Inhibitory Action Of Ca2 Research Articles

Disruption of Rab3-calmodulin interaction, but not other effector interactions, prevents Rab3 inhibition of exocytosis.

Rab GTPases regulate membrane traffic between the cellular compartments of eukaryotic cells. Rab3 is associated with secretory vesicles of neuronal and endocrine cells and controls the Ca(2+)-triggered release of neurotransmitters and hormones. To clarify the mode of action of Rab3 we generated mutants of the GTPase that do not interact efficiently with its putative effectors Rabphilin and RIM. Surprisingly, these mutants transfected in PC12 cells were still capable of inhibiting Ca(2+)-evoked secretion. Rab3 was shown previously to bind to calmodulin in a Ca(2+)-dependent manner. By replacing two arginines conserved between Rab3 isoforms, we generated a mutant with a reduced affinity for calmodulin. This mutant retained the capacity to interact with the Rab3 regulatory proteins, Rabphilin, RIM, Mss4 and RabGDI, and was correctly targeted to dense-core secretory granules. However, replacement of the two arginines abolished the ability of the GTP-bound form of Rab3 to inhibit exocytosis of catecholamine- and insulin-secreting cells. We propose that a Rab3-calmodulin complex generated by elevated Ca(2+) concentrations mediated at least some of the effects of the GTPase and limited the number of exocytotic events that occurred in response to secretory stimuli.

Calcium mediated cytokinin action on chlorophyll synthesis in isolated embryo of Scots pine

Chlorophyll (Chl) synthesis in isolated Scots pine embryos depended on exogenous application of cytokinin (CK) and Ca2+. At a constant benzyladenine (BA) level (4.4×10-5 M) 10-4 to 10-2 M Ca2+ concentrations in mineral medium were optimum for Chl biosynthesis under both light and dark. At a zero or very low (10-6 M) concentration of external Ca2+, Chl synthesis was relatively more Ca2+-dependent in embryos cultured in darkness than in the light, which suggested that the light: (a) stimulated the transport of Ca2+ from external sources to cytosol, and/or (b) interacted with Ca2+ directly in the pathway of Chl biosynthesis. The need of external Ca2+ was evidenced in experiments with modulators of Ca2+-transport systems. The reduction of the inward current of Ca2+ from readily accessible external sites by chelating agent (ethylene glycol-bis (beta-aminoethyl ether-N,N,N'N'-tetraacetic acid, EGTA) and Ca2+-channel blockers canceled the formation of Chl. The effect of EGTA depended on the level of external Ca2+. Inhibitory action of Ca2+-channel blockers depended on their kind and concentration: at the 10-5 M concentration La3+>verapamil>nifedipine inhibited Chl formation. In the presence of Ca2+, the Ca2+-agonist A 23187 mimicked the BA effect and about 92% of Chl was synthesized as compared with the BA variant. Low concentrations of calmodulin antagonists reduced the amounts of Chl. Calmodulin was included in a second messenger system for BA action in promoting Chl biosynthesis in isolated Scots pine embryos.

P-174 - Inhibitory actions of Ca2+ and Mn2+, on Ca-free contraction of uterine smooth muscle

P-174 - Inhibitory actions of Ca2+ and Mn2+, on Ca-free contraction of uterine smooth muscle

Effect of Organic and Inorganic Calcium Channel Blockers on γ‐Amino‐n‐Butyiic Acid Release Induced by Monensin and Veratrine in the Absence of External Calcium

The effects of two organic Ca2+ antagonists (verapamil and nitrendipine) and of two inorganic Ca2+ channel blockers (Co2+ and ruthenium red) on the Na+-dependent release of gamma-amino-n-butyric acid (GABA) triggered by veratrine and monensin in the absence of external Ca2+ were studied in mouse brain synaptosomes. Ca2+-independent release of GABA stimulated by the Na+ channel activator veratrine was inhibited with micromolar concentrations of verapamil and nitrendipine. In contrast, GABA release induced by the Na+ ionophore monensin was insensitive to the organic Ca2+ antagonists. Verapamil also failed to modify A23187-stimulated release of GABA in the presence of Ca2+ but inhibited high K+-induced release of the transmitter. Co2+ partially diminished veratrine-induced release but did not change monensin-induced release. Releasing responses to monensin and veratrine were insensitive to ruthenium red, which inhibited the Ca2+-dependent component of GABA release evoked by high K+ depolarization. These data demonstrate that the mechanism of inducing GABA release is different for veratrine and monensin, as evidenced by their differing sensitivities to inhibition by Ca2+ channel antagonists and organic Ca2+ blockers. It is concluded that voltage-sensitive Ca2+ channels of the presynaptic membrane are not involved in the inhibitory action of Ca2+ antagonists on the Na+-dependent, Ca2+-independent mechanism of GABA release.

Ca2+ channel activating function of maitotoxin, the most potent marine toxin known, in clonal rat pheochromocytoma cells.

Actions of maitotoxin, the most potent marine toxin known obtained from toxic dinoflagellate, Gambier-discus toxicus, were studied using clonal rat pheochromocytoma cells (PC12), rat liver mitochondria and liposomes. Maitotoxin induced a profound release of norepinephrine and dopamine from PC12 cells and the molar ratio of norepinephrine to dopamine was almost the same as that stored in the cells. This releasing action was apparent at a concentration of 5 X 10(-10) g/ml or more, the releasing rate increased with an increase in the concentration of applied maitotoxin and attained maximum at about 10(-6) g/ml. The [3H]norepinephrine release induced by maitotoxin was abolished in the absence of external Ca2+ and increased with increasing concentration of external Ca2+ up to 10 mM. The release gradually decreased as the external Na+ concentrations were reduced from 130 to 20 mM, but maitotoxin is still able to induce a profound release in the absence of external Na+. The releasing action of maitotoxin was markedly suppressed by various Ca2+ channel blockers, such as Mn2+, verapamil, and nicardipine, and by a local anesthetic, tetracaine. The inhibitory actions of Ca2+ channel blockers were antagonized by external Ca2+ and became less obvious in the higher Ca2+ concentration range. Maitotoxin did not exhibit any ionophoretic activities on rat mitochondrial and liposomal membranes. These results suggest that maitotoxin has the ability to activate voltage-dependent Ca2+ channels of PC12 cells.

Inhibitory action of Ca2+ on spontaneous transmitter release at motor nerve terminals in a high K+ solution.

The inhibitory effect of a high external Ca2+ ([Ca2+]o) on spontaneous transmitter release in a high K+ solution (Gage and Quastel 1966; Birks et al. 1968) was studied at the frog neuromuscular junction, based on the hypothesis that an increased intracellular free Ca2+ ([Ca2+]i) in the nerve terminal plays a key role in the depression. Three procedures were employed to increase [Ca2+]i; increasing [Ca2+]o, application of caffeine and tetanic nerve stimulation. All of these procedures increased m.e.p.p. frequency in normal Ringer. However, as the basic m.e.p.p. frequency was increased by raising the external K+ concentration (7--15 mM), their facilitatory effects on m.e.p.p. frequency decreased, dissapeared and eventually reversed to depressant actions. Since a rise in the external K+ concentration would increase the steady state level of [Ca2+]i, it is suggested that when the [Ca2+]i is preset at a high level, manipulations so as to further increase [Ca2+]i depress spontaneous release of transmitter. Possible mechanisms for this inhibition was discussed in relation to a question whether or not the rate of spontaneous transmitter release is a monotonic function of [Ca2+]i.