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It has been suggested that store-operated [Ca.sup.2+] entry (SOC) facilitates catecholamine secretion and synthesis in bovine adrenal medullary (AM) cells. However, there has been no experimental result clearly showing that cation channel activity is enhanced by store [Ca.sup.2+] depletion. Thus the present experiments were undertaken to address the issue of whether rat AM cells have SOC channels. Inhibition of the sarco(endo)plasmic reticulum [Ca.sup.2+] (SERCA) pump resulted in a sustained increase in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in rat AM cells. This increase was completely suppressed by 2 mM [Ni.sup.2+] but not by 100 [micro]M D600. A bath application of [Ni.sup.2+], but not D600, produced an outward current at -60 mV in rat AM cells, whereas exposure to a SERCA pump inhibitor did not affect either the whole cell current level or the [Ni.sup.2+]-induced outward current. The refilling of intracellular store sites was suppressed by the addition of [Ni.sup.2+] to the perfusate. RT-PCR revealed that transcripts for transient receptor potential channels 1 (TRPC1) and 5 (TRPC5) were present in rat adrenal medullas. Immunocytochemistry showed that TRPC1 channels, which have been implicated in SOC in certain types of cells, were mainly localized in the endoplasmic reticulum (ER) and not in the plasma membrane, and that STIM1, a [Ca.sup.2+] sensor in the ER, was not expressed in rat AM cells. On the basis of these results, we conclude that rat AM cells lack the SOC mechanism. calcium store sites; transient receptor potential channel; endoplasmic reticulum

It has been suggested that store-operated Ca2+ entry (SOC) facilitates catecholamine secretion and synthesis in bovine adrenal medullary (AM) cells. However, there has been no experimental result clearly showing that cation channel activity is enhanced by store Ca2+ depletion. Thus the present experiments were undertaken to address the issue of whether rat AM cells have SOC channels. Inhibition of the sarco(endo)plasmic reticulum Ca2+ (SERCA) pump resulted in a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) in rat AM cells. This increase was completely suppressed by 2 mM Ni2+ but not by 100 μM D600. A bath application of Ni2+ but not D600, produced an outward current at -60 mV in rat AM cells, whereas exposure to a SERCA pump inhibitor did not affect either the whole cell current level or the Ni2+-induced outward current. The refilling of intracellular store sites was suppressed by the addition of Ni2+ to the perfusate. RT-PCR revealed that transcripts for transient receptor potential channels 1 (TRPC1) and 5 (TRPC5) were present in rat adrenal medullas. Immunocytochemistry showed that TRPC1 channels, which have been implicated in SOC in certain types of cells, were mainly localized in the endoplasmic reticulum (ER) and not in the plasma membrane, and that STIM1, a Ca2+ sensor in the ER, was not expressed in rat AM cells. On the basis of these results, we conclude that rat AM cells lack the SOC mechanism. [ABSTRACT FROM AUTHOR]

Abstract: To study the role of mitochondrial Ca2+ clearance in stimulated cells, changes in free Ca2+ concentration in the cytosol, [Ca2+]c and that in mitochondria, [Ca2+]m along with secretory responses were observed using chromaffin cells co-loaded with Fura-2 and Rhod-2 in the perfused rat adrenal medulla. When the cells were stimulated with 40mM K+ in the perfusate, the duration of [Ca2+]m response markedly increased with prolongation of the stimulation period, exhibiting a mean half-decay time of 21min with 30s stimulation, whereas its amplitude was not altered with stimulations of 10–30s. A computer simulation analysis showed that such a mode of [Ca2+]m response can be produced if excess Ca2+ taken up by mitochondria precipitates as calcium phosphate (Pi) salt. In the presence of 5μM rotenone plus 10μM oligomycin, a decrease in the duration of [Ca2+]m response and a slight but significant increase (24%) in the secretory response to 30s stimulation with 40mM K+ were observed. Simulation analyses suggested that this effect of rotenone may be due to reduction in mitochondrial Ca2+ uptake induced by rotenone-elicited partial depolarization of the mitochondrial membrane potential. In chromaffin cells transsynaptically stimulated through the splanchnic nerve, the intensity of NAD(P)H autofluorescence changed with time courses similar to those of [Ca2+]m responses. The temporal profiles of those two responses were prolonged in a similar manner by application of an inhibitor of mitochondrial Na+/Ca2+ exchanger, CGP37157. Thus, due to the unique Ca2+ buffering mechanism, [Ca2+]m responses associated with massive mitochondrial Ca2+ uptake may occur within a limited concentration range in which Ca2+-sensitive dehydrogenases are activated to control the mitochondrial redox state in stimulated chromaffin cells. [Copyright &y& Elsevier]

Effects of toxins III and IV (ATX III and IV) from the sea anemone Anemonia sulcata on the Na current of crayfish giant axons were studied. Both toxins slowed the inactivation of Na channels, producing a maintained Na current during a depolarizing voltage pulse. Using the intensity of the toxin-induced maintained current as an index for the fraction of Na channels to which toxin is bound, the toxin association and dissociation kinetics were analyzed. The dissociation rate of ATX III was increased by two orders of magnitudes by depolarizing the membrane from −70 to −40mV. This increase of the dissociation rate caused a marked decrease in the binding rate of ATX III to Na channels in the same potential range. ATX IV exhibited association and dissociation kinetics that had a potential dependency quite similar to that of ATX III in spite of different ionic charge distribution in these two toxins. The results support the view that the potential-dependent kinetics of these toxins are not due to an electrostatic interaction between the ionic charges of toxins and the membrane potential but result from a modulation of the binding energy depending on the gate configuration of the Na channel. [ABSTRACT FROM AUTHOR]

Amino-acid sequence of a toxin from sea anemone, Parasicyonis actinostoloides, is determined. The toxin consists of 31 amino acid residues and is cross-linked with four disulphide bridges. The sequence has some similarity to that of toxin III and no similarity to those of toxin I and toxin II both from sea anemone, Anemonia sulcata, or to that of Anthopleurin A from Anthopleura xanthogrammica. [ABSTRACT FROM AUTHOR]