The myogenic response in isolated rat cerebrovascular arteries: smooth muscle cell model

Previous models of the cerebrovascular smooth muscle cell have not addressed the interaction between the electrical, chemical, and mechanical components of cell function during the development of active tension. These models are primarily electrical, biochemical or mechanical in their orientation, and do not permit a full exploration of how the smooth muscle responds to electrical or mechanical forcing. To address this issue, we have developed a new model that consists of two major components: electrochemical and chemomechanical subsystem models of the smooth muscle cell. Included in the electrochemical model are models of the electrophysiological behavior of the cell membrane, fluid compartments, Ca2+ release and uptake by the sarcoplasmic reticulum (SR), and cytosolic Ca2+ buffering, particularly by calmodulin (CM). With this subsystem model, we can study the mechanics of the production of intracellular Ca2+ transient in response to membrane voltage clamp pulses. The chemomechanical model includes models of: (a) the chemical kinetics of myosin phosphorylation, and the formation of phosphorylated (cycling) myosin cross-bridges with actin, as well as attached (non-cycling) latch-type cross-bridges; and (b) a model of force generation and mechanical coupling to the contractile filaments and their attachments to protein structures and the skeletal framework of the cell. The two subsystem models are tested independently and compared with data. Likewise, the complete (combined) cell model responses to voltage pulse stimulation under isometric and isotonic conditions are calculated and compared with measured single cell length-force and force-velocity data obtained from literature. This integrated cell model provides biophysically based explanations of electrical, chemical, and mechanical phenomena in cerebrovascular smooth muscle, and has considerable utility as an adjunct to laboratory research and experimental design.     

Cellular mechanisms of myogenic activity in gastric smooth muscle

In many regions of the intestine, a thin layer of interstitial cells of Cajal (ICC) lie in the myenteric region, between the circular and longitudinal muscle layers. ICC are connected by gap junctions to surrounding ICC and also with circular and longitudinal smooth muscle cells, forming a large electrical syncytium. Damage of the ICC causes a disorder in the patterns of rhythmic activity. Isolated ICC produce a rhythmic oscillation of the membrane potential. All these observations have led to the suggestion that ICC may be the pacemaker cell responsible for intestinal activity. Gastric smooth muscles generate slow oscillatory membrane potential changes (slow waves) and spike potentials. The activity is considered to be linked to the metabolism in the cell. Three types of cells located in the gastric wall (circular and longitudinal smooth muscle cells and ICC) produce synchronized electrical responses with different shapes. The electrical responses appear to originate in ICC and then spread to the smooth muscle layers, indicating that ICC may also be the pacemaker cells responsible for gastric activity. However, isolated circular smooth muscle tissues spontaneously generate regenerative potentials, suggesting that there are at least two sites for the initiation of spontaneous activity in the stomach. Regenerative potentials persist in the presence of Ca-antagonists and are inhibited by agents which disrupt intracellular Ca(2+) homeostasis. Depolarization of the membrane elicits regenerative potentials after a long delay and the potentials have long refractory periods. This suggests that an unidentified 2nd messenger may be formed during the delay between membrane depolarization and the initiation of a regenerative potential. In gastric muscles of mutant mice which do not express inositol trisphosphate (InsP(3)) receptors, spike potentials but not slow waves are generated, suggesting the possible involvement of InsP(3) in the initiation of spontaneous activity.     

Dual effects of cyclopiazonic acid on excitation of circular smooth muscle isolated from the guinea-pig gastric antrum.

The effects of cyclopiazonic acid (CPA), a known Ca2+-pump inhibitor at internal stores, were investigated on electrical responses of the membrane of smooth muscle cells in small segments (0.3-0.5 mm long) of circular smooth muscle isolated from the guinea-pig gastric antrum. In most preparations, the membrane was spontaneously active with the generation of unitary potentials and regenerative slow potentials. Low concentrations (< 1 microM) of CPA did not alter either the membrane potential or the amplitude and frequency of slow potentials. CPA at a concentration of 1 microM initially increased the frequency of slow potentials, but this was followed by a decrease in the frequency as a result of sustained exposure to CPA, with no alteration of either the membrane potential or the amplitude of slow potentials. Higher concentrations of CPA (2-5 microM) depolarized the membrane and decreased the amplitude and frequency of slow potentials. CPA at higher than 10 microM abolished slow potentials with depolarization of the membrane. Intracellular electrical responses recorded simultaneously from paired cells were synchronized, indicating electrical coupling of the cells. Depolarization of the membrane with current stimuli through one electrode evoked regenerative slow potentials superimposed on the electrotonic potentials. The evoked slow potential had a refractory period of about 7 s. CPA (up to 10 microM) did not prevent the synchronization of paired cells. The refractory period for slow potentials was reduced by low concentrations of CPA (< 1 microM) and increased by higher concentrations of CPA (2-10 microM). These results suggest that lower concentrations of CPA produce excitatory actions on gastric smooth muscles due to a secondary effect of increased intracellular [Ca2+], while higher concentrations of CPA produce inhibitory actions as a result of reduced release of Ca2+ from depleted internal stores.     

Increased smooth muscle contractility in mice deficient for neuropilin 2

Neuropilins (NRPs) are transmembrane receptors that bind class 3 semaphorins and VEGF family members to regulate axon guidance and angiogenesis. Although expression of NRP1 by vascular smooth muscle cells (SMCs) has been reported, NRP function in smooth muscle (SM) in vivo is unexplored. Using Nrp2(+/LacZ) and Nrp2(+/gfp) transgenic mice, we observed robust and sustained expression of Nrp2 in the SM compartments of the bladder and gut, but no expression in vascular SM, skeletal muscle, or cardiac muscle. This expression pattern was recapitulated in vitro using primary human SM cell lines. Alterations in cell morphology after treatment of primary visceral SMCs with the NRP2 ligand semaphorin-3F (SEMA3F) were accompanied by inhibition of RhoA activity and myosin light chain phosphorylation, as well as decreased cytoskeletal stiffness. Ex vivo contractility testing of bladder muscle strips exposed to electrical stimulation or soluble agonists revealed enhanced tension generation of tissues from mice with constitutive or SM-specific knockout of Nrp2, compared with controls. Mice lacking Nrp2 also displayed increased bladder filling pressures, as assessed by cystometry in conscious mice. Together, these findings identify Nrp2 as a mediator of prorelaxant stimuli in SMCs and suggest a novel function for Nrp2 as a regulator of visceral SM contractility.     

Physiological roles of K+ channels in vascular smooth muscle cells

In this review, we present the basic properties, physiological functions, regulation, and pathological alterations of four major classes of K+ channels that have been detected in vascular smooth muscle cells. Voltage-dependent K+ (Kv) channels open upon depolarization of the plasma membrane in vascular smooth muscle cells. The subsequent efflux of K+ through the channels induces repolarization to the resting membrane potential. Changes in the intracellular Ca2+ concentration and membrane depolarization stimulate large-conductance Ca2+-activated K+ (BKCa) channels, which are thought to play an important role in maintaining the membrane potential. ATP-sensitive K+ (K(ATP)) channels underscore the functional bond between cellular metabolism and membrane excitability. The blockade of KATP channel function results in vasoconstriction and depolarization in various types of vascular smooth muscle. Inward rectifier K+ (Kir) channels, which are expressed in smooth muscle of the small-diameter arteries, contribute to the resting membrane potential and basal tone. Kir channel activation has been shown to raise the extracellular K+ concentration to 10-15 mM, resulting in vasodilation. Each of K+ channels listed above is responsive to a number of vasoconstrictors and vasodilators, which act through protein kinase C (PKC) and protein kinase A (PKA), respectively. Impaired Kv, KATP, and Kir channel functions has been linked to a number of pathological conditions, which may lead to vasoconstriction.     

Evidence that action potential generation is not the exclusive determinant to trigger spontaneous myogenic contraction of guinea-pig urinary bladder smooth muscle

Urinary bladder smooth muscle (UBSM) exhibits spontaneous contraction. This spontaneous mechanical activity is myogenic and can be closely related to the UBSM cell action potential to facilitate Ca2+ influx through voltage-gated Ca2+ channels. In the present study, to know whether this membrane electrical event is the exclusive mechanism to trigger spontaneous smooth muscle contraction, we compared the inhibitory effects of Ca2+ channel blockers on the spontaneous action potential and mechanical activity in the isolated guinea-pig UBSM. Both action potential and rhythmic contraction were generated spontaneously in the presence of atropine (1 microM), phentolamine (1 microM), propranolol (1 microM), suramin (10 microM) and tetrodotoxin (1 microM), which suggest that both phenomena were myogenic in origin. Nisoldipine (100 nM) and diltiazem (10 microM) completely eliminated the generation of action potential whereas its frequency was dramatically increased by a dihydropyridine Ca2+ agonist, BayK 8644 (1 microM). In contrast to disappearance of action potential in the presence of Ca2+ channel blockers, spontaneous contraction of UBSM was inhibited only partly by nisoldipine or diltiazem and most of the mechanical components persisted in these channel blockers. These results indicate that spontaneous action potential in UBSM cell is generated through the activation of L-type voltage-gated Ca2+ channels. The subsequent elevation of intracellular Ca2+ concentrations during a burst of action potentials can be partly responsible for the induction of UBSM mechanical activity. In addition, the present study provides evidence that UBSM spontaneous mechanical activity is also attributable to the mechanism(s) other than the generation of Ca2+ spike.     

Transients in intracellular free calcium in subconfluent and confluent cultures of a rat smooth muscle cell line.

The Ca2+ mobilizing mechanisms in the smooth muscle cell line A7r5 were found to undergo changes related to the degree of confluence of the cultures. In sparse cultures resting calcium was stable and exposure to arginine vasopressin (AVP) resulted in a single transient increase in intracellular free calcium (Ca2+i). In confluent cultures the cells could be divided into two general groups, those with a stable resting Ca2+i and those which demonstrated spontaneous brief elevations in Ca2+i of variable frequency. Application of AVP elevated Ca2+i, induced oscillations in quiescent confluent cells, increased the frequency of oscillatory activity in cells which were already active and, in cells which exhibited high frequency spontaneous fluctuations, inhibited this activity. Isotonic K+ depolarizing solution and normal solutions containing Co2+ inhibited Ca2+ spikes. These data suggest that the mechanism underlying the transients involves cyclical electrical phenomena at the cell membrane possibly utilizing calcium channels. There is no indication that the mechanism involves cytoplasmic oscillators.     

Ultrastructural localization of calcium-activated adenosine triphosphatase (Ca2+-ATPase) in cerebral endothelium

There is increasing interest in the role of calcium in a variety of biological processes. One of the mechanisms that regulate intracellular calcium concentrations is the calcium-activated adenosine triphosphatases (Ca2+-ATPase). The availability of an histochemical method for ultrastructural localization of Ca2+-ATPase has led to a number of studies attempting to localize this enzyme in a variety of cell types. This ultrastructural study was undertaken to localize Ca2+-ATPase in walls of intracerebral cortical vessels of rats. Both capillary and arteriolar endothelium showed discontinuous deposits of Ca2+-ATPase along the outer plasma membrane including the junctional plasma membranes. Patchy distribution of Ca2+-ATPase was also observed on the outer plasma membranes of smooth muscle and adventitial cells. Focal deposits of reaction product were associated with the actin filaments in endothelium. Invaginating pinocytotic vesicles at the outer plasma membrane of endothelium and smooth muscle cells showed Ca2+-ATPase. Intracytoplasmic vesicles showed the enzyme along the inner plasma membrane. Localization of Ca2+-ATPase on endothelial plasma membranes suggests that Ca2+ may be involved in many endothelial reactions. Further studies are required to determine the role of this enzyme and Ca2+ in endothelial reactions in normal and abnormal states.     

腺苷受体与血管反应性调节

腺苷是严重创伤及组织缺血缺氧时释放的重要内源性调质,主要通过腺苷受体发挥作用。血管平滑肌上主要有4种腺苷受体亚型（A1AR,A2aAR,A2bAR及A3AR）,其中A1AR和/或A3AR活性及其偶联信号活性的抑制,或A2aAR和/或A2bAR活性及其偶联信号活性的上调,或两者同时发生,通过Rho激酶和/或PKC依赖信号通路,参与对血管MLC磷酸化及钙敏感性调节,并在休克血管低反应性中起重要作用。     

Regulation of Ca2+ channel and phosphatase activities by polyamines in intestinal and vascular smooth muscle--implications for cellular growth and contractility

Polyamines added extracellularly to intestinal and vascular smooth muscle cells cause relaxation through inhibition of Ca2+ channel activity. Intracellularly applied polyamines also affect Ca2+ channel properties. Polyamines do not readily pass over the plasma membrane because of their positive charges but in permeabilized smooth muscle preparations they have free access to the cytoplasm. In this system they increase sensitivity of the contractile machinery to Ca2+ through inhibition of myosin phosphatase activity. The magnitude of Ca2+ channel and phosphatase inhibition depends on the number of positive charges on the polyamine molecule. Polyamines have an obligatory, but yet undefined, role in regulation of cell growth and proliferation. Several groups of protein kinases, such as tyrosine and mitogen activated protein (MAP)-kinases transmit the growth signal from the plasma membrane to the cell nucleus where mitosis and protein synthesis are initiated. The data reviewed here show that polyamines may affect such signal transmission via inhibition of phosphatase activity.     

Excitation-induced Ca2+ influx and skeletal muscle cell damage.

Excessive exercise may lead to skeletal muscle cell damage with degradation of cellular components and leakage of intracellular enzymes. Calcium has repeatedly been proposed to be involved in these processes. Studies have shown that the resting level of cytoplasmic Ca2+ increases up to threefold during long-term low-frequency stimulation. We have shown that electrical stimulation produces a marked increase in Ca2+ uptake and Ca2+ content in rat skeletal muscle, both in vivo and in vitro. Continuous stimulation for 240 min at 1 Hz results in an increased release (18-fold) of lactate dehydrogenase (LDH) from extensor digitorum longus (EDL) muscle. This was associated with an increased total Ca2+ content (185%), was augmented at high [Ca2+]o and suppressed at low [Ca2+]o. The release of LDH may reflect partial loss of sarcolemmal integrity as a result of degradation of membrane components by Ca2+-activated enzymes (e.g. calpain or phospholipase A2). After cessation of stimulation the increased release of LDH continues for at least 120 min. This is associated with an up to sevenfold increase in 45Ca uptake. The increased permeability to Ca2+ may further activate calpain and phospholipase A2 and accelerate the loss of membrane integrity. Stimulation-induced uptake of Ca2+ and release of LDH is most pronounced in EDL (mainly composed of fast-twitch fibres at variance with soleus which is mainly composed of slow-twitch fibres). This may account for the observation that prolonged exercise leads to preferential damage to fast-twitch fibres. We hypothesize that excessive exercise may lead to an intracellular accumulation of Ca2+ and increased cytoplasmic Ca2+ causing activation of self-accelerating degradative pathways leading to muscle damage.     

Spontaneous rhythmicity in cultured cell clusters isolated from mouse small intestine

To investigate spontaneous rhythmicity in smooth muscle tissue, we have developed a cell cluster preparation. Cell clusters were enzymatically isolated from the muscle layer of mouse small intestine and cultured for several days. They included smooth muscle, neurones, and c-Kit-immunopositive interstitial cells. c-Kit-immunopositive cells in myenteric plexus, showing a networklike structure, are putative pacemaker cells. The cultured cell clusters routinely show spontaneous contraction and preserve characteristic features in this tissue: (1) high temperature dependency of contractile frequency; (2) spontaneous electrical activities measured with patch clamp techniques are insensitive to tetradotoxin (TTX) and dihydropyridine Ca(2+) antagonists. This preparation could therefore be used as a good model system to investigate the underlying mechanisms of intestinal motility and pacemaker function. The relationship between the frequency of electrical activity and cluster size suggests that the minimum unit of small intestine tissue to yield normal pacemaker activity is approximately 100 microm in diameter, or less. The applications of 100-120 microM Cd(2+) and Ni(2+) significantly suppressed the spontaneous activity. Ca(2+) influx pathways other than L-type and "classical" T-type voltage-sensitive Ca(2+) channels seem very likely to play an important role, such as nonselective cation channels and capacitative Ca(2+) entry. Furthermore, applications of heptanol reduced the amplitude and the frequency of the oscillating inward currents and eventually terminated them, suggesting that electrical cell-to-cell coupling may also make some contribution to the generation of spontaneous activity.     

Localization of cation interactions in the smooth muscle of the guinea-pig taenia coli

1. An electron microscopic method has been developed and used to study cation binding sites in smooth muscle.2. Uranyl cations normally bind to the external surface of the plasma membrane. However, uranyl also binds to the inner surface when it is accessible. Similar binding has been observed in skeletal muscle, nerve and red blood cells.3. Uranyl binds electrostatically, and the binding can be competitively reversed by other cations. By a quantitative procedure the relative affinities Ca(2+) approximately Mg(2+) > K(+) > Na(+) for the membrane sites have been determined. This sequence is in agreement with previous values determined analytically.4. The results support a counter-cation hypothesis for the plasma membrane surfaces of the taenia coli, and may explain features of the electrical activity in smooth muscle.     

Relaxation of contracted rabbit tracheal and human bronchial smooth muscle by Y-27632 through inhibition of Ca2+ sensitization.

The mechanism of Ca2+ sensitization of contraction has not been elucidated in airway smooth muscle (SM). To determine the role of a small G protein, rhoA p21, and its target protein, rho-associated coiled coil-forming protein kinase (ROCK), in receptor-coupled Ca2+ sensitization of airway SM, we studied the effect of (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexane carboxamide dihydrochloride, monohydrate (Y-27632), a ROCK inhibitor, on isometric contractions in rabbit tracheal and human bronchial SM. Y-27632 completely reversed 1 microM carbachol (CCh)-induced contraction of intact trachea with a concentration producing half-maximum inhibition of effect (IC50) of 1.29 +/- 0.2 microM (n = 5). Although 4beta-phorbol 12,13-dibutyrate (1 microM)-induced Ca2+ sensitization was relatively resistant to Y-27632 in alpha-toxin-permeabilized trachea, CCh (100 microM) plus guanosine triphosphate (GTP) (3 microM)- and guanosine 5'-O-(3'-thiotriphosphate) (10 microM)-induced contractions were relaxed completely by Y-27632 with IC50 of 1.44 +/- 0.3 (n = 6) and 1.15 +/- 0.3 microM (n = 6). Endothelin-1 (1 microM) plus GTP (3 microM)- developed force was also reversed by Y-27632 with IC50 of 4. 10 +/- 1.1 microM (n = 6) in the alpha-toxin-permeabilized bronchus. Both the rabbit and human SM expressed rhoA p21, ROCK I, and its isoform ROCK II. Collectively, rho/ROCK-mediated Ca2+ sensitization plays a central role in the sustained phase of airway SM contraction, and selective inhibition of this pathway may become a new strategy to resolve airflow limitation in asthma.     

Anoxia induces Ca2+ influx and loss of cell membrane integrity in rat extensor digitorum longus muscle

Anoxia can lead to skeletal muscle damage. In this study we have investigated whether an increased influx of Ca2+, which is known to cause damage during electrical stimulation, is a causative factor in anoxia-induced muscle damage. Isolated extensor digitorum longus (EDL) muscles from 4-week-old Wistar rats were mounted at resting length and were either resting or stimulated (30 min, 40 Hz, 10 s on, 30 s off) in the presence of standard oxygenation (95% O2, 5% CO2), anoxia (95% N2, 5% CO2) or varying degrees of reduced oxygenation. At varying extracellular Ca2+ concentrations ([Ca2+]o), 45Ca influx and total cellular Ca2+ content were measured and the release of lactic acid dehydrogenase (LDH) was determined as an indicator of cell membrane leakage. In resting muscles, incubated at 1.3 mM Ca2+, 15-75 min of exposure to anoxia increased 45Ca influx by 46-129% (P<0.001) and Ca2+ content by 20-50% (P<0.001). Mg2+ (11.2 mM) reduced the anoxia-induced increase in 45Ca influx by 43% (P<0.001). In muscles incubated at 20 and 5% O2, 45Ca influx was also stimulated (P<0.001). Increasing [Ca2+]o to 5 mM induced a progressive increase in both 45Ca uptake and LDH release in resting anoxic muscles. When electrical stimulation was applied during anoxia, Ca2+ content and LDH release increased markedly and showed a significant correlation (r2=0.55, P<0.001). In conclusion, anoxia or incubation at 20 or 5% O2 leads to an increased influx of 45Ca. This is associated with a loss of cell membrane integrity, possibly initiated by Ca2+. The loss of cell membrane integrity further increases Ca2+ influx, which may elicit a self-amplifying process of cell membrane leakage.     

Interaction of acetylcholine and cholecystokinin with dispersed smooth muscle cells.

Isolated gastric smooth muscle cells were prepared from the stomach of Bufo marinus by successive incubation in collagenase without added trypsin. Contraction was determined by image-splitting micrometry and expressed as the mean percentage decrease in cell length from control. Peak contractile response was attained within 30 s. Dose-response curves constructed from peak responses showed that the maximal responses to CCK-OP (37.2 +/- 3.8%), acetylcholine (35.3 +/- 2.5%), and Ca2+ (42.3 +/- 0.9%) were similar. The D50s for octapeptide of cholecystokinin (CCK-OP) and acetylcholine were around 10(-12) M and 10(-11) M, respectively. The response to a combination of submaximal concentrations of acetylcholine and CCK-OP exceeded the individual responses but did not exceed the maximal response to either agent alone. A low concentration of atropine (5 X 10(-10) M) inhibited specifically the maximal response to acetylcholine. A high concentration of atropine (5 X 10(-8) M) inhibited partially the maximal response to CCK-OP but had no effect on the maximal response to Ca2+. It was concluded that 1) dispersed gastric smooth muscle cells are highly sensitive to stimulation; 2) CCK-OP has a direct (myogenic) contractile effect on gastric smooth muscle; and 3) the effect of CCK-OP and acetylcholine are mediated by separate receptors.     

Relationship between global cytosolic Ca2+ concentration and Ca2+-activated K+ current in rabbit cerebral arterial myocyte.

In smooth muscle cells, the sarcoplasmic reticulum (SR) has been identified as the primary storage site for intracellular Ca2+. The peripheral SR is in close proximity with plasma membrane to make a narrow subsarcolemmal space. In this study, we investigated the regulation of subsarcolemmal [Ca2+] ([Ca2+]sl) and global cytosolic [Ca2+] ([Ca2+]c) of rabbit arterial smooth muscle using whole cell patch clamp technique and microspectrofluorimetry. The Ca2+-activated K+ current (IK(Ca)) and the ratio of fura-2 fluorescence (R340/380) were considered to reflect the [Ca2+]sl and [Ca2+]c, respectively. At a holding potential of 0 mV, extracellular application of 10 mM caffeine, a well known Ca2+-releasing agent, induced transient increase of IK(Ca) and R340/380 (IK(Ca)-transient and R340/380-transient, respectively). The increase and decay of IK(Ca) transient was faster than R340/380-transient. By repetitive application of caffeine, when the refilling state of SR was supposed to be lower than the control condition, IK(Ca)-transient and R340/380 transient were suppressed to different levels; e.g. the second application 20 sec after the first could induce smaller IK(Ca) transient than R340/380-transient. Dissociation of IK(Ca)-transient and R340/380-transient was removed by sufficient (>3 min) washout of caffeine. Recovery from the dissociation was also dependent upon the membrane potential; faster recovery was observed at negative (-40 mV) holding potential than at depolarized (0 mV) condition. Dissociation of IK(Ca) from [Ca2+]c was also partially prevented by perfusion with Na+-free (replaced by NMDG+) extracellular solution. These results suggest that, 1) there is prominent spatial inhomogeneity of [Ca2+] in cerebral arterial myocyte, 2) [Ca2+]Sl is preferentially affected by the interference from nearby plasmalemmal Ca2+ regulation mechanism which is partly dependent upon extracellular Na+.     

Membrane stretch increases the activity of Ca(2+)-activated K+ channels in rabbit coronary vascular smooth muscles

It has been proposed that Ca(2+)-activated K+ channels play an essential role in maintaining vascular tone during stretch of blood vessel. However, the underlying mechanism of stretch-induced change of Ca(2+)-activated K+ channel activities are still unknown. The present experiment was designed to investigate the effect of membrane stretch on these channels whose activity was measured from rabbit coronary smooth muscle cells using a patch clamp technique. Ca(2+)-activated K+ channel were identified by their Ca2+ and voltage dependencies and its large conductances as in other preparations. Perfusion of cells with a hypotonic solution, which mimics stretching the cell membrane by making a cell swelling, produced an increase in channel activity in cell-attached patch mode. The similar increase was observed when negative pressure was applied into the patch pipette for stretching the cell membrane within a patch area. In inside-out patch, stretch still increased channel activity even under the conditions which exclude the possible involvement of secondary messengers, or of transmembrane Ca2+ influx via stretch-activated cation channels. Pretreatment of arachidonic acid or albumin showed no effect on stretch-induced channel activation, excluding the possibility of fatty acids mediated channel activation during membrane stretch. These results indicate that the stretch may directly increase the activity of Ca(2+)-activated K+ channels in our experimental condition.     

Nonselective cation channels activated by the stimulation of muscarinic receptors in mammalian gastric smooth muscle.

Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptors were present in circular smooth muscle cells of the guinea-pig gastric antrum. Nonselective cation channels (NSCC) activated by ACh or CCh are coupled to pertussis toxin (PTX)-sensitive Go protein through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach. CCh-activated currents (I(CCh)), especially the steady-state I-V relationship of I(CCh) showed a chracteristic U-shaped curve; reversal potential of around 0 mV and inward rectification at around +15 mV and a negative slope conductance at negative potential range. Under physiological conditions, the measured single channel conductance of NSCC was approximately 25 pS. The single channel conductance was modulated by external monovalent and divalent cations including Na+, Cs+, Li+, and Ca2+ through changing both the open probability and unitary conductance. Through the NSCC, Ca2+ can move into the cell from extracellular solution as well as Na+. Calculated fractional Ca2+ current of I(CCh) (f(Ca)) was around 1% at the 2 mM [Ca2+]o and at the 4 mM [Ca2+]o, f(Ca) was 2.3%. Quinidine blocked I(CCh) potently in a reversible manner; IC50 was 0.25 microM. There were two kinds of I(CCh) modulations through Ca(2+)-dependent pathways in guinea-pig gastric smooth muscle cells; 1) Facilitation of I(CCh) via Ca2+/CaM-dependent MLCK pathway, 2) Desensitization of I(CCh) via Ca(2+)-dependent PKC pathway. In the mouse stomach, all seven types of TRPC mRNA were detected with RT-PCR. On the basis of electrophysiological, pharmacological, and molecular biological experiments, we reported the mTRPC5 as a candidate for the NSCC activated by muscarinic stimulation in mouse stomach.