Reversal of α1-receptor mediated relaxation in intestinal smooth muscle

The α₁-receptor agonist phenylephrine relaxed longitudinal rabbit jejunal muscle contracted in vitro by low concentrations of barium ions (1 mM). When the Ba²⁺ concentration was increased to 10–15 mM the response to phenylephrine was a contraction, and at Ba²⁺ concentrations in between the high and low range this response was biphasic—a relaxation followed by a contractile phase. The α₂-receptor agonist clonidine did not affect the tone of the Ba²⁺ contracted preparation. When the muscle preparation was contracted by Sr²⁺ (1–20 mM) in the presence of Ca²⁺ (2.5 mM), phenylephrine relaxed it, and no contractile response to phenylephrine was observed. In the absence of extracellular Ca²⁺, 5 mM Ba²⁺ caused a contraction. Under these conditions phenylephrine had no effect on the tissue tone. When Ca²⁺ was added in a low concentration (0.2-2 mM), phenylephrine elicited a gradually increasing contractile response. At 5 mM Ca²⁺ the contractile response was replaced by the normal relaxation. The contractile response to phenylephrine in the presence of 5 mM Ba²⁺ and 2.5 mM Ca²⁺ was partially blocked by low concentrations of verapamil. In higher concentrations verapamil abolished the tissue tonus completely. The contractile response to phenylephrine in the presence of 5 mM Ba²⁺ and 2.5 mM Ca²⁺ could be reverted to the normal relaxation by the addition of 20 mM Mg²⁺. Increasing the K⁺ concentration from the normal 5.9 to 62.9 mM blocked the phenylephrine-induced relaxation. No contractile response to phenylephrine occurred. It is concluded that Ba²⁺ could reverse the response of α₁ receptor stimulation in rabbit jejunum from a relaxation to a contraction and that this contractile response was dependent on the presence of Ca²⁺.     

Avian shell gland contractility: interaction of PGF2 alpha and arginine vasotocin with Ca2+

The effects of prostaglandin F2 alpha (PGF2 alpha) and arginine vasotocin (AVT) on the isometric contractile activity of avian shell gland longitudinal muscle strips were studied in relation to the role of extracellular Ca2+. PGF2 alpha and AVT stimulated contractile tension in a dose-related manner. This was attenuated when either of the Ca2+ channel blockers verapamil or R33956 was added to the muscle chamber baths. Ca2+-free solution containing 1 mM ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid completely prevented contraction in response to increasing doses of PGF2 alpha or AVT. Washing of the strips with Ca2+-free solution eliminated spontaneous contractile activity, but replenishment of CaCl2 to the medium (0.1-5.0 mM) restored it. Addition of PGF2 alpha to the Ca2+-free medium enhanced contractile tension during Ca2+ replenishment, whereas AVT had no effect on tension generation at low extracellular Ca2+ concentration (0.1-0.5 mM) but increased it at higher extracellular Ca2+ concentration (1.0-5.0 mM). PGF2 alpha stimulation was sensitive to extracellular Na+ concentration, whereas AVT-induced activity was not. Potassium depolarization (20 mM K) potentiated PGF2 alpha-stimulated activity, whereas the response to AVT was unaffected. At 127.5 mM K, AVT-stimulated activity was inhibited. PGF2 alpha-enhanced Ca2+-dependent tension generation was right shifted in a dose-related manner by AVT. These results suggest that extracellular Ca2+ is necessary for the full expression of PGF2 alpha- and AVT-stimulated muscle contraction and suggest that each agonist has a different mechanism of action.     

Cyclic AMP-mediated inhibition of noradrenaline-induced contraction and Ca2+ influx in guinea-pig vas deferens

The relaxation effects of forskolin and methylxanthines on noradrenaline (NA)-induced contractions were investigated by measuring isotonic contraction and intracellular calcium concentration ([Ca2+]i) in the epididymal side of guinea-pig vas deferens. NA (100 microM) and high K+ (55 mM) induced a biphasic contraction; fast, transient (phasic) and slow, sustained (tonic) phases. Both phases in either NA or high K+ stimulation were abolished in Ca2+-free solution. Pretreatment with 10 microM nifedipine, an L-type Ca2+ channel blocker, reduced both phasic and tonic contractions induced by high K+. In the case of NA-induced contraction, however, nifedipine reduced the phasic contraction but not the tonic contraction. The nifedipine-insensitive tonic contraction was relaxed by the application of polyvalent cations (Mn2+, Co2+, Cd2+ and La3+). These findings indicate that NA-induced biphasic contraction is mainly due to nifedipine-insensitive Ca2+ influx, especially in the tonic phase. Cyclic AMP-increasing agents such as forskolin (0.5-10 microM), IBMX (5-500 microM) and caffeine (1-20 mM) relaxed the NA-induced contraction extensively in a concentration-dependent manner. However, these agents only partially relaxed the high K+-induced contraction. Forskolin (10 microM) and IBMX (100 microM) reduced the [Ca2+]i response to NA, but had no effect on the [Ca2+]i response to high K+. These results suggest that an increase in intracellular cAMP may relax the NA-induced contraction by attenuating a nifedipine-insensitive Ca2+ influx and by a mechanism independent of a reduction in [Ca2+]i.     

Tension development in frog skeletal muscle induced by silver ions

In single fibers of frog toe muscles placed in a Cl- free MOPS solution containing 1.8 mM Ca2+, tension developed slowly in the presence of very low concentrations of Ag+. This tension was not blocked by the administration of Co2+ or Ni2+. On the other hand, two types of transient tensions developed with the application of 5 microM Ag+, in fibers pretreated with 0-Ca2+ MOPS solution, containing either 2 mM Co2+ or 1mM Ni2+, for 10 min. In the presence of divalent cations or TTX, the first repetitive twitch-like contraction disappeared, indicating this tension is induced by action potentials repeatedly generated by the lack of divalent cations. The 2nd subsequent transient tension was caused by 5 microM Ag+ in the presence of various kinds of divalent cations, or TTX. After reversion to the resting tension, the fiber was contracted by adding more than 0.1 mM of Ca2+ or 25 mM caffeine to the external medium. Even when placed in a Ca2+-free solution containing 3 mM EGTA and 3 mM Mg2+ for 30 min, the fiber still developed an appreciable tension in response to 5 microM Ag+. These findings suggest that a transient development of the Ag+-induced tension does not require the presence of external Ca2+. A specific sulfhydryl reagent, pCMPS, did not contract the muscle fiber. Therefore, Ag+ may develop tension by mediating unknown chemical reaction(s) other than the sulfhydryl group on T-tubular membrane proteins.     

Effects of Ca antagonist on the contractile force in glycerinated dog heart muscles

The effect of Ca antagonist on the contractile apparatus was investigated in glycerinated cardiac muscle preparations obtained from canine hearts. Each muscle preparation had three consecutive isometric contractions. The 1st and 3rd contractions were produced with a control contraction solution, and compared with the 2nd contraction which was induced with a contraction solution containing verapamil. The results showed that maximal developed tension (Po) was enhanced significantly by 1.02×10^–2 mM of verapamil, and the augmentation of contractility was dependent on the concentrations of verapamil. Thus, not only Po, but also dT/dt increased tremendously at 1.02 mM of verapamil. Such contractile potentiation by verapamil was also ascertained by another Ca antagonist, Diltiazem hydrochloride. The developed tension was maximum at pCa 4.0, and no developed tension was found at pCa 8.0. The relationship between pCa and tension with verapamil shifted to the left from that without verapamil, showing higher sensitivity to Ca²⁺. From these results, it was strongly indicated that Ca antagonist is a potentiating agent of the contractile force.     

Adenosine relaxation of isolated vascular smooth muscle.

Adenosine relaxed hog carotid media strips contracted with norepinephrine (NE) and potassium (K+). Adenosine (3 X 10(-6)M) was more effective in relaxing the NE contractures than those produced by K+. In both cases, adenosine's efficacy decreased with increasing concentrations of the stimulating agent. A high adenosine concentration (1 X 10(-3)M) was necessary to elicit relaxation of completely depolarized (124 mM K+) media strips and equimolar concentrations of aminophylline caused greater relaxation than did adenosine. Adenosine inhibited the Ca2+ dose-response curves of strips stimulated with 20 mM and 30 mM K+ and its effect was dependent on the Ca2+ concentration. Neither 1 X 10(-6)M nor 1 X 10(-4)M adenosine produced any change in the cAMP content of vascular strips. Only at high concentrations did adenosine increase the cAMP content of vascular strips, but the increase was signficantly more than that observed with the same dose of aminophylline. The present results are consistent with the possibility that adenosine relaxes vascular smooth muscle by directly altering Ca2+ permeability and/or membrane potential; they do not support a role for cAMP in the adenosine-induced relaxation of vascular smooth muscle.     

Effects of divalent cations on the inhibitory potentials in the duodenal smooth muscle cells of the guinea-pig

The effects of divalent cations and some Ca2+ antagonists on the non-adrenergic inhibitory potential (i.p.) in the duodenal smooth muscle cells of the guinea-pig were investigated intracellularly. The membrane potential was a function of the external Ca2+ (0.25-7.5 mM) and Mg2+ (1.2-24 mM) concentrations. The latency and the time to peak of the i.p. were prolonged by low Ca2+ and excess Mg2+. The amplitude and the rate of hyperpolarization of the i.p. were reduced by low Ca2+ and excess Mg2+. The effects of Mn2+ (5-10 mM) on the i.p. were similar to those by excess Mg2+ except for the small depolarization. The i.p. evoked in the Ca2+-free solution was considerably restored by the addition of Ba2+ (1.25 mM) and Sr2+ (2 mM). The actions of Ba2+ and Sr2+ on the i.p. were inhibited by verapamil (10(-4)g/ml). Verapamil (2 X 10(-5)-2 X 10(-4)g/ml) and gentamicin (10(-3)g/ml) reduced the amplitude and the rate of hyperpolarization of the i.p. The latency and the time to peak of the i.p. were prolonged by verapamil but not by gentamicin. In nifedipine (10(-4)g/ml), the two-peaked i.p. was evoked by a single stimulus. This potential was similar to that evoked by a paired pulse in normal solution. The results obtained suggest that the released of the non-adrenergic inhibitory substance requires Ca2+ which moves into the non-adrenergic inhibitory neurons through the Ca2+ conductance pathway.     

Temperature sensitive effects of alpha-adrenergic stimulation in mouse ventricular myocardia

The effects of alpha- and beta-adrenergic stimulation on the contractile force and L-type Ca2+ channel current were studied in mouse ventricular myocardia at two different temperatures. In ventricular tissue, at 35-36 degrees C, phenylephrine, in the presence of propranolol, produced a sustained negative inotropic response with no change in the time course of contraction and relaxation. These effects were greatly reduced at 22-23 degrees C. Isoproterenol produced marked positive inotropic responses accompanied by shortening of the time required for relaxation. In isolated ventricular myocytes, phenylephrine, in the presence of propranolol, significantly increased the L-type Ca2+ channel current (I(Ca)) at 35-36 degrees C, but the effect was small at 22-23 degrees C. Isoproterenol increased I(Ca) similarly at both temperatures. These results suggest that, in mouse ventricular myocardia, alpha-adrenoceptor mediated regulation of the contractile force and I(Ca) involve temperature-dependent mechanisms different from those of beta-adrenoceptor-mediated mechanisms.     

Effects of reduced oxygen tension on endothelium-dependent relaxation induced by acetylcholine differ in rabbit femoral artery and jugular vein

In intact rabbit femoral artery rings pre-contracted with phenylephrine, acetylcholine (ACh; 10(-9)-10(-6) M) produces endothelium-dependent relaxation, abolished after mechanical rubbing to remove the endothelium. The response to ACh was absent at low oxygen tension (less than or equal to 4 kPa) or in the presence of sodium cyanide (1 mM). Intact rabbit jugular veins relaxed to ACh in lower concentration than did the femoral artery, 10(-10)-10(-8) M; at sufficient ACh concentration the relaxation was complete. In veins with completely removed endothelium no relaxation to ACh occurred, and at concentrations above 3 x 10(-7) M the response was a contraction. The relaxation response to ACh of intact veins persisted during contraction at lower oxygen tension or in the presence of 1 mM sodium cyanide. In rubbed veins, cyanide consistently induced a transient contraction, which was absent in intact veins. The study demonstrates pronounced endothelium-dependent relaxation to ACh in a venous preparation, with a markedly lower sensitivity of the relaxation response to hypoxia than in a muscular artery of the same species.     

Penetration-induced hyperpolarization as evidence for Ca2+ activation of K+ conductance in isolated smooth muscle cells

Single smooth muscle cells, freshly isolated by enzymatic digestion of the stomach muscularis of the toad Bufo marinus were studied under direct microscopic observation using standard electrophysiological techniques. Following penetration with a microelectrode, a hyperpolarization lasting many seconds occurred before the membrane depolarized to a steady-state level. The following lines of evidence indicate that the penetration-induced hyperpolarization results from an increase in K+ conductance caused by Ca2+ that enters the cell at the time of penetration: 1) The cell contracted at the time of penetration indicating that [Ca2+]i was elevated even though no action potential had occurred; the cell subsequently relaxed. 2) The input resistance was much lower during the hyperpolarization than during the steady-state resting potential. In the steady state all cells displayed outward-going rectification. 3) At constant [Ca2+]0, the amplitude of the hyperpolarization varied with log[K+]0 (1.3-56 mM) to a much greater degree than did the steady-state potential. Tetraethylammonium chloride (TEA) (18.2 mM) reduced the hyperpolarization. 4) At constant [K+]0, the amplitude of the hyperpolarization increased as the [Ca2+]0 was raised (1.8-52.1 mM). 5) With [Ca2+]0 low (less than or equal to 0.16 mM), the hyperpolarization was almost completely abolished in the presence of a high concentration of Ba2+ (80 mM) or Mn2+ (79.2 mM); this was not the case with Sr2+.     

Relative contribution of superficially bound and extracellular calcium to activation of contraction in isolated rat portal vein.

The spontaneous electrical and mechanical activity of the isolated rat portal vein is abolished after only 2-3 min in nominally Ca-free medium, and after 5-6 min there is no contractile response to depolarizing (122 mM K+), Ca-free solution. In the present study we have examined the electrical and mechanical responses of the portal vein to depolarization with simultaneous readministration of Ca2+ (2.5 mM) after periods of variable length in Ca-free standard solution. After 30 to 60 min of Ca depletion a slow contracture occurred in response to the high-K+ solution with 2.5 mM Ca2+. When the period in Ca-free medium was reduced below 30 min an early, faster phase appeared in the contracture response, and this phase was more rapid the shorter the time of Ca depletion. It is suggested that the slow contracture obtained after 30 min or more uses mainly extracellular Ca for activation and that the faster phase seen after shorter periods of Ca depletion is due to release of superficially bound Ca. This latter pool of tissue bound Ca does not alone produce contraction in response to depolarization, suggesting that extracellular Ca is required to trigger the release perhaps through a regenerative process.     

Influence of extracellular calcium and nifedipine on alpha 1- and alpha 2-adrenoceptor-mediated contractile responses in isolated rat and cat cerebral and mesenteric arteries

The influence of extracellular Ca2+ and nifedipine on contractile responses to 10 microM noradrenaline (NA) was investigated in isolated rat and cat middle cerebral (RCA, CCA) and mesenteric (RMA, CMA) arteries. In the CCA (containing predominantly alpha 2-adrenoceptors), the NA-induced contractions developed considerably more slowly than in the RCA, RMA (containing mainly alpha 1-adrenoceptors) and CMA (sensitive to both alpha 1- and alpha 2-adrenoceptor selective antagonists). The tonic component of the NA-induced contraction in the four types of artery was substantially suppressed after only short periods in Ca2+-free solution. In each type of artery, excluding the CCA, the contractile response to 124 mM K+ was more sensitive to Ca2+ deprivation than that to NA. This suggests that NA, besides mobilizing extracellular Ca2+, can also release Ca2+ from an intracellular pool in the RCA, RMA and CMA, but not in the CCA. Thus, alpha 1-adrenoceptor-mediated contractions in the RCA and RMA seem to depend on both Ca2+ influx and intracellular Ca2+ release, whereas alpha 2-adrenoceptor-mediated contractile responses in the CCA appear to rely almost entirely on Ca2+ influx. Both the maximum response and the tonic component of the NA-induced contraction were significantly more sensitive to nifedipine in the CCA than in the RCA. In comparison with the NA-induced contractions in these arteries, those in the RMA and CMA were relatively resistant to nifedipine. In the CCA exposed to NA in Ca2+-free medium, nifedipine almost abolished the contraction induced by re-addition of Ca2+, whereas in the other types of artery, Ca2+ re-application evoked a significant contraction also in the presence of the drug. The differential effects of nifedipine presumably reflect differences between the arteries, not only in the relative contribution of Ca2+ influx and intracellular Ca2+ release to the contractile activation, but also in the nifedipine sensitivity of the Ca2+ entry pathways utilized by NA. It is concluded that the mechanisms through which NA induces contraction seem to be related both to the subtype of alpha-adrenoceptor stimulated by NA and to the type of vessel studied.     

Differential effects of divalent cations on spontaneous and evoked glycine release from spinal interneurons

The effects of Ca2+, Sr2+, and Ba2+ on spontaneous and evoked glycinergic inhibitory postsynaptic currents (mIPSCs and eIPSCs) were studied using the "synaptic bouton" preparation of rat spinal neurons and conventional whole cell recording under voltage-clamp conditions. In response to application of Ca2+-free solution, the frequency of mIPSC initially rapidly decreased to 40 approximately 50% of control followed by a gradual further decline in mIPSC frequency to approximately 30% of control. Once mIPSC frequency had significantly decreased in Ca2+-free solution, application of Ca2+, Sr2+, or Ba2+ increased mIPSC frequency. The rank order of effect in restoring mIPSCs was Ba2+>Ca2+>Sr2+. Moreover, the application of excess external [K+]o solution (30 mM) containing Sr2+ or Ba2+ after 2 h in Ca2+-free solution also increased mIPSC frequency in the order Sr2+>or==Ba2+>Ca2+. The mean mIPSC amplitude was not affected at all. In contrast, eIPSCs produced by focal stimulation of single boutons were completely abolished in Ca2+-free solution or when Ca2+ was replaced by Sr2+ or Ba2+ (2 mM each). However, eIPSCs were restored in increased concentrations of Sr2+ or Ba2+ (5 mM each). The results show that these divalent cations affect mIPSC and eIPSCs differently and indicate that the mechanisms underlying transmitter release that generates eIPSCs and mIPSC in presynaptic nerve terminals are different. The different mechanisms might be explained by the different sensitivity of synaptotagmin isoforms to Ca2+, Sr2+, and Ba2+.     

Characteristics of contractile activity in the renal artery of ovariectomized rats.

The incidence of cardiovascular disease is markedly lower in cycling, pre-menopausal women and post-menopausal women receiving estrogen than in men or untreated post-menopausal women. Clinical studies demonstrate a protective role of estrogen in hormone replacement therapy in terms of reducing cardiovascular risk. However, the benefits of hormone replacement therapy in cardiovascular disease remain unclear. We investigated the effects of estrogen on the contractile responses of the renal artery of ovariectomized Wistar rats (OVX) compared to both ovariectomized 17 beta-estradiol-treated rats (OVXE) and sham-operated (control) rats. Isometric contraction of renal artery was recorded with a strain gauge transducer. The maximum contractile response of the renal artery smooth muscle to KCl (80 mM) in the OVXE group was significantly higher than that in both the control and OVX groups. The phenylephrine (PE) concentration-response curves in all three groups indicated a greater sensitivity at lower concentrations of PE following treatment with 100 microM L-arginine methyl ester (L-NAME). The EC50 values for PE in the three groups were 2 times lower in the presence of L-NAME than those lacking exposure to L-NAME. The EC50 value for PE in the OVX group was approximately 3 times lower in the presence of L-NAME than in those lacking exposure to L-NAME and 100 nM BMY 7378, an alpha 1D-adrenoceptor antagonist. The rate of relaxation of the PE-induced contraction (T1/2) was significantly reduced in the OVX group relative to both the control and OVXE groups. T1/2 values after treatment with 100 microM L-NAME were slower than those lacking exposure to L-NAME in all groups. Further, the T1/2 value of the OVX group was 2 times greater than that of the control; this change was reversed in the OVXE group. In conclusion, our results suggest that estrogen regulates contraction and relaxation in the renal artery via NO synthase activity and alteration of the Ca2+ transport systems.     

Characteristics of contractile response of isolated portal veins from chronic portal hypertensive rats under altered levels of external K+, Ca2+, and norepinephrine concentrations: a comparison with normal Wistar rats

We studied contractile properties of portal veins isolated from chronic portal hypertensive rats (PHR) resulting from liver cirrhosis, a model obtained by repeated subcutaneous injections of CCl4 (2 mg/kg) twice weekly for over 45 weeks. Portal venous pressure in vivo was significantly higher in PHR (167.0 +/- 38.7 mmH2O) than in the control normal Wistar rats (NWR) (102.0 +/- 25.5 mmH2O). A pair of portal veins from PHR and NWR were mounted longitudinally in an organ bath and perfused with Tyrode's solution with different K+, Ca2+, and norepinephrine concentrations. The isometric tension was measured by a strain-gauge. Under control conditions, spontaneous phasic contractile force, corrected by cross-sectional area, was greater, and the frequency was lower in PHR than in NWR preparations. The averaged peak contractile force measured at different [K]o (5.4-86.4 mM) was also greater in PHR than in NWR. Force of the tonic contraction measured at different [Ca]o (0.45-5.4 mM), under conditions of 86.4 mM [K]o was significantly larger in PHR than in NWR preparations. However, the Ca2+ sensitivity of both preparations was the same. D-600 (greater than or equal to 0.1 microM) inhibited the tonic contraction in both preparations with an identical sensitivity to the drug. In the presence of norepinephrine (10 microM), the Ca2+ sensitivity of the tonic contraction increased both in PHR and NWR preparations. The increase was more pronounced in PHR and was completely reversed in the presence of the alpha 1-adrenoceptor blocker, prazosin (0.1 microM). The alpha 1-adrenoceptor sensitivity to norepinephrine was not altered in PHR preparations. The rate of Ca2+ release and uptake of intracellular Ca2+ seemed identical in both preparations. Thus, in the absence of norepinephrine, the phasic and tonic contractile forces of portal veins from PHR are larger than that of NWR, probably due to increased membrane Ca2+ permeability. The PHR preparations have a higher affinity for external Ca2+ in the presence of norepinephrine, an additional factor contributing to elevation of portal blood pressure in the presence of chronic liver cirrhosis.     

Actions of some vasodilators on isolated human hand veins

Vasospasm is a well recognized complication during microvascular surgery of the hand. In the search for new spasmolytic drug therapies, the effects of papaverine, nitroprusside, nimodipine and lidocaine on isolated human hand veins contracted by several postulated mediators of vasospasm were examined. Mechanical activity was recorded isometrically in ring segments of the vessels. Potassium ions, noradrenaline (NA), 5-hydroxytryptamine (5-HT) and prostaglandin F2 alpha (PGF2 alpha) all produced strong contractions that were highly dependent on the presence of extracellular Ca2+. Papaverine acted as a nonselective vasodilator, as it produced an almost identical inhibition of contractile responses to all examined stimulants. Nitroprusside inhibited contractions induced by agonists more than those evoked by K+, whereas the opposite was found for nimodipine. Nitroprusside also seemed to display a certain degree of selectivity among the agonist-induced responses (NA greater than PGF2 alpha greater than 5-HT). Lidocaine increased the contractile response to K+ and at high concentrations (greater than 10(-5) M) produced a contraction per se. The clinical efficacy of lidocaine as a vasodilator after topical application therefore seems to reflect an inhibitory action on vasoconstrictor nerves. Papaverine, nitroprusside, nimodipine and lidocaine differ considerably in their profiles of action and therefore deserve to be further evaluated in the treatment of vasospasm during microvascular surgery.     

Stimulation of amylase and sialic acid releases from dog submandibular gland slices by pilocarpine or high K+ medium: a possible role of calmodulin for their releases

The mechanism of amylase and sialic acid releases stimulated by pilocarpine or high K+ medium was investigated in the slices of dog submandibular glands. The release of both amylase and sialic acid was dose-dependently increased by pilocarpine and a considerable release was observed at pilocarpine concentrations of more than 1 microM. Similar effects were observed when K+ concentration in the medium was increased and the maximal response was observed at 75 mM K+. The release of amylase and sialic acid by pilocarpine or K+ considerably decreased by removing Ca2+ from the medium and the slices. The release of amylase in the Ca2+-deficient slices was nearly recovered by the addition of 2.5 and 5.0 mM Ca2+, whereas that of sialic acid was recovered by only 60-75%. Ca2+ inhibitors, La3+ and verapamil, and calmodulin inhibitors, trifluoperazine, prenylamine, and W-7, significantly inhibited the release of amylase and sialic acid induced by the stimulants. These results suggest that the release of amylase and sialic acid stimulated by pilocarpine or K+ is dependent on the presence of Ca2+, and that the activation of calmodulin is involved in the process of the release.     

The effect of alpha-latrotoxin on the neurosecretory PC12 cell line: studies on toxin binding and stimulation of transmitter release

alpha-Latrotoxin of black widow spider venom was found to bind with high affinity (KA = 1.8 X 10(9)M-1) to specific sites present in discrete number (approximately 6300/cell, approximately 12/micron2) at the surface membrane of PC12 cells. This binding correlated with (and therefore, probably caused) the secretory response produced by the toxin. Binding was enhanced (approximately 2-fold) in the presence of mM concentrations of various divalent cations (Ca2+, Mn2+ and Co2+) while Ba2+ and Sr2+ had a smaller effect and Mg2+ was inactive. Hypertonicity, concanavalin A and trypsin pretreatment of the cells blocked the binding interaction. The alpha-latrotoxin-induced stimulation of 3H-dopamine release was massive and occurred very rapidly when cells were exposed to the toxin in a Ca2+-containing Krebs-Ringer medium, whereas it occurred at a much slower rate in a Ca2+-free, Mg2+-containing Ringer. Introduction of Ca2+ into the latter medium resulted in a shift of the release rate from slow to fast. In contrast, in divalent cation-free medium the response was abolished. The toxin-induced secretory response was unaffected by Na+ and Ca2+ channel blockers (tetrodotoxin and D600) as well as by calmodulin inhibitors (calmidazolium and trifluoperazine). The effects of Ca2+ and Mg2+ were found to be concentration-dependent, with half maximal responses occurring at approximately 0.3 and 1.5 mM for the two divalent cations, respectively. Other divalent cations could substitute for Ca2+ and Mg2+, the relative efficacy being Sr2+ greater than Ca2+ greater than Ba2+ much greater than Mn2+ greater than Mg2+ greater than Co2+. Moreover, the response occurring at suboptimal concentration of Ca2+ (0.4 mM) was potentiated by the concomitant addition of either Mg2+, Mn2+ or Co2+. The effect(s) of divalent cations in supporting the alpha-latrotoxin-induced release response seem(s) to occur primarily at step(s) beyond toxin binding because (a) the stimulatory effects of the various cations on release were not matched by parallel effects on binding, and (b) Ca2+ maintained its ability to stimulate fast release even when toxin binding had occurred in a Ca2+-free medium. Delays in the release responses were observed when cells were exposed to alpha LTx in Na+-free, glucosamine or methylamine-based media, or depolarized with high K+ (in the presence of D600) before toxin treatment. Moreover, in these two conditions the ability of Mg2+ to support the alpha LTx response was considerably decreased.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in contractile force by barium in the frog skeletal muscle

Effects of Ba2+ ions on the contractility were investigated in the frog skeletal muscle under the current clamp condition. The membrane potential was depolarized by 10 to 20 mV by perfusing 2 mM Ba2+ for 5 to 10 min. Membrane resistance was first increased and then decreased. The mechanical threshold examined in TTX-containing solution was shifted to more positive potential by applying Ba2+. The electrically induced contracture in TTX-solution as well as twitch in normal Ringer were enhanced by superfusing Ba2+. In both types of contraction an afterpotentiation was observed on washing out of Ba2+ ions, reflecting the existence of an inhibitory action during Ba2+ perfusion which could be masked by potentiation. Caffeine was able to induce contracture even in muscles soaked in "zero" Ca2+ solution for 24 to 48 h. In these muscles Ba2+ was more effective than Ca2+ to potentiate the contracture tension presumably by releasing Ca2+ ions from the sarcoplasmic reticulum whereas Mg2+ inhibited it.     

Effects of prostanoids on isolated feline cerebral arteries. II. Roles of extra- and intracellular calcium for the prostaglandin F2 alpha-induced contraction

The roles of extra- and intracellular calcium for the contractile effects of PGF2 alpha in the feline basilar artery (BA) were investigated. Comparisons were made with contractions induced by K+ and noradrenaline (NA). Addition of nifedipine to PGF2 alpha- or K+ (124 mM)-contracted arteries resulted in an incomplete relaxation, whereas NA-contracted vessels were completely relaxed. Incubation of the preparations in a calcium-free medium containing 10(-5) M EGTA for 5-10 min almost abolished contractions induced by K+ and NA. In contrast, 63% of the response to PGF2 alpha remained after pretreatment of the arteries in a calcium-free solution for 40 min; PGF2 alpha produced a biphasic contraction in 17 out of 20 preparations consisting of a rapidly developing initial phase followed by a second increase in tension after 1-6 min. The second phase was absent if the EGTA-concentration was increased to 10(-4) M, or if the arteries were pre-treated with nifedipine. After incubation of the arteries in a calcium-free medium for 40-120 min and K+-depolarization, re-addition of calcium elicited contractions at lower concentrations in the presence of PGF2 alpha than in controls. The results suggest that PGF2 alpha-induced contractions in the feline BA are considerably less dependent on extracellular calcium than contractions evoked by K+ or NA. PGF2 alpha appears to be able to release calcium from two cellular stores, and may also promote calcium influx through the cell membrane.