Radioactive Calcium Research Articles

Calcium compartmentation and exchange rates in primary hepatocyte culture

We utilized a technique, previously used to study myocardial cells ( G. A. Langer, J. S. Frank, and L. M. Nudd, 1979, Amer. J. Physiol. 237, H239–H246), to study 45Ca 2+ isotope exchange kinetics in hepatocyte monolayers, cultured on scintillation disks, and perfused in a flow-through chamber. Isolated rat hepatocytes were plated directly on Primaria-coated disks impregnated with seintillation fluors which made up the walls of the perfusion chamber. Following the labeling of the cells with radioactive calcium ( 45Ca 2+), to apparent asymptote, the washout of 45Ca 2+ from the cells was measured. A large very fast turnover compartment, as well as small fast and slow turnover compartments, were identified in each experiment. Surface calcium (Ca 2+) was determined by its displacement with 1 m m La 3+ after asymptote had been reached during 45Ca 2+ labeling (1.59 mmol Ca 2+/kg dry wt). The rate constant for this compartment was faster than the washout of the chamber (>3.4 min −1 with a t 1 2 <12 s ). The rate constants for the fast and slow exchangeable compartments were 0.11 min −1 ( t 1 2 = 6.5 min ) and 0.013 min −1 ( t 1 2 = 56 min ), respectively. The fast compartment contained 0.40 mmol Ca 2+/kg dry wt and the slow compartment contained 0.27 mmol Ca 2+/kg dry wt. Neither the fast nor the slow compartment was lanthanum displaceable. Release of 45Ca 2+ in response to 100 μ m phenylephrine, 10 n m angiotensin II, and 100-μ m 2,5-ditert-butyl hydroquinone was measured during the washout phase. Ca 2+ released by these compounds was determined to be 0.50 mmol 0.44, and 0.43 mmol Ca 2+/kg dry cell wt, respectively. These agents had an effect only during the washout of the fast compartment. In conclusion, this novel technique of on-line measurement of 45Ca 2+ exchange in hepatocyte monolayers identified three exchangeable compartments: (1) a very rapidly exchangeable surface compartment, (2) a fast “microsomal” hormone-releasable compartment, and (3) a slow, non-hormone-releasable compartment.

Prevention of chemically induced synaptosomal changes

The ability of altered environmental conditions to modulate some properties of synaptosomes has been studied. Incubation conditions used included the presence of methyl mercury or an organochlorine insecticide: chlordecone. Other adverse chemical conditions during incubation were the absence of calcium salts from the incubation medium or the addition of agents bringing about enhanced oxidative conditions. Synaptosomal parameters studied were the cytosolic level of free, ionic calcium, [Ca2+]i, the extent of depolarization-induced uptake of radioactive calcium, and the permeability of the limiting membrane. In addition, peroxidative activity was estimated by quantitation of thiobarbituric acid-reactive material. All these facets of synaptosomal function were responsive to the presence of these potentially deleterious changes in the incubation medium. While the response of [Ca2+]i was potentially in either direction, all adverse conditions increased synaptosomal permeability as evaluated by leakage of fura-2 into the extracellular compartment. Pretreatment with ganglioside GM1 in some situations or alpha-tocopherol in others could either wholly or partially prevent the onset of such altered synaptosomal characteristics.

Reduced Gastrointestinal Absorption of Calcium in Dementia

Several reports have suggested that the neurodegenerative change in Alzheimer-type dementia (ATD) may be related to alterations in calcium homoeostasis. The absorption of radiocalcium (45Ca) in 26 ATD subjects and 11 patients with multi-infarct dementia (MID) was compared to 24 normal age- and sex-matched controls. The absorption of radiocalcium was significantly lower in both ATD and MID when compared to controls. The reduced 45Ca absorption in ATD occurred in the presence of normal plasma concentrations of PTH and vitamin D metabolites and the serum concentrations of calcium and aluminium were in the normal range. The data suggest that the reduced uptake of radioactive calcium observed in ATD is a non-specific derangement.

Otolith resorption induced by anaerobic stress in the goldfish, Carassius auratus

To examine otolith resorption induced by anaerobic stress, 45Ca‐prelabelled goldfish, Carassius auratus, were kept in oxygen‐deficient ambient water (O2 &lt; 0.5 .1 1−1, 26° C) for 2 days and otoliths (asterisci) were analysed for 45Ca retention. In a second experiment, fish were anaerobically stressed for 2 days, and then received a single intraperitoneal injection of 45Ca. They were maintained under stress for one more day and killed to examine 45Ca deposition in otoliths. Plasma was analysed for total and radioactive calcium. Otoliths (lapilli) were examined for stress‐induced check formation by scanning electron microscopy (SEM).Stress significantly reduced plasma calcium levels and the rate of calcium retention in otoliths, which was calculated from the 45Ca specific activity of the plasma. On the other hand, the rate of calcium deposition in otoliths was the same in the stressed as in the control fish. SEM observation revealed that the applied stress resulted in a check formation in otoliths. These results indicate that a 48‐h stress of oxygen deficiency induces calcium resorption in otoliths.

Inhibitory action of carvedilol, a novel α,β-adrenoceptor antagonist, on catecholamine secretion and calcium influx in cultured bovine adrenal chromaffin cells

The effect of carvedilol on the secretory function was studied using cultured bovine adrenal chromaffin cells. Carvedilol caused the concentration-dependent inhibition of catecholamine secretion evoked by carbamylcholine, high K + or veratridine. The drug also caused the inhibition of radioactive calcium uptake stimulated by these secretagogues into the cells, and the inhibition of calcium uptake was observed in parallel with that of catecholamine secretion. The inhibitory action of carvedilol on catecholamine secretion was shown to be similar to that caused by a classical β-adrenoceptor antagonist, propranolol. Furthermore, although the level of carbamylcholine-stimulated catecholamine secretion inhibited by diltiazem, a potent calcium channel antagonist, was significantly raised by elevating the calcium concentration in the reaction mixture, increasing the concentration of calcium ions in the mixture failed to induce any substantial influence on the secretion inhibited by carvedilol, as well as propranolol, under the same experimental conditions. These results seem to indicate that carvedilol may cause the inhibition of catecholamine secretion through its blocking action on calcium influx into the cells, and suggest the possibility that the inhibitory action of carvedilol on calcium influx is presumably based on its stabilizing action on the plasma membranes rather than its blocking action on the calcium channels in the chromaffin cell.

Intracellular calcium and neurotoxic events

Calcium is important in many intracellular regulatory processes. However, the maintenance of low levels of this cation within the cytosol is essential for maintenance of cell viability, in view of the large concentration gradient of ionic calcium across the plasma membrane. The expenditure of energy is needed to maintain intracellular calcium concentration [Ca 2+] i at normal levels. In addition, the integrity of the limiting membrane is also vital for this function. Thus, any disruption of membrane characteristics or of mitochondrial anabolic processes may lead to deleterious levels of [Ca 2+] i. The toxicity of a wide range of unrelated agents may, therefore, be in part due to elevation of cytosolic calcium. This general event may synergize with the more selective harmful properties of a compound, thus adversely affecting cell metabolism. The capacity now exists to measure levels of [Ca 2+] i in isolated cells or organelles such as synaptosomes. The use of such in vitro models can be of value in the evaluation of the neurotoxic potential of compounds. This method, in conjunction with the use of pharmacological agents known to act at specific sites, and with the use of radioactive calcium in translocation studies, also has utility in the delineation of the biochemical mode of action of neurotoxic agents.

Receptor occupation, calcium mobilization, and amylase release in pancreatic acini: effect of CCK-JMV-180

We examined the relationships between receptor occupation, calcium mobilization, and stimulated amylase release for cholecystokinin octapeptide (CCK-8) and for CCK-JMV-180, an analogue of the COOH-terminal heptapeptide of CCK having the structure Boc-Tyr(SO3)-Nle-Gly-Trp-Nle-Asp-2-phenylethyl ester using dispersed acini from rat pancreas. CCK-8 and CCK-JMV-180 each bind to two classes of CCK receptors: one class has a high affinity for CCK-8 and CCK-JMV-180 and the other class has a low affinity for CCK-8 and CCK-JMV-180. Mobilization of cellular calcium was assessed by measuring cytosolic calcium with a fluorescent indicator and by measuring outflux of radioactive calcium from preloaded cells. In terms of causing an increase in cytosolic calcium or an increase in calcium outflux, CCK-JMV-180 was 50-60% as efficacious as CCK-8. Analysis of the relationship between receptor occupation and calcium mobilization caused by CCK-8 and CCK-JMV-180 in combination indicates that calcium mobilization is caused by occupation of low-affinity CCK receptors. Comparison of the dose-response curve for calcium mobilization and amylase release stimulated by CCK-8 or CCK-JMV-180 indicates that very low concentrations of each peptide stimulate amylase release without causing detectable calcium mobilization. At these very low concentrations, CCK-8 or CCK-JMV-180 do not cause potentiation of amylase release when combined with vasoactive intestinal peptide.

Interleukin‐2 stimulates osteoclastic activity; Increased acid production and radioactive calcium release

Recombinant human interleukin-2 (IL-2) was studied to determine effects on acid production by individual osteoclasts in situ on mouse calvarial bones. This analysis was performed using a microspectrofluorimetric technique to quantify acid production in individual cells. Radioactive calcium release was determined using calvarial bones in a standard tissue culture system. This allowed us to correlate changes in acid production with a measure of bone resorption. IL-2 stimulated acid production and bone resorbing activity. Both effects were inhibited by calcitonin. No stimulation of bone resorption occurred when IL-2-containing test media was incubated with a specific anti-IL-2 antibody and ultrafiltered. Our data demonstrated a correlation between acid production and bone resorbing activity in mouse calvaria exposed to parathyroid hormone (PTH). The data obtained from cultured mouse calvaria exposed to IL-2 demonstrated similar stimulatory effects to those seen during PTH exposure. These data suggest that calvaria exposed to IL-2 in vitro have increased osteoclastic acid production corresponding with increased bone resorption.

Multiple power‐density windows and their possible origin

We have previously reported that in vitro exposure of chick forebrain tissue to 50-MHz radiofrequency (RF) electromagnetic radiation, amplitude modulated (AM) at 16 Hz, would enhance the efflux of calcium ions within only two power-density ranges: one from 1.44 to 1.67 mW/cm2, and the other including 3.64 mW/cm2. No effect on efflux occurred at 0.37, 0.72, 2.17, and 4.32 mW/cm2. We confirmed and extended these results by testing at another set of power densities, which included the range of the previous study. Forebrain tissue from 1-7-day-old chickens was labeled in vitro with radioactive calcium ions (30 min, at 37 degrees C), rinsed, placed in a physiological salt solution, and then exposed for 20 min to 50-MHz radiation, AM at 16 Hz, in a transverse electric and magnetic field (TEM) cell maintained at 37 degrees C. The solution was then assayed for radioactive calcium activity. A power-density series was tested. An enhanced efflux of calcium ions was found at 1.75, 3.85, 5.57, 6.82, 7.65, 7.77, and 8.82 mW/cm2; no change was observed at 0.75, 2.30, 4.50, 5.85, 7.08, 8.19, 8.66, 10.6, and 14.7 mW/cm2. Power density is converted to specific absorption rate (SAR) by 0.36 mW/kg per mW/cm2. Even the highest SAR tested (0.005 W/kg) is much too low to result in generalized heating of the sample and thus to be the underlying cause of the enhanced response. A hypothetical mechanism is proposed involving dynamic systems that may account for the power-density dependency as well as for part of the frequency dependency observed with both modulated RF radiation and extremely-low-frequency (ELF) fields.

Calcium ion uptake, somatotropin release, and fine structure of somatotrophs in cultures of the rat anterior pituitary upon the action of an oligopeptide (Boc-Gln-Leu-Lysinal).

Cultured cells from the anterior pituitary glands of adult rats were treated with the tripeptide aldehyde proteinase inhibitor, Boc-Gln-Leu-Lys-H. The oligopeptide had a profound releasing effect on growth hormone, whereas the prolactin release remained unchanged at 10(-3) mol/l drug concentration after an incubation for 2 h. In the presence of the oligopeptide a time- and dose-dependent calcium influx into cultured cells has been shown which was proved to be almost completely antagonized with magnesium ions but not with Nifedipine. In addition, radioactive calcium ions could be detected in a number of cells by light microscopic autoradiography when cultures were treated with Boc-Gln-Leu-Lys-H for short periods. The selective Gel action of the oligopeptide on growth hormone producing cells has been demonstrated also in fine structural investigations: multigranular and single exocytotic profiles have been observed. Accordingly, we have postulated that Boc-Gln-Leu-Lysinal mimics the effects of the known ionophores. Its mode of action needs, however, further studies especially on isolated somatotrophs.

Inhibition of bone resorption in vitro by serine-esterase inhibitors

The effect of two synthetic serine esterase inhibitors, N- α-dansyl( p-guanidino)phenylalaninepiperidine hydrochloride (I 2581) and d-phenylalanyl- l-prolyl- l-arginine chloromethyl ketone ( d-Phe-Pro-Arg-CH 2Cl), on bone resorption in organ cultured mouse calvaria from neonatal mice has been examined. Mineral mobilization was assessed by analyzing the release of 45Ca, stable calcium (Ca 2+) and inorganic phosphate (P i). Organic matrix degradation was studied by analyzing the release of 3H from [ 3H]proline-labelled bones, and by quantifying the amounts of hydroxyproline in bone after culture. It was found that I 2581, at and above 30 μmol/l, dose-dependently inhibited 45Ca release induced by thrombin, parathyroid hormone (PTH), prostaglandin E 2 and 1-α-hydroxyvitamin D-3. I 2581 (50 μmol/l) inhibited PTH-stimulated release of 3H from [ 3H]proline-labelled bones, and this effect was reversible after withdrawal of I 2581. I 2581 (50 μmol/l) inhibited the release of Ca 2+, P i, β-glucuronidase and β- N-acetylglucosaminidase in bones stimulated by PTH and 1-α-hydroxyvitamin D-3, without affecting the release of lactate dehydrogenase. In parallel, I 2581 decreased PTH and 1-α-hydroxyvitamin D-3 induced reduction of hydroxyproline levels in bones after culture. I 2581 (50 μmol/l) did not affect the basal release of 45Ca, Ca 2+, β-glucuronidase and β- N-acetylglucosaminidase, nor the basal amounts of hydroxyproline in bones after culture. d-Phe-Pro-Arg-CH 2Cl (100 μmol/l) significantly inhibited PTH- and PGE 2-induced release of 45Ca without affecting basal release of radioactive calcium. These data indicate that activation of serine proteinase(s) may be a necessary step in the mechanism of action of several stimulators of bone resorption.

1,25-Dihydroxycholecalciferol-dependent calcium uptake by mouse mammary gland in culture.

Mammary explants from pregnant mice cultured in the presence of lactogenic hormones express their differentiated function by producing milk components in vitro. In the present paper radioactive calcium uptake was studied in the mammary gland during terminal differentiation, when lactogenesis was initiated. Under these conditions a relevant accumulation of calcium in the tissue was clearly demonstrable. We have characterized the calcium uptake by the tissue as a saturable and energy dependent process. When 1,25-dihydroxycholecalciferol [1,25-(OH)2D3], the major calcium regulator hormone, was added to the incubation medium the secosteroid strongly stimulated this process by increasing the maximal velocity without significantly altering the Michaelis Menten constant (Km). Detectable increases in calcium uptake could be measured after the explants were cultured with 1,25-(OH)2D3 for 30 min, with the response continuing to increase sharply over time and reaching a plateau at 3 h. The increase was not affected by the presence of actinomycin D or cycloheximide suggesting that the 1,25-(OH)2D3 stimulation of calcium uptake may be independent of de novo protein synthesis. These results provide evidence for early and direct action of the hormonal form of vitamin D in affecting calcium transport across membranes of functionally differentiated epithelial cells of the mammary gland.

Distribution of calcium and phosphate in cells of the enamel organ in the rat lower incisor.

The distribution of calcium and phosphate in the cells of the enamel organ of the rat lower incisors was investigated by autoradiography and energy-dispersive x-ray spectrometry (EDS). Radioactive calcium or phosphate was injected i.p. into seven-day-old rats of the Wistar strain. The animals were frozen 0.5, 1, and 10 min after injection, and embedded in 5% carboxymethyl cellulose. Sagittal sections of 10 μm thickness were made in which the lower incisor was included as a part of the whole-body section. For autoradiography, the sections were freeze-dried and placed in contact with dry thin films prepared from autoradiographic emulsion. For EDS, sections were mounted on carbon stubs, freeze-dried, coated with carbon, and examined by EDS in a SEM. 45Ca and 32P autoradiograms showed that the radioactivity was located over the papillary layer cells adjacent to the secretory stage ameloblasts and was much higher here than in the ameloblastic layer. On the other hand, there was no significant difference between the amount of radioactivity of these two cell layers in the maturation stage, although higher radioactivity was detectable in the maturation stage enamel than in the secretory stage enamel. Pronounced Ka x-ray peaks were obtained for P, S, Cl, and K originating from the cells of the papillary and ameloblastic layers in the secretory stage, but only very low peaks were obtained for Ca. On the other hand, in addition to these elements, remarkably high Ca and Fe peaks could be detected in the ameloblastic layer of the maturation stage.

Interrelationships between motility, c-AMP, respiration and calcium uptake of ram and boar sperm

Uptake of radioactive calcium by washed ejaculated ram and epididymal boar spermatozoa was inhibited by theophylline and dibutyryl cyclic AMP (diBc-AMP) which increases cyclic AMP (c-AMP) levels in cells, by the uncouplers 2,4-dinitrophenol (2,4-DNP) and carbonylcyanide m-chlorophenylhydrazone (CCP), by sodium azide, rotenone and antimycin A and by ruthenium red and La 3+. Nicotine, eserine and ouabain stimulated the calcium uptake of caput boar sperm and decamethonium inhibited it. Inhibition of calcium uptake was accompanied by a decrease in oxygen uptake in the case of the metabolic inhibitors (rotenone, antimycin A and sodium azide) and substances that interfere with cell calcium transport (ruthenium red and La 3+). Respiration was increased in the presence of 2,4-DNP and CCP, and theophylline and diBc-AMP. The phosphodiesterase inhibitors (caffeine and theophylline), diBc-AMP, 2,4-DNP, bicarbonate ion and ouabain increased the motility of caput boar sperm. Most other substances depressed motility. The ionophore A23187 severely inhibited the motility of ram and boar sperm with an accompanying increase in their calcium and oxygen uptake which was largely unaffected by addition of inhibitors, activators and surfactants. This may be explained by the operation of competing mitochondrial and plasma membrane pumps. However, a selective increase in the permeability of the sperm plasma membrane on addition of the polyene antibiotic filipin released calcium accumulated in the presence of A23187. This provides further evidence for the lack of mitochondrial involvement in the influx of calcium produced by the ionophore.

Prostaglandin E2 causes a transient inhibition of mineral mobilization, matrix degradation, and lysosomal enzyme release from mouse calvarial bonesIn Vitro

The effect of prostaglandin E2 (PGE2) on the kinetic of bone resorption in vitro was assessed by following the release of minerals and degradation of matrix in cultured mouse calvarial bones. PGE2 (1 and 3 mumol/liter) caused an initial inhibition of the release of 45Ca, stable calcium, and inorganic phosphate from unstimulated calvarial bones. The effect was transient and after 24 and 48 hours the release of 45Ca, stable calcium, and inorganic phosphate from PGE2-treated bones was enhanced. 0.3 mumol/liter of PGE2 stimulated the release of 45Ca after 24 hours, but at this concentration no initial inhibition was observed. The initial inhibitory effect of PGE2 (1 mumol/liter) could be further increased by three structurally different inhibitors of cyclic AMP breakdown. PGE2 (1 mumol/liter) caused not only an initial inhibition of mineral release but also an initial inhibition of matrix degradation, as assessed by the release of 3H from [3H]-proline labeled bones. In addition, PGE2 (3 mumol/liter), in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, caused a rapid (6 hours) inhibition of the release of the lysosomal enzymes beta-glucuronidase and beta-N-acetyl-glucosaminidase, without affecting the release of the cytosolic enzyme lactate dehydrogenase. Similar specific initial inhibition of lysosomal enzyme release was also seen in the presence of calcitonin and dibutyryl cyclic AMP, but not in the presence of parathyroid hormone (PTH). Neither PGE2 nor the phosphodiesterase inhibitors rolipram and Ro 20.1724, could inhibit the initial stages of PTH-induced 45Ca release. Nor did PGE2 inhibit the stimulation of radioactive calcium mobilization induced by 1 alpha (OH)-vitamin D3.(ABSTRACT TRUNCATED AT 250 WORDS)

Human gingival fibroblasts secrete non-dialyzable, prostanoid-independent products which stimulate bone resorption in vitro.

Conditioned media from human gingival fibroblasts stimulate release of radioactive calcium, stable calcium and inorganic phosphate from mouse calvarial bones in organ culture. In addition, human gingival fibroblast‐conditioned media decrease the hydroxyproline content in the bones. Stimulation of mineral mobilization could be reduced by calcitonin, bisphosphonates and glucocorticoids, but not by cyclooxygenase inhibitors. The production of stimulating factors by fibroblasts was unaffected by indomethacin, meclofenamic acid and naproxen. Dialysis of human gingival fibroblast‐conditioned media showed that the product(s) had a molecular weight larger than 3000 daltons. Bone resorbing activity in human fibroblast‐conditioned media was heat‐labile. These data show that human gingival fibroblasts can secrete non‐prostanoid products with the ability to stimulate osteoclastic bone resorption in vitro.

Effects of calcium antagonists on isolated bovine cerebral arteries: inhibition of constriction and calcium-45 uptake induced by potassium or serotonin.

The purpose of this study was to determine the mechanisms by which organic calcium channel blockers inhibit cerebral vasoconstriction. Isolated bovine middle cerebral arteries were cut into rings to measure contractility or into strips to measure radioactive calcium (45Ca) influx and efflux. Calcium channel blockers (10(-5) M verapamil or 3.3 X 10(-7) M nifedipine) and calcium-deficient solutions all produced near-maximal inhibition of both potassium- and serotonin-induced constriction. In calcium-deficient solutions containing potassium or serotonin, verapamil and nifedipine each blocked subsequent calcium-induced constriction in a competitive manner. Potassium and serotonin significantly increased 45Ca uptake into cerebral artery strips during 5 minutes of 45Ca loading; for potassium 45Ca uptake increased from 62 to 188 nmol/g, and for serotonin from 65 to 102 nmol/g. Verapamil or nifedipine had no effect on basal 45Ca uptake but significantly blocked the increase in 45Ca uptake induced by potassium or serotonin. Potassium, and to a lesser extent serotonin, each induced a brief increase in the rate of 45Ca efflux into calcium-deficient solutions. Verapamil or nifedipine had no effect on basal or potassium-stimulated 45Ca efflux. The results demonstrate that verapamil and nifedipine block 45Ca uptake through both potential-operated (potassium) and receptor-operated (serotonin) channels in bovine middle cerebral arteries.

Calcium transport by permeabilised rabbit small intestinal epithelial cells.

Intestinal epithelial cells isolated from rabbit small intestine and whose plasma membrane had been rendered highly permeable were used to study the role of intracellular structures in Ca2+ buffering. Monitoring free Ca2+ with a selective electrode revealed that the cells could reduce Ca2+ concentration in the medium to a level of 3.6 X 10(-7) M independently, within a certain range, of the initial Ca2+ concentration or amount of cells used. Ca2+ buffering by permeabilised enterocytes was Mg2+- and ATP-dependent and was abolished in the presence of the Ca2+ ionophore A23187. The rapidity of Ca2+ buffering, but not the final Ca2+ level attained, was reduced in the combined presence of the mitochondrial inhibitors azide and oligomycin or in the presence of ruthenium red. Buffering of Ca2+ was abolished in the presence of vanadate, although some uptake was still observed. Complete blocking occurred in the presence of both vanadate and mitochondrial inhibitors. Measurement of initial rates of uptake with radioactive calcium revealed that mitochondrial uptake plays a role at relatively high Ca concentrations but that at the presumably physiological levels most of the uptake is into a non-mitochondrial compartment. Non-differentiated crypt cells seemed to handle intracellular Ca2+ in a similar way as mature villus cells, although they appeared to buffer at a level about 2 X 10(-7) M lower.

Rapid filtration study of the phosphorylation-dependent dissociation of calcium from transport sites of purified sarcoplasmic reticulum ATPase and ATP modulation of the catalytic cycle

Time-resolved filtration measurements using radioactive calcium were conducted to investigate with leaky preparations the kinetic features of the dissociation of transported calcium to the luminal side of the sarcoplasmic reticulum calcium pump, which occurs concomitantly with isomerization of the phosphorylated ATPase. At pH 6 and 20 degrees C, Ca2+ dissociation was moderately fast in the absence of potassium (3-5 s-1 at 0.05 mM ATP), implying that the dephosphorylation step (about 1.5 s-1) was the main contributor to rate limitation under these conditions. Potassium slowed down Ca2+ release but stimulated dephosphorylation, so that in its presence Ca2+-releasing isomerization did contribute to rate limitation, especially at neutral pH. At pH 6 in the absence of potassium and in the presence of magnesium, millimolar concentrations of ATP doubled the rate of Ca2+ dissociation, as also shown by dual-wavelength detection of fast changes in the absorbance of the Ca2+-sensitive dye Antipyrylazo III. Under the same conditions, low-affinity binding of ATP to phosphoenzyme was demonstrated. It is suggested that this low-affinity acceleration by ATP of the crucial step leading to dissociation of transported Ca2+ is the specific interaction responsible for the low-affinity acceleration of overall ATPase activity generally observed in the presence of potassium at neutral pH. Hydrolysis of the Ca2+-deprived phosphoenzyme was accelerated by ATP in the absence but not in the presence of Mg2+ in the dephosphorylation medium. We suggest that metal-free ATP is a more potent activator than Mg X ATP for transitions involving phosphoenzyme.

Inhibitory action of hydralazine on catecholamine secretion from cultured bovine adrenal chromaffin cells

Hydralazine caused a concentration-dependent inhibition of the secretion of catecholamines induced by carbamylcholine or high K + from cultured bovine adrenal chromaffin cells, and also caused the significant inhibition of radioactive calcium uptake induced by carbamylcholine into the cells. However, hydralazine failed to inhibit the secretion of catecholamines evoked by the calcium-ionophore, A23187. The inhibitory action of hydralazine on catecholamine secretion induced by carbamylcholine was not affected by increasing the concentration of calcium ion in the reaction mixture. These observations therefore seem to indicate that the inhibitory action of hydrazaline is not due to either the blocking of receptors for carbamylcholine or the disruption of the secretory machinery, and suggest that the drug may cause the inhibition of catecholamine secretion through its blocking action on calcium influx into the cells.