Regional differences in cholinergic regulation of potassium current in feline esophageal circular smooth muscle

Potassium channels are important contributors to membrane excitability in smooth muscles. There are regional differences in resting membrane potential and K(+)-channel density along the length of the feline circular smooth muscle esophagus. The aim of this study was to assess responses of K(+)-channel currents to cholinergic (ACh) stimulation along the length of the feline circular smooth muscle esophageal body. Perforated patch-clamp technique assessed K(+)-channel responses to ACh stimulation in isolated smooth muscle cells from the circular muscle layer of the esophageal body at 2 (distal)- and 4-cm (proximal) sites above the lower esophageal sphincter. Western immunoblots assessed ion channel and receptor expression. ACh stimulation produced a transient increase in outward current followed by inhibition of spontaneous transient outward currents. These ACh-induced currents were abolished by blockers of large-conductance Ca(2+)-dependent K(+) channels (BK(Ca)). Distal cells demonstrated a greater peak current density in outward current than cells from the proximal region and a longer-lasting outward current increase. These responses were abolished by atropine and the specific M(3) receptor antagonist 4-DAMP but not the M(1) receptor antagonist pirenzipine or the M(2) receptor antagonist methoctramine. BK(Ca) expression along the smooth muscle esophagus was similar, but M(3) receptor expression was greater in the distal region. Therefore, ACh can differentially activate a potassium channel (BK(Ca)) current along the smooth muscle esophagus. This activation probably occurs through release of intracellular calcium via an M(3) pathway and has the potential to modulate the timing and amplitude of peristaltic contraction along the esophagus.     

Direct contractile effect of motilin on isolated smooth muscle cells of guinea pig small intestine.

We examined the direct effect of motilin on longitudinal and circular smooth muscle cells isolated from the guinea pig small intestine. In addition, the effects of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8, an inhibitor of intracellular Ca(2+)-release), verapamil (a voltage-dependent Ca(2+)-channel blocker), and removal of extracellular Ca2+ were investigated to evaluate the role of intracellular Ca2+ stores and extracellular Ca2+ on the muscle contraction induced by motilin. The effects of atropine (a muscarinic receptor antagonist), spantide (a substance P receptor antagonist) and loxiglumide (a CCK-receptor antagonist) were also examined to determine whether the motilin-induced contraction was independent of those receptors. Motilin induced a contraction of the longitudinal and circular smooth muscle cells in a dose-dependent manner with the maximal effect attained after 30 seconds of incubation. The ED50 values were 0.3 nM and 0.05 nM, respectively. TMB-8 suppressed completely the motilin-induced contraction of both types of smooth muscle cells. Verapamil had only a slight suppressive effect. Removal of extracellular Ca2+ did not have any significant influence on motilin-induced contraction. The contractile response to motilin was not affected by atropine, spantide or loxiglumide. Our findings showed that:1) motilin has a direct contractile effect on both longitudinal and circular smooth muscle cells; 2) this contractile effect is not evoked via muscarinic, substance P or CCK receptors, and 3) the intracellular release of Ca2+ plays an important role in the contractile response to motilin on both types of smooth muscle cells.     

Electrical behavior of guinea pig tracheal smooth muscle

Intracellular recordings were taken from the smooth muscle of the guinea pig trachea, and the effects of intrinsic nerve stimulation were examined. Approximately 50% of the cells had stable resting membrane potentials of -50 +/- 1 mV. The remaining cells displayed spontaneous oscillations in membrane potential, which were abolished either by blocking voltage-dependent Ca(2+) channels with nifedipine or by depleting intracellular Ca(2+) stores with ryanodine. In quiescent cells, stimulation with a single impulse evoked an excitatory junction potential (EJP). In 30% of these cells, trains of stimuli evoked an EJP that was followed by oscillations in membrane potential. Transmural nerve stimulation caused an increase in the frequency of spontaneous oscillations. All responses were abolished by the muscarinic-receptor antagonist hyoscine (1 microM). In quiescent cells, nifedipine (1 microM) reduced EJPs by 30%, whereas ryanodine (10 microM) reduced EJPs by 93%. These results suggest that both the release of Ca(2+) from intracellular stores and the influx of Ca(2+) through voltage-dependent Ca(2+) channels are important determinants of spontaneous and nerve-evoked electrical activity of guinea pig tracheal smooth muscle.     

Critical intracellular Ca2+ concentration for all-or-none Ca2+ spiking in single smooth muscle cells.

Neurotransmitters induce contractions of smooth muscle cells initially by mobilizing Ca2+ from intracellular Ca2+ stores through inositol 1,4,5-trisphosphate (InsP3) receptors. Here we studied roles of the molecules involved in Ca2+ mobilization in single smooth muscle cells. A slow rise in cytoplasmic Ca2+ ([Ca2+]i) in agonist-stimulated smooth muscle cells was followed by a wave of rapid regenerative Ca2+ release as the local [Ca2+]i reached a critical concentration of approximately 160 nM. Neither feedback regulation of phospholipase C nor caffeine-sensitive Ca(2+)-induced Ca2+ release was found to be required in the regenerative Ca2+ release. These results indicate that Ca(2+)-dependent feedback control of InsP3-induced Ca2+ release plays a dominant role in the generation of the regenerative Ca2+ release. The resulting Ca2+ release in a whole cell was an all-or-none event, i.e. constant peak [Ca2+]i was attained with agonist concentrations above the threshold value. This finding suggests a possible digital mode involved in the neural control of smooth muscle contraction.     

Hypertonicity triggers RhoA-dependent assembly of myosin-containing striated polygonal actin networks in endothelial cells

Electrical slow waves determine the timing and force of peristaltic contractions in the stomach. Slow waves originate from a dominant pacemaker in the orad corpus and propagate actively around and down the stomach to the pylorus. The mechanism of slow-wave propagation is controversial. We tested whether Ca(2+) entry via a voltage-dependent, dihydropyridine-resistant Ca(2+) conductance is necessary for active propagation in canine gastric antral muscles. Muscle strips cut parallel to the circular muscle were studied with intracellular electrophysiological techniques using a partitioned-chamber apparatus. Slow-wave upstroke velocity and plateau amplitude decreased from the greater to the lesser curvature, and this corresponded to a decrease in the density of interstitial cells of Cajal in the lesser curvature. Slow-wave propagation velocity between electrodes impaling cells in two regions of muscle and slow-wave upstroke and plateau were measured in response to experimental conditions that reduce the driving force for Ca(2+) entry or block voltage-dependent Ca(2+) currents. Nicardipine (0.1-1 microM) did not affect slow-wave upstroke or propagation velocities. Upstroke velocity, amplitude, and propagation velocity were reduced in a concentration-dependent manner by Ni(2+) (1-100 microM), mibefradil (10-30 microM), and reduced extracellular Ca(2+) (0.5-1.5 mM). Depolarization (by 10-15 mM K(+)) or hyperpolarization (10 microM pinacidil) also reduced upstroke and propagation velocities. The higher concentrations (or lowest Ca(2+)) of these drugs and ionic conditions tested blocked slow-wave propagation. Treatment with cyclopiazonic acid to empty Ca(2+) stores did not affect propagation. These experiments show that voltage-dependent Ca(2+) entry is obligatory for the upstroke phase of slow waves and active propagation.     

Functional TRPV4 channels are expressed in human airway smooth muscle cells

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.     

Phospholamban knockout increases CaM kinase II activity and intracellular Ca2+ wave activity and alters contractile responses of murine gastric antrum

Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA), and this inhibition is relieved by Ca(2+) calmodulin-dependent protein kinase II (CaM kinase II) phosphorylation. We previously reported significant differences in contractility, SR Ca(2+) release, and CaM kinase II activity in gastric fundus smooth muscles as a result of PLB phosphorylation by CaM kinase II. In this study, we used PLB-knockout (PLB-KO) mice to directly examine the effect of PLB absence on contractility, CaM kinase II activity, and intracellular Ca(2+) waves in gastric antrum smooth muscles. The frequencies and amplitudes of spontaneous phasic contractions were elevated in antrum smooth muscle strips from PLB-KO mice. Bethanecol increased the amplitudes of phasic contractions in antrum smooth muscles from both control and PLB-KO mice. Caffeine decreased and cyclopiazonic acid (CPA) increased the basal tone of antrum smooth muscle strips from PLB-KO mice, but the effects were less pronounced compared with control strips. The CaM kinase II inhibitor KN-93 was less effective at inhibiting caffeine-induced relaxation in antrum smooth muscle strips from PLB-KO mice. CaM kinase II autonomous activity was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. Similarly, the intracellular Ca(2+) wave frequency was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. These findings suggest that PLB is an important modulator of gastric antrum smooth muscle contractility by modulation of SR Ca(2+) release and CaM kinase II activity.     

Capsaicin inhibits the voltage-operated calcium channels intracellularly in the antral circular myocytes of guinea-pig stomach

Studies of the effect of capsaicin (CAP) on the smooth muscle contractions have shown both contraction and relaxation in various preparations. The direct effect of CAP on gastric smooth muscle itself has not yet been reported, though CAP was reported to relax the isolated guinea-pig stomach by releasing nitric oxide from the CAP-sensitive sensory neurons. Here we showed an evidence that CAP evokes a prolonged relaxation of gastric antral circular smooth muscle (CAP-induced relaxation) by blocking the voltage-operated Ca2+ channels (VOCC) from inside of the cell. CAP suppressed dose-dependently the spontaneous contractions of guinea-pig gastric circular muscle strip under the condition without neural influence (IC50 = 5.8 microM). The inhibitory effects of CAP both on the high K+ contracture induced by 50 mM K+ Tyrode solution and on the slow waves recorded using a conventional intracellular microelectrode technique were similar to those of Ca2+ channel antagonists, indicating that Ca2+ influx through the VOCC is decreased by CAP. Ca2+ channel current (I(Ba)) decreased in a concentration-dependent manner on superfusing the physiological salt solution containing various concentrations of CAP. The steady-state activation and inactivation curves of I(Ba) were not affected by the treatment with CAP. The experiment using a synthetic water-soluble analog of CAP, DA-5018 x HCl, suggested that the acting site of CAP is present in the intracellular side. Spontaneous transient outward K+ currents (STOCs) recorded at a holding potential of 0 mV were also inhibited by CAP and verapamil, Ca channel blocker. Taken together, these results indicate that CAP-induced relaxation is associated with the direct inhibitory action on the VOCC from inside of the cell.     

P2X1 receptors mediate sympathetic postjunctional Ca2+ transients in mesenteric small arteries

Brief, spatially localized Ca(2+) transients occur in the smooth muscle adjacent to perivascular nerves of small arteries during neurogenic contractions. We named these "junctional Ca(2+) transients" (jCaTs) and postulated that they arose from Ca(2+) entering smooth muscle cells through P2X(1) receptors activated by neurally released ATP. Nevertheless, the lack of potent, subtype-selective P2X-receptor antagonists made determining the exact molecular identity of the channels difficult. Here we used small, pressurized mesenteric arteries from P2X(1)-receptor-deficient mice (KO) to test the hypothesis that jCaTs arise from Ca(2+) entering the smooth muscle cell via P2X(1) receptors. In wild-type (WT) arteries, confocal microscopy of fluo-4 fluorescence during electrical field stimulation (EFS) of perivascular sympathetic nerves revealed jCaTs in the smooth muscle cells adjacent to the perivascular nerves, similar to those reported previously in rat arteries, and alpha-latrotoxin (2.5 nM) markedly increased the frequency of "spontaneous" jCaTs. In the KO arteries, however, neither EFS nor alpha-latrotoxin elicited any jCaTs. A potent P2X-receptor agonist, alpha,beta-methylene ATP (10.0 microM), elicited strong contractions and increased intracellular Ca(2+) concentration in WT arteries but elicited neither in KO arteries. A biphasic vasoconstriction in response to EFS was observed in WT arteries. In KO arteries, however, the initial rapid, transient component of the biphasic vasoconstriction was absent. The data support the hypothesis that jCaTs represent Ca(2+) that enters the smooth muscle cells through P2X(1) receptors activated by neurally released ATP and that this Ca(2+) is involved in the initial rapid component of the sympathetic neurogenic contraction.     

Stoichiometry of contraction and Ca2+ mobilization by inositol 1,4,5-trisphosphate in isolated gastric smooth muscle cells

Smooth muscle cells were isolated from the circular muscle layer of guinea pig stomach and permeabilized by brief exposure to saponin. Both permeabilized and intact muscle cells contracted in response to cholecystokinin octapeptide (CCK-8) and acetylcholine, but only permeabilized muscle cells contracted in response to inositol 1,4,5-trisphosphate (InsP3). The contractile response to InsP3 was prompt (peak less than 5 s), concentration-dependent (EC50-0.3 microM), and insensitive to antimycin or oligomycin. Contraction induced by either InsP3 or CCK-8 was accompanied by a concentration-dependent increase in free Ca2+ that was directly correlated with the magnitude of contraction. Both InsP3 and CCK-8 caused rapid net efflux of Ca2+ from cells preloaded with 45Ca2+. Contraction, increase in free Ca2+ concentration, and net 45Ca2+ efflux elicited by a combination of maximal concentrations of InsP3 and CCK-8 were not significantly different from those elicited by maximal concentrations of either agent alone. Repeated stimulation of single muscle cells with either InsP3 or CCK-8 in Ca2+-free medium caused eventual loss of the contractile response to all agents. The response to all agents was restored upon re-exposure of the cell to a cytosol-like concentration of Ca2+, implying equal access of InsP3 and receptor-linked agonists to the same intracellular Ca2+ store. The results demonstrate that InsP3 mimics the effects of receptor-linked agonists on contraction and mobilization of intracellular Ca2+ in permeabilized smooth muscle cells that retain the functional properties of intact smooth muscle cells and support a role for InsP3 as membrane-derived messenger responsible for mobilization of intracellular Ca2+ in smooth muscle cells.     

The role of Ca2+ in the contractility of rabbit small intestine in vitro.

This study evaluated the role of Ca2+ in spontaneous and ACh- and KCl-induced contractions in longitudinal and circular smooth muscle from rabbit small intestine in vitro. In the first experiment, the amplitude, frequency and tone of spontaneous contractions in longitudinal and circular smooth muscle of small intestine were determined and, in the second experiment, the ACh- and KCl-induced responses of longitudinal and circular smooth muscle were measured. Atropine and guanethidine reduced the amplitude and tone of contractions in longitudinal and circular muscle, but reduced the frequency of contractions in circular muscle, only. TTX attenuated the amplitude of contractions and decreased the tone of contractions in longitudinal muscle, but increased the tone in circular muscle. Ca2+-free solutions, verapamil, nifedipine and caffeine diminished the three parameters of spontaneous contractions. Thapsigargin and cyclopiazonic acid increased the amplitude and tone of contractions in ileum longitudinal muscle, only, and cyclopiazonic acid increased the amplitude of contractions in circular muscle. Ca2+-free solutions, verapamil, nifedipine, thapsigargin, cyclopiazonic acid, and caffeine diminished ACh- and KCl-induced contractions. Those results suggest that extracellular Ca2+ plays a role in spontaneous contractions, and extracellular and intracellular Ca2+ participate in the ACh- and KCl-induced contractions of rabbit small intestine.     

Regulation of slow wave frequency by IP(3)-sensitive calcium release in the murine small intestine

Slow waves determine frequency and propagation characteristics of contractions in the small intestine, yet little is known about mechanisms of slow wave regulation. We propose a role for intracellular Ca(2+), inositol 1,4,5,-trisphosphate (IP(3))-sensitive Ca(2+) release, and sarcoplasmic reticulum (SR) Ca(2+) content in the regulation of slow wave frequency because 1) 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, a cytosolic Ca(2+) chelator, reduced the frequency or abolished the slow waves; 2) thapsigargin and cyclopiazonic acid (CPA), inhibitors of SR Ca(2+)-ATPase, decreased slow wave frequency; 3) xestospongin C, a reversible, membrane-permeable blocker of IP(3)-induced Ca(2+) release, abolished slow wave activity; 4) caffeine and phospholipase C inhibitors (U-73122, neomycin, and 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate) inhibited slow wave frequency; 5) in the presence of CPA or thapsigargin, stimulation of IP(3) synthesis with carbachol, norepinephrine, or phenylephrine acting on alpha(1)-adrenoceptors initially increased slow wave frequency but thereafter increased the rate of frequency decline, 6) thimerosal, a sensitizing agent of IP(3) receptors increased slow wave frequency, and 7) ryanodine, a selective modulator of Ca(2+)-induced Ca(2+) release, had no effect on slow wave frequency. In summary, these data are consistent with a role of IP(3)-sensitive Ca(2+) release and the rate of SR Ca(2+) refilling in regulation of intestinal slow wave frequency.     

Phosphatidylinositol 3,5-bisphosphate increases intracellular free Ca2+ in arterial smooth muscle cells and elicits vasocontraction

Phosphoinositide (3,5)-bisphosphate [PI(3,5)P(2)] is a newly identified phosphoinositide that modulates intracellular Ca(2+) by activating ryanodine receptors (RyRs). Since the contractile state of arterial smooth muscle depends on the concentration of intracellular Ca(2+), we hypothesized that by mobilizing sarcoplasmic reticulum (SR) Ca(2+) stores PI(3,5)P(2) would increase intracellular Ca(2+) in arterial smooth muscle cells and cause vasocontraction. Using immunohistochemistry, we found that PI(3,5)P(2) was present in the mouse aorta and that exogenously applied PI(3,5)P(2) readily entered aortic smooth muscle cells. In isolated aortic smooth muscle cells, exogenous PI(3,5)P(2) elevated intracellular Ca(2+), and it also contracted aortic rings. Both the rise in intracellular Ca(2+) and the contraction caused by PI(3,5)P(2) were prevented by antagonizing RyRs, while the majority of the PI(3,5)P(2) response was intact after blockade of inositol (1,4,5)-trisphosphate receptors. Depletion of SR Ca(2+) stores with thapsigargin or caffeine and/or ryanodine blunted the Ca(2+) response and greatly attenuated the contraction elicited by PI(3,5)P(2). The removal of extracellular Ca(2+) or addition of verapamil to inhibit voltage-dependent Ca(2+) channels reduced but did not eliminate the Ca(2+) or contractile responses to PI(3,5)P(2). We also found that PI(3,5)P(2) depolarized aortic smooth muscle cells and that LaCl(3) inhibited those aspects of the PI(3,5)P(2) response attributable to extracellular Ca(2+). Thus, full and sustained aortic contractions to PI(3,5)P(2) required the release of SR Ca(2+), probably via the activation of RyR, and also extracellular Ca(2+) entry via voltage-dependent Ca(2+) channels.     

Calcium signaling in smooth muscle

Changes in intracellular Ca(2+) are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca(2+) signal is a reflection of the source of Ca(2+) (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca(2+) entry may be detected optically as graded elevations in intracellular Ca(2+), junctional Ca(2+) transients, Ca(2+) flashes, or Ca(2+) sparklets, whereas release of Ca(2+) from intracellular stores may manifest as Ca(2+) sparks, Ca(2+) puffs, or Ca(2+) waves. These diverse Ca(2+) signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression).     

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.     

Propagation of calcium waves along endothelium of hamster feed arteries

An increase in tissue blood flow requires relaxation of smooth muscle cells along entire branches of the resistance vasculature. Whereas the spread of hyperpolarization along the endothelium can coordinate smooth muscle cell relaxation, complementary signaling events have been implicated in the conduction of vasodilation. We tested the hypothesis that Ca(2+) waves propagate from cell to cell along the endothelium of feed arteries exhibiting spontaneous vasomotor tone. Feed arteries of the hamster retractor muscle were isolated, pressurized to 75 mmHg at 37 degrees C, and developed myogenic tone spontaneously. Smooth muscle cells and endothelial cells were loaded with the Ca(2+) indicator Fluo-4. An acetylcholine stimulus was delivered locally using microiontophoresis (1-microm pipette tip, 1 microA, 1 s), and Ca(2+) signaling within and along respective cell layers was determined using laser-scanning confocal microscopy. Acetylcholine triggered an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) of endothelial cells at the site of stimulation that preceded two distinct events: 1) a rapid synchronous decrease in smooth muscle [Ca(2+)](i) along the entire vessel and 2) an ensuing Ca(2+) wave that propagated bidirectionally along the endothelium at approximately 111 microm/s for distances exceeding 1 mm. Maximal dilation of vessels with either nifedipine (1 microM) or sodium nitroprusside (SNP, 100 microM) reduced the distance that Ca(2+) waves traveled to approximately 300 microm (P < 0.05). Thus Ca(2+) waves propagate along the endothelium of resistance vessels with vasomotor tone, and this signaling pathway is compromised during maximal dilation with nifedipine or SNP.     

Excitatory regulation of angiotensin II on gastric motility and its mechanism in guinea pig

In the present study, we investigated the effect of Ang II on gastric smooth muscle motility and its mechanism using intracellular recording and whole-cell patch clamp techniques. Ang II dose-dependently increased the tonic contraction and the frequency of spontaneous contraction in the gastric antral circular smooth muscles of guinea pig. ZD7155, an Ang II type 1 receptor (AT(1)R) blocker, completely blocked the effect of Ang II on the spontaneous contraction of gastric smooth muscle. In contrast, TTX, a sodium channel blocker, failed to block the effect. Furthermore, nicardipine, a voltage-gated Ca(2+)-channel antagonist, did not block the effect of Ang II on the tonic contraction of gastric smooth muscle, but external free-calcium almost completely blocked this effect. Both ryanodine, an inhibitor of calcium-induced Ca(2+) release (CICR) from ryanodine-sensitive calcium stores, and thapsigargin, which depletes calcium in calcium stores, almost completely blocked the effect of Ang II on tonic contraction. However, 2-APB, an inositol trisphosphate (IP(3)) receptor blocker, significantly, but not completely, blocked the Ang II effect on tonic contraction. We also determined that Ang II depolarized membrane potential and increased slow wave frequency in a dose-dependent manner. It also inhibited delayed rectifying potassium currents in a dose-dependent manner, but did not affect L-type calcium currents or calcium-activated potassium currents. These results suggest that Ang II plays an excitatory regulation in gastric motility via AT(1)R-IP(3) and the CICR signaling pathway. The Ang II-induced inhibition of delayed rectifying potassium currents that depolarize membrane potential is also involved in the potentiation of tonic contraction and the frequency of spontaneous contraction in the gastric smooth muscle of guinea pig.     

Whole cell current and membrane potential regulation by a human smooth muscle mechanosensitive calcium channel

Mechanotransduction is required for a wide variety of biological functions. The aim of this study was to determine the effect of activation of a mechanosensitive Ca(2+) channel, present in human jejunal circular smooth muscle cells, on whole cell currents and on membrane potential. Currents were recorded using patch-clamp techniques, and perfusion of the bath (10 ml/min, 30 s) was used to mechanoactivate the L-type Ca(2+) channel. Perfusion resulted in activation of L-type Ca(2+) channels and an increase in outward current from 664 +/- 57 to 773 +/- 72 pA at +60 mV. Membrane potential hyperpolarized from -42 +/- 4 to -50 +/- 5 mV. In the presence of nifedipine (10 microM), there was no increase in outward current or change in membrane potential with perfusion. In the presence of charybdotoxin or iberiotoxin, perfusion of the bath did not increase outward current or change membrane potential. A model is proposed in which mechanoactivation of an L-type Ca(2+) channel current in human jejunal circular smooth muscle cells results in increased Ca(2+) entry and cell contraction. Ca(2+) entry activates large-conductance Ca(2+)-activated K(+) channels, resulting in membrane hyperpolarization and relaxation.     

Paracrine effects of endothelial cells in a diabetic mouse model: capacitative calcium entry stimulated thromboxane release

Augmented vasoconstriction contributes to arterial stiffness associated with diabetes. It has been shown that capacitative calcium entry induced by sarcoplasmic-endoplasmic reticulum calcium ATPase blocker cyclopiazonic acid (CPA) in endothelial cells stimulates production of constrictor prostaglandins, which causes contractions of vascular smooth muscle cells. The aim of the work was to study the effect of diabetes on the vasoconstrictor response induced by calcium entry into endothelial and smooth muscle cells. Force was measured in isolated aortae of diabetic ob/ob and control C57BL/6J mice under isometric conditions. Contractions caused by 10 mumol/l CPA in diabetic mouse aortae featured higher amplitudes and longer durations in comparison with nondiabetic aortae. These contractions were abolished by a COX inhibitor indomethacin (10 mumol/l) or a specific thromboxane A2 receptor blocker SQ 29548 (1 mumol/l) and were not observed in denuded aortae. The contractions were sensitive to extracellular Ca (2+) and store-operated channel blockers. All together this suggests that vasoconstriction was caused by thromboxane A2 synthesis in endothelial cells induced by Ca (2+) entry through store-operated channels. Higher concentrations of CPA (30 mumol/l) or thapsigargin (1 mumol/l) elicited indomethacin-resistant tonic contractions of aortae with 2-fold amplitude in diabetic mice compared to their nondiabetic littermates, which were sensitive to store-operated channel blockers, but not to indomethacin, SQ 29548, or denudation. In conclusions, increases in intracellular Ca (2+) cause augmented vasoconstriction in diabetic vasculature through endothelial synthesis of contractile prostaglandins. In addition capacitative Ca (2+) entry is enhanced in diabetic vascular smooth muscle. These mechanisms indicate possible targets for clinical applications.