Caffeine effects on buccal muscles of Aplysia

1. Caffeine (35-70 mM) elicited contractions of Aplysia buccal muscle El. In a Ca2+-free medium, in which ACh-elicited contractions rapidly fail, caffeine elicited contractions of approximately the same size as in normal medium. 2. 5-HT (10(-8) M and 10(-7) M) did not enhance caffeine-elicited contractions. 3. Lower concentrations (1-10 mM) of caffeine inhibited ACh-elicited contractions. Caffeine (7 mM) reduced the contraction by 80%. 4. Caffeine (7 mM) reduced ACh-elicited depolarization by 60%. 5. Caffeine (7 mM) increased 45Ca2+ influx into Aplysia buccal muscle I5. The stimulation of influx of 45Ca2+ by 10(-3) M ACh was non-additive with the stimulation caused by caffeine, and 7 mM caffeine reduced the influx caused by 10(-3) M ACh.     

Sources of activator calcium ions in the contraction of smooth muscles in Aplysia kurodai

The physiological and pharmacological properties of contraction and the ultrastructure of buccal mass retractor muscle (I4) and gill-pinnule closure muscle (GPCM) in Aplysia kurodai were studied to learn more about the sources of activator Ca2+ in molluscan smooth muscle. Acetylcholine (ACh) and high K+-induced contractions were reduced by lowering the external Ca2+ concentration, and eliminated by the removal of extracellular Ca2+. Nifedipine appreciably reduced ACh- and high K+-induced contractions, while amiloride decreased only ACh-induced contractions and had no significant effect on high K+-induced contractions. When nifedipine and amiloride were applied together, either type of contraction was still appreciable. Serotonin (5-HT) could potentiate subsequent ACh- and high K+-induced contractions in I4; potentiated tension was significantly reduced by nifedipine and amiloride, whereas 5-HT inhibited ACh-and high K+-induced contractions in GPCM. The potentiating effects of 5-HT may be mediated by the activation of the Ca2+-channel to increase the influx from extracellular Ca2+. Caffeine caused contractions in Ca2+-free solution in both muscles. Electron microscopy revealed sarcolemmal vesicles underneath the plasma membrane in both muscle fibers. Electron microscopical cytochemistry demonstrated that pyroantimonate precipitates were localized in the sarcolemmal vesicles and in the inner surface of plasma membranes in the resting fibers. Present results indicate that the contractions of I4 and GPCM fibers are caused not only by Ca2+-influx but also by Ca2+ release from the intracellular storage sites, such as the sarcolemmal vesicles and the inner surface of plasma membranes.     

Regulation of calcium influx into buccal muscles of Aplysia by acetylcholine and serotonin

Acetylcholine (ACh) causes contraction of Aplysia buccal muscles E1 and I5, and serotonin (5-hydroxytryptamine, 5-HT) enhances ACh-elicited contractions of these muscles. Possible roles of calcium influx in mediating these responses were examined by studying influx of 45Ca++. 5-HT increased calcium influx into both I5 and E1. Maximal influx occurred at 10(-6) M 5-HT and the increased influx could be sustained in the presence of 5-HT for at least 10 min. ACh also caused calcium influx, and calcium influx increased approximately in proportion to log[ACh] from 10(-5) M to 10(-3) M ACh. 5-HT and ACh probably bring about calcium influx by different mechanisms since the effect of ACh was additive to a maximal 5-HT response, and 10(-4) M hexamethonium bromide inhibited the increased influx caused by ACh but did not affect influx caused by 5-HT. Cyclic AMP analogues and forskolin neither caused an increase in calcium influx nor an increase in the influx caused by ACh. The data support a model in which ACh-elicited contractions of I5 and E1 are due primarily to calcium entry across the extracellular membrane, and 5-HT can "load" an intracellular site by a mechanism different from that activated by ACh. The data do not support a role for cyclic AMP in mediating the calcium influx response to 5-HT.     

Contribution of Na+ and membrane depolarization to contraction induced by adrenaline in the guinea pig vas deferens

The contribution of Na+ and membrane depolarization to biphasic contractions induced by adrenaline were investigated in the smooth muscle of guinea pig vas deferens. Adrenaline (5 X 10(-6) M) produced an initial small contraction (first contraction) followed by a large tonic contraction (second contraction) with subsequent rhythmic activity. The entire response to adrenaline was largely inhibited by phentolamine (5 X 10(-6) M). By adding an appropriate concentration of Mn2+ (2 X 10(-4) M) or nifedipine (3 X 10(-7) M), a Ca2+ blocker, the second contraction was strongly reduced, accompanied by abolishment of the rhythmic contraction, whereas the first contraction was virtually unaffected. However, the first contraction was markedly suppressed by a higher concentration of Mn2+. All contractions produced by adrenaline were greatly reduced in Ca2+-free solution containing 0.5 mM EGTA. By lowering external Na+ concentration, the first contraction was markedly increased without greatly affecting the second contraction. By exposure to Na+-free isotonic high K+ solution, which elicited a greater depolarization of the membrane, the first contraction produced by adrenaline was also greatly potentiated, while the second and rhythmic contractions were eliminated. These results suggest that the adrenaline-evoked first contraction may be due to an influx of membrane bound Ca2+ which is independent of membrane depolarization, while the second (rhythmic) contraction is due to an influx of extracellular Ca2+ which is dependent upon depolarization.     

The ATP4- receptor-operated ion channel of human lymphocytes: inhibition of ion fluxes by amiloride analogs and by extracellular sodium ions.

Extracellular ATP is known to increase the membrane permeability of a variety of cells. Addition of ATP to human leukemic lymphocytes loaded with the Ca2+ indicator, fura-2, induced a rise in cytosolic Ca2+ concentration which was attenuated or absent in NaCl media compared with KCl, choline Cl, or NMG Cl media. In contrast, anti-immunoglobulin antibody gave similar Ca2+ transients in NaCl and KCl media. A half-maximal inhibition of peak ATP-induced Ca2+ response was observed at 10-16 mM extracellular Na+. Basal 45Ca2+ influx into lymphocytes was stimulated 9.6-fold by ATP added to cells in KCl media, but the effect of ATP was greatly reduced for cells in NaCl media. Hexamethylene amiloride blocked 74% of the ATP-stimulated Ca45 uptake of cells in KCl media. Flow cytometry measurements of fluo-3-loaded cells confirmed that the ATP-induced rise in cytosolic Ca2+ was inhibited either by extracellular Na+ or by addition of hexamethylene amiloride. Extracellular ATP stimulated 86Rb efflux from lymphocytes 10-fold and this increment was inhibited by the amiloride analogs in a rank order of potency 5-(N-methyl-N-isobutyl)amiloride greater than 5-(N,N-hexamethylene)amiloride greater than 5-(N-ethyl-N-isopropyl)amiloride greater than amiloride. ATP-induced 86Rb efflux showed a sigmoid dependence on the concentration of ATP and Hill analysis gave K1/2 of 90 and 130 microM and n values of 2.5 and 2.5 for KCl and NaCl media, respectively. However, the maximal ATP-induced 86Rb efflux was 3-fold greater in KCl than in NaCl media. Raising extracellular Na+ from 10 to 100 mM increased ATP-induced Na+ influx from a mean of 2.0 to 3.7 nEq/10(7) cells/min, suggesting either saturability or self-inhibition by Na+ of its own influx. These data suggest that ATP opens a receptor-operated ion channel which allows increased Ca2+ and Na+ influx and Rb+ efflux and these fluxes are inhibited by extracellular Na+ ions as well as by the amiloride analogs.     

Na+-H+ exchange in the process of glucose-induced insulin release from the pancreatic B-cell. Effects of amiloride on 86Rb, 45Ca fluxes and insulin release

The effect of amiloride, an inhibitor of Na+-H+ exchange, on intracellular pH (pHi), 86Rb outflow, 45Ca outflow and insulin release from pancreatic rat islets was examined. In the 0.1-1 mM range, amiloride transiently reduced pHi of glucose-deprived islets and allowed glucose to induce a sustained decrease in pHi of the islet cells. Amiloride reproduced the effect of glucose to decrease 86Rb and 45Ca outflow. In the presence of glucose (5.6 mM or more), amiloride (100 microM) acted synergistically with the sugar to reduce K+ outflow, and to stimulate 40Ca inflow and insulin release from perifused islets. These results add strong support to the view that the generation of protons through the metabolism of glucose represents an important step in the process of glucose-induced release. The stimulation by glucose of Na+-H+ exchange apparently masks and even overcomes the glucose-induced decrease in pHi otherwise expected from the increase in catabolic fluxes.     

Postsynaptic alpha adrenoceptors on vascular smooth muscle

A heterogeneous population of alpha adrenoceptors mediates vasoconstriction in the canine saphenous vein (CSV). Studies with isolated strips of venous smooth muscle incubated with selective alpha-adrenoceptor agonists and antagonists revealed that both alpha 1 and alpha 2 adrenoceptors exist independently in this tissue and both subtypes mediate a contractile response. Measurement of contractile responses in reduced or zero external calcium conditions indicates that stimulation of alpha 1 adrenoceptors induces contractions by influx of extracellular calcium and release of calcium from internal stores. In contrast, 45Ca uptake studies suggest that activation of the postsynaptic alpha 2 adrenoceptor produces vasoconstriction dependent only on influx of extracellular calcium. The influx of calcium produced by the selective alpha 2-adrenoceptor agonist BHT-920 is inhibited by calcium entry blockers. Measurements of transmembrane potentials from smooth muscle cells of the CSV suggest that alpha 1-adrenoceptor activation produces depolarization and contraction (electromechanical coupling) whereas alpha 2-adrenoceptor stimulation does not result in concentration-dependent depolarization of the smooth muscle cells (pharmacomechanical coupling).     

Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.     

Antagonists of cholinergic and serotonergic responses of Aplysia buccal muscle

Cholinergic and serotonergic receptors of Aplysia californica buccal muscles were characterized pharmacologically by determining compounds that effectively inhibited contractile responses to acetylcholine (ACh) and modulatory effects of serotonin (5-HT), respectively. pA50 for ACh to elicit contraction averaged 4.7 ± 0.1 (mean ± SE, equivalent to 2 × 10⁻⁵ M). Both hexamethonium bromide and atropine inhibited ACh-elicited contractions, but neither inhibited the response completely, nor were the two together able to antagonize the response completely. Curare caused inhibition only at low ACh doses, and muscarinic antagonists pirenzapine and 4-diphenylacetoxy-N-methylpiperidine methiodide caused partial inhibition. The most effective blocker of ACh-elicited contractions was the nicotinic antagonist mecamylamine. 10⁻⁴M mecamylamine completely blocked the cholinergic response. ACh contractions were inhibited 90% within 10 min and took >40 min to recover from mecamylamine. Specificity was indicated by the lack of effect of mecamylamine on potassium-elicited contraction. NAN-190 blocked the potentiating effect of 5-HT without having inhibitory or potentiating effects by itself on ACh-elicited contractions. NAN-190 blocked the potentiating effect of 8-OH-DPAT. Cholinergic receptors on Aplysia buccal muscles are most effectively inhibited by mecamylamine and may have mixed nicotinic/muscarinic character. Serotonergic receptors have pharmacological similarities to vertebrate 5-HT_1A receptors and may be closely related to the gastropod 5-HTlym receptor.     

Electro- and pharmacomechanical coupling in the smooth muscle cells of the rabbit ear artery

A contraction of the rabbit ear artery can be induced by depolarizing the cells with a K-rich solution if Ca is present. 10(-9)-10(-6) M noradrenaline and 10(-8)-10(-7) M histamine cause a contraction of this tissue without modifying the membrane potential. If the histamine concentration exceeds 10(-7) M some depolarization of the membrane also occurs. Both noradrenaline and histamine also induce a contraction in Ca-free medium, even if La is present. None of these stimuli produces action potentials or fluctuations of the membrane potential. Besides these tonic contractions, the ear artery can also produce phasic contractions when 10 mM TEA is added to the medium. Such contractions are caused by the appearance of action potentials which are Ca dependent and which are similar to those appearing in visceral smooth muscle. A study of 45Ca fluxes has revealed that K depolarization and noradrenaline cause only a small increase in 45Ca uptake by the cells, while noradrenaline also releases cellular Ca, even in Ca-free medium. A comparison of tension development and 45Ca release induced by noradrenaline in Ca-free medium suggests that Ca extrusion could be very efficient in the rabbit ear artery and that it could play a direct role in its relaxation.     

Contribution of transient and sustained calcium influx, and sensitization to depolarization-induced contractions of the intact mouse aorta

ABSTRACT: BACKGROUND: Electrophysiological studies of L-type Ca2+ channels in isolated vascular smooth muscle cells revealed that depolarization of these cells evoked a transient and a time-independent Ca2+ current. The sustained, non-inactivating current occurred at voltages where voltage-dependent activation and inactivation overlapped (voltage window) and its contribution to basal tone or active tension in larger multicellular blood vessel preparations is unknown at present. This study investigated whether window Ca2+ influx affects isometric contraction of multicellular C57Bl6 mouse aortic segments. RESULTS: Intracellular Ca2+ (Cai2+, Fura-2), membrane potential and isometric force were measured in aortic segments, which were clamped at fixed membrane potentials by increasing extracellular K+ concentrations. K+ above 20 mM evoked biphasic contractions, which were not affected by inhibition of IP3- or Ca2+ induced Ca2+ release with 2-aminoethoxydiphenyl borate or ryanodine, respectively, ruling out the contribution of intracellular Ca2+ release. The fast force component paralleled Cai2+ increase, but the slow contraction coincided with Cai2+ decrease. In the absence of extracellular Ca2+, basal tension and Cai2+ declined, and depolarization failed to evoke Cai2+ signals or contraction. Subsequent re-introduction of external Ca2+ elicited only slow contractions, which were now matched by Cai2+ increase. After Cai2+ attained steady-state, isometric force kept increasing due to Ca2+- sensitization of the contractile elements. The slow force responses displayed a bell-shaped voltage-dependence, were suppressed by hyperpolarization with levcromakalim, and enhanced by an agonist of L-type Ca2+ channels (BAY K8644). CONCLUSION: The isometric response of mouse aortic segments to depolarization consists of a fast, transient contraction paralleled by a transient Ca2+ influx via Ca2+ channels which completely inactivate. Ca2+ channels, which did not completely inactivate during the depolarization, initiated a second, sustained phase of contraction, which was matched by a sustained non-inactivating window Ca2+ influx. Together with sensitization, this window L-type Ca2+ influx is a major determinant of basal and active tension of mouse aortic smooth muscle.     

Calcium channels and excitation-contraction coupling in cardiac cells. I. Two components of contraction in guinea-pig papillary muscle

Biphasic contractions have been obtained in guinea-pig papillary muscle by inducing partial depolarization in K+-rich solution (17 mM) containing 0.3 microM isoproterenol; whereas in guinea-pig atria, the same conditions led to monophasic contractions corresponding to the first component of contraction in papillary muscle. The relationships between the amplitude of the two components of the biphasic contraction and the resting membrane potential were sigmoidal curves. The first component of contraction was inactivated for membrane potentials less positive than those for the second component. In Na+-low solution (25 mM), biphasic contraction became monophasic subsequent to the loss of the second component, but tetraethylammonium unmasked the second component of contraction. The relationship between the amplitude of the first component of contraction and the logarithm of extracellular Ca2+ concentration was complex, whereas for the second component it was linear. When Ca2+ ions were replaced by Sr2+ ions, only the second component of contraction was observed. It is suggested that the first component of contraction may be triggered by a Ca2+ release from sarcoplasmic reticulum, induced by the fast inward Ca2+ current and (or) by the depolarization. The second component of contraction may be due to a direct activation of contractile proteins by Ca2+ entering the cell along with the slow inward Ca2+ current and diffusing through the sarcoplasm. These results do not exclude the existence of a third "tonic" component, which could possibly be mixed with the second component of contraction.     

Increased lung uptake of liposomes coated with polysaccharides

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na+), or in a medium containing only 25 mM Na+. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin ( PzO ) is not affected. Amiloride also inhibits the carbachol-induced 45Ca efflux from rabbit pancreatic acini, but again not that induced by PzO . The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and 45Ca efflux are 40 and 80 microM, respectively. Amiloride also competitively inhibits the specific binding of [3H]quinuclidinyl benzylate ( [3H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.     

Properties of chloride-stimulated 45Ca flux in skinned muscle fibers

Isometric force and 45Ca loss from fiber to bath were measured simultaneously in skinned fibers from frog muscle at 19 degrees C. In unstimulated fibers, 45Ca efflux from the sarcoplasmic reticulum (SR) was very slow, with little or no dependence on EGTA (0.1-5 mM) or Mg++ (20 micrometer-1.3 mM). Stimulation by high [Cl] at 0.11 mM Mg++ caused rapid force transients (duration approximately 10 s) and 45Ca release. This response was followed for 55 s, with 5 mM EGTA added to chelate myofilament space (MFS) Ca either (a) after relaxation, (b) near the peak of the force spike, or (c) before or with the stimulus. When EGTA was present during Cl application, stimulation of 45Ca release was undetectable. Analysis of the time-course of tracer loss during the three protocols showed that when EGTA was absent, 16% of the fiber tracer was released from the SR within approximately 3 s, and 70% of the tracer still in the MFS near the peak of the force spike was subsequently reaccumulated. The results suggest that (a) the Cl response is highly Ca-dependent; (b) stimulation increases 45Ca efflux from the SR at least 100-200-fold; and (c) the rate of reaccumulation is much slower than the influx predicted from published data on resting fibers, raising the possibility that depolarization inhibits active Ca transport by the SR.     

Calcium influx into dendrites of the leech Retzius neuron evoked by 5-hydroxytryptamine

5-Hydroxytryptamine (5-HT) is a ubiquitous neurotransmitter and neuromodulator that affects neural circuits and behaviours in vertebrates and invertebrates. In the present study, we have investigated 5-HT-induced Ca(2+) transients in subcellular compartments of Retzius neurons in the leech central nervous system using confocal laser scanning microscopy, and studied the effect of 5-HT on the electrical coupling between the Retzius neurons. Bath application of 5-HT (50mM) induced a Ca(2+) transient in axon, dendrites and cell body of the Retzius neuron. This Ca(2+) transient was significantly faster and larger in dendrites than in axon and cell body, and was half-maximal at a 5-HT concentration of 5-12mM. The Ca(2+) transient was suppressed in the absence of extracellular Ca(2+) and by methysergide (100mM), a non-specific antagonist of metabotropic 5-HT receptors, and was strongly reduced by bath application of the Ca(2+) channel blocker Co(2+) (2mM). Injection of the non-hydrolysable GTP analogue GTPgammaS increased and prolonged the dendritic 5-HT-induced Ca(2+) transient. The non-selective protein kinase inhibitor H7 (100mM) and the adenylate cyclase inhibitor SQ22536 (500 mM) did not affect the Ca(2+) transient, and the membrane-permeable cAMP analogue dibutyryl-cAMP (500 mM) did not mimic the effect of 5-HT application. 5-HT reduced the apparent electrical coupling between the two Retzius neurons, whereas suppression of the Ca(2+) influx by removal of external Ca(2+) improved the transmission of action potentials at the electrical synapses which are located between the dendrites of the adjacent Retzius neurons. The results indicate that 5-HT induces a Ca(2+) influx through calcium channels located primarily in the dendrites, and presumably activated by a G protein-coupled 5-HT receptor. The dendritic Ca(2+) increase appears to modulate the excitability of, and the synchronization between, the two Retzius neurons.     

Amiloride is a cholinergic antagonist in the rabbit pancreas

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na⁺), or in a medium containing only 25 mM Na⁺. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin (PzO) is not affected. Amiloride also inhibits the carbachol-induced ⁴⁵Ca efflux from rabbit pancreatic acini, but again not that induced by PzO. The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and ⁴⁵Ca efflux are 40 and 80 μM, respectively. Amiloride also competitively inhibits the specific binding of [³H]quinuclidinyl benzylate ([³H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.     

Contractile responses of the human umbilical artery to KCl and serotonin in Ca-free medium and the effects of levcromakalim

It is known that K(ATP) channel openers inhibit the release and refilling of Ca(2+) from intracellular stores. The present study was designed to test the effects of levcromakalim in human umbilical artery (HUA) rings stimulated by serotonin (5-HT) and KCl in Ca-free medium. Umbilical cords were obtained at vaginal or cesarean deliveries from healthy, term pregnancies. After the isolation, HUA rings were placed in organ baths in solution with indomethacin (10(-5) M) and N(G)-nitro-L-arginine methyl ester (L-NAME) (10(-3) M) at 37 degrees C and aerated with 95% O(2) and 5% CO(2) for the measurement of isometric force. In Ca-free solution with Ethylene glycol-bis (ss-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (2 mM) the contractions produced by 5-HT (10(-6) M) and KCl (40 mM) decreased significantly. Afterwards, HUA rings were treated with 5-HT and KCl in repeated manner in Ca-free medium. In contrast to KCl, 5-HT induced contractions reduced in each application, progressively. Levcromakalim (10(-4) M) abolished the contractions elicited by 5-HT. On the other hand, levcromakalim had a little but significant inhibitory effect on KCl induced contraction in Ca-free medium. These results suggest that Ca(2+) is not the only transduction pathway in KCl produced contractions of HUA smooth muscle cells.     

Dinitrofluorobenzene-induced rigor in ileal longitudinal smooth muscle of the guinea-pig.

1. The administration of 2,4-dinitrofluorobenzene (DFB) (0.1-1 mM) to the ileal longitudinal muscle produced contractions within seconds of its administration. 2. A component of the first 2 min duration of the phasic phase of 1 or 0.5 mM DFB contraction and the first phase of 0.35 or 0.1 mM DFB contraction was inhibited by Ca2+ antagonists, 1 x 10(-6) M D-600. 3. The DFB contraction resistant to D-600 began to develop when the tissue ATP concentration rapidly reduced. 4. The DFB contraction in ileum consists of two components; an initial fast contraction which is sensitive to Ca2+ antagonists, and a late contraction referred to as a rigor which is resistant to it.     

Calcium channels and excitation-contraction coupling in cardiac cells. II. A pharmacological study of the biphasic contraction in guinea-pig papillary muscle

Biphasic contractions were obtained in guinea-pig papillary muscle by inducing partial depolarization in K+-rich solution (17 mM) in the presence of 0.3 microM isoproterenol. Mn2+ ions inhibited the two components of contraction in a similar way. Nifedipine and particularly Cd2+ ions specifically inhibited the second component of contraction. Isoproterenol and BAY K 8644 markedly increased the amplitude of the second component (P2) of contraction. Nevertheless, a moderate positive inotropic effect of isoproterenol was found on the first component (P1) of contraction when excitability was restored by 0.2 mM Ba instead of isoproterenol. Acetylcholine and hypoxia decreased the amplitude of the second component of contraction to a greater extent. In the presence of digoxin or Na+-free solution, P1 was strongly increased. When sarcoplasmic reticular function was hindered by 1mM caffeine or in the presence of Ca2+-free Sr2+ solution, digoxin always induced a negative inotropic effect on P2. Inversely in these conditions the transient positive inotropic effect of Na+-free solution was strongly reduced. These results are consistent with the hypothesis that the late component of contraction is triggered by the slow inward Ca2+ current and that the early component is due to Ca2+ release from the sarcoplasmic reticulum.     

Src modulates serotonin-induced calcium signaling by regulating phosphatidylinositol 4,5-bisphosphate

We tested the hypothesis that, in airway smooth muscle cells, stimulation of G-protein-coupled receptors by contractile agonists activates Src kinase and that this kinase modulates cell contractility and Ca(2+) signaling by affecting the levels of the phospholipase C substrate phosphatidylinositol 4,5-bisphosphate (PIP(2)). Stimulation of cultured rat tracheal smooth muscle cells with serotonin (5-HT) induced an increase in Src activity, Ca(2+) mobilization, and contraction (decrease in cell area). 5-HT-evoked cell contraction was reduced by a specific inhibitor of Src family kinases, 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1). Peak Ca(2+) responses to 5-HT were attenuated by PP1 and an anti-Src-blocking antibody and augmented by expression of constitutively activated Y529F Src. Sustained phases of Ca(2+) responses to 5-HT and Ca(2+) influx resulting from emptying of Ca(2+) stores in the endoplasmic reticulum by thapsigargin were also decreased after PP1 treatment. PP1 significantly reduced the turnover of inositol phosphates produced on 5-HT stimulation and the amount of PIP(2) in the Triton X-100-insoluble lipid fraction. Overall, these data demonstrate that, in rat tracheal smooth muscle cells, Src kinase modulates 5-HT-evoked cell contractility and Ca(2+) signaling by regulating PIP(2) levels and Ca(2+) influx.