Rabbit Portal Vein Research Articles

Role of intracellular stores in the regulation of rhythmical [Ca 2+] i changes in interstitial cells of Cajal from rabbit portal vein

Interstitial cells of Cajal (ICCs) freshly isolated from rabbit portal vein and loaded with the Ca 2+-sensitive indicator fluo-3 revealed rhythmical [Ca 2+] i changes occurring at 0.02–0.1 Hz. Each increase in [Ca 2+] i originated from a discrete central region of the ICC and propagated as a [Ca 2+] i wave towards the cell periphery, but usually became attenuated before reaching the ends of the cell. In about 40% of ICCs each rhythmical change in [Ca 2+] i consisted of an initial [Ca 2+] i increase (phase 1) followed by a faster rise in [Ca 2+] i (phase 2) and then a decrease in [Ca 2+] i (phase 3); the frequency correlated with the rate of rise of [Ca 2+] i during phase 1, but not with the peak amplitude. Rhythmical [Ca 2+] i changes persisted in nicardipine, but were abolished in Ca 2+-free solution as well as by SK&F96365, cyclopiazonic acid, thapsigargin, 2-APB, xestospongin C or ryanodine. Intracellular Ca 2+ stores visualised with the low-affinity Ca 2+ indicator fluo-3FF were found to be enriched with ryanodine receptors (RyRs) detected with BODIPY TR-X ryanodine. Rhythmical [Ca 2+] i changes originated from a perinuclear S/ER element showing the highest RyR density. Immunostaining with anti-TRPC3,6,7 antibodies revealed the expression of these channel proteins in the ICC plasmalemma. This suggests that these rhythmical [Ca 2+] i changes, a key element of ICC pacemaking activity, result from S/ER Ca 2+ release which is mediated via RyRs and IP 3 receptors and is modulated by the activity of S/ER-Ca 2+-ATPase and TRP channels but not by L-type Ca 2+ channels.

A New Insight Into the Pathogenesis of Coronary Vasospasm

See related article, pages 682–689 To elucidate the control mechanisms of coronary vascular tone is one of the central interests in cardiac pathophysiology, because ischemic heart disease is one of the major causes of death in many countries. Although the coronary vascular tone is finally determined by the contractile state of smooth muscle, exo-smooth muscle mechanisms including autonomic nerves, endothelial cells, and blood cells have been shown indispensable for the control. Particular types of receptors, ion channels, and intracellular signal-cascades exist in coronary smooth muscle cells and mediate the controls by exo-smooth muscle elements (Figure). For example, stimulation of α1-adrenergic receptor, which is physiologically achieved by noradrenaline (NA) released from sympathetic presynapses, increases the intracellular Ca2+ (Ca2+i) via phosphatidylinositol (PI)-turnover and also probably via opening of receptor-activated Ca2+-permeable TRP channel. Membrane depolarization does so via opening voltage-dependent Ca2+ channels. The augmentation of Ca2+i results in contraction of the smooth muscle cells and thus constriction of the coronary artery.1 Activation of β2-adrenergic receptor, in turn, inhibits the coronary smooth muscle contraction through protein-kinase A pathway.2 The contraction is also suppressed by protein-kinase G activated by a dilating factor, nitric oxide (NO), which is produced in the endothelial cells. The mechanism for control of vascular tone . Activation of voltage-gated Ca2+ channel at the presynapse of sympathetic nerve increases intracellular Ca2+, which triggers a release of norepinephrine (NE). NE stimulates adrenergic α1 receptor (α1R) in the smooth muscle cells and drives PI-turnover as described in this scheme. The intracellular Ca2+, which flows into the cells via smooth-muscle’s Ca2+ channel or moves from endoplasmic reticulum (ER), contracts the cells. The constriction of smooth muscle cells is prevented by activation …

Adenosine A1 receptor antagonist improves intradialytic hypotension

Intradialytic hypotension is a most frequent complication of hemodialysis and may contribute to cardiovascular events and high mortality. There is a hypothesis that an increase in adenosine generation during hemodialysis may cause vasodilation and a decrease in cardiac output, which results in systemic hypotension. We studied whether this can be blocked by an adenosine A1 receptor antagonist. We investigated the effects of an A1 antagonist, FK352, injection in 30 chronic hemodialysis patients with frequent intradialytic hypotension by a prospective, multicenter, double-blind placebo-controlled study for 4 weeks after 4 weeks of the observation period. Intradialytic hypotension was defined as systolic blood pressure (SBP) less than 110 mmHg, with SBP drop of more than 30 mmHg from the predialysis level. The efficacy of FK352 was primarily assessed by the reduction rate of dialysis hypotension between the FK352 and placebo groups. Incidence of emergency treatments caused by hypotension was evaluated. FK352 (50 mg, intravenous) or an equivalent placebo was injected into the dialysis circuit 1 h after starting dialysis. Blood pressure and heart rate were monitored every 30 min during dialysis. FK352 significantly improved intradialytic hypotension (P=0.046), in that the reduction rates of intradialytic hypotension in the FK352 and placebo groups were -12.8% (Q1 (first quantile), Q3 (third quantile): -27.5, -1.7), and +8.3% (Q1, Q3: -16.6, +16.7), respectively. The frequency of discontinuation of dialysis was significantly reduced by FK352. No apparent side effects were observed from treatment with FK352. In conclusion, the A1 antagonist FK352 may offer a novel therapeutic option for chronic dialysis patients associated with intradialytic hypotension.

Inactivation of Calcium Channels in Vascular Smooth Muscle Myocytes

Many of the structural domains involved in Ca2+ channel (CACN) inactivation are also involved in determining their sensitivity to antagonist inhibition. We hypothesize that differences in inactivation properties and their structural determinants may suggest candidate domains as targets for the development of novel, selective antagonists. The characteristics of Ca2+ current (ICa) inactivation, steady-state inactivation (SSIN), and recovery from inactivation were studied in freshly dispersed smooth muscle cells from rabbit portal vein (RPV) using whole-cell, voltage-clamp methods. The time course of inactivation could be represented by two time constants. Increasing ICa by increasing [Ca2+]o or with more negative holding potentials decreased both time constants. With Sr2+, Ba2+, or Na+ as the charge carrier, ICa inactivation was also represented by two time constants, both of which were larger than those found with Ca2+. With Ca2+, Sr2+, or Ba2+ as the charge carrier, both time constants had minimum values near the voltage associated with maximum current. When Na+ (140 mM) was the charge carrier, voltages for Imax (-20 mV) or taumin (0 mV) did not correspond. SSIN of ICa had a half-maximum voltage of -32 +/- 4 mV for Ca2+, -43 mV +/- 5 mV for Sr2+, -41 +/- 5 mV for Ba2+, and -68 +/- 6 mV for Na+. The slope factor for SSIN per e-fold voltage change was 6.5 +/- 0.2 mV for Ca2+, 6.8 +/- 0.3 for Sr2+, and 6.6 +/- 0.2 for Ba2+, representing four equivalent charges. When Na+ or Li+ was the charge carrier, the slope factor was 13.5 +/- 0.7 mV, representing two equivalent charges. For ICa in rat left ventricular (rLV) myocytes, there was no difference in the slope factor of SSIN for Ca2+ and Na+. The rate of recovery of ICa from inactivation varied inversely with recovery voltage and was independent of the charge carrier. These results suggest that inactivation of ICa in PV myocytes possess an intrinsic voltage dependence that is modified by Ca2+. For RPV but not rLV ICa, the charge of the permeating ion confers the voltage-dependency of SSIN.

Angiogenesis inhibitor TNP-470 prevents implanted liver metastases after partial hepatectomy in an experimental model without impairing wound healing.

The ability of the angiogenesis inhibitor TNP-470 to prevent liver metastasis after partial hepatectomy, and whether TNP-470 impairs liver regeneration or skin wound healing, was evaluated. Following the injection of VX2 carcinoma cells into the portal vein of rabbits, half of the animals underwent resection of the middle hepatic lobe (hepatectomized group) and half did not (non-hepatectomized group). TNP-470 (50 mg) was infused continuously into the portal vein in both groups for 7 days, while controls received only water. The hepatectomized TNP-470-treated group had significantly fewer tumours (mean(s.e.m.) 23.3(12.3)) than the hepatectomized control group (123.7(24.4)). There was no significant difference in the 5-bromo-2'-deoxyuridine labelling index of regenerated hepatocytes between the TNP-470-treated and control groups. Wound healing in TNP-470-treated animals was not impaired. Intraportal infusion of TNP-470 prevents the recurrence of liver metastasis after partial hepatectomy without impairing healing or liver regeneration.

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.

Interstitial cells in the vasculature

Interstitial cells of Cajal are believed to play an important role in gastrointestinal tissues by generating and propagating electrical slow waves to gastrointestinal muscles and/or mediating signals from the enteric nervous system. Recently cells with similar morphological characteristics have been found in the wall of blood vessels such as rabbit portal vein and guinea pig mesenteric artery. These non-contractile cells are characterised by the presence of numerous processes and were easily detected in the wall of the rabbit portal vein by staining with methylene blue or by antibodies to the marker of Interstitial Cells of Cajal c-kit. These vascular cells have been termed "interstitial cells" by analogy with interstitial cells found in the gastrointestinal tract. Freshly dispersed interstitial cells from rabbit portal vein and guinea pig mesenteric artery displayed various Ca2+-release events from endo/sarcoplasmic reticulum including fast localised Ca2+ transients (Ca2+ sparks) and longer and slower Ca2+ events. Single interstitial cells from the rabbit portal vein, which is a spontaneously active vessel, also demonstrated rhythmical Ca2+ oscillations associated with membrane depolarisations, which suggests that in this vessel interstitial cells may act as pacemakers for smooth muscle cells. The function of interstitial cells from the mesenteric arteries is yet unknown. This article reviews some of the recent findings regarding interstitial cells from blood vessels obtained by our laboratory using electron microscopy, immunohistochemistry, tight-seal patch-clamp recording, and fluorescence confocal imaging techniques.

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.

Role of Protein Phosphatase Type 1 in Contractile Functions: Myosin Phosphatase

Role of Protein Phosphatase Type 1 in Contractile Functions: Myosin Phosphatase

Function of interstitial cells of Cajal in the rabbit portal vein.

Interstitial cells of Cajal (ICCs) were identified in the intact fixed media of the rabbit portal vein (RPV) using c-kit staining. The following experiments were performed using single cell preparations of the enzyme-dispersed vessel. Surviving contacts between the processes of single ICCs and the bodies of smooth muscle cells (SMCs) were observed in electron micrographs and by confocal microscopy. Spontaneous rhythmical [Ca2+]i oscillations were observed in ICCs after loading with the calcium indicator fluo-3 and were associated with depolarizations of the ICCs recorded by tight-seal patch pipette. To investigate signal transmission from ICCs to SMCs in dispersed cell pairs, or within small surviving fragments of the ICC network, an ICC was stimulated under voltage-clamp, while changes in [Ca2+]i in the stimulated cell as well as in a closely adjacent SMC or ICCs were monitored using fast x-y confocal imaging of fluo-3 fluorescence. After stimulation of single voltage-clamped ICC by a depolarizing step similar in duration to depolarizations associated with spontaneous [Ca2+]i oscillations, a depolarization and transient elevation of [Ca2+]i was observed in a closely adjacent SMCs after a delay of up to 4 seconds. In contrast, signal transmission from ICC to ICC was much faster, the delay being less than 200 ms. These results suggest that the an ICC may, in addition to generating an electrical signal (such as a slow wave) and thereby acting as a pacemaker for vascular SMCs of RPV, also release some unknown diffusible substance, which depolarizes the SMCs.

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.

Muscarinic M2 Receptor Stimulation of Cav1.2b Requires Phosphatidylinositol 3-Kinase, Protein Kinase C, and c-Src

This study investigated regulation of L-type calcium channels (Cav1.2b) by acetylcholine (ACh) in rabbit portal vein myocytes. Whole-cell currents were recorded using 5 mmol/L barium as charge carrier. ACh (10 micromol/L) increased peak currents by 40%. This effect was not reversed by the selective muscarinic M3 receptor antagonist 4-DAMP (100 nmol/L) but was blocked by the M2 receptor antagonist methoctramine (5 micromol/L). The classical and novel protein kinase C (PKC) antagonist calphostin C (50 nmol/L) abolished ACh responses, whereas the classical PKC antagonist Gö6976 (200 nmol/L) had no effect. ACh responses were also abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (20 micromol/L), by the c-Src inhibitor PP2 (10 micromol/L) (but not the inactive analogue PP3), and by dialyzing cells with an antibody to the G-protein subunit Gbetagamma. Cells dialyzed with c-Src had significantly greater currents than control cells. Current enhancement persisted in the presence of LY294002, suggesting that c-Src is downstream of PI3K. Phorbol 12,13-dibutyrate (PDBu, 0.1 micromol/L) increased currents by 74%. This effect was abolished by calphostin C and reduced by Gö6976. The PDBu response was also reduced by PP2, and the PP2-insensitive component was blocked by Gö6976. In summary, these data suggest that ACh enhances Cav1.2b currents via M2 receptors that couple sequentially to Gbetagamma, PI3K, a novel PKC, and c-Src. PDBu stimulates the novel PKC/c-Src pathway along with a second pathway that is independent of c-Src and involves a classical PKC.

Synergism between inositol phosphates and diacylglycerol on native TRPC6-like channels in rabbit portal vein myocytes.

In rabbit portal vein myocytes noradrenaline activates a non-selective cation current (Icat) which involves a transient receptor potential protein (TRPC6). Previously we have shown that the diaylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulates Icat via a protein kinase C (PKC)-independent mechanism, and in the present study we have investigated the interaction between inositol phosphates (InsPs) and OAG on Icat. With whole-cell recording of Icat from freshly isolated rabbit portal vein myocytes the amplitude and rate of activation of noradrenaline-evoked Icat were much greater than those of OAG-induced Icat. Inclusion of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in the pipette solution did not evoke Icat but greatly potentiated the amplitude and rate of activation of OAG-induced Icat. With isolated outside-out patches Ins(1,4,5)P3 markedly increased the rate of activation and the open probability of OAG-evoked channel activity, with no change in unitary conductance, channel mean open times or burst durations. The effects of Ins(1,4,5)P3 were mimicked by Ins(2,4,5)P3, 3-F-Ins(1,4,5)P3 and Ins(1,4)P2 but not by Ins(1,3,4,5)P4 and the potentiating effects of InsPs were not inhibited by heparin. Therefore it is concluded that both DAG and InsPs are necessary for full activation of Icat by noradrenaline and the effect of InsPs is via a heparin-insensitive mechanism and represents a novel action of InsPs.

Identification of Interstitial Cells of Cajal in the Rabbit Portal Vein

Abstract Two layers of interstitial cells (ICs) of Cajal were detected by c-kit and methylene blue staining in the media of the rabbit portal vein in subendothelial intramuscular and deeper intramuscular positions, displaced radially from each other by about 40–70 μm. Two morphologically distinct types of ICs were found among enzymatically dispersed cells from this vessel: small multipolar cells with stellate-shaped bodies not exceeding 20 μm, and spindle-shaped cells from 40 to 300 μm in length with numerous branching processes. Relaxed smooth muscle cells (SMCs) had a more constant length (90–150 μm). The cell membrane capacitance was 46.5±2.2 pF in SMCs, 39.7±2.4 pF in spindle-shaped ICs and 27.8±0.7 pF in multipolar ICs. Although darker under phase contrast, after loading with fluo-4 AM, single isolated ICs of both types usually had brighter fluorescence than SMCs and displayed various spontaneous calcium events, including Ca 2+ sparks and Ca 2+ waves. Ca 2+ waves were usually followed by contraction of SMCs but no change in shape of ICs. In some ICs spontaneous [Ca 2+ ] i transients (lasting about 2 s) which propagated towards the end of the processes were observed. Physical contacts between the processes of ICs and the body of one or more SMCs survived the isolation procedure. Application of noradrenaline (1–10 μM), caffeine (1–10 mM) or high-K + solution (60 mM) led to a rise of [Ca 2+ ] i in both SMCs and ICs evoking contraction of SMCs but not ICs. No differences in electrophysiological characteristics between single enzymatically isolated IC and SMC were detected; thus, the resting membrane potential estimated under current–clamp conditions was −46.5±2.0 mV in spindle-shaped ICs and −45.6±2.7 mV in SMCs. Under voltage–clamp, both ICs and SMCs revealed a well-developed voltage-gated nifedipine-sensitive L-type Ca 2+ current, a set of K + currents, including spontaneous transient outward currents (STOCs) but no Na + current. This study for the first time directly demonstrated the presence in vascular tissue of ICs. Possible roles for ICs including their involvement in spontaneous activity of the vessel were discussed.

Store-operated Ca 2+-permeable non-selective cation channels in smooth muscle cells

Over twenty years ago it was shown that depletion of the intracellular Ca 2+ store in smooth muscle triggered a Ca 2+ influx mechanism. The purpose of this review it to describe recent electrophysiological data which indicate that Ca 2+ influx occurs through discrete ion channels in the plasmalemma of smooth muscle cells. The effect of external Ca 2+ on the amplitude and reversal potential of whole-cell and single channel currents suggests that there are at least two, and probably more, distinct store-operated channels (SOCs) which have markedly different permeabilities to Ca 2+ ions. Two activation mechanisms have been identified which involve Ca 2+ influx factor and protein kinase C (PKC) activation via diacylglycerol. In addition, in rabbit portal vein cells there is evidence that stimulation of α-adrenoceptors can stimulate SOC opening via PKC in a store-independent manner. There is at present little knowledge on the molecular identity of SOCs but it has been proposed that TRPC1 may be a component of the functional channel. We also summarise the data showing that SOCs may be involved in contraction and cell proliferation of smooth muscle. Finally, we highlight the similarities and differences of SOCs and receptor-operated cation channels that are present in native rabbit portal vein myocytes.

Identification of interstitial cells of Cajal in the rabbit portal vein

Two layers of interstitial cells (ICs) of Cajal were detected by c-kit and methylene blue staining in the media of the rabbit portal vein in subendothelial intramuscular and deeper intramuscular positions, displaced radially from each other by about 40–70 μm. Two morphologically distinct types of ICs were found among enzymatically dispersed cells from this vessel: small multipolar cells with stellate-shaped bodies not exceeding 20 μm, and spindle-shaped cells from 40 to 300 μm in length with numerous branching processes. Relaxed smooth muscle cells (SMCs) had a more constant length (90–150 μm). The cell membrane capacitance was 46.5±2.2 pF in SMCs, 39.7±2.4 pF in spindle-shaped ICs and 27.8±0.7 pF in multipolar ICs. Although darker under phase contrast, after loading with fluo-4 AM, single isolated ICs of both types usually had brighter fluorescence than SMCs and displayed various spontaneous calcium events, including Ca 2+ sparks and Ca 2+ waves. Ca 2+ waves were usually followed by contraction of SMCs but no change in shape of ICs. In some ICs spontaneous [Ca 2+] i transients (lasting about 2 s) which propagated towards the end of the processes were observed. Physical contacts between the processes of ICs and the body of one or more SMCs survived the isolation procedure. Application of noradrenaline (1–10 μM), caffeine (1–10 mM) or high-K + solution (60 mM) led to a rise of [Ca 2+] i in both SMCs and ICs evoking contraction of SMCs but not ICs. No differences in electrophysiological characteristics between single enzymatically isolated IC and SMC were detected; thus, the resting membrane potential estimated under current–clamp conditions was −46.5±2.0 mV in spindle-shaped ICs and −45.6±2.7 mV in SMCs. Under voltage–clamp, both ICs and SMCs revealed a well-developed voltage-gated nifedipine-sensitive L-type Ca 2+ current, a set of K + currents, including spontaneous transient outward currents (STOCs) but no Na + current. This study for the first time directly demonstrated the presence in vascular tissue of ICs. Possible roles for ICs including their involvement in spontaneous activity of the vessel were discussed.

Phosphorylation of the myosin phosphatase targeting subunit and CPI-17 during Ca2+ sensitization in rabbit smooth muscle.

Myosin phosphatase (MLCP) plays a critical regulatory role in the Ca(2+) sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr(695) of the MLCP regulatory subunit (MYPT1) and at Thr(38) of the MLCP inhibitor protein CPI-17 results in inhibition of MLCP activity. We have previously demonstrated that CPI-17 Thr(38) phosphorylation plays an important role in G-protein-mediated inhibition of MLCP in tonic arterial smooth muscle. Here, we attempted to evaluate the function of MYPT1 in phasic rabbit portal vein (PV) and vas deferens (VD) smooth muscles. Using site- and phospho-specific antibodies, phosphorylation of MYPT1 Thr(695) and CPI-17 Thr(38) was examined along with MYPT1 Thr(850), which is a non-inhibitory Rho-kinase site. We found that both CPI-17 Thr(38) and MYPT1 Thr(850) were phosphorylated in response to agonists or GTPgammaS concurrently with contraction and myosin phosphorylation in alpha-toxin-permeabilized PV tissues. In contrast, phosphorylation of MYPT1 Thr(695) did not increase. Comparable results were also obtained in both permeabilized and intact VD. The Rho-kinase inhibitor Y-27632 and the protein kinase C (PKC) inhibitor GF109203X suppressed phosphorylation of MYPT1 Thr(850) and CPI-17 Thr(38), respectively, in intact VD while MYPT1 Thr(695) phosphorylation was insensitive to both inhibitors. These results indicate that phosphorylation of MYPT1 Thr(695) is independent of stimulation of G-proteins, Rho-kinase or PKC. In the phasic PV, phosphorylation of CPI-17 Thr(38) may contribute towards inhibition of MLCP while the phasic visceral VD, which has a low CPI-17 concentration, probably utilizes other Ca(2+) sensitizing mechanisms for inhibiting MLCP besides phosphorylation of MYPT1 and CPI-17.

Force maintenance in smooth muscle: analysis using sinusoidal perturbations

The “latch state” or force maintenance may be due to the emergence of a distinct set of dephosphorylated, slowly cycling “latch” cross-bridges, slowing of the overall cross-bridge cycling rate, or a non-cross-bridge contribution. This was investigated by sinusoidally oscillating strips of intact rabbit portal vein or aorta. Tissue strips were activated with KCl depolarization, resulting in a sustained increase of MLC 20 phosphorylation or 10 μM phenylephrine, resulting in a transient increase in MLC 20 phosphorylation. Stiffness was calculated from the force response to a small, sine-wave oscillation in muscle length (1–100 Hz). The results produced a 3-dimensional plot of stiffness versus the frequency of oscillation (Hz) versus time (s), or stiffness distribution profile. During KCl depolarization, the stiffness distribution profile displayed a shift toward lower frequencies, suggesting a general slowing in the overall cross-bridge cycling rate during force maintenance. On the other hand, phenylephrine stimulation did not display a significant change in the stiffness distribution profile, suggesting that the overall cross-bridge cycling rate did not significantly change during force maintenance.

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.

Crosstalk between ryanodine receptors and IP(3) receptors as a factor shaping spontaneous Ca(2+)-release events in rabbit portal vein myocytes.

In smooth muscle cells freshly isolated from rabbit portal vein, there was only one site discharging the majority of spontaneous Ca(2+)-release events; the activity of this single site was studied using laser scanning confocal imaging after loading the cells with the fluorescent Ca(2+) indicator fluo-4 acetoxymethyl ester. Localised spontaneous Ca(2+)-release events visualised by line-scan imaging revealed two predominant spatiotemporal patterns: (i) small-amplitude, fast events similar to Ca(2+) sparks in cardiomyocytes and (ii) larger and slower events. The sum of two Gaussian profiles was well fitted to the amplitude histogram (peak frequencies at 1.8 and 3.2 F/F(0)) and spatial spread (full width at half-maximal amplitude) histogram (peak frequencies at 2 and 3.8 microm) for the 230 localised Ca(2+)-release events analysed. The existence of two populations of Ca(2+)-release events was also supported by the histograms of the rise times and half-decay times, which revealed modes at 38 and 65 ms, respectively. Shifting the scan line along the z-axis during imaging from a single discharge site suggested that the appearance of two populations of Ca(2+)-release events is not due to out-of-focus imaging. Both small and large events persisted upon 3-5 min exposure to 1-5 microM nicardipine, but were abolished after 10-15 min exposure to 50-100 microM ryanodine, 0.1 microM thapsigargin or 10 microM cyclopiazonic acid. Only small-amplitude, fast events persisted in the presence of inhibitors of inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, 10 microM xestospongin C or 30 microM 2-aminoethoxy-diphenylborate (2-APB), or in the presence of 2.5 microM U-73122 (a phospholipase C (PLC) inhibitor). Coupling between neighbouring Ca(2+)-release domains giving rise to spontaneous [Ca(2+)](i) waves was abolished in the presence of 2-APB. Examination of the saltatory propagation of the waves suggested that the critical factor that determines propagation between domains is a time-dependent change in the sensitivity of ryanodine receptors and/or IP(3) receptors to Ca(2+), which can give rise to 'loose coupling' between release sites. These results suggest that activation of IP(3) receptors (due to the tonic activity of PLC and ongoing production of IP(3)) recruits neighbouring domains of ryanodine receptors, leading to larger Ca(2+) releases and saltatory propagation of [Ca(2+)](i) waves in portal vein myocytes.