Capacitative calcium entry in guinea pig gallbladder smooth muscle in vitro

This study investigates the involvement of capacitative Ca2+ entry in excitation-contraction coupling in guinea pig gallbladder smooth muscle. Thapsigargin (0.1 nM-1 microM, a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor) produced slowly developing sustained tonic contractions in guinea pig isolated gallbladder strips. All contractions approached 50% of the response to carbachol (10 microM) after 55 min. Contractile responses to thapsigargin (1 microM) were abolished in a Ca(2+)-free medium. Subsequent re-addition of Ca2+ (2.5 mM) produced a sustained tonic contraction (99 +/- 6% of the carbachol response). The contractile response to Ca2+ re-addition following incubation of tissues in a Ca(2+)-free bathing solution in the absence of thapsigargin was significantly less than in its presence (79 +/- 4 % vs 100 +/- 7 % of carbachol; p < 0.05). Contractile responses to Ca2+ re-addition following treatment with thapsigargin were attenuated by (a) the L-type voltage-operated Ca2+ channel antagonist, nifedipine (10 microM) and (b) the general inhibitor of Ca2+ entry channels including store-operated channels, SK&F96365 (50 microM and 100 microM). In separate experiments, responses to Ca2+ re-addition were essentially abolished by the tyrosine kinase inhibitor, genistein (100 microM). These results suggest that capacitative Ca2+ entry provides a source of activator Ca2+ for guinea pig gallbladder smooth muscle contraction. Contractile responses to Ca2+ re-addition following depletion of sarcoplasmic reticulum Ca2+ stores with thapsigargin, are mediated in part by Ca2+ entry through voltage-operated Ca2+ channels and by capacitative Ca2+ entry through store-operated Ca2+ channels which can be blocked by SK&F96365. Furthermore, capacitative Ca2+ entry in this tissue may be modulated by tyrosine kinase.     

Role of mitochondria in spontaneous rhythmic activity and intracellular calcium waves in the guinea pig gallbladder smooth muscle

Mitochondrial Ca(2+) handling has been implicated in spontaneous rhythmic activity in smooth muscle and interstitial cells of Cajal. In this investigation we evaluated the effect of mitochondrial inhibitors on spontaneous action potentials (APs), Ca(2+) flashes, and Ca(2+) waves in gallbladder smooth muscle (GBSM). Disruption of the mitochondrial membrane potential with carbonyl cyanide 3-chlorophenylhydrazone, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, rotenone, and antimycin A significantly reduced or eliminated APs, Ca(2+) flashes, and Ca(2+) waves in GBSM. Blockade of ATP production with oligomycin did not alter APs or Ca(2+) flashes but significantly reduced Ca(2+) wave frequency. Inhibition of mitochondrial Ca(2+) uptake and Ca(2+) release with Ru360 and CGP-37157, respectively, reduced the frequency of Ca(2+) flashes and Ca(2+) waves in GBSM. Similar to oligomycin, cyclosporin A did not alter AP and Ca(2+) flash frequency but significantly reduced Ca(2+) wave activity. These data suggest that mitochondrial Ca(2+) handling is necessary for the generation of spontaneous electrical activity and may therefore play an important role in gallbladder tone and motility.     

Changes in guinea pig gallbladder smooth muscle Ca2+ homeostasis by acute acalculous cholecystitis

Impaired smooth muscle contractility is a hallmark of acute acalculous cholecystitis. Although free cytosolic Ca2+ ([Ca2+]i) is a critical step in smooth muscle contraction, possible alterations in Ca2+ homeostasis by cholecystitis have not been elucidated. Our aim was to elucidate changes in the Ca2+ signaling pathways induced by this gallbladder dysfunction. [Ca2+]i was determined by epifluorescence microscopy in fura 2-loaded isolated gallbladder smooth muscle cells, and isometric tension was recorded from gallbladder muscle strips. F-actin content was quantified by confocal microscopy. Ca2+ responses to the inositol trisphosphate (InsP3) mobilizing agonist CCK and to caffeine, an activator of the ryanodine receptors, were impaired in cholecystitic cells. This impairment was not the result of a decrease in the size of the releasable pool. Inflammation also inhibited Ca2+ influx through L-type Ca2+ channels and capacitative Ca2+ entry induced by depletion of intracellular Ca2+ pools. In addition, the pharmacological phenotype of these channels was altered in cholecystitic cells. Inflammation impaired contractility further than Ca2+ signal attenuation, which could be related to the decrease in F-actin that was detected in cholecystitic smooth muscle cells. These findings indicate that cholecystitis decreases both Ca2+ release and Ca2+ influx in gallbladder smooth muscle, but a loss in the sensitivity of the contractile machinery to Ca2+ may also be responsible for the impairment in gallbladder contractility.     

Identification of a spontaneously active, Na+-permeable channel in guinea pig gallbladder smooth muscle

The action potential in gallbladder smooth muscle (GBSM) is caused by Ca2+ entry through voltage-dependent Ca2+ channels (VDCC), which contributes to the GBSM contractions. Action potential generation in GBSM is critically dependent on the resting membrane potential (about -50 mV), which is approximately 35 mV more positive of the K+ equilibrium potential. We hypothesized that a tonic, depolarizing conductance is present in GBSM and contributes to the regulation of the resting membrane potential and action potential frequency. GBSM cells were isolated from guinea pig gallbladders, and the whole cell patch-camp technique was used to record membrane currents. After eliminating the contribution of VDCC and K+ channels, we identified a novel spontaneously active cation conductance (I(cat)) in GBSM. This I(cat) was mediated predominantly by influx of Na+. Na+ substitution with N-methyl-D-glucamine (NMDG), a large relatively impermeant cation, caused a negative shift in the reversal potential of the ramp current and reduced the amplitude of the inward current at -50 mV by 65%. Membrane potential recordings with intracellular microelectrodes or in current-clamp mode of the patch-clamp technique indicated that the inhibition of I(cat) conductance by NMDG is associated with membrane hyperpolarization and inhibition of action potentials. Extracellular Ca2+, Mg2+, and Gd3+ attenuated the I(cat) in GBSM. Muscarinic stimulation did not activate the I(cat). Our results indicate that, in GBSM, an Na+-permeable channel contributes to the maintenance of the resting membrane potential and action potential generation and therefore plays a critical role in the regulation of GBSM excitability and contractility.     

Spontaneous electrical rhythmicity and the role of the sarcoplasmic reticulum in the excitability of guinea pig gallbladder smooth muscle cells

Spontaneous action potentials and Ca(2+) transients were investigated in intact gallbladder preparations to determine how electrical events propagate and the cellular mechanisms that modulate these events. Rhythmic phasic contractions were preceded by Ca(2+) flashes that were either focal (limited to one or a few bundles), multifocal (occurring asynchronously in several bundles), or global (simultaneous flashes throughout the field). Ca(2+) flashes and action potentials were abolished by inhibiting sarcoplasmic reticulum (SR) Ca(2+) release via inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] channels with 2-aminoethoxydiphenyl borate and xestospongin C or by inhibiting voltage-dependent Ca(2+) channels (VDCCs) with nifedipine or diltiazem or nisoldipine. Inhibiting ryanodine channels with ryanodine caused multiple spikes superimposed upon plateaus of action potentials and extended quiescent periods. Depletion of SR Ca(2+) stores with thapsigargin or cyclopiazonic acid increased the frequency and duration of Ca(2+) flashes and action potentials. Acetylcholine, carbachol, or cholecystokinin increased synchronized and increased the frequency of Ca(2+) flashes and action potentials. The phospholipase C (PLC) inhibitor U-73122 did not affect Ca(2+) flash or action potential activity but inhibited the excitatory effects of acetylcholine on these events. These results indicate that Ca(2+) flashes correspond to action potentials and that rhythmic excitation in the gallbladder is multifocal among gallbladder smooth muscle bundles and can be synchronized by excitatory agonists. These events do not depend on PLC activation, but agonist stimulation involves activation of PLC. Generation of these events depends on Ca(2+) entry via VDCCs and on Ca(2+) mobilization from the SR via Ins(1,4,5)P(3) channels.     

Calcium channels in smooth muscle cells

In smooth muscle cells, the electrophysiological properties of potential-dependent calcium channels are similar to those described in other excitable cells. The calcium current is dependent on the extracellular calcium concentration; it is insensitive to external sodium removal and tetrodotoxin application. Other ions (Ba2+, Sr2+, Na+) can flow through the calcium channel. This channel is blocked by Mn2+, Co2+, Cd2+ and by organic inhibitors. The inactivation mechanism is mediated by both the membrane potential and the calcium influx. Ca2+ ions can also penetrate into the cell through receptor-operated channels. These channels show a low ionic selectivity and are generally less sensitive to organic Ca-blockers than the potential-dependent calcium channels. The finding of specific channel inhibitors as well as the study of the biochemical pathways between receptor activation and channel opening are prerequisites to further characterization of receptor-operated channels.     

Cooling-induced contraction of guinea pig tracheal smooth muscle

Cooling of isolated guinea pig tracheal smooth muscle from 38 to 28 degrees C over 2.25 min produced a transient contraction followed by sustained relaxation. The cooling-induced contraction was blocked either by pretreatment with ouabain at concentrations of 10(-5) M or greater or by substitution of normal physiological salt solution with K-free solution. In contrast, the contractile response to cooling was not inhibited by pretreatment with phentolamine (10(-5) M), atropine (10(-5) M), tetrodotoxin (3 X 10(-7) M), diphenhydramine (10(-5) M), cromolyn sodium (10(-3) M), indomethacin (3 X 10(-7) M), nifedipine (10(-7) M), or verapamil (3 X 10(-6) M). Addition of NaHCO3 to the bath during cooling, preventing a change in pH of the physiological salt solution, did not affect the cooling-induced contraction. It is concluded that cooling of isolated guinea pig trachea produces a transient ouabain-sensitive contraction, and that the data suggest the contraction is mediated by inhibition of Na-K-ATPase in the smooth muscle rather than through neuronal stimulation or chemical mediator release.     

Vagal innervation of guinea pig bronchial smooth muscle

We isolated the guinea pig right bronchus with the vagus nerves intact and evaluated the changes in isometric tension of the smooth muscle in response to nerve stimulation. Brief (10-s) trains of electrical field stimulation or vagus nerve stimulation caused a biphasic contraction: the "first phase" sensitive to atropine and the "second phase" sensitive to capsaicin. The two phases could be dissociated by adjusting the stimulus intensity; greater stimulus intensities (pulse durations or voltage) were required to evoke the capsaicin-sensitive phase. When stimulated at 30-min intervals, the magnitude of both phases of the contractions declined over a 2-h period of repeated stimulation; however, this was prevented by indomethacin. Stimulation of the left vagus nerve resulted in a monophasic contraction of the right bronchus, with little evidence of a capsaicin-sensitive phase. Blocking neurotransmission through the bronchial ganglion, as monitored by intracellular recording techniques, abolished the first-phase contraction but had no effect on the capsaicin-sensitive phase. Selective blockade of muscarinic M1 receptors had no effect on vagus nerve-mediated contractions. The results demonstrate that the left and right vagus nerves carry preganglionic fibers to the right bronchial ganglion. The right but not the left vagus nerve also carries capsaicin-sensitive afferent fibers that, when stimulated, result in a persistent contraction of the right bronchus. Finally, we provide functional and electrophysiological evidence supporting the hypothesis that capsaicin-sensitive afferent neurons communicate with postganglionic motoneurons within the bronchus.     

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.     

Inward current in single smooth muscle cells of the guinea pig taenia coli

Using the tight-seal voltage-clamp method, the ionic currents in the enzymatically dispersed single smooth muscle cells of the guinea pig taenia coli have been studied. In a physiological medium containing 3 mM Ca2+, the cells are gently tapering spindles, averaging 201 (length) x 8 microns (largest diameter in center of cell), with a volume of 5 pl. The average cell capacitance is 50 pF, and the specific membrane capacitance 1.15 microF/cm2. The input impedance of the resting cell is 1-2 G omega. Spatially uniform voltage-control prevails after the first 400 microseconds. There is much overlap of the inward and outward currents, but the inward current can be isolated by applying Cs+ internally to block all potassium currents. The inward current is carried by Ca2+. Activation begins at approximately -30 mV, maximum ICa occurs at +10-+20 mV, and the reversal potential is approximately +75 mV. The Ca2+ channel is permeable to Sr2+ and Ba2+, and to Cs+ moving outwards, but not to Na+ moving inwards. Activation and deactivation are very rapid at approximately 33 degrees C, with time-constants of less than 1 ms. Inactivation has a complex time course, resolvable into three exponential components, with average time constants (at 0 mV) of 7, 45, and 400 ms, which are affected differently by voltage. Steady-state inactivation is half-maximal at -30 mV for all components combined, but -36 mV for the fast component and -26 and -23 mV for the other two components. The presence of multiple forms of Ca2+ channel is inferred from the inactivation characteristics, not from activation properties. Recovery of the fast channel occurs with a time-constant of 72 ms (at +10 mV). Ca2+ influx during an action potential can transfer approximately 9 pC of charge, which could elevate intracellular Ca2+ concentration adequately for various physiological functions.     

Outward current in single smooth muscle cells of the guinea pig taenia coli

In single myocytes of the guinea pig taenia coli, dispersed by enzymatic digestion, the late outward current is carried by K+. It has both a Ca2+-activated component and a voltage-dependent component which is resistant to external Co2+. The reversal potential is -84 mV, and the channel(s) for it are highly selective to K+. At 33 degrees C, the activation follows n2 kinetics, with a voltage-dependent time constant of 10.6 ms at 0 mV, which shortens to 1.7 ms at +70 mV. Deactivation follows a single-exponential time course, with a voltage-dependent time constant of 11 ms at -50 mV, which lengthens to 33 ms at -20 mV. During a 4.5-s maintained depolarization, IK inactivates, most of it into two exponential components, but there is a small noninactivating residue. It is surmised that during an action potential under physiological conditions, there is sufficient IK to cause repolarization.     

Calcium antagonists and contractile responses in rat vas deferens and guinea pig ileal smooth muscle.

The effect of depletion of extracellular Ca2+ (Ca2+ext) on the loss of responsiveness of the guinea pig ileal longitudinal muscle (g.p.i.l.m.) and the rat vas deferens (r.v.d.) to K+ and cis-2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide (CD), and K+ and noradrenaline (NA), has been examined and compared with the effects of a variety of local anesthetics and calcium antagonists. The results indicate that qualitative similarities are apparent with respect to the dependence of agonist-induced activity on Ca2+ext in both the g.p.i.l.m. and r.v.d. Distinct differences, however, in the Ca2+ translocation processes in these two tissues, in response to the different agonists, can be shown by the use of a variety of 'calcium antagonists' thus indicating that such translocation processes are both tissue and agonist selective. It is thus noted that, contrary to the Ca2+ depletion studies, D 600 and the usually more potent BAY-1040 showed no discrimination of action or potency in their ability to inhibit components of the NA response in the r.v.d. In contrast, D 600 and the more potent BAY-1040 selectively inhibited the tonic component of the K+ response. Treatment with SKF 525A and parethoxycaine (PC) in the g.p.i.l.m. and SKF 525A in the r.v.d. resulted in a nonselective inhibition of responses of the tissues to all stimulants. However, in the r.v.d. PC potentiated NA action, and its methobromide (MeBr) derivative potentiated both NA and K+ action and also, like PC, partially shifted to the left the dose-response curve to Ca2+ in NA-depolarizing Ca-free Tyrode's. The quaternary MeBr and the tertiary 2-chloroethyl (2Cl) derivatives of SKF 525A and PC were selectively more effective against CD- than K+ supported contractile activity in the g.p.i.l.m. and the 2Cl derivatives were more effective against NA than K+ responses in the r.v.d. The 2Cl derivative of PC also was more effective in antagonizing the Ca2+ dose-response curve in high-CD or high-NA than in high-K+ Ca2+-free Tyrode's.     

Atp-activated ionic permeability in smooth muscle cells isolated from the guinea pig urinary bladder

Smooth muscle cells from the guinea pig urinary bladder were investigated by voltage clamping at the plasma membrane and using an intracellular perfusion technique. Applying adenosine triphosphate (ATP) at a concentration greater than 3 × 10^–8 M and at a membrane potential of –100 to –30 mV produced a rise in fast inward transmembrane current. A similar effect was exerted by adenosine diphosphate (ADP) and -, -, and ,-methylene ATP. Application of guanosine triphosphate, inosine triphosphate, adenosine monophosphate (AMP), and adenosine failed to activate this current. It was found that AMP blocks ATP receptors competitively. No pharmacological differences were found between the latter ATP receptors and those of rat sensory neurons. The ATP receptors were rapidly desensitized and recovered their sensitivity to agonists extremely slowly. Speed of desensitization was reduced by a decrease in ATP concentration.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 95–100, January–February, 1987.     

Desensitization of isolated smooth muscle cells from guinea pig taenia caecum to acetylcholine

The role of tissue organization of smooth muscle in short-term desensitization to acetylcholine (ACh) was examined by studying the desensitization of isolated single cells from guinea pig taenia caecum. Cells were isolated by collagenase digestion. The conditions during cell isolation were adjusted to obtain cells that showed repeated contractions. The cells contracted on treatment with 10(-7)-10(-6) M ACh, showing an all-or-none response. Desensitized cells also showed an all-or-none response but required a higher concentration of ACh for induction of contraction; i.e., the magnitude of their maximal response was not changed appreciably but the threshold concentration of ACh for their contraction was raised. Incubation of the whole tissue with 10(-4) M ACh for 10 min also caused desensitization. This desensitization was accompanied by reduction of the contractile response at intermediate concentrations. The mode of desensitization of isolated cells determined from the average response of the isolated cells was almost the same as that of whole muscle. It is concluded that the desensitization occurred in each cell irrespective of its tissue organization and that the desensitization was due to an increase of the threshold for contraction to ACh of each cell.     

Inhibitory synaptic potentials of the smooth muscle cells of guinea pig taenia coli

A single submaximal intramural application of rectangular stimuli (duration 0.2–0.5 msec) to an atropine-treated taenia coli muscle band evoked inhibitory postsynaptic potentials (IPSP) and a marked relaxation of the muscle band in the vast majority of muscle cells. The latency period of the IPSP was 122±16 msec; the times for a rise and fall of amplitude were 96±8 and 370±60 msec, respectively. The mean latency period of muscle relaxation was 800 msec. The latency period, and especially the amplitude of the IPSP depended on the intensity of the intramural stimulation. This indicates that one muscle cell is inhibited by several nerve fibers. IPSP evoked by threshold stimuli displayed a tendency toward summation, while the amplitude of the second and of subsequent IPSP evoked by low-frequency maximal stimuli was always less than that of the first IPSP. After periodic stimulation (frequency 10–60 impulses/min) was discontinued, a posttetanic decrease in IPSP amplitude was observed. Anodic polarization of the muscle band with a direct current raised the effectiveness of synaptic transmission, as was evidenced by the considerable increase in IPSP amplitude. When the muscle membrane was hyperpolarized with noradrenaline, IPSP inhibition was reversible. This is evidence that the unknown mediator and noradrenaline have a common ionic inhibitory mechanism.A. A. Bogomol'ts Institute of Physiology of the Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 5, pp. 544–551, September–October, 1970.