Actions of a tumor-promoting phorbol ester in a cloned rat pituitary cell

Tumor promoters, such as phorbol myristate acetate (PMA), facilitate carcinogenesis by mechanisms that may involve changes in intracellular Ca²⁺ metabolism and distribution of Ca²⁺, as well as activation of a Ca²⁺-and phospholipid-dependent protein kinase, referred to as protein kinase C. We compared the actions of PMA on GH₃ cloned pituitary cells with those of thyrotropin releasing hormone (TRH), an established Ca²⁺-mobilizing agent. The TRH treatment produced a⁴⁵Ca efflux, inhibited⁴⁵Ca uptake, diminished chlortetracycline fluorescence, and stimulated cAMP accumulation and protein synthesis in a Ca²⁺-dependent manner. Like TRH, PMA produced an efflux of⁴⁵Ca and inhibited⁴⁵Ca uptake; however, the phorbol ester stimulated cAMP accumulation and protein synthesis in the absence of external calcium and failed to alter chlortetracycline fluorescence. The TMB-8, a putative inhibitor of the mobilization of membrane-associated Ca²⁺, did not alter PMA-induced stimulation of protein synthesis. The results suggest that PMA-induced changes in Ca²⁺ metabolism are not caused by the mobilization of membrane-associated calcium. Alternative proposals are that PMA (1) inhibits Ca²⁺ influx and/or (2) mobilizes calcium from nonmembranous storage sites. Further study is needed to characterize the mechanism through which tumor-promoting phorbol esters influence Ca²⁺ metabolism and to ascertain the significance of changes in Ca²⁺ metabolism to cellular processes affected by these substances.     

Inhibition of dibutyryl cyclic AMP induced steroidogenesis in rat adrenocortical cells by the putative calcium antagonist TMB-8

A significant proportion of the steroidogenic response of isolated rat adrenocortical cells to dibutyryl cyclic AMP does not require extracellular calcium, and this component is profoundly depressed by low concentrations of the putative calcium antagonist, TMB-8. The inhibition is reversed by either the readdition of calcium or the calcium ionophore A23187. The steroidogenic response to pregnenolone, whose mode of action does not require calcium, was not depressed by TMB-8. Corticotropin (ACTH)-induced steroidogenesis, which requires extracellular calcium, was markedly depressed by TMB-8, although enhanced cyclic AMP formation is only slightly depressed by this drug. Adrenal cortical microsomes possess an ATP-dependent 45calcium (45Ca2+) uptake system which responded to EGTA with a rapid efflux of 45Ca2+; EGTA-induced calcium efflux from this microsomal fraction was markedly reduced by a concentration of TMB-8 that blocked dibutyryl cyclic AMP-evoked steroidogenesis. TMB-8 produced a smaller but significant reduction of EGTA-facilitated 45Ca2+ efflux from a mitochondrial-enriched fraction. We interpret these results to mean that TMB-8 blocks the steroidogenic effect of dibutyryl cyclic AMP by interfering with the mobilization of a cellular pool of calcium that is probably localized to the endoplasmic reticulum. The physiological implications of these findings in relation to the complex interactions between calcium and cyclic AMP in adrenal steroidogenesis are discussed.     

Effect of inhibition of Na+-K+ ATPase on the prostacyclin generation of cultured human vascular endothelial cells

Prostacyclin (PGI2) generation of cultured human vascular endothelial cells (VEC) was observed coincidentally with the increase of 45Ca net influx. Ca ionophore A23187 enhanced not only PGI2 generation and 45Ca net influx but also 45Ca efflux. PGI2 generation was completely abolished by the pretreatment with Ca++ immobilizer, TMB-8. A Na+-K+ ATPase inhibitor, ouabain increased 45Ca net influx, but decreased 45Ca efflux, and enhanced PGI2 generation. These observation indicate that PGI2 generation of VEC may be regulated by not only Ca++ but also Na+, and it was suggested that enhanced PGI2 generation by ouabain might be derived from the increased cytosolic Ca++concentration by the decreased Ca++ efflux, and it was considered to be originated from the suppression of Na+-Ca++ exchange systems by the increased intracellular Na+ concentration via inhibition of Na+-K+ ATPase activity by ouabain. Enhancement of PGI2 generation of VEC by the increased ouabain like substances (OLS) in hypertension is suspected to be beneficial on the maintenance of vascular homeostasis.     

TMB-8 can block twitches without blocking high K+ or caffeine induced contractures in frog's skeletal muscle

TMB-8 [8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate] is known to inhibit calcium ion dependent processes in several tissues by stabilizing some intracellular stores of membrane-bound calcium. TMB-8 was used to study the excitation-contraction (E-C) coupling process in frog's skeletal muscle. TMB-8 (5 X 10(-5) - 10(-4) M) blocked electrically evoked twitches but not high K+ (123 mM)- or caffeine (2.36 mM)-induced contractures in isolated, curarized toe muscles. TMB-8 (10(-4) M) produced a small decrease (16%) in the action potential of frog's sartorius muscle fibres. However, reducing extracellular Na+ to 44.7 mM produced a similar reduction (17%) in action potential amplitude but did not suppress the twitch; i.e. it produced only a small increase (about 10%) in twitch amplitude. It is known that potassium contractures are produced by extracellular Ca++ ions which enter through calcium channels in the t-tubules and that caffeine produces contractures by sensitizing the sarcoplasmic reticulum to Ca++-induced Ca++ release. The present results suggest that TMB-8 blocks twitches by preventing the release of Ca++ ions bound to the intracellular surface of the t-tubular membrane which is often called the store of 'trigger-calcium' ions.     

Phorbol myristate acetate-induced release of granule enzymes from human neutrophils: inhibition by the calcium antagonist, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride

Phorbol myristate acetate (PMA) stimulated the extracellular release of the granule-associated enzyme lysozyme from human neutrophils. The extrusion of lysozyme was not accompanied by the release of β-glucuronidase or the cytosol enzyme lactate dehydrogenase. A time dependent PMA-induced release of lysozyme occurred in the absence of extracellular calcium and when neutrophils were preincubated with EGTA. 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an antagonist of intracellular calcium, caused a dose-dependent inhibition of lysozyme release from neutrophils exposed to PMA in a calcium-free medium. This effect of TMB-8 could be reversed by the addition of calcium to the extracellular medium. These studies indicate that TMB-8 represents a valuable pharmacologic tool used to define the dependence of a secretagogue such as PMA on intracellular as opposed to extracellular calcium.     

Mitogen and co-mitogen stimulation of lymphocytes inhibited by three Ca++ antagonists

The Ca++ requirement for in vitro lymphocyte stimulation by lectins is well known and can be demonstrated by the use of Ca++ chelators. In this study, three Ca++ antagonists were examined for their effects on lymphocyte proliferation. [3H]-thymidine incorporation was employed to measure DNA synthesis in several systems. Stimulation and proliferation were achieved by the addition of one of the following: the mitogenic lectin concanavalin A (ConA); the combination of two co-mitogens, the calcium ionophore A23187 and the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), neither of which is mitogenic alone; or the non-mitogenic lectin, wheat germ agglutinin (WGA) with TPA. These mitogenic systems were tested for their sensitivity to the Ca++ channel blockers verapamil and nicardipine and the intracellular Ca++ antagonist TMB-8. We found that the ConA and WGA plus TPA treated cells were inhibited approximately 50% by 10 microM verapamil, nicardipine or TMB-8. The stimulation caused by A23187 and TPA was only inhibited by TMB-8 and nicardipine. The inhibitory effects caused by the Ca++ antagonists could not be reversed by the addition of exogenous Ca++ (0.1-1.5 mM), but were reversed by repeated washings in antagonist free media. Using TMB-8 we saw an apparent intracellular Ca++ dependence throughout the G1 phase. Previous studies using Ca++ chelators or Ca++ antagonists suggested an endpoint at about halfway through this period.     

Mechanism of inhibitory action of TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] on aldosterone secretion in adrenal glomerulosa cells.

The mechanism of 8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) action was evaluated in isolated adrenal glomerulosa cells. TMB-8 inhibits both angiotensin II- and K+-stimulated aldosterone secretion in a dose-dependent manner. The ID50 for angiotensin II- and K+-stimulated aldosterone secretion is 46 and 28 microM, respectively. In spite of the fact that 100 microM-TMB-8 inhibits angiotensin II-stimulated aldosterone secretion almost completely, TMB-8 (100 microM) does not inhibit angiotensin II-induced 45Ca2+ efflux from prelabelled cells nor does it affect inositol 1,4,5-trisphosphate-induced calcium release from non-mitochondrial pool(s) in saponin-permeabilized cells. TMB-8 has no inhibitory effect on A23187-induced aldosterone secretion, but 12-O-tetradecanoylphorbol 13-acetate-induced aldosterone secretion is completely abolished. TMB-8 effectively inhibits both angiotensin II- and K+-induced increases in calcium influx but has no effect on A23187-induced calcium influx. TMB-8 inhibits the activity of protein kinase C dose-dependently. These results indicate that TMB-8 inhibits aldosterone secretion without inhibiting mobilization of calcium from an intracellular pool. The inhibitory effect of TMB-8 is due largely to an inhibition of plasma membrane calcium influx, but this drug also inhibits the activity of protein kinase C directly.     

Implications of a rise in cytosolic free calcium in the activation of RAW-264 macrophages for tumor cell killing

The concentration of cytosolic-free calcium (Ca2+i) was determined with aequorin in RAW-264 macrophage-like cells activated in vitro for tumor cell killing with lymphokine (LK) and lipopolysaccharide (LPS). Treatments of these cells with optimal doses of stimulants, which evoked the development of cytolytic activity, also induced a rise in their Ca2+i. No rise in Ca2+i could be observed under treatments which failed to activate cells. The presence of both stimulants was an absolute requirement for evoking cytolytic activity and also a rise in Ca2+i. There was an apparent parallelism between the rate of activation and the rate of rise in Ca2+i. Cells which slowly developed their cytolytic activity exhibited a slow rise in Ca2+i, while macrophages which acquired their cytolytic activity at the faster rate also showed a more rapid increase in Ca2+i. The development of cytolytic activity in RAW-264 macrophages was inhibited by two intracellular calcium antagonists, TMB-8 and ruthenium red. This inhibition could be reversed by high concentrations of extracellular calcium. TMB-8, at the concentrations which were effective in inhibiting the activation process, also completely blocked the associated rise in Ca2+i. These results suggested that Ca2+i might play a role in the mechanism of tumoricidal transformation of RAW-264 macrophages.     

Does endothelin mobilize calcium from intracellular store sites in rat aortic vascular smooth muscle cells in primary culture?

In the presence of endothelin, there was a rapid increase in the 45Ca++ efflux from primary cultured rat vascular smooth muscle cells, both in physiological salt solution and in calcium free medium containing 2 mM EGTA. The 45Ca++ influx was not affected. The endothelin-induced, transient increase in cytosolic calcium concentration is probably mainly due to release of calcium from the intracellular store in vascular smooth muscle cells.     

Carbamylcholine, TRH, PGF2 alpha and fluoride enhance free intracellular Ca++ and Ca++ translocation in dog thyroid cells

Effects on Ca++ translocation and [Ca++]i were studied in dog thyro?d cell monolayers using both 45Ca++ efflux and the indicator quin-2. Carbamylcholine, a non hydrolysable analog of acetylcholine, through muscarinic receptors, and to a lesser extent TRH and PGF2 alpha increased both these parameters. [Ca++]i increased by 171, 100 and 75% respectively over a basal level of 66 +/- 17 nM (mean +/- SD). The response to carbamylcholine was biphasic. A transient increase in [Ca++]i was followed by a more sustained phase where the [Ca++]i was slightly higher than the basal level. Only the first phase was insensitive to extracellular Ca++ depletion. This phase is probably due to a release of Ca++ from an intracellular store. NaF also induced a sustained rise in [Ca++]i dependent on extracellular Ca++ and affected 45Ca++ efflux. Our data provide direct evidence of an implication of intracellular Ca++ in the response of dog thyro?d cells to all these agents.     

Cortical granule exocytosis in sea urchin eggs is inhibited by drugs that alter intracellular calcium stores

In sea urchin eggs fertilization is accompanied by cortical granule exocytosis, a secretory event thought to be initiated by release of intracellularly sequestered calcium. We have examined the effect of two drugs on this process: chlortetracycline (CTC), a known chelator of intracellular calcium, and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an antagonist of intracellular calcium release in both skeletal and smooth muscle. Preincubation of eggs for 10 min with either CTC or TMB-8 blocked sperm entry, inhibited the burst of 45Ca2+ efflux normally seen postinsemination, and prevented fertilization envelope elevation. Half-maximal inhibition occurred with 200 microM CTC and 60 microM TMB-8. Electron microscopy confirmed that cortical granule exocytosis had been blocked, although inhibition was not due to a direct effect on exocytosis. CTC and TMB-8 had no effect on Ca2+-stimulated granule fusion in isolated egg cortices. Rather, these drugs block the early events in egg activation: sperm incorporation and triggering of exocytosis. These two effects appear to be independent since addition of either drug just before insemination permits sperm entry but inhibits calcium release and cortical granule exocytosis.     

Role of intracellular calcium concentration and protein kinase C activation in IFN-gamma stimulation of U937 cells

IFN-gamma enhances many monocyte functions, including oxidative metabolism and Ag presentation. IFN-gamma has been reported to increase the intracellular concentration of calcium ([Ca2+]i) and modulate protein kinase C activity in murine macrophages, but the signal transduction pathways induced by IFN-gamma in human cells and their functional significance are poorly understood. Our study examined the hypothesis that an increases in [Ca2+]i and protein kinase C activation are required for functional responses to IFN-gamma. The U937 cell line was used as a model of an IFN-gamma responsive cell. IFN-gamma caused a rapid and concentration-dependent increase in [Ca2+]i, which was partly inhibited by calcium-free medium, diltiazem, and TMB-8. IFN-gamma induced a fourfold increase in the concentration of inositol 1,4,5-trisphosphate. Induction of HLA-DR, Fc gamma R, CR3, and Mo3e Ag expression by IFN-gamma was blocked by concentrations of TMB-8 that inhibited an increase in [Ca2+]i, but not by protein kinase C inhibition by H-7 or inhibition of calmodulin with W-7. Ionomycin did not enhance Ag expression and PMA induced the expression of only the Mo3e Ag. We conclude that IFN-gamma induces antigenic expression on human U937 cells by a mechanism dependent on, but not limited to, an increase in intracellular calcium, which is likely due to inositol 1,4,5-trisphosphate generation.     

Ca2+ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca2+ activation of the enzyme was studied and the Ca2+ concentrations required to activate the enzyme were compared to free cytosolic Ca2+ concentrations in resting and activated polymorphonuclear leukocytes. The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca2+ concentrations corresponding to the intracellular free Ca2+ concentration under resting conditions. However, at similar Ca2+ concentrations (less than 2.5 X 10(-7) M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca2+ concentration. K0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K0.5 for PMA stimulation of superoxide (O-2) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca2+ antagonism nor by a direct inhibition of protein kinase C activity.     

Na,K-ATPase in excitation-contraction coupling of vascular smooth muscle from cattle.

Lowering the extracellular K+ content from 6 to 0.6 mM causes a rise, and elevation from 6 to 8.5 mM a fall of 45Ca++ efflux from the vascular smooth muscle cells of the arteria carotis communis of cattle. In contrast, a level of 17 mM K+ has no influence. Removal of extracellular calcium does not block these effects. 10(-4) M ouabain also induces a rise in Ca++ efflux, additional potassium reduction then being without effect; 10(-9) M ouabain is of no influence. The 45Ca++ efflux kinetics correlates with the activity of the isolated Na,K-ATPase. Tonus increases of the vascular strips by 10(-4) M ouabain and potassium deficiency cannot be blocked by 4 mM lanthanum or removal of extracellular calcium. Unlike sodium, potassium stimulates the active Ca++ binding and the activity of the Ca-ATPase of the microsomal fraction. The ative Ca++ binding of the mitochondria is stimulated by both ions. It is postulated that the activity of the plasma membrane Na,K-pump is able to regulate the tonus of big arteries through alteration of Ca++ storage processes.     

Action of TMB-8 (8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate) on cytoplasmic free calcium in adrenal glomerulosa cell

To clarify the action of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on cellular calcium handling, changes in cytoplasmic free calcium concentration ([Ca2+]c) were studied in adrenal glomerulosa cell with a calcium-sensitive photoprotein, aequorin. Results of our previous study demonstrate that 100 microM TMB-8 almost completely blocks aldosterone response to angiotensin II (Biochem. J. 232 (1985) 87-92). At 50 or 100 microM, TMB-8 decreased basal [Ca2+]c significantly; however, these doses of TMB-8 had little effect on an angiotensin-induced increase in [Ca2+]c. When angiotensin-induced calcium release from an intracellular pool(s) was assessed by measuring changes in [Ca2+]c in the presence of 1 microM extracellular Ca2+, 100 microM TMB-8 had little inhibitory effect on angiotensin-induced calcium release. A higher dose of TMB-8 (250 microM) slightly inhibited calcium release. Additionally, TMB-8 did not affect exogenous arachidonic acid-induced calcium release. In contrast, 50 microM TMB-8 markedly inhibited 8 mM potassium-induced increase in [Ca2+]c. These results indicate that a major action of TMB-8 on cellular calcium is an inhibition of calcium influx but not of calcium release. We suggest that TMB-8 should not be used as an 'inhibitor of calcium release'.     

Protein kinase C activation stimulates plasma membrane Ca2+ pump in cultured vascular smooth muscle cells

We examined the effect of phorbol myristate acetate (PMA), a potent activator of protein kinase C, on Ca2+ extrusion from cultured vascular smooth muscle cells (VSMCs) incubated in the absence of added extracellular Na+ (Na+o). Previously, strong experimental evidence was presented that the Na+o-independent Ca2+ extrusion from VSMCs is effected by the plasma membrane Ca2+ pump (Furukawa, K.-I., Tawada, Y., and Shigekawa, M. (1988) J. Biol. Chem. 263, 8058-8065). Brief (2 min) pretreatment of VSMCs with 30-300 nM PMA suppressed the intracellular Ca2+ transient induced with 1 microM ionomycin to about 60% of the control, whereas it accelerated the concomitant Na+o-independent 45Ca2+ extrusion by up to 20%. When the Ca2+ transient was induced with 0.1 microM angiotensin II, the PMA pretreatment markedly suppressed it and reduced also the rate of 45Ca2+ efflux from cells slightly. These effects of PMA were mimicked by 1-oleoyl-2-acetylglycerol, another protein kinase C activator, but were abolished by prior treatment of cells with staurosporine, an inhibitor of protein kinase C, or prior long incubation of cells with PMA. Analysis of the effect of PMA on [Ca2+]i dependence of the rate of Na+o-independent 45Ca2+ efflux revealed that PMA increased the maximum Ca2+ efflux rate without a significant change in the affinity for Ca2+. These results strongly suggest that the plasma membrane Ca2+ pump in VSMCs can be stimulated by PMA and that protein kinase C is involved in regulation of [Ca2+]i in intact VSMCs.     

Phorbol myristate acetate stimulates pinocytosis and membrane spreading in mouse peritoneal macrophages

Phorbol myristate acetate (PMA) at a concentration of 0.01 microgram/ml causes an approximately threefold increase in surface area of resident, proteose-peptone-elicited, and thioglycolate-broth-elicited mouse peritoneal macrophages. Resident and proteose-peptone-elicited macrophages, cultured for 24 h in the presence of PMA, increase their pinocytic rate twofold in response to addition of PMA (0.01 microgram/ml) to the medium. Thioglycolate-broth-elicited macrophages, cultured for 24 h in the absence of PMA, immediately increase their pinocytic rate 2- to 3.5-fold in response to a single challenge with PMA (0.01 microgram/ml). Cytochalasin B, colchicine, and podophyllotoxin have only modest inhibitory effects on the basal rate of pinocytosis and on PMA-induced cellular spreading, but completely block the stimulatory effects of PMA on pinocytosis in thioglycolate- broth-elicited macrophages. Cytochalasin D markedly inhibits both basal and PMA-stimulated pinocytosis in these cells. Thus, PMA is a useful tool for studying mechanisms of macrophage spreading and for enhancing the overall rate of pinosome formation.     

Stimulus-secretion coupling in the developing exocrine pancreas: secretory responsiveness to cholecystokinin

We have studied the onset of secretory responsiveness to cholecystokinin (CCK) during development of the rat exocrine pancreas. Although acinar cells of the fetal pancreas (1 d before birth) are filled with zymogen granules containing the secretory protein, alpha- amylase, the rate of amylase secretion from pancreatic lobules incubated in vitro was not increased in response to CCK. In contrast, the rate of CCK-stimulated amylase discharge from the neonatal pancreas (1 d after birth) was increased four- to eightfold above that of the fetal gland. The postnatal amplification of secretory responsiveness was not associated with an increase in the number or cell surface expression of 125I-CCK binding sites. When 125I-CCK-33 binding proteins were analyzed by affinity crosslinking, two proteins of Mr 210,000 and 100,000-160,000 were labeled specifically in both fetal and neonatal pancreas. To determine if cell surface receptors for CCK in the fetal pancreas are functional and able to generate a rise in the cytosolic [Ca++], we measured 45Ca++ efflux from tracer-loaded lobules. 45Ca++ efflux from both fetal and neonatal pancreas was comparably increased by CCK, indicating CCK-induced Ca++ mobilization and elevated cytosolic [Ca++]. The Ca++ ionophore A23187 also stimulated the rate of 45Ca++ extrusion from pancreas of both ages. Increased amylase secretion occurred concurrently with A23187-stimulated 45Ca++ efflux in neonatal pancreas, but not in the fetal gland. A23187 in combination with dibutyryl cAMP potentiated amylase release from the neonatal gland, but not from fetal pancreas. Similarly, the protein kinase C activator, phorbol dibutyrate, did not increase the rate of secretion from the fetal gland when added alone or in combination with A23187 or CCK. We suggest that CCK-receptor interaction in the fetal pancreas triggers intracellular Ca++ mobilization. However, one or more signal transduction events distal to Ca++ mobilization have not yet matured. The onset of secretory response to CCK that occurs postnatally may depend on amplification of these transduction events.     

45Ca-efflux in embryonic chick heart and its modification by caffeine and ryanodine.

Ontogenic changes in the kinetics of exchangeable cellular calcium were studied in embryonic (ECV) and post-hatch (PHCV) chick ventricular tissue by monitoring 45Ca-efflux. The isolated whole ventricle (5 & 7 days ECV) or ventricular strips (12 & 18 days ECV and 1-2 days PHCV) were "loaded" with 45Ca (37 degrees C) and then passed through a series of tubes containing efflux solution (4 degrees C) to determine 45Ca-efflux. Curve 'peeling' of the efflux curve indicated existence of 3 kinetically distinct components of exchangeable cellular Ca2+ compartments: C1, C2 & C3. The size of C1, which was the largest in 5 & 7 days ECV decreased significantly to become minimum in 18 days ECV & PHCV. The rate constant of this compartment, however, reduced with the age of the embryo. In contrast, the size of C3 increased with the embryonic development to become the largest in 18 days ECV & PHCV. An increase in the rate constant of this compartment was also observed during embryogenesis. The size and rate constant of C2 remained unaltered during development. However, the increase in size of C3 during embryonic development indicates differentiation of Ca2+ storage sites, like sarcoplasmic reticulum (SR), during the later stages. Caffeine (10 mM) and ryanodine (10 microM) enhanced fractional escape rate during slow phase (ie 120-180 min) of efflux at all developmental stages. The magnitude of enhancement increased during later stages of development indicating greater prominence of SR with the age of embryo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium transport and exchange in mouse 3T3 and SV40-3T3 cells

The kinetics of Ca++ uptake have been evaluated in 3T3 and SV40-3T3 mouse cells. The data reveal at least two exchangeable cellular compartments in the 3T3 and SV40-3T3 cell over a 50-min exposure to 45Ca++. A rapidly exchanging compartment may represent surface-membrane-localized Ca++ whereas a more slowly exchanging compartment is presumably intracellular. The transition of the 3T3 cell from exponential growth (at 3 day's incubation) to quiescence (at 7 days) is characterized by a 7.5-fold increase in the size of the fast component. Quiescence of the 3T3 cell is also characterized by a 3.2-fold increase in the unidirectional Ca++ influx into the slowly exchanging compartment and a 3.6-fold increase in its size. The increase in size of the slow compartment at quiescence may result from a redistribution of intracellular Ca++ to a more readily exchangeable compartment, possibly reflecting a release of previously bound Ca++. In contrast, no significant change in any of these parameters is observed in the proliferatively active SV40-3T3 cells after corresponding period of incubation, even though these cells attained higher growth densities and underwent postconfluence.