A new technique for continuous monitoring of the cellular calcium was developed and used for studying the effects of external and internal Na (Nao and Nai), external Ca (Cao), Ca ionophore A23187, and electrical activity on membrane-bound and intracellular Ca in mammalian nonmyelinated nerve fibers. Increasing Cao increased both the membrane-bound and the intracellular Ca. Lowering Nao increased the membrane-bound fraction of Ca indicating that lack of Nao enhanced the capacity of the plasma membrane to bind Ca, and produced an increase of the internal Ca pool. Increasing Nai by treatment with ouabain enhanced the Ca inflow in both, the presence and absence of Nao, presumably by stimulating the Cao/Nai exchange. The Ca ionophore A23187 produced a large and irreversible increase in the intracellular Ca without affecting the membrane-bound fraction. On the other hand, electrical activity, which is known to produce a large increase of the total Ca in squid axon, had no measurable effect on the total calcium content in our preparation. It is concluded that in mammalian nerve fibers a Ca load by exposition to Na-free solution or to A23187 produces an accumulation of Ca into the intracellular Ca stores, whereas during electrical activity the membrane-associated extrusion mechanisms are able to maintain the intracellular Ca2+ below the threshold for intracellular sequestration. Furthermore, the results indicate that the intracellular sequestration mechanisms are dependent on the internal concentration of Na.

The localization, distribution, elimination and metabolism of 14C-Sulpiride administered in doses of 20 mg/kg by i. p. and oral route in rats is described. Whole-body autoradiography and quantification in organs and tissues as a function of time showed that the distribution of radioactivity throughout the body is general with the highest level in the kidineys, pelvis, liver and hypophysis I hour after administration. The blood radioactivity level reaches its maximum 5 minutes after i. p. administration with a value of II Jlg/ml, of which 85 % is due to unchanged Sulpiride. At later determinations, the amount drops to 60-65 % of unchanged Sulpiride. In the bile, the maximum radioactivity is measured between I and I hour 1/2 in either extra-corporal circuit or choledocal fistula. After i. p. administration, urinary elimination is an average of 64 %of the administered dose in 72 hours, of which 70 %to 55 %, depending on the period, is due to unchanged Sulpiride. Fecal elimination after i. p. administration is an average of 26 % of the administered dose in the 72 hours period. Conversely, after oral administration, urinary elimination represents an average of 18 % of the administered dose in 72 hours and fecal elimination represents an average of 74 %for the same period. Aside from the unchanged product, 7 to 8 metabolites were separated and quantified at the plasmatic, urinary and biliary level. Very low amounts of radioactivity were recovered as "CO. indicating that the labelled position is very stable.

Phosphate efflux was measured as the fractional rate of loss of radioactivity from desheathed rabbit vagus nerves after loading with radiophosphate . The effects of strategies designed to increase intracellular calcium were investigated. At the same time, the exchangeable calcium content was measured using 45Ca. Application of calcium ionophore A23187 increased phosphate efflux in the presence of external calcium in parallel with an increase in calcium content. In the absence of external calcium, there was only a late, small increase in phosphate efflux. For nerves already treated with the calcium ionophore, the phosphate efflux was sensitive to small changes in external calcium, in the range 0.2 to 2 mM calcium, whereas similar increases in calcium in absence of ionophore gave much smaller increases in phosphate efflux. Removal of external sodium (choline substitution) produced an initial increase in phosphate efflux followed by a fall. The initial increase in phosphate efflux was much larger in the presence of calcium, than in its absence. The difference was again paralleled by an increase in calcium content of the preparation, thought to be due to inhibition of Na/Ca exchange by removal of external sodium. Measurements of ATP content and ATP, ADP, phosphate and creatine phosphate ratios did not indicate significant metabolic changes when the calcium content was increased. Stimulation of phosphate efflux by an increase in intracellular calcium may be due to stimulation of phospholipid metabolism. Alternatively, it is suggested that stimulation of phosphate efflux is associated with the stimulation of calcium efflux, possibly by cotransport of calcium and phosphate.

The effects of muscarinic agonists and depolarizing agents on inositol phospholipid hydrolysis in the rabbit vagus nerve were assessed by the measurement of [3H]inositol monophosphate production in nerves that had been preincubated with [3H]inositol. After 1 h of drug action, carbachol, oxotremorine, and arecoline increased the inositol monophosphate accumulation, though the maximal increase induced by these agonists differed. Addition of the muscarinic antagonists atropine or pirenzepine shifted the carbachol dose-response curves to the right, without decreasing the carbachol maximal stimulatory effects. The KB for pirenzepine was 35 nM, which is characteristic of muscarinic high-affinity binding sites coupled to phosphoinositide turnover and often associated with the M1 receptor subtype. On the other hand, agents known to depolarize or to increase the intracellular Ca2+ concentration, e.g., elevated extracellular K+, ouabain, Ca2+, and the Ca2+ ionophore A23187, also increased inositol monophosphate accumulation. These effects were not mediated by the release of acetylcholine, as suggested by the fact that they could not be potentiated by the addition of physostigmine nor inhibited by the addition of atropine. The Ca(2+)-channel antagonist Cd2+, also known to inhibit the Na+/Ca2+ exchanger, was able to block the effects of K+ and ouabain, but did not alter those of carbachol. These results suggest that depolarizing agents increase inositol monophosphate accumulation in part through elevation of the intracellular Ca2+ concentration and that muscarinic receptors coupled to phosphoinositide turnover are present along the trunk of the rabbit vagus nerve.

Calcium efflux was measured in desheathed rabbit vagus nerves loaded with 45Ca2+. The effects of extracellular calcium, sodium, phosphate, potassium and lanthanum ions on the calcium efflux were investigated and the distribution of intracellular calcium determined by kinetic analysis of 45Ca2+ efflux profiles. The 45Ca2+ desaturation curve can be adequately described by three exponential terms. The rate constant of the first component (0.2 min-1) corresponds to an efflux from an extracellular compartment. The two slow components had rate constants of 0.03 and 0.08 min-1 and represent the efflux from two intracellular pools. The amounts of exchangeable calcium in these two pools, after a loading period of 150 min, were 0.170 and 0.102 mmol/kg wet weight, respectively. The total calcium efflux in physiological conditions amounted to about 24 fmol cm-2 sec-1. The magnitude of the two intracellular compartments as well as the total calcium efflux were markedly affected by extracellular phosphate, sodium and lanthanum, whereas the corresponding rate constants remained almost unchanged. Phosphate reversed the effect of sodium withdrawal on the calcium efflux: in the absence of phosphate, sodium withdrawal increased the calcium efflux to 224%, but in the presence of phosphate, sodium withdrawal decreased calcium efflux to 44%. Phosphate also affected the increase in calcium efflux produced by inhibitors of mitochondrial calcium uptake, suggesting that two different mitochondrial pools contribute to the control and regulation of intracellular calcium and of the transmembrane calcium transport.