Ca(2+)-dependent aggregation of rabbit platelets induced by maitotoxin, a potent marine toxin, isolated from a dinoflagellate.

1. Administration of maitotoxin (MTX), a dinoflagellate toxin, caused aggregation of rabbit washed platelets. The cytosolic Ca2+ concentration ([Ca2+]i), measured by fura-2 fluorescence technique, was also increased by the presence of MTX. Rates of aggregation response and [Ca2+]i-increase were dependent on tested concentrations (3-100 ng ml-1) of the toxin. 2. The MTX-induced platelet aggregation and [Ca2+]i-increase were totally abolished in a Ca(2+)-free solution. The successive administration of Ca2+ in the presence of MTX elicited the aggregation and increase in [Ca2+]i. 3. Ba2+ was capable of substituting for Ca2+ in the MTX-induced platelet aggregation. In the presence of external Ca2+, transition metals, Co2+, Cd2+ and Ni2+, inhibited the aggregation response to MTX. 4. Organic calcium antagonists (verapamil and nifedipine) as well as a cyclo-oxygenase-inhibitor (aspirin) did not apparently inhibit the aggregation response to MTX, except for a high concentration (10(-5) M) of verapamil, while procaine (10 mM) reduced the rate of platelet aggregation. 5. MTX also elicited a release of ATP from platelets, which was abolished in the absence of external Ca2+. 6. In contrast, thrombin 0.5 unit ml-1 could elicit platelet shape change, [Ca2+]i-increase and ATP-release in the absence of external Ca2+. 7. These results suggest that the MTX-induced platelet activation is caused by an enhanced Ca(2+)-influx presumably through voltage-independent Ca2+ channels on the plasma membrane.     

A novel action of the antianginal drug bepredil: induction of internal Ca(2+) release and external Ca(2+) influx in Madin-Darby canine kidney (MDCK) epithelial cells

The effect of the antianginal drug bepridil on Ca(2+) signaling in Madin-Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Bepridil at 10-50 microM evoked a significant rise in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in a dose-dependent manner. The [Ca(2+)](i) rise consisted of an immediate initial rise and a slow decay. Removal of external Ca(2+) partly inhibited the Ca(2+) signals by reducing both the initial rise and the decay phase, suggesting that bepridil activated both external Ca(2+) influx and internal Ca(2+) release. In Ca(2+)-free medium, pretreatment with 50 microM bepridil nearly abolished the Ca(2+) release induced by thapsigargin (1 microM), an endoplasmic reticulum Ca(2+) pump inhibitor, and vice versa, pretreatment with thapsigargin inhibited most of the bepridil-induced Ca(2+) release, suggesting that the thapsigargin-sensitive Ca(2+) store was the main source of bepridil-induced Ca(2+) release. Bepridil (50 microM) induced considerable Mn(2+) quench of fura-2 fluorescence at an excitation wavelength of 360 nm, which was partly inhibited by La(3+) (0.1 mM). Consistently, La(3+) (0.1 mM) pretreatment significantly inhibited the bepridil-induced [Ca(2+)](i) rise. Addition of 3 mM Ca(2+) induced a significant [Ca(2+)](i) rise after prior incubation with 10-50 microM bepridil in Ca(2+)-free medium, suggesting that bepridil induced dose-dependent capacitative Ca(2+) entry. However, 50 microM bepridil inhibited 1 microM thapsigargin-induced capacitative Ca(2+) entry by 38%. Pretreatment with aristolochic acid (40 microM) so as to inhibit phospholipase A(2) inhibited 50 microM bepridil-induced internal Ca(2+) release by 42%, but inhibition of phospholipase C with U73122 (2 microM) or inhibition of phospholipase D with propranolol (0.1 mM) had little effect, suggesting that bepridil induced internal Ca(2+) release in an inositol 1,4,5-trisphosphate-independent manner that could be modulated by phospholipase A(2)-coupled events. This is the first report providing evidence that bepridil, currently used as an antianginal drug, induced a rise in [Ca(2+)](i) in a non-excitable cell line.     

Inhibition of alpha-receptor-induced Ca2+ release and Ca2+ influx by Mn2+ and La3+

The influence of Mn2+ and La3+ on alpha-receptor-stimulated Ca2+ movements was examined in arterial smooth muscle of the rabbit aorta. Both cations cause an inhibition of phenylephrine (PE) contractile response which exhibits a different pattern at low and high cation concentrations. At 0.1-1.0 mM inhibition by Mn2+ and La3+ was predominately due to a reduction in Ca2+ influx reflected as inhibition of the slow phase of contraction and reduction in PE-stimulated 45Ca uptake. PE log dose-response curves were shifted to the right in a non-parallel manner by 1 mM Mn2+ such that responses to lower PE concentrations were more inhibited. However, in the presence of 10 mM Mn2+ PE responses are equally inhibited at all PE levels. At 10 mM both Mn2+ and La3+ also inhibited PE-stimulated Ca2+ release resulting in a reduction in both the rapid phase of contraction and in the magnitude of PE stimulation of 45Ca efflux. The effects of Nm2+ (1 or 10 mM) on contraction and 45Ca efflux were rapidly reversible, while the effect of La3+ was not. Inhibition of Ca2+ release by 10 mM Mn2+ and La3+ was not caused by displacement of releasable Ca2+, but appeared to reflect their occupation of a superficially located receptor modulating site. The inhibition of Ca2+ influx by lower concentrations of Mn2+ may illustrate the functional consequence of configurational changes in the alpha 2-form of the receptor which have been recently described at lower concentrations of divalent cations.     

Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca(2+)-pump, reduces Ca(2+)-dependent K+ currents in guinea-pig smooth muscle cells.

1. Effects of cyclopiazonic acid (CPA), a specific inhibitor of the Ca(2+)-ATPase in sarcoplasmic reticulum (SR), on membrane ionic currents were examined in single smooth muscle cells freshly isolated from ileal longitudinal strips and urinary bladder of the guinea-pig. 2. Under whole-cell clamp, CPA (1-10 microM) reduced peak outward current elicited by depolarization in a concentration-dependent manner. The concentration of CPA required for 50% decrease in the peak outward current was approximately 3 microM in ileal cells under these conditions. The current reduced by CPA recovered by more than 70% after washout. 3. The transient outward current elicited by application of 5 mM caffeine at a holding potential of -50 mV in Ca2+ free solution was almost abolished, when the preceding Ca(2+)-loading of the cell in a solution containing 2.2 mM Ca2+ was performed in the presence of 3 microM CPA. 4. When the Ca(2+)-dependent K+ current (IK-Ca) and Ca2+ current (ICa) were inhibited by addition of Ca2+, the remaining delayed rectifier type K+ current was not affected by 10 microM CPA. When outward currents were blocked by replacement of K+ by Cs+ in the pipette solution, the remaining ICa was not affected by 10 microM CPA. 5. CPA (10 microM) did not affect the conductance of single maxi Ca(2+)-dependent K+ channels or the Cd(2+)-dependence of their open probability in both inside- and outside-out configurations. 6. These results indicate that IK-Ca is selectively and strongly suppressed by CPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ release induced by myotoxin alpha, a radio-labellable probe having novel Ca2+ release properties in sarcoplasmic reticulum.

1. Myotoxin alpha (MYTX), a polypeptide toxin purified from the venom of prairie rattlesnakes (Crotalus viridis viridis) induced Ca2+ release from the heavy fraction (HSR) but not the light fraction of skeletal sarcoplasmic reticulum at concentrations higher than 1 microM, followed by spontaneous Ca2+ reuptake by measuring extravesicular Ca2+ concentrations using the Ca2+ electrode. 2. The rate of 45Ca2+ release from HSR vesicles was markedly accelerated by MYTX in a concentration-dependent manner in the range of concentrations between 30 nM and 10 microM, indicating the most potent Ca2+ releaser in HSR. 3. The Ca2+ dependency of MYTX-induced 45Ca2+ release has a bell-shaped profile but it was quite different from that of caffeine, an inducer of Ca(2+)-induced Ca2+ release. 4. 45Ca2+ release induced by MYTX was remarkable in the range of pCa between 8 and 3, whereas that by caffeine was prominent in the range of pCa, i.e., between 7 and 5.5. 5. MYTX-induced 45Ca2+ release consists of both early and late components. The early component caused by MYTX at low concentrations (30-300 nM) completed within 20 s, while the late component induced by it at higher concentrations (> 0.3 microM) was maintained for at least 1 min. 6. Both the components were almost completely inhibited by inhibitors of Ca2+ such as Mg2+, ruthenium red and spermine. 7. 45Ca2+ release induced by caffeine or beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) was completely inhibited by high concentrations of procaine. Procaine abolished the early component but not the late one, suggesting that at least the early component is mediated through Ca(2+)-induced Ca2+ release channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

4,6-Dibromo-3-hydroxycarbazole (an analogue of caffeine-like Ca2+ releaser), a novel type of inhibitor of Ca(2+)-induced Ca2+ release in skeletal muscle sarcoplasmic reticulum.

1. 4,6-Dibromo-3-hydroxycarbazole (DBHC) was synthesized as an analogue of bromoeudistomin D (BED), a powerful Ca2+ releaser, and its pharmacological properties were examined. 2. In Ca2+ electrode experiments, DBHC (100 microM) markedly inhibited Ca2+ release from the heavy fraction of sarcoplasmic reticulum (HSR) induced by caffeine (1 mM) and BED (10 microM). 3. DBHC (0.1 to 100 microM) inhibited 45Ca2+ release induced by Ca2+ from HSR in a concentration-dependent manner. 4. DBHC (100 microM) abolished 45Ca2+ release induced by caffeine (1 mM) and BED (10 microM) in HSR. 5. Inhibitory effects of calcium-induced calcium release (CICR) blockers such as procaine, ruthenium red and Mg2+ on 45Ca2+ release were clearly observed at Ca2+ concentrations from pCa 7 to pCa 5.5, and were decreased at Ca2+ concentrations higher than pCa 5.5 or lower than pCa 7. However, DBHC decreased Ca2+ release induced by Ca2+ over the wide range of extravesicular Ca2+ concentrations. 6. [3H]-ryanodine binding to HSR was suppressed by ruthenium red, Mg2+ and procaine, but was not affected by DBHC up to 100 microM. 7. [3H]-ryanodine binding to HSR was enhanced by caffeine and BED. DBHC antagonized the enhancement in a concentration-dependent manner. 8. 9-[3H]-Methyl-7-bromo-eudistomin D, an 3H-labelled analogue of BED, specifically bound to HSR. Both DBHC and caffeine increased the KD value without affecting the Bmax value, indicating a competitive mode of inhibition. 9. These results suggest that DBHC binds to the caffeine binding site to block Ca2+ release from HSR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free Ca2+ requirements of agonist-induced thromboxane A2 synthesis in human platelets

In quin2-loaded human platelets ionomycin raised cytosolic free calcium to greater than 1 microM and generated less than 1 ng thromboxane. Collagen alone or in the presence of EPO92 generated up to 32 and 16 ng thromboxane respectively; in the latter case at calcium levels around 120 nM. Thrombin maximally raised calcium to greater than 1 microM and generated up to 27 ng thromboxane, although in the presence of 1 mM EGTA these calcium and thromboxane levels were reduced to 200 nM and 5 ng respectively.     

Ca(2+)-dependent inhibition of Ca(2+)-independent contraction in uterine smooth muscle.

Uterine smooth muscle of the rat shows Ca(2+)-independent contraction in response to oxytocin in Ca(2+)-free medium. Micromolar Ca2+ inhibits this contraction. We now tested whether Ca2+ itself is the cause of this inhibition. The ratio of fura-2 fluorescence, the indicator of the intracellular level of Ca2+, was increased in parallel with the degree of inhibition by Ca2+. When inhibition was elicited by Ca2+, EGTA released the inhibition. Comparison of the dose-response curve for oxytocin in Ca(2+)-free solution and that in the medium with 1 microM Ca2+ showed that the inhibition by Ca2+ is non-competitive. EGTA chelation of the intracellular Ca2+ by loading of EGTA as its acetoxymethylester resulted in diminution of inhibition by Ca2+. EGTA suppressed the Ca(2+)-induced contraction but did not affect Ca(2+)-independent contraction. It is concluded that the inhibition is induced by intracellular Ca2+ itself.     

Ca(2+)-induced translocation of protein kinase C during Ca(2+)-dependent histamine release from beta-escin-permeabilized rat mast cells.

When rat mast cells were cultured for a short period in plastic dishes and adhering cells were permeabilized with beta-escin and exposed to Ca2+ at concentrations higher than 10(-7) mol/l, histamine release was induced dose-dependently. Protein kinase C (PKC) activity in the crude extracts obtained from adhering mast cells was induced in the presence of Ca2+, phospholipid and diacylglycerol. The apparent Km value of PKC for Ca2+ was 0.33 mumol/l, and this Ca2+ concentration was equivalent to that which can elicit half the maximum of the Ca(2+0-induced histamine release. After permeabilization, approximately 80% of the total PKC activity remained in the cytosolic fraction. In the resting state, 95% of the total PKC activity was detected in the soluble fraction, and the rest was detected in the membrane fraction. When permeabilized mast cells were incubated with Ca2+ at micromolar concentrations, which are effective in releasing histamine, the total PKC activity did not change. However, the translocation of PKC took place from the cytosolic fraction to the membrane fraction, corresponding to Ca2+ concentrations in the medium. When the crude PKC extract of mast cells was incubated with phospholipid vesicles and centrifuged, the PKC activity in the supernatant was diminished; the amount of PKC binding to the vesicles was dependent upon Ca2+ concentrations in the medium. Calphostin C, a potent PKC inhibitor, interacts with PKC in a noncompetitive manner, and it does not inhibit Ca(2+)-induced translocation of PKC. It can be concluded that PKC is translocated into the cell membrane along with an increase in intracellular Ca2+ concentrations and the subsequent activation of PKC may be crucial for the process leading to histamine release.     

Inhibition of T-type and L-type Ca(2+) currents by aranidipine, a novel dihydropyridine Ca(2+) antagonist

The effects of aranidipine, a novel dihydropyridine Ca(2+) channel antagonist, on membrane currents in guinea pig ventricular myocytes and on action potentials in rabbit sinoatrial node tissue were examined. In myocytes, aranidipine (10 nmol/l to 1 micromol/l) concentration-dependently decreased T-type and L-type Ca(2+) currents. Aranidipine (1 micromol/l) had little effect on K(+) currents. In the sinoatrial node, 0.1 micromol/l aranidipine increased cycle length, and decreased +V(max) and the slope of the phase 4 depolarization. Thus, inhibition of both T-type and L-type Ca(2+) currents by aranidipine may partly explain its potent negative chronotropic activity.     

异紫堇啡碱对血管平滑肌钙内流和钙释放的影响

目的研究异紫堇啡碱（ＩＳＯＣ）对血管平滑肌钙内流和钙释放的影响，以初步阐明其作用方式。方法利用兔胸主动脉螺旋条标本观察ＩＳＯＣ对去甲肾上腺素（ＮＡ）及ＫＣｌ量效曲线的影响和对无钙液中ＮＡ、ＣａＣｌ２复钙、咖啡因缩血管效应的影响。结果ＩＳＯＣ１０μｍｏｌ·Ｌ－１对ＮＡ的量效曲线呈非竞争性拮抗作用，而对高钾的作用则不明显。在无钙液中，ＩＳＯＣ１００μｍｏｌ·Ｌ－１能明显抑制ＮＡ所致的收缩及复钙后外钙内流诱发的收缩，ＩＳＯＣ１０及３０μｍｏｌ·Ｌ－１则只作用于前者；各实验浓度的ＩＳＯＣ对咖啡因在无钙液中的缩血管作用均无影响。结论ＩＳＯＣ抑制受体中介的钙释放和钙内流，但不是典型的钙拮抗剂。     

Fluoxetine inhibits ATP-induced [Ca(2+)](i) increase in PC12 cells by inhibiting both extracellular Ca(2+) influx and Ca(2+) release from intracellular stores

Fluoxetine, a widely used antidepressant, has additional effects, including the blocking of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells using fura-2-based digital calcium imaging, an assay for [3H]-inositol phosphates (IPs) and whole-cell patch clamping. Treatment with ATP (100 microM) for 2 min induced increases in intracellular free Ca(2+) concentrations ([Ca(2+)](i)). Treatment with fluoxetine (100 nM to 30 microM) for 5 min inhibited the ATP-induced [Ca(2+)](i) increases in a concentration-dependent manner (IC(50) = 1.85 microM). Treatment with fluoxetine (1.85 microM) for 5 min significantly inhibited the ATP-induced responses following the removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) stores. Whereas treatment for 10 min with nimodipine (1 microM) significantly inhibited the ATP-induced [Ca(2+)](i) increase, treatment with fluoxetine further inhibited the ATP-induced response. Treatment with fluoxetine significantly inhibited [Ca(2+)](i) increases induced by 50 mM K(+). In addition, treatment with fluoxetine markedly inhibited ATP-induced inward currents in a concentration-dependent manner. However, treatment with fluoxetine did not inhibit ATP-induced [3H]-IPs formation. Therefore, we conclude that fluoxetine inhibits ATP-induced [Ca(2+)](i) increases in PC12 cells by inhibiting both the influx of extracellular Ca(2+) and the release of Ca(2+) from intracellular stores without affecting IPs formation.     

Staurosporine, a protein kinase inhibitor, attenuates intracellular Ca(2+)-dependent contractions of strips of rabbit aorta.

The protein kinase inhibitor staurosporine was found to be a potent relaxant of rabbit aortic strips contracted by various agonists. This relaxing effect was slow, long-lasting and in a non-competitive fashion against various agonists. The effect of staurosporine on the KCl-induced contraction was not altered by atropine, propranolol, theophylline or indomethacin. Staurosporine (5-200 nM) inhibited the contractile responses to prostaglandin F2 alpha (3 microM), endothelin (0.1 microM), phenylephrine (3 microM) and KCl (30 mM), with ED50 values of 10.8 +/- 0.6, 24.6 +/- 8.7, 48.8 +/- 7.0, 54.0 +/- 12.7 nM, respectively. Even in Ca(2+)-free physiological salt solution, staurosporine potently antagonized the contractile responses elicited by 10 microM prostaglandin F2 alpha and 0.1 microM endothelin; in fact it was more effective than it was in normal Ca2+ solution: ED50 of 11.3 +/- 3.8 vs. 21.0 +/- 4.4 nM (P less than 0.02), and of 10.6 +/- 3.6 vs. 24.6 +/- 4.3 nM (P less than 0.01), respectively. Thus, staurosporine has an equipotent inhibitory action on intracellular and extracellular calcium-dependent contractions of aortic tissues.     

普鲁托品对兔血小板内钙的影响

AIM: To study the influence of protopine (Pro) on the cytoplasmic free Ca2+ concentration ([Ca2+]i) in rabbit platelets. METHODS: Measurement of [Ca2+]i of platelets in vitro by Fura 2-AM fluorescence technique. RESULTS: In the presence of CaCl2 1 mmol.L-1, Pro 10, 20, and 40 mumol.L-1 attenuated the rise in [Ca2+]i evoked by ADP from (420 +/- 57) to (320 +/- 26), (264 +/- 21), and (180 +/- 14) nmol.L-1, respectively, by arachidonic acid (AA) from (280 +/- 36) to (210 +/- 17), (184 +/- 21), and (143 +/- 16) nmol.L-1, respectively, and by platelet-activating factor (PAF) from (350 +/- 42) to (282 +/- 31), (223 +/- 30), and (165 +/- 15) nmol.L-1, respectively. In the presence of egtazic acid 1 mmol.L-1, Pro 10, 20, and 40 mumol.L-1 reduced the Ca2+ release induced by ADP, AA, and PAF, respectively. Pro 10, 20, and 40 mumol.L-1 also decreased ADP-, AA-, and PAF-induced Ca2+ influx. CONCLUSION: Pro inhibited not only Ca2+ release but also the influx of Ca2+.     

Inhibitory effect of 8-bromo cyclic GMP on an extracellular Ca2+-dependent arachidonic acid liberation in collagen-stimulated rabbit platelets

The inhibitory effect of cyclic GMP on collagen-induced platelet activation was studied using 8-bromo cyclic GMP (8brcGMP) in washed rabbit platelets. Addition of collagen (1 micrograms/ml) to platelet suspension caused shape change and aggregation associated with thromboxane (TX) A2 formation. 8brcGMP (10-1000 microM) inhibited collagen-induced platelet aggregation and TXA2 formation in a concentration-dependent manner. 8brcGMP did not affect platelet cyclooxygenase pathways, but markedly inhibited collagen-induced arachidonic acid (AA) liberation from membrane phospholipids in [3H]AA-prelabeled platelets, indicating that the inhibitory effect of 8brcGMP on collagen-induced aggregation is due to an inhibition of AA liberation. In [32P]orthophosphate-labeled platelets, collagen stimulated phosphorylation of a 20,000 dalton (20-kD) and 40-kD proteins. 8BrcGMP stimulated phosphorylation of a specific protein having molecular weight of 46-kD and inhibited collagen-induced both 20- and 40-kD protein phosphorylation. Collagen could stimulate the AA liberation without activation of phospholipase C or Na+-H+ exchange, but could not in the absence of extracellular Ca2+. These findings suggest that cyclic GMP inhibits collagen-induced AA liberation which is mediated by an extracellular Ca2+-dependent phospholipase A2. However, cyclic GMP seems to inhibit the Ca2+-activated phospholipase A2 indirectly, since 8brcGMP had no effect on Ca2+ ionophore A23187-induced platelet aggregation or AA liberation. It is therefore suggested that cyclic GMP may regulate collagen-induced increase in an availability of extracellular Ca2+ which is responsible for phospholipase A2 activation in rabbit platelets.     

Ca(2+)-dependent sensitization of adenylyl cyclase activity.

It has been shown that intracellular Ca(2+) concentrations have multiple modulatory influences on hormone-stimulated adenylyl cyclase activity. Here, we report that increasing intracellular Ca(2+) concentration by treating cells with the Ca(2+) ionophore A23187 leads to a sensitization of the beta-adrenoceptor-stimulated adenylyl cyclase activity in Ltk(-) cells expressing the human beta(2)-adrenoceptor. The ionophore treatment led to a 20+/-5% increase of the maximal isoproterenol-stimulated cyclase activity that could be prevented by chelation of the extracellular Ca(2+) using EGTA. A similar Ca(2+)-mediated sensitization (20+/-6%) was observed when the adenylyl cyclase was directly stimulated by the diterpene forskolin indicating that the catalytic activity of the enzyme itself is modulated by the change in Ca(2+) concentration. Sensitization of both the isoproterenol- and forskolin-stimulated adenylyl cyclase activities were completely blocked by treatment with KN-62[1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine], an inhibitor of the Ca(2+)-calmodulin-dependent protein kinase (CamKinase). Taken together, our data reveal the existence of a CamKinase-dependent sensitization process acting at the level of the adenylyl cyclase catalytic moiety.     

External Ca2+-dependent Ca2+ release in directly stimulated diaphragm muscles of mice

Intracellular Ca2+ release in directly stimulated diaphragm muscles of mice was found to be dependent on external Ca2+, using the intracellular Ca2+ aequorin luminescence transient technique. The Ca2+ mobilization into the cells may operate via a voltage- and external Ca2+-dependent mechanism, and via a caffeine- and external Ca2+-independent one, from the Ca2+ pools in plasma membranes or in the terminal cisternae of sarcoplasmic reticulum.     

Different pathways for Ca2+ influx and intracellular release of Ca2+ mediated by muscarinic receptors in ileal longitudinal smooth muscle.

Muscarinic receptor-mediated elevations in intracellular Ca2+ concentration ([Ca2+]i) in the longitudinal smooth muscle of guinea pig ileum were studied by the use of fura-2 fluorescence. Dose-response analysis indicated a difference in the potencies of carbachol (CCh) to increase [Ca2+]i in the presence and absence of extracellular Ca2+. For the increase in [Ca2+]i due to Ca2+ release from intracellular stores in the absence of extracellular Ca2+, the ED50 value of CCh was 3 x 10(-5) M. On the other hand, in the presence of Ca2+, the ED50 value was 2.5 x 10(-7) M, indicating that a low concentration of CCh (less than 10(-7) M) caused influx of extracellular Ca2+ without Ca2+ release. Oxotremorine and pilocarpine induced Ca2+ influx, but were less potent inducers of Ca2+ release. CCh also stimulated the formation of inositol trisphosphates (IP3) with an ED50 value of (4.5 x 10(-5) M), which was similar to that for Ca2+ release from intracellular stores. Treatment of the smooth muscle with neomycin (1 mM), a phospholipase C inhibitor, abolished both CCh-induced IP3 formation and Ca2+ release from intracellular stores, but did not affect CCh-induced Ca2+ influx. These results suggest that the pathway for muscarinic stimulation of Ca2+ influx through plasma membranes is different from that for Ca2+ release from intracellular stores, which seems to be coupled with IP3 formation.     

Excitatory amino acid-elicited tonic convulsions in mice and N-methyl-D-aspartate receptor activation: role of Ca(2+) influx and involvement of intracellular Ca(2+)-dependent biochemical processes

Intravenously administered nimodipine (an L-type Ca(2+) antagonist) as well as dizocilpine (an N-methyl-D-aspartate--NMDA--antagonist) showed a wide spectrum of anticonvulsant activity in intracerebroventricular mouse models for excessive activation of excitatory amino acid receptors. The duration of Bay k-8644 (L-type Ca(2+) agonist; intracerebroventricular administration) caused seizures was significantly reduced by intravenously administered nimodipine. Intracisternal administration of Bay k-8644 lowered the convulsion threshold of an intracerebroventricular injection of NMDA. Intracisternal administration of omega-conotoxin GVIA (N-type Ca(2+) antagonist) only tended to inhibit the NMDA-induced tonic convulsions. Intracisternal administration of staurosporine (a protein kinase C inhibitor) or calmidazolium (a calmodulin antagonist) was effective in inhibiting the NMDA-induced tonic convulsions. Calmidazolium, unlike staurosporine, produced side effects at a dose showing its anticonvulsant activity. From these results, it is suggested that excessive activation of excitatory amino acid receptors results in tonic convulsions by virtue of a massive increase of Ca(2+) influx mainly through NMDA receptor channels, and at least in part through L-type Ca(2+) channels, and in subsequent activation of protein kinase C and possibly calmodulin.