Molecular mechanisms of activation and regulation of ANO1-Encoded Ca2+-Activated Cl- channels.

Ca2+-activated Cl- channels (CaCCs) perform a multitude of functions including the control of cell excitability, regulation of cell volume and ionic homeostasis, exocrine and endocrine secretion, fertilization, amplification of olfactory sensory function, and control of smooth muscle cell contractility. CaCCs are the translated products of two members (ANO1 and ANO2, also known as TMEM16A and TMEM16B) of the Anoctamin family of genes comprising ten paralogs. This review focuses on recent progress in understanding the molecular mechanisms involved in the regulation of ANO1 by cytoplasmic Ca2+, post-translational modifications, and how the channel protein interacts with membrane lipids and protein partners. After first reviewing the basic properties of native CaCCs, we then present a brief historical perspective highlighting controversies about their molecular identity in native cells. This is followed by a summary of the fundamental biophysical and structural properties of ANO1. We specifically address whether the channel is directly activated by internal Ca2+ or indirectly through the intervention of the Ca2+-binding protein Calmodulin (CaM), and the structural domains responsible for Ca2+- and voltage-dependent gating. We then review the regulation of ANO1 by internal ATP, Calmodulin-dependent protein kinase II-(CaMKII)-mediated phosphorylation and phosphatase activity, membrane lipids such as the phospholipid phosphatidyl-(4,5)-bisphosphate (PIP2), free fatty acids and cholesterol, and the cytoskeleton. The article ends with a survey of physical and functional interactions of ANO1 with other membrane proteins such as CLCA1/2, inositol trisphosphate and ryanodine receptors in the endoplasmic reticulum, several members of the TRP channel family, and the ancillary Κ+ channel ß subunits KCNE1/5.

Molecular and functional characterization of Kv 7 channels in penile arteries and corpus cavernosum of healthy and metabolic syndrome rats.

BACKGROUND AND PURPOSE: KCNQ-encoded voltage-dependent potassium channels (Kv 7) are involved in the regulation of vascular tone. In this study we evaluated the influence of Kv 7 channel activation on smooth muscle relaxation in rat penile arteries and corpus cavernosum from normal and spontaneously hypertensive, heart failure-prone (SHHF) rats - a rat model of human metabolic syndrome. EXPERIMENTAL APPROACH: Quantitative PCR and immunohistochemistry were used to determine the expression of KCNQ isoforms in penile tissue. Isometric tension was measured in intracavernous arterial rings and corpus cavernosum strips isolated from normal and SHHF rats. KEY RESULTS: Transcripts for KCNQ3, KCNQ4 and KCNQ5 were detected in penile arteries and corpus cavernosum. KCNQ1 was only found in corpus cavernosum. Immunofluorescence signals to Kv 7.4 and Kv 7.5 were found in penile arteries, penile veins and corpus cavernosum. The Kv 7.2-7.5 activators, ML213 and BMS204352, relaxed pre-contracted penile arteries and corpus cavernosum independently of nitric oxide synthase or endothelium-derived hyperpolarization. Relaxations to sildenafil, a PDE5 inhibitor, and sodium nitroprusside (SNP), an nitric oxide donor, were reduced by blocking Kv 7 channels with linopirdine in penile arteries and corpus cavernosum. In SHHF rat penile arteries and corpus cavernosum, relaxations to ML213 and BMS204352 were attenuated, and the blocking effect of linopirdine on sildenafil-induced and SNP-induced relaxations reduced. KCNQ3, KCNQ4 and KCNQ5 were down-regulated, and KCNQ1 was up-regulated in corpus cavernosum from SHHF rats. KCNQ1-5 transcripts remained unchanged in penile arteries from SHHF rats. CONCLUSIONS AND IMPLICATIONS: These data suggest that Kv 7 channels play a role in erectile function and contribute to the pathophysiology of erectile dysfunction, an early indicator of cardiovascular disease.

Vasorelaxant effects of novel Kv 7.4 channel enhancers ML213 and NS15370.

BACKGROUND AND PURPOSE: The KCNQ-encoded voltage-gated potassium channel family (Kv 7.1-Kv 7.5) are established regulators of smooth muscle contractility, where Kv 7.4 and Kv 7.5 predominate. Various Kv 7.2-7.5 channel enhancers have been developed that have been shown to cause a vasorelaxation in both rodent and human blood vessels. Recently, two novel Kv 7 channel enhancers have been identified, ML213 and NS15370, that show increased potency, particularly on Kv 7.4 channels. The aim of this study was to characterize the effects of these novel enhancers in different rat blood vessels and compare them with Kv 7 enhancers (S-1, BMS204352, retigabine) described previously. We also sought to determine the binding sites of the new Kv 7 enhancers. KEY RESULTS: Both ML213 and NS15370 relaxed segments of rat thoracic aorta, renal artery and mesenteric artery in a concentration-dependent manner. In the mesenteric artery ML213 and NS15370 displayed EC50 s that were far lower than other Kv 7 enhancers tested. Current-clamp experiments revealed that both novel enhancers, at low concentrations, caused significant hyperpolarization in mesenteric artery smooth muscle cells. In addition, we determined that the stimulatory effect of these enhancers relied on a tryptophan residue located in the S5 domain, which is the same binding site for the other Kv 7 enhancers tested in this study. CONCLUSIONS AND IMPLICATIONS: This study has identified and characterized ML213 and NS15370 as potent vasorelaxants in different blood vessels, thereby highlighting these new compounds as potential therapeutics for various smooth muscle disorders.

Inhibitory role of phosphatidylinositol 4,5-bisphosphate on TMEM16A-encoded calcium-activated chloride channels in rat pulmonary artery.

BACKGROUND AND PURPOSE: Calcium-activated chloride channels (CaCCs) are key depolarizing mechanisms that have an important role in vascular smooth muscle contraction. Here, we investigated whether these channels are regulated by phosphatidylinositol (4,5) bisphosphate [P(4,5)P2 ], a known regulator of various ion channels. EXPERIMENTAL APPROACH: Calcium-activated Cl(-) currents (IClCa ) were recorded by patch clamp electrophysiology of rat isolated pulmonary artery smooth muscle cells. TMEM16A protein-phosphoinositide interaction was studied by co-immunoprecipitation and phosphoinositide binding arrays on protein lysates from whole pulmonary arteries and HEK293 cells overexpressing TMEM16A, the molecular correlate. KEY RESULTS: PI(4,5)P2 and other phospholipids were shown to bind directly to TMEM16A isolated from whole pulmonary artery (PA) and TMEM16A-eGFP expressed in HEK293 cells. Agents that reduced PI(4,5)P2 levels through different routes [PLC activation, PI4K inhibition, PI(4,5)P2 scavenging and absorption] all increased IClCa evoked by solutions containing clamped-free [Ca(2+) ], whereas enrichment of activating solutions with PI(4,5)P2 inhibited IClca in PA smooth muscle cells with approximately 50% reduction at 1 µM. CONCLUSIONS AND IMPLICATIONS: These data are the first to show a negative regulation of TMEM16A-encoded CaCCs by PI(4,5)P2 and propose that control of PI(4,5)P2 levels is a key determinant of arterial physiology.

One man's side effect is another man's therapeutic opportunity: targeting Kv7 channels in smooth muscle disorders.

Retigabine is a first in class anticonvulsant that has recently undergone clinical trials to test its efficacy in epileptic patients. Retigabine's novel mechanism of action - activating Kv7 channels - suppresses neuronal activity to prevent seizure generation by hyperpolarizing the membrane potential and suppressing depolarizing surges. However, Kv7 channels are not expressed exclusively in neurones and data generated over the last decade have shown that Kv7 channels play a key role in various smooth muscle systems of the body. This review discusses the potential of targeting Kv7 channels in the smooth muscle to treat diseases such as hypertension, bladder instability, constipation and preterm labour.

Phosphorylation alters the pharmacology of Ca(2+)-activated Cl channels in rabbit pulmonary arterial smooth muscle cells.

BACKGROUND AND PURPOSE: Ca(2+)-activated Cl(-) currents (I(Cl(Ca))) in arterial smooth muscle cells are inhibited by phosphorylation. The Ca(2+)-activated Cl(-) channel (Cl(Ca)) blocker niflumic acid (NFA) produces a paradoxical dual effect on I(Cl(Ca)), causing stimulation or inhibition at potentials below or above 0 mV respectively. We tested whether the effects of NFA on I(Cl(Ca)) were modulated by phosphorylation. EXPERIMENTAL APPROACH: I(Cl(Ca)) was elicited with 500 nM free internal Ca(2+) in rabbit pulmonary artery myocytes. The state of global phosphorylation was altered by cell dialysis with either 5 mM ATP or 0 mM ATP with or without an inhibitor of calmodulin-dependent protein kinase type II, KN-93 (10 microM). KEY RESULTS: Dephosphorylation enhanced the ability of 100 microM NFA to inhibit I(Cl(Ca)). This effect was attributed to a large negative shift in the voltage-dependence of block, which was converted to stimulation at potentials <-50 mV, approximately 70 mV more negative than cells dialysed with 5 mM ATP. NFA dose-dependently blocked I(Cl(Ca)) in the range of 0.1-250 microM in cells dialysed with 0 mM ATP and KN-93, which contrasted with the stimulation induced by 0.1 microM, which converted to block at concentrations >1 microM when cells were dialysed with 5 mM ATP. CONCLUSIONS AND IMPLICATIONS: Our data indicate that the presumed state of phosphorylation of the pore-forming or regulatory subunit of Cl(Ca) channels influenced the interaction of NFA in a manner that obstructs interaction of the drug with an inhibitory binding site.

Inhibition of vascular calcium-gated chloride currents by blockers of KCa1.1, but not by modulators of KCa2.1 or KCa2.3 channels.

BACKGROUND AND PURPOSE: Recent pharmacological studies have proposed there is a high degree of similarity between calcium-activated Cl(-) channels (CaCCs) and large conductance, calcium-gated K(+) channels (K(Ca)1.1). The goal of the present study was to ascertain whether blockers of K(Ca)1.1 inhibited calcium-activated Cl(-) currents (I(ClCa)) and if the pharmacological overlap between K(Ca)1.1 and CaCCs extends to intermediate and small conductance, calcium-activated K(+) channels. EXPERIMENTAL APPROACHES: Whole-cell Cl(-) and K(+) currents were recorded from murine portal vein myocytes using the whole-cell variant of the patch clamp technique. CaCC currents were evoked by pipette solutions containing 500 nM free [Ca(2+)]. KEY RESULTS: The selective K(Ca)1.1 blocker paxilline (1 microM) inhibited I(ClCa) by approximately 90%, whereas penitrem A (1 microM) and iberiotoxin (100 and 300 nM) reduced the amplitude of I(ClCa) by approximately 20%, as well as slowing channel deactivation. Paxilline also abolished the stimulatory effect of niflumic acid on the CaCC. In contrast, an antibody against the Ca(2+)-binding domain of murine K(Ca)1.1 had no effect on I(ClCa) while inhibiting spontaneous K(Ca)1.1 currents. Structurally different modulators of small and intermediate conductance calcium-activated K(+) channels (K(Ca)2.1 and K(Ca)2.3), namely 1-EBIO, (100 microM); NS309, (1 microM); TRAM-34, (10 microM); UCL 1684, (1 microM) had no effect on I(ClCa). CONCLUSIONS AND IMPLICATIONS: These data show that the selective K(Ca)1.1 blockers also reduce I(ClCa) considerably. However, the pharmacological overlap that exists between CaCCs and K(Ca)1.1 does not extend to the calcium-binding domain or to other calcium-gated K(+) channels.

Loss of functional K+ channels encoded by ether-à-go-go-related genes in mouse myometrium prior to labour onset.

There is a growing appreciation that ion channels encoded by the ether-à-go-go-related gene family have a functional impact in smooth muscle in addition to their accepted role in cardiac myocytes and neurones. This study aimed to assess the expression of ERG1-3 (KCNH1-3) genes in the murine myometrium (smooth muscle layer of the uterus) and determine the functional impact of the ion channels encoded by these genes in pregnant and non-pregnant animals. Quantitative RT-PCR did not detect message for ERG2 and 3 in whole myometrial tissue extracts. In contrast, message for two isoforms of mERG1 were readily detected with mERG1a more abundant than mERG1b. In isometric tension studies of non-pregnant myometrium, the ERG channel blockers dofetilide (1 microM), E4031 (1 microM) and Be-KM1 (100 nM) increased spontaneous contractility and ERG activators (PD118057 and NS1643) inhibited spontaneous contractility. In contrast, neither ERG blockade nor activation had any effect on the inherent contractility in myometrium from late pregnant (19 days gestation) animals. Moreover, dofetilide-sensitive K(+) currents with distinctive 'hooked' kinetics were considerably smaller in uterine myocytes from late pregnant compared to non-pregnant animals. Expression of mERG1 isoforms did not alter throughout gestation or upon delivery, but the expression of genes encoding auxillary subunits (KCNE) were up-regulated considerably. This study provides the first evidence for a regulation of ERG-encoded K(+) channels as a precursor to late pregnancy physiological activity.

KCNQ-encoded channels regulate Na+ transport across H441 lung epithelial cells.

H441 cells are a model of absorptive airway epithelia that are characterised by a pronounced apical Na+ flux through amiloride-sensitive Na+ channels. The flux of Na+ is intimately linked to Na+ handling by the cell as well as the membrane potential across the apical membrane. As KCNQ-encoded K+ channels influence chloride secretion in gastrointestinal epithelia, the goal of the present study was to ascertain the expression of KCNQ genes in H441 cells and determine the functional role of the expression products. Message for KCNQ3 and KCNQ5 was detected by RT-polymerase chain reaction and the translated proteins were observed by immunocytochemistry. Ussing experiments showed that the pan-KCNQ channel blocker XE991, but not KCNQ1 selective blockers, reduced the short circuit current and the amiloride-sensitive component. These data show for the first time that potassium channels encoded by KCNQ3 or KCNQ5 are crucial determinants of epithelial Na+ flux.

Molecular expression and pharmacological identification of a role for K(v)7 channels in murine vascular reactivity.

BACKGROUND AND PURPOSE: This study represents a novel characterisation of KCNQ-encoded potassium channels in the vasculature using a variety of pharmacological and molecular tools to determine their role in contractility. EXPERIMENTAL APPROACH: Reverse transcriptase polymerase chain reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse aorta, carotid artery, femoral artery and mesenteric artery using primers specific for all known KCNQ genes. RNA isolated from mouse heart and brain were used as positive controls. Pharmacological experiments were undertaken on segments from the same blood vessels to determine channel functionality. Immunocytochemical experiments were performed on isolated myocytes from thoracic aorta. KEY RESULTS: All blood vessels expressed KCNQ1, 4 and 5 with hitherto 'neuronal' KCNQ4 being, surprisingly, the most abundant. The correlated proteins K(v)7.1, K(v)7.4 and K(v)7.5 were identified in the cell membranes of aortic myocytes by immunocytochemistry. Application of three compounds known to activate K(v)7 channels, retigabine (2 -20 microM), flupirtine (20 microM) and meclofenamic acid (20 microM), relaxed vessels precontracted by phenylephrine or 1 mM 4-aminopyridine but had no effect on contractions produced by 60 mM KCl or the K(v)7 channel blocker XE991 (10 microM). All vessels tested contracted upon application of the K(v)7 channel blockers XE991 and linopirdine (0.1-10 microM). CONCLUSIONS AND IMPLICATIONS: Murine blood vessels exhibit a distinctive KCNQ expression profile with 'neuronal' KCNQ4 dominating. The ion channels encoded by KCNQ genes have a crucial role in defining vascular reactivity as K(v)7 channel blockers produced marked contractions whereas K(v)7 channel activators were effective vasorelaxants.

Pharmacological and molecular evidence for the involvement of Kv4.3 in ultra-fast activating K+ currents in murine portal vein myocytes.

BACKGROUND AND PURPOSE: The aim of this study was to determine the molecular identity of a transient K+ current (termed IUF) in mouse portal vein myocytes using pharmacological and molecular tools. EXPERIMENTAL APPROACH: Whole cell currents were recorded using the ruptured patch con from either acutely dispersed single smooth muscle cells from the murine portal vein or human embryonic kidney cells. Reverse transcriptase polymerase reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse portal vein using primers specific for various voltage-dependent K+ channels, auxillary subunits and calcium-binding proteins. Immunocytochemistry was undertaken using an antibody specific for Kv4.3. KEY RESULTS: IUF had a mean amplitude at +40 mV of 558 +/- 50 pA (n = 32) with a mean time to peak at +40 mV of approximately 4 ms. IUF activated and inactivated with a half maximal voltage of -12 +/- 2 mV and -85 +/- 2 mV, respectively. IUF was relatively resistant to 4-aminopyridine (5 mM produced 30 +/- 6 % block at +20 mV) but was inhibited effectively by flecainide (IC50 value was 100 nM) and phrixotoxin II. This pharmacological profile is consistent with IUF being comprised of Kv4.x proteins and this is supported by the results from the quantitative PCR and immunocytochemical experiments. CONCLUSIONS AND IMPLICATIONS: These data represent a rigorous investigation of the molecular basis of vascular transient K+ currents and implicates Kv4.3 as a major component of the channel complex.

Modulation of volume-sensitive chloride current by noradrenaline in rabbit portal vein myocytes.

The effect of noradrenaline on the volume-sensitive chloride current (I(Cl(swell))) was studied with conventional whole-cell recording techniques in freshly dispersed isolated smooth muscle cells of the rabbit portal vein. In the absence of receptor antagonists, noradrenaline produced an increase in the amplitude of I(Cl(swell)) in some cells and a decrease in others. In the presence of the beta-adrenoceptor antagonist propranolol, noradrenaline increased I(Cl(swell)) and in the presence of the alpha(1)-adrenoceptor antagonist prazosin, noradrenaline reduced I(Cl(swell).) The phospholipase C (PLC) inhibitor U73122 reduced the amplitude of I(Cl(swell)) whereas the inactive analogue U73343 had no effect. The phorbol esters phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu) increased the amplitude of I(Cl(swell)) by approximately 60 and 100 %, respectively, in a voltage-independent fashion. Inhibitors of protein kinase C (PKC) chelerythrine and calphostin-C decreased the amplitude of I(Cl(swell)) in a concentration-dependent but voltage-independent manner. Bath application of 8-Br-cAMP decreased I(Cl(swell)) by about 60 % whereas the inhibitor of protein kinase A (PKA) KT5720 increased the amplitude of I(Cl(swell)) by approximately 80-90 %. In the presence of propranolol, chelerythrine prevented the increase of I(Cl(swell)) by noradrenaline; in the presence of prazosin, KT5720 blocked the inhibitory action of noradrenaline. The results show that in rabbit portal vein myocytes noradrenaline enhances I(Cl(swell)) by acting on alpha(1)-adrenoceptors and reduces I(Cl(swell)) by stimulating beta-adrenoceptors. The data suggest that the potentiating and inhibitory effects of noradrenaline are mediated, respectively, by PKC and PKA.

Characteristics of hyperpolarization-activated cation currents in portal vein smooth muscle cells.

Voltage-clamp studies of freshly isolated smooth muscle cells from rabbit portal vein revealed the existence of a time-dependent cation current evoked by membrane hyperpolarization (termed I(h)). Both the rate of activation and the amplitude of I(h) were enhanced by membrane hyperpolarization. Half-maximal activation of I(h) was about -105 mV with conventional whole cell and -80 mV when the perforated patch technique was used. In current clamp, injection of hyperpolarizing current produced a marked depolarizing "sag" followed by rebound depolarization. Activation of I(h) was augmented by an increase in the extracellular K(+) concentration and was blocked rapidly by externally applied Cs(+) (1-5 mM). The bradycardic agent ZD-7288 (10 microM), a selective inhibitor of I(h), produced a characteristically slow inhibition of the portal vein I(h). The depolarizing sag recorded in current clamp was also abolished by application of 5 mM Cs(+). Cs(+) significantly decreased the frequency of spontaneous contractions in both whole rat portal vein and rabbit portal vein segments. Multiplex RT-PCR of rabbit portal vein myocytes using primers derived from existing genes for hyperpolarization-activated cation channels (HCN1-4) revealed the existence of cDNA clones corresponding to HCN2, 3, and 4. The present study shows that portal vein myocytes contain genes shown to encode for hyperpolarization-activated channels and exhibit an endogenous current with characteristics similar to I(h) in other cell types. This conductance appears to determine, in part, the rhythmicity of this vessel.

Dual effect of blocking agents on Ca2+-activated Cl(-) currents in rabbit pulmonary artery smooth muscle cells.

The effects of the Cl- channel antagonists, niflumic acid (NFA), dichloro-diphenylamine 2-carboxylic acid (DCDPC) and diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) on Ca2+-activated Cl- current (I(Cl(Ca))) evoked by adding fixed intracellular calcium concentrations ([Ca2+]i) to the pipette solution were studied in rabbit pulmonary artery myocytes. With 250 and 500 nM [Ca2+]i bath application of NFA (100 microM) increased inward current at negative potentials, but inhibited outward current at positive potentials. On wash out of NFA, I(Cl(Ca)) was greatly enhanced at all potentials. When external Na+ ions were replaced by N-methyl-D-glucamine (NMDG+) NFA still enhanced I(Cl(Ca)) at negative potentials but the increase of I(Cl(Ca)) on wash out was blocked. When the mean reversal potential (E(r)) of I(Cl(Ca)) was shifted to negative potentials by replacing external Cl- with SCN-, NFA increased inward current but blocked outward current suggesting that the effect of NFA is dependent on current flow. Inclusion of NFA in the pipette solution had no effect on I(Cl(Ca)). Voltage jump experiments indicated that I(Cl(Ca)) displayed characteristic outward current relaxations at +70 mV and inward current relaxations at -80 mV that were abolished by NFA. DCDPC (100 microM) produced similar effects to NFA but 1 mM DIDS produced inhibition of I(Cl(Ca)) at both positive and negative potentials and there was no increase in current on wash out of DIDS. These results suggest that NFA and DCDPC, but not DIDS, simultaneously enhance and block I(Cl(Ca)) by binding to an external site, probably close to the mouth of the chloride channel.

Differential regulation of Ca(2+)-activated Cl(-) currents in rabbit arterial and portal vein smooth muscle cells by Ca(2+)-calmodulin-dependent kinase.

1. Ca(2+)-activated chloride currents (I(Cl(Ca))) were recorded from smooth muscle cells isolated from rabbit pulmonary (PA) and coronary artery (CA) as well as rabbit portal vein (PV). The characteristics and regulation by Ca(2+)-calmodulin-dependent kinase II (CaMKII) were compared between the three cell types. 2. In PA and CA myocytes dialysed and superfused with K+ -free media, pipette solutions containing fixed levels of free Ca(2+) in the range of 250 nM to 1 microM evoked well sustained, outwardly rectifying I(Cl(Ca)) currents in about 90 % of cells. The CaMKII inhibitor KN-93 (5 microM) increased the amplitude of I(Cl(Ca)) in PA and CA myocytes. However, the threshold intracellular Ca(2+) concentration for detecting this effect was different in the two arterial cell types. KN-93 also enhanced the rate of activation of the time-dependent current during depolarising steps, slowed the kinetics of the tail current following repolarisation, and induced a negative shift of the steady-state activation curve. 3. In PA myocytes, the effects of KN-93 were not mirrored by its inactive analogue KN-92 but were reproduced by the inclusion of autocamtide-2-related CaMKII inhibitory peptide (ARIP) in the pipette solution. Cell dialysis with constitutively active CaMKII (30 nM) significantly reduced I(Cl(Ca)) evoked by 500 nM Ca(2+). 4. In PV myocytes, I(Cl(Ca)) was evoked by pipette solutions containing up to 1 microM free Ca(2+) in less than 40 % of cells. Application of KN-93 to cells where I(Cl(Ca)) was sustained produced a small inhibition (approximately 25%) of the current in 70 % of the cells. 5. The present study shows that regulation of Ca(2+)-dependent Cl(-) channels by CaMKII differs between arterial and portal vein myocytes.

Direct visualization of sarcoplasmic reticulum regions discharging Ca(2+)sparks in vascular myocytes.

Localized Ca(2+)-release events, Ca(2+)sparks, have been suggested to be the 'elementary building blocks' of the calcium signalling system in all types of muscles. In striated muscles these occur at regular intervals along the fibre corresponding to the sarcomeric structures which do not exist in smooth muscle. We showed previously that in visceral and vascular myocytes Ca(2+)sparks occurred much more frequently at certain sites (frequent discharge sites [FDSs]). In this paper, we have related the position of FDSs to the distribution of the sarcoplasmic reticulum in the same living myocyte. The three-dimensional distribution of the SR in freshly isolated rabbit portal vein myocytes was visualized by means of high-resolution confocal imaging after staining with DiOC(6)and/or BODIPY TR-X ryanodine. Both fluorochromes revealed a similar staining pattern indicating a helical arrangement of well-developed superficial SR which occupied about 6% of the cell volume. Computing the frequency of spontaneous Ca(2+)sparks detected by means of fluo-4 fluorescence revealed that in about 70% of myocytes there was only one major FDS located on a prominent portion of superficial SR network usually within 1-2 microm of the nuclear envelope, although a few sparks occurred at other sites scattered generally in superficial locations throughout the cell. Polarized mitochondria were readily identified by accumulation of tetramethylrhodamine ethyl ester (TMRE). These were closely associated with the SR network in extra-nuclear regions. TMRE staining, however, failed to reveal any mitochondria near the FDS-related SR element. When observed, propagating [Ca(2+)](i)waves and associated myocyte contractions were initiated at FDSs. This study provide first insight into the three-dimensional arrangement of the SR in living smooth muscle cells and relates the peculiarity of the structural organization of the myocyte to the features of Ca(2+)signalling at subcellular level.

Tonic regulation of vascular tone by nitric oxide and chloride ions in rat isolated small coronary arteries.

We have investigated the involvement of Cl(-) in regulating vascular tone in rat isolated coronary arteries mounted on a small vessel myograph. Mechanical removal of the endothelium or inhibition of nitric oxide (NO) synthase with N(omega)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) led to contraction of rat coronary arteries, and these contractions were sensitive to nicardipine (10(-6) M). This suggests that release of NO tonically inhibits a contractile mechanism that involves voltage-dependent Ca(2+) channels. In arteries contracted with L-NAME, switching the bathing solution to physiological saline solution with a reduced Cl(-) concentration potentiated the contraction. DIDS (5 x 10(-6)-3 x 10(-4) M) caused relaxation of L-NAME-induced tension (IC(50) = 55 +/- 10 microM), providing evidence for a role of Cl(-). SITS (10(-5)-5 x 10(-4) M) did not affect L-NAME-induced tension, suggesting that DIDS is not acting by inhibition of anion exchange. Mechanical removal of the endothelium led to contraction of arteries, which was sensitive to DIDS (IC(50) = 50 +/- 8 microM) and was not affected by SITS. This study suggests that, in rat coronary arteries, NO tonically suppresses a contractile mechanism that involves a Cl(-) conductance.

Dual modulation of swelling-activated chloride current by NO and NO donors in rabbit portal vein myocytes.

1. The effects of authentic NO and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) on swelling-activated chloride currents (Iswell) were investigated in freshly dispersed rabbit portal vein smooth muscle cells. Iswell was recorded with the perforated patch configuration of the whole-cell patch clamp technique. 2. In approximately 50 % of cells NO and SNAP inhibited the amplitude of Iswell by about 45 % in a voltage-independent manner. Iswell was also inhibited by an inhibitor of NO-sensitive guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and by KT5823, an inhibitor of cGMP-dependent protein kinase. 3. In other cells both NO and SNAP enhanced Iswell by about 40 % in a voltage-independent manner. A similar increase was produced by application of the cell-permeable cGMP analogue 8-bromo-guanosine 3', 5'-cyclic monophosphate (8-Br-cGMP). However, 8-Br-cGMP had no effect on current amplitude in cells pre-treated with KT5823. In contrast 8-Br-cGMP increased the amplitude of Iswell in cells which had been pre-treated with ODQ. 4. SNAP also modulated Iswell recorded in the conventional whole-cell configuration with internal solutions containing 10 mM EGTA to rule out any contribution from Ca2+-activated Cl- currents. 5. These data suggest that the amplitude of Iswell can be enhanced by NO via a cGMP-dependent phosphorylation and inhibited by NO in a cGMP-independent manner.

Modulation of the decay of Ca2+-activated Cl- currents in rabbit portal vein smooth muscle cells by external anions.

1. The effects of external anions on the decay kinetics of Ca2+-activated Cl- currents (ICl(Ca)) were studied in smooth muscle cells isolated from rabbit portal vein using the perforated patch whole-cell voltage clamp technique. 2. In normal NaCl-containing external solution the decay of spontaneous Ca2+-activated Cl- currents (STICs) and Ca2+-activated Cl- 'tail' currents (Itail) was described by a single exponential with a time constant (tau) that was prolonged by external anions which are more permeable than Cl- (Br-, I- and SCN-) and accelerated by less permeant anions. However, intracellular I- did not affect the tau of STICs and Itail. 3. There was a positive correlation between the ability of an external anion to affect the decay tau of ICl(Ca) and its permeability relative to Cl-. 4. The voltage dependence of STIC and Itail decay was not affected by external or internal anions. 5. External permeating anions were not obligatory for activation of ICl(Ca) and STIC tau was not altered in Cl--free external solution. 6. Modulation of tau by mole fractions of SCN- and Cl- ions was fitted by a logistic curve, suggesting competition between SCN- and Cl- ions for a binding site. 7. In conclusion, external anions affect the decay of ICl(Ca) by a mechanism compatible with an interaction with a binding site which modulates Cl- channel kinetics.