembled that induced by the Ca(2+) channel blocker nifedipine and were reversed specifically by the Ca(2+) channel activator BAY K 8644. We speculate that a major component of the relaxant action of PDBu in IAS smooth muscle is caused by the inhibition of Ca(2+) influx and of PKC translocation to the membrane. The specific role of PKC downregulation and other factors in the phorbol ester-mediated fall in basal IAS smooth muscle tone remain to be determined.