Functional evidence for Na+-activated K+ channels in circular smooth muscle of the opossum lower esophageal sphincter

Na(+) reduction induces contraction of opossum lower esophageal sphincter (LES) circular smooth muscle strips in vitro; however, the mechanism(s) by which this occurs is unknown. The purpose of the present study was to investigate the electrophysiological effects of low Na(+) on opossum LES circular smooth muscle. In the presence of atropine, quanethidine, nifedipine, and substance P, conventional intracellular electrodes recorded a resting membrane potential (RMP) of -37.5 +/- 0.9 mV (n = 4). Decreasing [Na(+)] from 144.1 to 26.1 mM by substitution of equimolar NaCl with choline Cl depolarized the RMP by 7.1 +/- 1.1 mV. Whole cell patch-clamp recordings revealed outward K(+) currents that began to activate at -60 mV using 400-ms stepped test pulses (-120 to +100 mV) with increments of 20 mV from holding potential of -80 mV. Reduction of [Na(+)] in the bath solution inhibited K(+) currents in a concentration-dependent manner. Single channels with conductance of 49-60 pS were recorded using cell-attached patch-clamp configurations. The channel open probability was significantly decreased by substitution of bath Na(+) with equimolar choline. A 10-fold increase of [K(+)] in the pipette shifted the reversal potential of the single channels to the positive by -50 mV. These data suggest that Na(+)-activated K(+) channels exist in the circular smooth muscle of the opossum LES.     

Role of HERG-like K+ currents in opossum esophageal circular smooth muscle

An inwardlyrectifying K⁺ conductance closelyresembling the human ether-a-go-go-related gene (HERG) current wasidentified in single smooth muscle cells of opossum esophageal circularmuscle. When cells were voltage clamped at 0 mV, in isotonicK⁺ solution (140 mM), stephyperpolarizations to 120 mV in 10-mV increments resulted inlarge inward currents that activated rapidly and then declined slowly(inactivated) during the test pulse in a time- and voltage- dependentfashion. The HERG K⁺ channelblockers E-4031 (1 µM), cisapride (1 µM), andLa³⁺ (100 µM) strongly inhibitedthese currents as did millimolar concentrations ofBa²⁺. Immunoflourescence stainingwith anti-HERG antibody in single cells resulted in punctate stainingat the sarcolemma. At membrane potentials near the resting membranepotential (50 to 70 mV), thisK⁺ conductance did not inactivatecompletely. In conventional microelectrode recordings, both E-4031 andcisapride depolarized tissue strips by 10 mV and also induced phasiccontractions. In combination, these results provide direct experimentalevidence for expression of HERG-likeK⁺ currents in gastrointestinalsmooth muscle cells and suggest that HERG plays an important role inmodulating the resting membrane potential.

     

MAPK mediates PKC-dependent contraction of cat esophageal and lower esophageal sphincter circular smooth muscle

Esophageal (ESO) circular muscle contraction and lower esophageal sphincter (LES) tone are PKC dependent. Because MAPKs may be involved in PKC-dependent contraction, we examined ERK1/ERK2 and p38 MAPKs in ESO and LES. In permeabilized LES muscle cells, ERK1/2 antibodies reduced 1,2-dioctanoylglycerol (DG)- and threshold ACh-induced contraction, which are PKC dependent, but not maximal ACh, which is calmodulin dependent. LES tone was reduced by the ERK1/2 kinase inhibitor PD-98059 and by the p38 MAPK inhibitor SB-203580. In permeable ESO cells, ACh contraction was reduced by ERK1/ERK2 and p38 MAPK antibodies and by PD-98059 and SB-203580. ACh increased MAPK activity and phosphorylation of MAPK and of p38 MAPK. The 27-kDa heat shock protein (HSP27) antibodies reduced ACh contraction. HSP27 and p38 MAPK antibodies together caused no greater inhibition than either one alone. p38 MAPK and HSP27 coprecipitated after ACh stimulation, suggesting that HSP27 is linked to p38 MAPK. These data suggest that PKC-dependent contraction in ESO and LES is mediated by the following two distinct MAPK pathways: ERK1/2 and HSP27-linked p38 MAPK.     

PGF(2alpha)-induced contraction of cat esophageal and lower esophageal sphincter circular smooth muscle

Lower esophageal sphincter (LES) tone depends on PGF(2alpha) and thromboxane A(2) acting on receptors linked to G(i3) and G(q) to activate phospholipases and produce second messengers resulting in muscle contraction. We therefore examined PGF(2alpha) signal transduction in circular smooth muscle cells isolated by enzymatic digestion from cat esophagus (Eso) and LES. In Eso, PGF(2alpha)-induced contraction was inhibited by antibodies against the alpha-subunit of G(13) and the monomeric G proteins RhoA and ADP-ribosylation factor (ARF)1 and by the C3 exoenzyme of Clostridium botulinum. A [(35)S]GTPgammaS-binding assay confirmed that G(13), RhoA, and ARF1 were activated by PGF(2alpha). Contraction of Eso was reduced by propranolol, a phospholipase D (PLD) pathway inhibitor and by chelerythrine, a PKC inhibitor. In LES, PGF(2alpha)-induced contraction was inhibited by antibodies against the alpha-subunit of G(q) and G(i3), and a [(35)S]GTPgammaS-binding assay confirmed that G(q) and G(i3) were activated by PGF(2alpha). PGF(2alpha)-induced contraction of LES was reduced by U-73122 and D609 and unaffected by propranolol. At low PGF(2alpha) concentration, contraction was blocked by chelerythrine, whereas at high concentration, contraction was blocked by chelerythrine and CGS9343B. Thus, in Eso, PGF(2alpha) activates a PLD- and protein kinase C (PKC)-dependent pathway through G(13), RhoA, and ARF1. In LES, PGF(2alpha) receptors are coupled to G(q) and G(i3), activating phosphatidylinositol- and phosphatidylcholine-specific phospholipase C. At low concentrations, PGF(2alpha) activates PKC. At high concentration, it activates both a PKC- and a calmodulin-dependent pathway.     

Opposing roles of K(+) and Cl(-) channels in maintenance of opossum lower esophageal sphincter tone

The ionic basis underlying the maintenance of myogenic tone of lower esophageal sphincter circular muscle (LES) was investigated in opossum with the use of standard isometric tension and conventional intracellular microelectrode recordings in vitro. In tension recording studies, nifedipine (1 microM) reduced basal tone to 27.7 +/- 3.8% of control. The K(+) channel blockers tetraethylammonium (TEA, 2 mM), charybdotoxin (100 nM), and 4-aminopyridine (4-AP, 2 mM) enhanced resting tone, whereas apamin and glibenclamide were without affect. Cl(-) channel blockers DIDS (500 microM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (500 microM), as well as niflumic acid (0.1-300 microM), decreased basal tone, but tamoxifen was without effect. Intracellular microelectrode recordings revealed ongoing, spontaneous, spike-like action potentials (APs). Nifedipine abolished APs and depolarized resting membrane potential (RMP). Both TEA and 4-AP significantly depolarized RMP and augmented APs, whereas niflumic acid dose-dependently hyperpolarized RMP and abolished APs. These data suggest that, in the opossum, basal tone is associated with continuous APs and that K(+) and Ca(2+)-activated Cl(-) channels have important opposing roles in the genesis of LES tone.     

IL-1beta signaling in cat lower esophageal sphincter circular muscle

In a cat model of acute experimental esophagitis, resting in vivo lower esophageal sphincter (LES) pressure and in vitro tone are lower than in normal LES, and the LES circular smooth muscle layer contains elevated levels of IL-1beta that decrease the LES tone of normal cats. We now examined the mechanisms of IL-1beta-induced reduction in LES tone. IL-1beta significantly reduced acetylcholine-induced Ca(2+) release in Ca(2+)-free medium, and this effect was partially reversed by catalase, demonstrating a role of H(2)O(2) in these changes. IL-1beta significantly increased the production of H(2)O(2), and the increase was blocked by the p38 MAPK inhibitor SB-203580, by the cytosolic phospholipase A(2) (cPLA(2)) inhibitor AACOCF3, and by the NADPH oxidase inhibitor apocynin, but not by the MEK1 inhibitor PD-98059. IL-1beta significantly increased the phosphorylation of p38 MAPK and cPLA(2). IL-1beta-induced cPLA(2) phosphorylation was blocked by SB-203580 but not by AACOCF3, suggesting sequential activation of p38 MAPK-phosphorylating cPLA(2). The IL-1beta-induced reduction in LES tone was partially reversed by AACOCF3 and by the Ca(2+)-insensitive PLA(2) inhibitor bromoenol lactone (BEL). IL-1beta significantly increased cyclooxygenase (COX)-2 and PGE(2) levels. The increase in PGE(2) was blocked by SB-203580, AACOCF3, BEL, and the COX-2 inhibitor NS-398 but not by PD-98059 or the COX-1 inhibitor valeryl salicylate. The data suggested that IL-1beta reduces LES tone by producing H(2)O(2), which may affect Ca(2+)-release mechanisms and increase the synthesis of COX-2 and PGE(2). Both H(2)O(2) and PGE(2) production depend on sequential activation of p38 MAPK and cPLA(2). cPLA(2) activates NADPH oxidases, producing H(2)O(2), and may produce arachidonic acid, converted to PGE(2) via COX-2.     

Inwardly rectifying K(+) channels in esophageal smooth muscle

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K(+) (K(ir)) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K(+) equilibrium potential (E(k)) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential (E(rev)) of -76.5 mV. Elevation of external K(+) increased the inward current amplitude and positively shifted its E(rev) after the E(k), suggesting that potassium ions carry this current. External Ba(2+) and Cs(+) inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC(50) for Ba(2+) and Cs(+) at -60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba(2+) of 10 microM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that K(ir) channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.     

Functional and molecular analysis of L-type calcium channels in human esophagus and lower esophageal sphincter smooth muscle

Excitation of human esophageal smooth muscle involves the release of Ca(2+) from intracellular stores and influx. The lower esophageal sphincter (LES) shows the distinctive property of tonic contraction; however, the mechanisms by which this is maintained are incompletely understood. We examined Ca(2+) channels in human esophageal muscle and investigated their contribution to LES tone. Functional effects were examined with tension recordings, currents were recorded with patch-clamp electrophysiology, channel expression was explored by RT-PCR, and intracellular Ca(2+) concentration was monitored by fura-2 fluorescence. LES muscle strips developed tone that was abolished by the removal of extracellular Ca(2+) and reduced by the application of the L-type Ca(2+) channel blocker nifedipine (to 13 +/- 6% of control) but was unaffected by the inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). Carbachol increased tension above basal tone, and this effect was attenuated by treatment with CPA and nifedipine. Voltage-dependent inward currents were studied using patch-clamp techniques and dissociated cells. Similar inward currents were observed in esophageal body (EB) and LES smooth muscle cells. The inward currents in both tissues were blocked by nifedipine, enhanced by Bay K8644, and transiently suppressed by acetylcholine. The molecular form of the Ca(2+) channel was explored using RT-PCR, and similar splice variant combinations of the pore-forming alpha(1C)-subunit were identified in EB and LES. This is the first characterization of Ca(2+) channels in human esophageal smooth muscle, and we establish that L-type Ca(2+) channels play a critical role in maintaining LES tone.     

Role of Ca2+-activated Cl- channels and MLCK in slow IJP in opossum esophageal smooth muscle

The possible contribution of Ca2+-activated Cl- channel [I(Cl(Ca))] and myosin light-chain kinase (MLCK) to nonadrenergic, noncholinergic slow inhibitory junction potentials (sIJP) was studied using conventional intracellular microelectrode recordings in circular smooth muscle of opossum esophageal body and guinea pig ileum perfused with Krebs solution containing atropine (3 microM), guanethidine (3 microM), and substance P (1 microM). In opossum esophageal circular smooth muscle, resting membrane potential (MP) was -51.9 +/- 0.7 mV (n = 89) with MP fluctuations of 1-3 mV. A single square-wave nerve stimulation of 0.5 ms duration and 80 V induced a sIJP with amplitude of 6.3 +/- 0.2 mV, half-amplitude duration of 635 +/- 19 ms, and rebound depolarization amplitude of 2.4 +/- 0.1 mV (n = 89). 9-Anthroic acid (A-9-C), niflumic acid (NFA), wortmannin, and 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) abolished MP fluctuations, sIJP, and rebound depolarization in a concentration-dependent manner. A-9-C and NFA but not wortmannin and ML-9 hyperpolarized MP. In guinea pig ileal circular smooth muscle, nerve stimulation elicited an IJP composed of both fast (fIJP) and slow (sIJP) components, followed by rebound depolarization. NFA (200 microM) abolished sIJP and rebound depolarization but left the fIJP intact. These data suggest that in the tissues studied, activation of I(Cl(Ca)), which requires MLCK, contributes to resting MP, and that closing of I(Cl(Ca)) is responsible for sIJP.     

Evidence for altered circular smooth muscle cell function in lower esophageal sphincter of W/Wv mutant mice

Nitrergic neurotransmission to gut smooth muscle is impaired in W/W(v) mutant mice, which lack intramuscular interstitial cells of Cajal (ICC-IM). In addition, these mice have been reported to have smaller amplitude unitary potentials (UPs) and a more negative resting membrane potential (RMP) than control mice. These abnormalities have been attributed to absence of ICC-IM, but it remains possible that they are due to alterations at the level of the smooth muscle itself. Amphotericin-B-perforated patch-clamp recordings and Ca(2+) imaging (fura 2) were compared between freshly isolated single circular smooth muscle cells (CSM) from W/W(v) mutant and control mice lower esophageal sphincter (LES). There was no significant difference in seal resistance, capacitance, or input resistance in response to applied electrotonic current pulses between CSM cells from W/W(v) mutants and controls. Compared with control mice, RMP was more negative and UPs significantly smaller in CSM cells from mutant mice LES. Administration of caffeine induced an inward current in cells from both mutant and control mice, but the current density was significantly larger in cells from W/W(v) mutants. Membrane potential hyperpolarization induced by sodium nitroprusside was larger in cells from control mice vs. W/W(v) mutants. In addition, intracellular Ca(2+) transients induced by caffeine were significantly increased in cells from mutants. These findings indicate that LES CSM is abnormal in W/W(v) mutant mice. Thus some physiological functions attributed to ICC-IM based on experiments in smooth muscle of ICC deficient mice may need to be reconsidered.     

Calcium source diversity in feline lower esophageal sphincter circular and sling muscle

Within muscular equivalents of cat lower esophageal sphincter (LES), the circular muscle develops greater spontaneous tone, whereas the sling muscle is more responsive to cholinergic stimulation. Smooth muscle contraction involves a combination of calcium release from stores and of calcium entry via several pathways. We hypothesized that there are differences in the sources of Ca(2+) used for contraction in sling and circular muscles and that these differences could contribute to functional asymmetry observed within LES. Contraction of muscle strips from circular and sling regions of LES was assessed in the presence of TTX. In Ca(2+)-free Krebs, tone was inhibited to a greater degree in circular than sling muscle. L-type Ca(2+) channel blockade with nifedipine or verapamil inhibited tone in LES circular but not sling muscle. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA) caused greater increase in tone in sling than in circular muscle. The phospholipase C inhibitor U-73122 and the SR inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptor blocker 2-aminoethoxydiphenyl borate (2-APB) inhibited tone in circular and sling muscles, demonstrating that continuous release of Ca(2+) from Ins(1,4,5)P(3)-sensitive stores is important in tone generation in both muscles. In Ca(2+)-free Krebs, ACh-induced contractions (AChC) were inhibited to a greater degree in sling than circular muscles. However, nifedipine and verapamil greatly inhibited AChC in the circular but not sling muscle. Depletion of SR Ca(2+) stores with CPA or inhibition of Ins(1,4,5)P(3)-mediated store release with either U-73122 or 2-APB inhibited AChC in both muscles. We demonstrate that LES circular and sling muscles 1) use intracellular and extracellular Ca(2+) sources to different degrees in the generation of spontaneous tone and AChC and 2) use different Ca(2+) entry pathways. These differences hold the potential for selective modulation of LES tone in health and disease.     

Atypical localization of myenteric neurons in the opossum lower esophageal sphincter

This study investigated sphincter-body differences in neuronal density and morphometry between the esophageal sphincter and body with a view to determining whether previously reported differences are authentic. The anatomical limits of the opossum lower esophageal sphincter were correlated with its physiological behavior by manometric demarcation. Following this, peeled whole mounts and paraffin and cryosections were used to study the morphology and morphometry of the esophageal myenteric plexus. Thirty animals were used and seven quantitated. The plexus of the esophageal body was located as usual in a plane between the longitudinal and circular muscle, which coincided with the plane of cleavage when these muscle layers were peeled apart for studying the plexus in whole mounts. In contrast, the plexus was located in several planes in the lower esophageal sphincter, which had no cleavage plane. Therefore, peeling the sphincter removes neurons and yields falsely low counts, making peel preparations of this region unsuitable for neuronal quantitation. In paraffin sections, the neuron density in the esophageal body 7 cm above the sphincter was 6,353 +/- 850/cm2, but decreased significantly to 2,254 +/- 353/cm2 at the 1-cm segment. In the lower esophageal sphincter, the neuronal count increased again to 8,530 +/- 1,606/cm2. Flash-frozen cryosections, which produced neuronal morphology similar to the in vivo condition, showed that there was no difference in neuronal size between esophageal body and sphincter. These studies show that atypical myenteric plexus localization causes spuriously low neuronal counts reported in the lower esophageal sphincter and that reported neuronal size differences are technique-dependent.     

Ca2+-induced contraction of cat esophageal circular smooth muscle cells

ACh-induced contraction of esophageal circular muscle (ESO) depends on Ca2+ influx and activation of protein kinase Cepsilon (PKCepsilon). PKCepsilon, however, is known to be Ca2+ independent. To determine where Ca2+ is needed in this PKCepsilon-mediated contractile pathway, we examined successive steps in Ca2+-induced contraction of ESO muscle cells permeabilized by saponin. Ca2+ (0.2-1.0 microM) produced a concentration-dependent contraction that was antagonized by antibodies against PKCepsilon (but not by PKCbetaII or PKCgamma antibodies), by a calmodulin inhibitor, by MLCK inhibitors, or by GDPbetas. Addition of 1 microM Ca2+ to permeable cells caused myosin light chain (MLC) phosphorylation, which was inhibited by the PKC inhibitor chelerythrine, by D609 [phosphatidylcholine-specific phospholipase C inhibitor], and by propranolol (phosphatidic acid phosphohydrolase inhibitor). Ca2+-induced contraction and diacylglycerol (DAG) production were reduced by D609 and by propranolol, alone or in combination. In addition, contraction was reduced by AACOCF(3) (cytosolic phospholipase A(2) inhibitor). These data suggest that Ca2+ may directly activate phospholipases, producing DAG and arachidonic acid (AA), and PKCepsilon, which may indirectly cause phosphorylation of MLC. In addition, direct G protein activation by GTPgammaS augmented Ca2+-induced contraction and caused dose-dependent production of DAG, which was antagonized by D609 and propranolol. We conclude that agonist (ACh)-induced contraction may be mediated by activation of phospholipase through two distinct mechanisms (increased intracellular Ca2+ and G protein activation), producing DAG and AA, and activating PKCepsilon-dependent mechanisms to cause contraction.     

Activation of K+ channels induces apoptosis in vascular smooth muscle cells

Intracellular K⁺ playsan important role in controlling the cytoplasmic ion homeostasis formaintaining cell volume and inhibiting apoptotic enzymes in thecytosol and nucleus. Cytoplasmic K⁺ concentration is mainlyregulated by K⁺ uptake viaNa⁺-K⁺-ATPase and K⁺ efflux throughK⁺ channels in the plasma membrane. Carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP), a protonophorethat dissipates the H⁺ gradient across the inner membraneof mitochondria, induces apoptosis in many cell types. In ratand human pulmonary artery smooth muscle cells (PASMC), FCCP opened thelarge-conductance, voltage- and Ca²⁺-sensitiveK⁺ (maxi-K) channels, increased K⁺ currentsthrough maxi-K channels [I_K(Ca)], and inducedapoptosis. Tetraethylammonia (1 mM) and iberiotoxin (100 nM)decreased I_K(Ca) by blocking the sarcolemmalmaxi-K channels and inhibited the FCCP-induced apoptosis inPASMC cultured in media containing serum and growth factors.Furthermore, inhibition of K⁺ efflux by raisingextracellular K⁺ concentration from 5 to 40 mM alsoattenuated PASMC apoptosis induced by FCCP and theK⁺ ionophore valinomycin. These results suggest thatFCCP-mediated apoptosis in PASMC is partially due to anincrease of maxi-K channel activity. The resultant K⁺ lossthrough opened maxi-K channels may serve as a trigger for cellshrinkage and caspase activation, which are major characteristics ofapoptosis in pulmonary vascular smooth muscle cells.

     

ATP inhibits smooth muscle Ca2(+)-activated K+ channels

There has been much recent interest in the roles played by smooth-muscle K+ channels in protecting cells against ischemic and anoxic insults and in therapeutic vaso- and bronchodilation (Buckingham 1990; Longmore & Weston 1990). A K+ channel, which is uniquely sensitive to cytoplasmic ATP (KATP), has been identified as a likely candidate for mediating these important functions (Standen et al. 1989). We now show, by using electrophysiological techniques in three different types of smooth muscle, that a large-conductance voltage and Ca2(+)-sensitive channel, otherwise indistinguishable from the the large-conductance Ca2(+)-activated K+ channel (BK channel), is also sensitive to cytoplasmic ATP and cromakalim. ATP, in a dose-dependent manner, decreased the probability of channel opening (Po) of rabbit aortic, rabbit tracheal and pig coronary artery BK channels with a Ki of 0.2-0.6 mM. Cromakalim, 10 microM, partially reversed the ATP induced inhibition and increased Po. Our observations raise the possibility that the ubiquitous BK channel may play a role during pathophysiological events.     

Large-conductance Ca2+-activated K+ channels in freshly dissociated smooth muscle cells

Freshly dissociated cells from the stomach muscularis of the toad Bufo marinus have been employed to carry out a systematic set of electrophysiological studies on the membrane properties of smooth muscle. The existence of Ca2+-activated K+ channels became apparent during the first studies under current clamp. In subsequent studies under voltage clamp, a Ca2+-activated. TEA-sensitive outward current was evident, and it was more than an order of magnitude larger than any other current observed in the cells. The channel responsible, at least in part, for this large outward current has been identified on the basis of single-channel records, and some of its main characteristics have been studied. It is similar in many respects to the large-conductance, Ca2+-activated K+ channel seen in other preparations. This channel has now been found in a considerable diversity of smooth muscle types.     

Role of cADPR in sodium nitroprusside-induced opossum esophageal longitudinal smooth muscle contraction

Nitric oxide (NO) relaxes most smooth muscle, including the circular smooth muscle (CSM) of the esophagus, whereas in the adjacent longitudinal smooth muscle (LSM), it causes contraction. The second messenger pathways responsible for this NO-induced LSM contraction are unclear, given that these opposing effects of NO are both cGMP dependent. In intestinal LSM, but not CSM, cADP ribose (cADPR)-dependent pathways participate in Ca(2+) mobilization and muscle contraction; whether similar differences exist in the esophagus is unknown. The purpose of this study was to determine whether cADPR plays a role in the NO-mediated contraction of opossum esophageal LSM. Standard isometric tension recordings were performed using both LSM and CSM strips from opossum distal esophagus that were hung in 10-ml tissue baths perfused with oxygenated Krebs solution. cADPR produced concentration-dependent contraction of LSM strips with an EC(50) of 1 nM and peak contraction of 57 +/- 18% of the 60 mM KCl-induced contraction. cADPR had no effect on CSM strips at concentrations up to 10(-6) M. The EC(50) of cADPR caused contraction (18 +/- 2% from initial resting length) of isolated LSM cells. Sodium nitroprusside (SNP; 300 muM) induced contraction of LSM strips that averaged 67 +/- 5% of the KCl response. cADPR antagonists 8-bromo-cADPR and 8-amino-cADPR, as well as ryanodine receptor antagonists ryanodine and tetracaine, significantly inhibited the SNP-induced contraction. In conclusion, in the opossum esophagus, 1) cADPR induces contraction of LSM, but not CSM, and 2) NO-induced contraction of LSM appears to involve a cADPR-dependent pathway.     

Parathyroid hypertensive factor inhibits voltage-gated K+ channels in vascular smooth muscle cells

Parathyroid hypertensive factor (PHF) has been implicated in regulation of vascular smooth muscle tone and pathogenesis of several forms of hypertension. Earlier studies have suggested that PHF enhances the actions of other vasoconstrictors, while it has no in vitro vasoconstrictor property of its own. PHF was previously found to enhance the L-type Ca channel currents and intracellular Ca responses to depolarization in vascular smooth muscle cells (VSMCs). The present study examined whether PHF might act on K channels in the plasma membrane of VSMCs. Primary cultured VSMCs from rat tail artery were used. The whole-cell version of the patch-clamp technique was used under conditions in which there was no contribution of Ca-activated K channels to the outward current. Both purified and semipurified PHF inhibited the delayed rectifier type potassium current in a dose-dependent manner. The effect was time dependent and was first significantly different from the control current after 30 min. The inhibition of the delayed rectifier K channel was associated with a time-dependent decrease in the resting membrane potential. Therefore, PHF may alter VSMC cellular Ca responses by reducing the membrane potential to a level closer to the activation potential of Ca channels.