Inhibition of 45Ca2+ uptake and catecholamine secretion by phenothiazines and pimozide in adrenal medulla cells cultures

The inhibition by several phenothiazine drugs and pimozide of the uptake of 45Ca2+ and secretion of catecholamines by cultured adrenal medulla cells stimulated with nicotine, veratridine, 50 mM K+, ionomycin and Ba2+ was studied. The inhibition of 45Ca2+ uptake, except for ionomycin, closely parallelled the inhibition of catecholamine secretion. The nicotine-and veratridine-stimulated effects were several fold more sensitive to inhibition by the drugs than were those stimulated by 50mM K+, ionomycin and Ba2+; the ionomycin-stimulated effects were least sensitive to inhibition. These studies indicate that the drugs have multiple effects on stimulus-secretion coupling in adrenal medulla cells. It is suggested that inhibition of the veratridine- and nicotine-stimulated events is due to membrane perturbations caused by the drugs, that inhibition of the 50mM K+- and Ba2+-stimulated events is due to alterations in the voltage sensitive membrane Ca2+ channel, and that inhibition of secretion elicited by ionomycin may be due to inhibition of Ca2+-calmodulin reactions or to more profound non specific membrane effects.     

Effect of divalent cations and chelators on metaphase to telophase progression and nuclear envelope formation in Chinese hamster cells

Chinese hamster DON cells in log phase were treated with Colcemid in the G₂ period with or without divalent cation chelating agents. The metaphase cells were isolated and incubated in two ways: 1) without Colcemid but with chelating agents or La³⁺ and observed for metaphase to telophase progression, and 2) with Colcemid, with or without chelating agents and the rate of micronuclei formation in the absence of anaphase monitored. The effect of the chelating agents on cellular ⁴⁵Ca²⁺ during metaphase to telophase progression was also studied.The results indicate that Ca²⁺ and possibly Mg²⁺ ions are involved in the regulation of certain segments of mitosis. The reduction of environmental and plasma membrane associated Ca²⁺ with the chelators and La³⁺ promoted the metaphase to telophase progression as well as nuclear envelope and micronuclei formation.     

The dependence of fluid secretion by mandibular salivary gland and pancreas on extracellular calcium

Acetylcholine-stimulated fluid secretion from the perfused rabbit mandibular salivary gland was inhibited in a biphasic manner when extracellular calcium concentration was reduced in the range 5 X 10(-4) - 10(-5)M. An initial rapid inhibition was followed by partial recovery to a plateau, the level of which depended upon the calcium concentration. Since no recovery was observed during substitution of calcium by strontium, recovery may depend upon an increased membrane permeability to calcium. It is concluded that acetylcholine evokes fluid secretion in this gland by enhancing calcium entry from the extracellular space, an action which can be mimicked by the calcium ionophore A23187. Changes in the electrolyte composition of saliva during calcium-depletion were such as to suggest that ductal reabsorption of sodium and chloride, and secretion of potassium are inhibited as extracellular calcium concentration is reduced. Secretin-stimulated fluid secretion from the cat pancreas was unaffected when perfusate calcium concentration was reduced to 2.5 X 10(-6)M and carbachol-stimulated amylase secretion was only slightly reduced. Since the latter is a calcium-dependent process, the source of calcium is presumably intracellular. In both glands, reducing calcium to 1 X 10(-6)M caused rapid and irreversible inhibition of fluid secretion.     

The calcium uptake in smooth muscle microsomal vesicles is reduced by centrifugation

A membrane fraction was isolated from the smooth muscle of the pig stomach by density gradient centrifugation. It was observed that the ATP-dependent Ca uptake in this fraction was diminished if the microsomes were pelleted by differential centrifugation. The decrease of the oxalate-independent Ca uptake was relatively small, but the oxalate-stimulated Ca uptake was reduced dramatically. Evidence is presented which indicates that the selective decrease of the oxalate-stimulated Ca uptake is mainly caused by mechanical damage of the vesicles. Since the oxalate-stimulated Ca uptake can be largely preserved by avoiding pelleting during the membrane fractionation, this observation may be very useful for the further study of Ca transport in subcellular fractions of smooth muscle.     

Molecular properties of the red cell calcium pump: I. Effects of calmodulin,proteolytic digestion and drugs on the kinetics of active calcium uptake in inside-out red cell membrane vesicles

In calmodulin-stripped inside-out human red cell membrane vesicles /IOV/ ATP + Mg²⁺-dependent active calcium uptake is stimulated by the addition of calmodulin. Calmodulin increases the maximum calcium transport rate /V_max/, decreases K_Ca, and does not affect K_ATP of calcium uptake. The action of both membrane bound and external calmodulin is competitively inhibited by phenothiazines. Drugs reacting with SH groups of proteins reversibly inhibit calcium pumping by decreasing V_max and not affecting K_Ca and K_ATP. The relative magnitude of calmodulin stimulation of calcium transport is unaltered by SH reagents.Mild proteolytic digestion of IOVs stimulates active calcium uptake and mimics the effects of calmodulin on the kinetic parameters — that is converts the system to a “high calcium-affinity” state. Proteolysis eliminates calcium-dependent calmodulin binding to IOV membranes and any further stimulation of calcium uptake by calmodulin. Based on these results the presence of a calmodulin-binding regulatory subunit of the red cell calcium pump at the internal membrane surface is postulated.     

Effects of calcium chelators, divalent cations and sulfhydryl reagents on calcium uptake and motility of bovine spermatozoa

Addition of 1 mM Ca/EGTA complex (1:1 ratio) to an incubation medium containing 1.5 mM Ca2+ produced a notable increase in the Ca2+ cycling in ejaculated bovine spermatozoa. Similar results were also obtained with the Ca/EDTA and Ca/EDTA complexes or with the heavy metal chelator DTPA (50 microM). Ba2+, Ni2+ or Co2+ added at 0.1 mM concentration abolished the stimulatory effect of the Ca/EGTA complex on Ca2+ cycling, whereas it did not affect the calcium movement in the absence of the calcium chelator complex. It is concluded that small amounts of these cations should be bound to the plasma membrane of bovine spermatozoa and inhibit the cellular calcium influx. 0.1 mM Cd2+ and NEM or 1 mM diamide produced a calcium efflux from the spermatozoa together with an inhibition of cellular motility and an increase in glutamate-oxaloacetate transaminase release. Conversely the impermeant sulfhydryl reagent mersalyl caused a net calcium efflux but did not alter the cellular motility nor the transaminase release. It is suggested that the permeant thiol reagents could decrease the spermatozoal mobility by impairing the mitochondrial ATP-synthesis.     

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 mediated calcium uptake in ATP depleted human red cells

The A23187 induced calcium uptake in ATP depleted cells was determined at pH 6.9 in the presence of trifluoperazine (TFP, 0.30 mM), compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). Apart from verapamil the drugs all increased the maximum rate of ionophore-mediated calcium flux by 50-60 per cent. After the ionophore addition some time elapsed before the calcium flux attained the maximum value, and this time dependence could be interpreted as a slow uptake of A23187 into the membrane: five seconds after the addition of A23187 half of the added ionophore was able to transport calcium through the membrane. The effect of pH on the ionophore-mediated calcium uptake was determined in the absence and presence of TFP. At pH 7.4 the maximum rate of calcium flux in the absence of TFP was two to three times higher than that at pH 6.9 and TFP increased the uptake rate by 98 per cent.     

Calcium uptake and release by skeletal-muscle mitochondria

THE EGTA — ruthenium red quench technique was used to obtain initial-velocity plots of Ca²⁺ uptake by skeletal-muscle mitochondria. The K_m was 5 μM and the Hill coefficient 1.9 at both 0° and 10°C. Inorganic phosphate stimulated and Mg²⁺ inhibited initial rates of transport. In experiments on Ca²⁺ release, the $\text{Na}{}^{\mathrm{+}}\text{Ca}{}^{\mathrm{2+}}$ exchange was demonstrated. Factors influencing Ca²⁺ release during anaerobiosis include phosphate concentration and extent of Ca²⁺ loading. The results are discussed in relation to the possible participation of mitochondria in the calcium-ion regulation of muscle.     

Effects of ionophore A23187 on calcium flux and amylase release in isolated mouse pancreatic acini

The divalent cation ionophore A23187 has been used extensively to demonstrate the importance of Ca²⁺ in the control of pancreatic enzyme secretion. The relative importance, however, of the ability of the ionophore to facilitate Ca²⁺ movement across plasma and intracellular membranes in the stimulation of amylase release is not clear. We therefore studied these relationships in isolated pancreatic acini, a preparation in which it is possible to precisely measure both ⁴⁵Ca²⁺ fluxes, Ca²⁺ content and amylase release. A23187 increased the initial rates of both ⁴⁵Ca²⁺ uptake and washout. In addition, the content of both exchangeable ⁴⁵Ca²⁺ and total Ca²⁺ were reduced. These results indicated, therefore, that A23187 increases Ca²⁺ fluxes across both plasma and intracellular membranes. Consistent with this observation, the initial stimulation of amylase release by A23187 was independent of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, however, A23187 caused a rapid fall in acinar Ca²⁺ and subsequent amylase release was abolished. Depletion of intracellular Ca²⁺ by the ionophore also blocked the subsequent stimulation by cholecystokinin (CCK). The results indicate certain similarities in the actions of A23187 and CCK on pancreatic acini; both the agonists have striking effects on intracellular Ca²⁺ which in turn mediates their actions.     

Evidence for the mobilization of cellular calcium by prostacyclin in cat adrenocortical cells: The effect of TMB-8

Stimulation of steroid production by isolated cat adrenocortical cells is partially dependent upon the presence of extracellular Ca²⁺ when elicited by prostacyclin (PGI₂) and completely dependent upon extracellular Ca²⁺ when elicited by corticotropin. TMB-8, an intracellular Ca²⁺ antagonist, completely blocked PGI₂-evoked steroid output in the absence of external Ca²⁺; this inhibition was partially reversed by the addition of Ca²⁺. The increase in secretion caused by corticotropin or PGI₂ in the presence of Ca²⁺ was also reduced in a dose-dependent manner by TMB-8. The steroidogenic action of pregnenolone, which is induced by a Ca²⁺ independent mechanism, was not blocked by TMB-8, either in the presence or absence of Ca²⁺. Corticotropin significantly potentiated the Ca²⁺-independent aspect of PGI₂ action. These studies provide evidence for an internal, PGI₂-sensitive Ca²⁺ store in cat adrenocortical cells.     

Inhibition by sodium of A23187-mediated calcium translocation

Sodium inhibits in a dose-related fashion the translocation of calcium from an aqueous milieu into an organic phase containing the divalent-cation ionophore A23187. This inhibitory effect is reproduced by other monovalent cations, modulated by the nature of the anion in the sodium halide, and inversely related to the absolute amount of calcium translocated. The inhibitory effect cannot be attributed to a change in osmolarity or ionic strength, to sequestration of the ionophoretic molecule at the interface between the aqueous and organic phases, or to translocation of sodium or chloride. These findings indicate that sodium may directly affect the handling of calcium by ionophoretic systems specifically mediating the transport of divalent cations.     

Ionophoretic Ca2+ mobilization in rat gonadotropes and bovine adrenomedullary cells

Stimulation of luteinizing hormone (LH) release from the pituitary gonadotrope and catecholamine release from the adrenomedullary cell are Ca²⁺ dependent processes (for reviews, see 1, and 2, respectively). In both systems, extracellular Ca²⁺ is requisite for stimulation of release by the naturally occurring secretogogue (gonadotropin releasing hormone, GnRH, for the pituitary gonadotrope; acetylcholine, Ach, for the adrenomedullary cell). Inhibitors of Ca²⁺ movement are also effective blockers of GnRH and Ach action on the respective release systems. The observation that ionophores including A23187 (Lilly) and X537A (Roche) as well as K⁺ depolarization in the presence of extracellular Ca²⁺ evoke release from both systems, suggests that Ca²⁺ may actually mediate the responses in these systems. In the present study we have examined the effect of Ca · Ionomycin (Squibb) and shown it to be a particularly potent secretogogue whose action is coupled to its ability to transfer Ca²⁺ from the extracellular medium across the cell membrane.     

Estimation of the levels of calcium and other electrolytes during various phases of contraction of the longitudinal smooth muscle of the guinea pig ileum

A method is described for measuring calcium and other electrolyte levels during various phases of contraction of the longitudinal smooth muscle of the guinea pig ileum. Muscles are immersed in a solution of 160 mM Tris-Cl containing 10 mM lanthanum for 30 min to reduce the contribution of extracellularly bound cations, while the temperature is reduced to 4°C to prevent active cation fluxes. The muscle cells gained calcium and lost magnesium during the phasic and tonic phases of contractions by the muscarinic agent, CD (cis-2-methyl-4-dimethylaminomethyl-1, 3-dioxolane methiodide) and during the tonic phase of contractions by KCl. Muscles lost potassium during contractions to CD, but gained potassium during contractions to 60 mM KCl. The potassium lost during contractions to CD was regained slowly over a period of 30 min when the CD was washed out. This recovery corresponded to the rate of recovery of spontaneous activity and normal responsiveness of the muscle to further doses of stimulants. Calcium levels were reduced rapidly to below normal levels on washout of the CD and were regained in a similar time course to that of potassium. Sodium levels were not significantly changed with either stimulant.     

In situ accumulation of calcium by organelles of squid axoplasm

Existing morphological and physiological evidence indicates that axoplasm of squid axons sequesters calcium by both mitochondrial and non-mitochondrial buffers. The present work demonstrates that essentially all of the non-mitochondrial component is located in organelles. Extruded axoplasm was loaded with varying amounts of calcium by mixing with small volumes of solutions containing pH buffered ⁴⁵Ca. Ethyleneglycol-bis(β-amino-ethyl ether)N,N′-tetraacetic acid (EGTA) or diethylenetriamine pentaacetic acid (DTPA) was used to stabilize the free calcium. The axoplasm was then sucked up in a polyethylene tube and centrifuged at 100,000 g for 2–3 hours to produce a loose pellet comprising 10–20% of the axoplasm volume. After centrifugation, the tube was frozen, sliced into segments, and counted by liquid scintillation. No significant pellet accumulation of exogenous calcium occurred at physiological concentrations of free calcium (ca. 50 nM); however, a threshold for accumulation existed at 150–200 nM. Essentially complete pellet sequestration of the exogenous load occurred at a free calcium concentration above 1 μM. About half of the pellet buffering capacity was sensitive to carbonyl cyanide, p-trifluoromethoxy phenylhydrazone (FCCP). Variation of exogenous load between 0.1 – 3 mmole/kg axoplasm did not affect the buffering capacity of either the FCCP sensitive or insensitive components when the free calcium concentration was above threshold.     

Effects of divalent cations on phosphatase secretion in cultured tobacco cells

Some differences were found between Mg²⁺- and Ca²⁺-stimulated phosphatase secretion in cultured tobacco cells. The effect of Mg²⁺ ions was greater than that of Ca²⁺ ions, and Ca²⁺ ions at below 1 mM rather depressed the secretion. Upon the addition of Mg²⁺ ions plus Ca²⁺ ions, a synergistic stimulation of the secretion occurred. Different influences on the effects of Mg²⁺ and Ca²⁺ ions on the secretion were exerted by treating cells with metabolic inhibitors that reduced the level of cellular metabolic energy. Phosphate (Pi) and arsenate did not depress the secretion in the presence of Mg²⁺ ions, but did depress it in the presence of Ca²⁺ ions. These results strongly suggested that the secretion of phosphatase involved at least two different steps affected by divalent cations.     

Antagonism of calmodulin and phosphodiesterase by nifedipine and related calcium entry blockers

Nifedipine, a 1,4-dihydropyridine Ca2+ entry blocker, partially inhibits calmodulin-activated and, to a lesser extent, basal (non-activated) cyclic AMP phosphodiesterase activity at 10-440 microM. The inhibition of calmodulin-activated phosphodieserase does not parallel Ca2+ entry blockade, since analogs of nifedipine, which are 500-fold less potent than nifedipine as Ca2+ entry blockers (Bolger et al. (1982) Biochemical and Biophysical Research Communications 104, 1604-1609), are equal in potency to nifedipine as calmodulin-activated phosphodiesterase inhibitors. Furthermore, the inhibition of calmodulin-activated phosphodiesterase by nifedipine is about 500-fold less potent than its inhibition of Ca2+ entry blockade. It is suggested that the low affinity interaction of nifedipine and related 1,4-dihydropyridines with calmodulin and phosphodiesterase is also of low specificity and therefore is unlikely to contribute to the cardiac and vascular muscle relaxant actions of these drugs at normal pharmacological concentrations.     

Molecular properties of the red cell calcium pump: II. Effects of calmodulin,proteolytic digestion and drugs on the calcium-induced membrane phosphorylation by ATP in inside-out red cell membrane vesicles

In inside-out human red cell membrane vesicles /IOV/, in the absence of Mg²⁺, the only calcium-induced labelling by γ³²P-ATP occurs in a 140–150 000 molecular weight protein fraction, representing the hydroxylamine-sensitive phosphorylated intermediate /EP/ of the calcium pump. In the presence of Mg²⁺ calcium-induced phosphorylation is accelerated but several other membrane proteins are also phosphorylated through protein kinase action forming hydroxylamine-insensitive bonds. Addition of calmodulin accelerates EP formation both in the absence and presence of Mg²⁺.Treatment of the membrane with SH-group reagents significantly reduces EP formation. Mild trypsin digestion of IOVs, stimulating active calcium transport, eliminates calmodulin action and decreases the steady-state level of EP. In trypsin-digested IOVs the molecular weight of the ³²P-labelled EP is shifted to lower values /110–120 000/ We suggest that trypsin digestion cleaves off a 20–40 000 molecular weight calmodulin-binding regulatory subunit of the calcium pump molecule.     

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 induced calcium transients in human red cells

A transient increase of cellular calcium was induced by addition of the divalent cation ionophore A23187 to human red cells in the absence or presence of drugs. The peak height of the calcium transient was increased about five times at pH 6.9 and up to eighteen times at pH 7.4 by trifluoperazine (0.30 mM), and two to three times at pH 6.9 by compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). The time-dependent changes of cellular calcium were analysed by the aid of a pump-leak model based partly on the calcium dependent parameters obtained from calcium ATPase experiments, partly on the A23187 induced calcium fluxes determined in experiments with ATP depleted cells. The transient increase of cellular calcium induced within few minutes after the addition of ionophore A23187 could be explained satisfactorily by the model both in the absence and presence of the four drugs, whereas the final level of cellular calcium in the drug experiments was more difficult to predict from the pump-leak model. Comparison of experimental and model calcium transients suggested that trifluoperazine and TMB-8 affected both pump and leak, whereas compound 48/80, probably due to low membrane-permeability, mainly affected the leak and verapamil affected the pump only.