Portal Vein Smooth Muscle Cells Research Articles

Increased TMEM16A-Mediated Ca2+-Activated Cl- Currents in Portal Vein Smooth Muscle Cells of Caveolin 1-Deficient Mice.

Ca2+-activated Cl- (ClCa) channels regulate membrane excitability and myogenic tone in vascular smooth muscles. TMEM16A-coding proteins are mainly responsible for functional ClCa channels in vascular smooth muscles, including portal vein smooth muscles (PVSMs). Caveolae are cholesterol-rich and Ω-shaped invaginations on the plasma membrane that structurally contributes to effective signal transduction. Caveolin 1 (Cav1) accumulates in caveolae to form functional complexes among receptors, ion channels, and kinases. The present study examined the functional roles of Cav1 in the expression and activity of ClCa channels in the portal vein smooth muscle cells (PVSMCs) of wild-type (WT) and Cav1-knockout (KO) mice. Contractile experiments revealed that the amplitude of spontaneous PVSM contractions was larger in Cav1-KO mice than WT mice. Under whole-cell patch-clamp configurations, ClCa currents were markedly inhibited by 1 µM Ani9 (a selective TMEM16A ClCa channel blocker) in WT and Cav1-KO PVSMCs. However, Ani9-sensitive ClCa currents were significantly larger in Cav1-KO PVSMCs than in WT PVSMCs. Expression analyses showed that TMEM16A expression levels were higher in Cav1-KO PVSMs than in WT PVSMs. Therefore, the caveolar structure formed by Cav1 negatively regulates the expression and activity of TMEM16A-mediated ClCa channels in vascular smooth muscle cells.

Downregulation of Ca2+-Activated Cl- Channel TMEM16A Mediated by Angiotensin II in Cirrhotic Portal Hypertensive Mice.

Portal hypertension is defined as an increased pressure in the portal venous system and occurs as a major complication in chronic liver diseases. The pathological mechanism underlying the pathogenesis and development of portal hypertension has been extensively investigated. Vascular tone of portal vein smooth muscles (PVSMs) is regulated by the activities of several ion channels, including Ca2+-activated Cl− (ClCa) channels. TMEM16A is mainly responsible for ClCa channel conductance in vascular smooth muscle cells, including portal vein smooth muscle cells (PVSMCs). In the present study, the functional roles of TMEM16A channels were examined using two experimental portal hypertensive models, bile duct ligation (BDL) mice with cirrhotic portal hypertension and partial portal vein ligation (PPVL) mice with non-cirrhotic portal hypertension. Expression analyses revealed that the expression of TMEM16A was downregulated in BDL-PVSMs, but not in PPVL-PVSMs. Whole-cell ClCa currents were smaller in BDL-PVSMCs than in sham- and PPVL-PVSMCs. The amplitude of spontaneous contractions was smaller and the frequency was higher in BDL-PVSMs than in sham- and PPVL-PVSMs. Spontaneous contractions sensitive to a specific inhibitor of TMEM16A channels, T16Ainh-A01, were reduced in BDL-PVSMs. Furthermore, in normal PVSMs, the downregulation of TMEM16A expression was mimicked by the exposure to angiotensin II, but not to bilirubin. This study suggests that the activity of ClCa channels is attenuated by the downregulation of TMEM16A expression in PVSMCs associated with cirrhotic portal hypertension, which is partly mediated by increased angiotensin II in cirrhosis.

Role of transmembrane protein 16A in vascular remodeling in portal hypertension induced by bile duct ligation of SD rats

Objective To investigate the role of transmembrane protein 16A(TMEM16A)in murine portal vein remodeling in cirrhotic portal hypertension induced by bile duct ligation(BDL). Methods Sixty male SD rats were randomly divided into the model group(n= 40)and the control group(n= 20).Rats in the model group were subjected to BDL.Animals were sacrificed at 4th week.Hematoxylin and eosin(HE)staining was used to observe the liver cirrhosis in rats, and the portal vein pressure was measured to determine portal hypertension in rats.The change of histological structure of the portal vein was observed by HE staining.In addition, immunohistochemistry and Western blotting were applied to detect the expression of TMEM16A, phosphorylated extracellular signal- regulated kinase 1/2(p- ERK1/2)and extracellular signal - regulated kinase 1/2(ERK1/2) proteins. Results Compared with sham control group, the liver of rats in model group had false lobules and the portal vein pressure significantly increased[(16.79±2.65) mm Hg (1 mm Hg= 0.133 kPa)vs.(8.86±0.53)mm Hg,P<0.05].And the intima- media thickness of portal vein in model group was increased as compared with sham control group[(43.27±9.62)μm vs.(21.75±5.56) μm,P<0.05].The protein expression of TMEM16A was significantly decreased, and that of p- ERK1/2 was significantly increased in model group as compared with sham control group.The protein expression of ERK1/2 almost had no changes Conclusion TMEM16A may regulate the proliferation of portal vein smooth muscle cells by influencing the activity of mitogen- activated protein kinase(MAPK)/ERK signal pathway. Downregulation of TMEM16A may play an important role in the occurrence of portal vein remodeling induced by portal hypertension. Key words: Transmembrane protein 16A; Portal hypertension; Vascular remodeling; Cell proliferation; Mitogen-activated protein kinase/extracellular signal-regulated kinase signal pathway

Heterogeneity in the Proliferative Capacity of Smooth Muscle Cells (SMCs)

In cardiovascular disease, artery walls remodel through an increase in SMC numbers. The predominant hypothesis for this is that SMCs in the tunica media undergo phenotypic modulation into a proliferative cell type. However, direct evidence for SMC phenotypic modulation is scant. We therefore exploited time‐lapse microscopy methods to track the fate of freshly isolated, contractile SMCs. As SMCs isolated from different smooth muscle (SM) tissues are heterogeneous in nature, we have used time lapse microscopy in combination with immunocytochemistry to investigate the proliferative capacity of SMCs from portal vein (PV), carotid artery (CA) and distal colon.The adventia/endothelium and mucosa/serosa were mechanically removed before isolating cells by enzymatic digestion and trituration. Highly elongated, contractile SMCs that stained for both SM α‐actin (SMA) and SM myosin heavy chain (SM‐MHC) were obtained from all tissues. Significantly, both colon and PV tissues also contained large numbers of spherical cells that did not stain for SMA or SM‐MHC (nonSMCs). In standard culture conditions, the SMCs showed limited proliferative capacity: with the exception of one SMC that divided once (out of 15 tracked cells), colon SMCs did not proliferate. Of 11 PV SMCs tracked, 7 SMCs did divide, though none progressed beyond the 3rd generation (daughter of daughter) by confluency. Conversely, the nonSMCs proliferated rapidly (reaching the 5th generation in 5 days) and dominated the resulting cultures. In contrast, all CA cells stained for both SMA and SM‐MHC i.e. no nonSMCs were present. Whilst most CA SMCs underwent apoptosis early in culture (27 of 31 cells), those that survived went on to proliferate at varying rates (up to the 6th generation in 5 days). These results illustrate the complexities involved in creating models of SMC proliferation.

Role of hydrogen sulfide in portal hypertension and esophagogastric junction vascular disease

To investigate the association between endogenous hydrogen sulfide (H₂S) and portal hypertension as well as its effect on vascular smooth muscle cells. Portal hypertension patients were categorized by Child-Pugh score based on bilirubin and albumin levels, prothrombin time, ascites and hepatic encephalopathy. Plasma H₂S concentrations and portal vein diameters (PVDs) were compared between portal hypertension patients and control participants, as well as between portal hypertension patients with varying degrees of severity. In addition, we established a rabbit hepatic schistosomiasis portal hypertension (SPH) model and analyzed liver morphology, fibrosis grade, plasma and liver tissue H₂S concentrations, as well as cystathionine γ-lyase (CSE) activity and phosphorylated extracellular signal-regulated kinase (pERK)1/2, B cell lymphoma (Bcl)-2 and Bcl-XL expression in portal vein smooth muscle cells, in addition to their H₂S-induced apoptosis rates. In portal hypertension patients, endogenous H₂S levels were significantly lower than those in healthy controls. The more severe the disease was, the lower were the H₂S plasma levels, which were inversely correlated with PVD and Child-Pugh score. Liver tissue H₂S concentrations and CSE expression were significantly lower in the SPH rabbit livers compared with the control animals, starting at 3 wk, whereas pERK 1/2 expressions gradually increased 12-20 wk after SPH model establishment. In portal vein smooth muscle cells, increasing H₂S levels led to increased apoptosis, while Bcl-2 and Bcl-XL expression decreased. H₂S prevents vascular restructuring caused by excessive proliferation of smooth muscle cells via apoptosis induction, which helps to maintain normal vascular structures.

Modulation of TMEM16A-Channel Activity as Ca2+ Activated Cl− Conductance via the Interaction With Actin Cytoskeleton in Murine Portal Vein

TMEM16A is a major component of Ca(2+)-activated Cl(-) channel (CaCC) conductance in murine portal vein smooth muscle cells (mPVSMCs). Here, the regulation of CaCC activity by the actin cytoskeleton was examined in mPVSMCs. Actin disruption by cytochalasin D did not affect the current density, but increased the deactivation time constant in mPVSMCs. The elongated deactivation was recovered by jasplakinolide. When murine TMEM16A was transfected into HEK293 cells that have a poorly developed actin cytoskeleton, electrophysiological properties of CaCC currents were not changed by cytochalasin D. In conclusion, the CaCC activity in mPVSMCs is modified by the interaction of TMEM16A with abundant actin cytoskeleton.

Lysophosphatidylcholine Increases Ca2+Current via Activation of Protein Kinase C in Rabbit Portal Vein Smooth Muscle Cells

Lysophosphatidylcholine (LPC), a metabolite of membrane phospholipids by phospholipase A(2), has been considered responsible for the development of abnormal vascular reactivity during atherosclerosis. Ca(2+) influx was shown to be augmented in atherosclerotic artery which might be responsible for abnormal vascular reactivity. However, the mechanism underlying Ca(2+) influx change in atherosclerotic artery remains undetermined. The purpose of the present study was to examine the effects of LPC on L-type Ca(2+) current (I(Ca(L))) activity and to elucidate the mechanism of LPC-induced change of I(Ca(L)) in rabbit portal vein smooth muscle cells using whole cell patch clamp. Extracellular application of LPC increased I(Ca(L)) through whole test potentials, and this effect was readily reversed by washout. Steady state voltage dependency of activation or inactivation properties of I(Ca(L)) was not significantly changed by LPC. Staurosporine (100 nM) or chelerythrine (3 microM), which is a potent inhibitor of PKC, significantly decreased basal I(Ca(L)), and LPC-induced increase of I(Ca(L)) was significantly suppressed in the presence of PKC inhibitors. On the other hand, application of PMA, an activator of PKC, increased basal I(Ca(L)) significantly, and LPC-induced enhancement of I(Ca(L)) was abolished by pretreatment of the cells with PMA. These findings suggest that LPC increased I(Ca(L)) in vascular smooth muscle cells by a pathway that involves PKC, and that LPC-induced increase of I(Ca(L)) might be, at least in part, responsible for increased Ca(2+) influx in atherosclerotic artery.

Pharmacological and biophysical isolation of K+ currents encoded by ether-à-go-go-related genes in murine hepatic portal vein smooth muscle cells

Previous studies have shown that murine portal vein myocytes express ether-à-go-go related genes (ERGs) and exhibit distinctive currents when recorded under symmetrical K(+) conditions. The aim of the present study was to characterize ERG channel currents evoked from a negative holding potential under conditions more pertinent to a physiological scenario to assess the possible functional impact of this conductance. Currents were recorded with ruptured or perforated patch variants of the whole cell technique from a holding potential of -60 mV. Application of three structurally distinct and selective ERG channel blockers, E-4031, dofetilide, and the peptide toxin BeKM-1, all inhibited a significant proportion of the outward current and abolished inward currents with distinctive "hooked" kinetics recorded on repolarization. Dofetilide-sensitive currents at negative potentials evoked by depolarization to +40 mV had a voltage-dependent time to peak and rate of decay characteristic of ERG channels. Application of the novel ERG channel activator PD-118057 (1-10 microM) markedly enhanced the hooked inward currents evoked by membrane depolarization and hyperpolarized the resting membrane potential recorded by current clamp and the perforated patch configuration by approximately 20 mV. In contrast, ERG channel blockade by dofetilide (1 microM) depolarized the resting membrane potential by approximately 8 mV. These data are the first record of ERG channel currents in smooth muscle cells under quasi-physiological conditions that suggest that ERG channels contribute to the resting membrane potential in these cells.

Facilitatory effect of Ins(1,4,5)P3 on store‐operated Ca2+‐permeable cation channels in rabbit portal vein myocytes

In rabbit portal vein smooth muscle cells, store-operated Ca2+-permeable cation channels (SOCs) display multi-modal gating mechanisms. SOCs are activated by depletion of intracellular Ca2+ stores but also may be stimulated in a store-independent manner by noradrenaline acting on alpha-adrenoceptors and by diacylglycerol (DAG) via protein kinase C (PKC). In the present study we have investigated whether inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) modulates SOC activity in freshly dispersed rabbit portal vein myocytes with patch pipette recording techniques. Inclusion of 1 mum Ins(1,4,5)P3 in the patch pipette solution increased whole-cell currents evoked by the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) by about 3-fold at -80 mV. In the cell-attached configuration the cell-permeable Ca2+ chelator BAPTA-AM stimulated SOC activity and after excision of an isolated inside-out patch bath application of 1 mum Ins(1,4,5)P3 increased open channel probability (NP(o)) by approximately 3-fold. Ins(1,4,5)P3 also produced a similar increase in NP(o) of SOCs stimulated by the phorbol ester, phorbol 12,13-dibutyrate (PDBu) in inside-out patches and these channel currents had a unitary conductance of about 2 pS. The equilibrium constant of Ins(1,4,5)P3 on increasing PDBu-evoked SOC activity was about 0.4 mum. The facilitatory effect of Ins(1,4,5)P3 was also manifest as markedly increasing the rate of activation of SOCs. The synergistic effect of Ins(1,4,5)P3 was mimicked by the metabolically stable analogue 3-fluoro-Ins(1,4,5)P3 and Ins(1,4)P2, a metabolite of Ins(1,4,5)P3, but was not inhibited by the classical Ins(1,4,5)P3 receptor antagonist heparin. Finally Ins(1,4,5)P3 also increased NP(o) of SOCs activated by a PKC catalytic subunit. It is concluded that Ins(1,4,5)P3 facilitates SOC opening via a heparin-insensitive mechanism at, or close to, the channel protein.

Stimulation of β‐adrenoceptors inhibits store‐operated channel currents via a cAMP‐dependent protein kinase mechanism in rabbit portal vein myocytes

Previously we have described the properties of store-operated channel currents (SOCs) in freshly dispersed rabbit portal vein smooth muscle cells. In addition to Ca(2+) store depletion these SOCs could also be activated by alpha-adrenoceptor stimulation and diacylglycerol (DAG) via a protein kinase C (PKC)-dependent mechanism. In the present study we have investigated the effect of beta-adrenoceptor stimulation on SOCs in rabbit portal vein myocytes. With whole-cell recording the selective beta-adrenoceptor agonist isoprenaline reduced the current evoked by cyclopiazonic acid (CPA, sarcoplasmic/endoplasmic reticulum ATPase inhibitor) by over 85%. With cell-attached patch recording, bath application of isoprenaline produced a pronounced inhibition of SOC activity evoked by either CPA or the acetoxymethyl ester form of BAPTA (BAPTA-AM). SOC activity evoked by CPA, the DAG analogue, 1-oleoyl-acetyl-sn-glycerol (OAG) or the phorbol ester, phorbol-12,13-dibutyrate (PDBu) was also markedly inhibited by the adenylate cyclase activator, forskolin, and the cell-permeable non-hydrolysable analogue of cyclic adenosine monophosphate (cAMP), 8-Br-cAMP. With inside-out patches, bath application of PDBu evoked channel currents with similar properties to SOCs which were inhibited by over 90% by a catalytic subunit of protein kinase A (PKA) and by 8-Br-cAMP. Moreover bath application of PKA inhibitors, H-89, KT5720 and an inhibitory peptide to quiescent cell-attached or inside-out patches, activated channel currents with similar properties to SOCs. These data suggest that in rabbit portal vein myocytes, stimulation of beta-adrenoceptors inhibits SOC activity via a cAMP-dependent protein kinase signal transduction cascade. In addition it is concluded that constitutive PKA activity has a profound inhibitory effect on SOC activity in this vascular preparation.

Evidence for the involvement of the cyclooxygenase-metabolic pathway in diclofenac-induced inhibition of spontaneous contraction of rat portal vein smooth muscle cells

The effects of diclofenac, a cyclooxygenase (COX) inhibitor, were investigated on spontaneous phasic contractions of longitudinal preparations of the rat portal vein. Diclofenac produced a concentration-dependent decrease in the amplitude of these spontaneous phasic contractions. Diclofenac (30 microM) decreased the amplitude of the spontaneous phasic increase in the F340/F380 ratio of Fura PE3, an indicator of intracellular Ca2+ concentration. It also reduced the number of action potentials in each burst discharge without changing the resting membrane potential of longitudinal smooth muscle cells. The extent of the distribution of Lucifer Yellow injected into a smooth muscle cell was decreased in the presence of diclofenac (30 microM). Both AH6809, a prostanoid EP receptor antagonist, and SQ22536, an adenylate cyclase inhibitor, decreased the amplitude of the spontaneous contractions. On the other hand, neither ozagrel, a thromboxane synthase inhibitor, nor SQ29548, a prostanoid TP receptor antagonist, significantly affected spontaneous contractions. These results indicate that diclofenac inhibits the amplitude of spontaneous contractions of the rat portal vein through inhibition of electrical activity, which may be related to an inhibition of the cyclooxygenase pathway.

Activation of chloride currents in murine portal vein smooth muscle cells by membrane depolarization involves intracellular calcium release.

The present study describes the first characterization of Ca(2+)-activated Cl(-) currents (I(ClCa)) in single smooth muscle cells from a murine vascular preparation (portal veins). I(ClCa) was recorded using the perforated patch version of the whole cell voltage-clamp technique and was evoked using membrane depolarization. Generation of I(ClCa) relied on Ca(2+) entry through dihydropyridine-sensitive Ca(2+) channels because I(ClCa) was abolished by 1 microM nicardipine and enhanced by raising external Ca(2+) concentration or by application of BAY K 8644. I(ClCa) was characterized by the sensitivity to Cl(-) channel blockers and the effect of altering the external anion on reversal potential. Activation of I(ClCa) after membrane depolarization was dependent on Ca(2+) release from intracellular stores. Thus the amplitude of I(ClCa) was diminished by the SR-ATPase inhibitor cyclopiazonic acid, the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate (2-APB), and the ryanodine receptor blocker tetracaine. The degree of inhibition produced by the application of 2-APB and tetracaine together was significantly greater than the effect of each agent applied alone. In current-clamp mode, injection of depolarizing current elicited a biphasic action potential, with the later depolarization being sensitive to niflumic acid (NFA; 10 microM). In isometric tension recordings, NFA inhibited spontaneous contractions. These data support a role for this conductance in portal vein excitability.

Heme oxygenase-2 products activate IKCa: role of CO and iron in guinea pig

Hemin (10 microM) and carbon monoxide (CO) increased iberiotoxin-blockable IKCa in portal vein smooth muscle cells. CO-induced IKCa activation was abolished by 10 microM ODQ, 10 microM cyclopiazonic acid and 1 microM KT5823. The hemin-induced effect on IKCa was abolished by pretreatment with Sn-protoporphyrin IX, a heme oxygenase inhibitor and Fe2+ chelator but was insensitive to inhibitors of soluble guanylate cyclase (GC) and cGMP-dependent protein kinase (PKG). There was no effect of hemin on IKCa in the presence of 3 microM dithiotreitol into the bath or 3 mM glutathione into the pipette solution. Superoxide dismutase (1000 U/ml) or catalase (3000 U/ml) added into the pipette solution also abolished the effect of hemin on IKCa in this tissue. Additionally, 10 microM hemin could not influence IKCa in Ca2+-free external solution or in the presence of 30 microM SKF 95356. It was concluded that CO increases IKCa via its "conventional" signaling pathway, which involves soluble GC and PKG activation and subsequent stimulation of sarcoplasmic reticulum Ca2+ pump activity resulting in Ca2+-dependent activation of IKCa due to the accumulation of Ca2+ into the space near the plasma membrane. On the other hand, internally produced CO could not yield the same IKCa increase, while Fe2+ derived from heme oxygenase 2-dependent degradation of hemin in portal vein smooth muscle cells gives rise to reactive oxygen species namely hydroxyl and superoxide radicals. Both radicals are responsible for the SKF 95356-sensitive non-selective cation channel activation, the Ca2+ influx and the subsequent increase of Ca2+ concentration near the plasma membrane that augments the KCa channel activity.

Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC.

It has been suggested that L-type Ca(2+) channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca(2+) channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca(2+) channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba(2+) currents (I(Ba)) through L-type Ca(2+) channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca(2+) channel activity but did not alter the voltage-dependent characteristics of Ca(2+) channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31-8425 or Go-6983, prevented I(Ba) enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I(Ba) under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I(Ba) when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca(2+) channel activity in rabbit portal vein smooth muscle cells through activation of PKC.

Vasodilation by banhabackchulchunmatang, a Chinese medicine, is associated with negative modulation of PKCα activation and NO production

Banhabackchulchunmatang (BCT) is a widely used herbal medicine with vasodilatory actions. In the present study, we investigated the subcellular mechanisms of its vascular actions. Both in the presence and absence of endothelium, BCT relaxed vascular strips precontracted with phenylephrine, but the magnitude of relaxation was greater in the presence of endothelium. The relaxation was inhibited by either L-NAME, an NOS inhibitor, or methylene blue, a cGMP inhibitor, indicating the involvement of nitric oxide (NO). The involvement of NO was supported by the increased formation of nitrite from human umbilical vein endothelial cells in the presence of BCT. In vascular strips, BCT lowered the phosphorylation level of the 20 kDa myosin light chains. BCT also directly inhibited phenylephrine-induced protein kinase Cα (PKCα) translocation in freshly isolated single ferret portal vein smooth muscle cells. Together, these effects are likely to contribute to the vasodilatory actions of BCT.

Activation of store-operated channels by noradrenaline via protein kinase C in rabbit portal vein myocytes.

In the present study we have investigated the role of diacylglycerol (DAG) and protein kinase C (PKC) in mediating activation of Ca(2+)-permeable store-operated channels (SOCs) by noradrenaline in rabbit portal vein smooth muscle cells. With cell-attached recording, bath application of noradrenaline, 1-oleoyl-acetyl-sn-glycerol (OAG) and phorbol 12,13-dibutyrate (PDBu) evoked single channel currents. The biophysical properties of these channel currents were similar to those of the channel currents activated by depletion of internal Ca(2+) stores with cyclopiazonic acid (CPA). The activation of SOCs in cell-attached recording by noradrenaline, OAG, PDBu, CPA and the acetoxymethyl ester form of BAPTA (BAPTA-AM) was markedly inhibited by the PKC inhibitors chelerythrine and RO-31-8220. In isolated outside-out patches CPA did not evoke SOCs but noradrenaline stimulated SOC activity, which was reduced by about 90 % by PKC inhibitors. The addition of the serine/threonine phosphatase inhibitors calyculin A and microcystin also stimulated SOCs in isolated outside-out patches. It is concluded that in rabbit portal vein myocytes, noradrenaline activates SOCs via DAG and PKC, possibly by a store-independent mechanism. In addition in this cell type it appears that PKC and phosphorylation may play an important role in stimulating SOC activity in response to depletion of internal Ca(2+) stores by CPA and BAPTA-AM.

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.

The effect of external divalent cations on spontaneous non-selective cation channel currents in rabbit portal vein myocytes.

1. The effects of external divalent cations on spontaneous single non-selective cation channel currents were studied in outside-out patches from rabbit portal vein smooth muscle cells in K+-free conditions. 2. In an external medium containing 1.5 mM Ca2+ (Ca2+o) the majority of spontaneous channel currents had a unitary conductance of 23 pS, reversal potential (Vr) of +10 mV and a low open probability (Po) at negative patch potentials. Some channels opened to a lower conductance state of about 13 pS suggesting that the cation channels have two conductance states. Open time and burst duration distributions could both be described by two exponentials with time constants of about of 1 ms and 7 ms for open times and 3 ms and 16 ms for burst durations. 3. In 0 Ca2+o the majority of spontaneous cation channels had a unitary conductance of 13 pS and Vr was shifted to +4 mV. Moreover the longer open time and longer burst duration time constants were both reduced to approximately half the values in 1.5 mM Ca2+o. 4. Compared to 0 Ca2+o the single channel currents in 3 microM and 100 microM Ca2+o had a 5- to 6-fold increase in Po which was accompanied by increases in both open times and burst durations. In 3 microM and 100 microM Ca2+o the unitary conductance of the single channel currents was between 22 and 26 pS. 5. At positive membrane potentials the single channel currents had an increased Po compared to negative potentials which was associated with increased open times and burst durations but these values were similar in 3 microM, 100 microM and 1.5 mM Ca2+o. 6. In 1.5 mM Sr2+o and 1.5 mM Ba2+o channels opened to the higher conductance state of about 22-25 pS and had a 3- to 7-fold greater Po than in 0 Ca2+o. 7. In conclusion, external divalent cations have marked effects on the unitary conductance and kinetic behaviour of non-selective cation channels in rabbit portal vein smooth muscle cells.

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.

Beta-Adrenergic receptor stimulation of L-type Ca2+ channels in rabbit portal vein myocytes involves both alphas and betagamma G protein subunits.

1. Previous studies have shown that purified G protein alphas and betagamma subunits stimulate vascular L-type Ca2+ channels through protein kinase A and C (PKA and PKC), respectively. The present study tested whether activation of endogenous G proteins via beta-adrenergic receptor binding also stimulates vascular Ca2+ channels through both Galphas and Gbetagamma and the subsequent activation of PKA and PKC. 2. Peak Ba2+ current (IBa) in freshly isolated rabbit portal vein smooth muscle cells was significantly increased by bath application of 0.5 microM isoproterenol (isoprenaline; ISO) when measured using the whole-cell patch clamp method (53 +/- 3 % increase, n = 15). Stimulation of IBa by ISO was partially reversed by a PKA inhibitor, KT 5720, or a PKC inhibitor, calphostin C, and completely blocked when cells were pretreated with both KT 5720 and calphostin C. 3. Dialysis of cells with polyclonal antibody to Galphas significantly reduced but did not completely eliminate ISO-induced stimulation of IBa. The remaining stimulation was abolished by calphostin C. Dialysis of cells with a polyclonal antibody to Gbeta also significantly reduced ISO-induced stimulation and the remaining stimulation was abolished by KT 5720. Dialysis of cells with both antibodies completely prevented the stimulation of IBa by ISO. 4. ISO-induced stimulation of IBa was reversed by ICI-118,551, a specific beta2-adrenoceptor antagonist, but not by CGP 20712A, a specific beta1-adrenoceptor antagonist. In addition, the beta2-adrenoceptor agonist zinterol significantly increased peak IBa while the beta1-adrenoceptor agonist dobutamine and beta3-adrenoceptor agonist BRL 37344A had little effect on peak IBa. 5. These data suggest that beta-adrenergic receptor stimulation of vascular L-type Ca2+ channels involves both alphas and betagamma G-protein subunits, which exert their effects through PKA and PKC, respectively.