Cadmium uptake and toxicity via voltage-sensitive calcium channels

The mechanism of cellular uptake of cadmium, a highly toxic metal ion, is not known. We have studied cadmium uptake and toxicity in an established secretory cell line, GH4C1, which has well characterized calcium channels. Nimodipine, an antagonist of voltage-sensitive calcium channels, protected cells against cadmium toxicity by increasing the LD50 for CdCl2 from 15 to 45 microM, whereas the calcium channel agonist BAY K8644 decreased the LD50. Organic calcium channel blockers of three classes protected cells from cadmium toxicity at concentrations previously shown to block high K+-induced 45Ca2+ influx and secretion. Half-maximal protective effects were obtained at 20 nM nifedipine, 4 microM verapamil, and 7 microM diltiazem. Increasing the extracellular calcium concentration from 20 microM to 10 mM also protected cells from cadmium by causing a 5-fold increase in the LD50 for CdCl2. Neither the calcium channel antagonist nimodipine nor the agonist BAY K8644 altered intracellular metallothionein concentrations, while cadmium caused a 9-20-fold increase in metallothionein over 18 h. Cadmium was a potent blocker of depolarization-stimulated 45Ca2+ uptake (IC50 = 4 microM), and the net uptake of cadmium measured with 109Cd2+ was less than 0.3% that of calcium. Although the rate of cadmium uptake was low relative to that of calcium, entry via voltage-sensitive calcium channels appeared to account for a significant portion of cadmium uptake; 109Cd2+ uptake at 30 min was increased 57% by high K+/BAY K8644, which facilitates entry through channels. Furthermore, calcium channel blockade with 100 nM nimodipine decreased total cell 109Cd2+ accumulation after 24 h by 63%. These data indicate that flux of cadmium through dihydropyridine-sensitive, voltage-sensitive calcium channels is a major mechanism for cadmium uptake by GH4C1 cells, and that pharmacologic blockade of calcium channels can afford dramatic protection against cadmium toxicity.     

Pathways of cadmium influx in mammalian neurons

The influx of the toxic cation Cd2+ was studied in fura 2-loaded rat cerebellar granule neurons. In cells depolarized with Ca2(+)-free, high-KCI solutions, the fluorescence emission ratio (R) increased in the presence of 100 microM Cd2(+). This increase was fully reversed by the Cd2+ chelator tetrakis(2-pyridylmethyl)ethylenediamine, indicating a cadmium influx into the cell. The rate of increase, dR/dt, was greatly reduced (67+/-5%) by 1 microM nimodipine and enhanced by 1 microM Bay K 8644. Concurrent application of nimodipine and omega-agatoxin IVA (200 nM) blocked Cd2+ permeation almost completely (88+/-5%), whereas omega-conotoxin MVIIC (2 microM) reduced dR/dt by 24+/-8%. These results indicate a primary role of voltage-dependent calcium channels in Cd2+ permeation. Stimulation with glutamate or NMDA and glycine also caused a rise of R in external Cd2+. Simultaneous application of nimodipine and omega-agatoxin IVA moderately reduced dR/dt (25+/-3%). NMDA-driven Cd2(+) entry was almost completely prevented by 1 mM Mg2+, 50 microM memantine, and 10 microM 5,7-dichlorokynurenic acid, suggesting a major contribution of NMDA-gated channels in glutamate-stimulated Cd2+ influx. Moreover, perfusion with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate caused a slow increase of R. These results suggest that Cd2+ permeates the cell membrane mainly through the same pathways of Ca2+ influx.     

Characterization of voltage-sensitive calcium channels in a clonal pituitary cell line

We have pharmacologically characterized voltage sensitive calcium channels (VSCCs) in GH3 cells, an anterior pituitary clonal cell line known to secrete prolactin and growth hormone. Raising the medium K+ concentration from 5 to 50 mM caused an immediate increase in net 45Ca2+ uptake which remained apparent over a 15 minute time course. 45Ca2+ uptake was maximally stimulated nearly 10-fold over basal levels. This K+-induced stimulation of Ca2+ uptake was not prevented by 10-5M tetrodotoxin or by replacing sodium with choline in the assay medium. Ca2+ uptake was, however, inhibited by several VSCC antagonists: nitrendipine, D-600, diltiazem and Cd2+. Further, the novel dihydropyridine VSCC agonists, BAY K8644 and CGP 28392, enhanced 50 mM K+-stimulated 45Ca2+ uptake and these effects were blocked by nitrendipine.     

Dihydropyridine modulators of voltage-sensitive Ca2+ channels specifically regulate prolactin production by GH4C1 pituitary tumor cells

To determine whether hormone synthesis by the GH4C1 pituitary cell line could be regulated by specifically modulating the movement of Ca2+ through voltage-sensitive channels, we have compared the effects of the dihydropyridine Ca2+ channel agonist BAY K8644 and the antagonist nimodipine on hormone production and Ca2+ current in these cells. BAY K8644 elicited, after a 10-15-h lag, a dose-dependent increase in prolactin (PRL) production as determined by measurements of total intracellular and secreted hormone. Over a 72-h period, GH4C1 cells incubated with 300 nM BAY K8644 produced 2-3 times as much total PRL as control cells. The effect on PRL was specific, since BAY K8644 did not increase growth hormone production, cell growth rate, or total cell protein. Exposing GH4C1 cells to BAY K8644 for short periods, up to 90 min, did not induce the delayed increase in PRL production observed with longer incubations. The effects of nimodipine were opposite to those of the Ca2+ channel agonist. PRL production was reduced 85% during 48-h treatment with 200 nM nimodipine, whereas growth hormone production was decreased less than 15%, and cell growth and total protein were unaffected. The actions of these two drugs on PRL production were well correlated with their effects on GH4C1 Ca2+ currents as measured by whole-cell patch-clamp recordings. BAY K8644 enhanced the magnitude of the peak Ca2+ current and shifted the current-voltage relationship such that Ca2+ channels were activated at less depolarized potentials. Nimodipine potently inhibited Ca2+ movement through the non-inactivating channel, while it antagonized the increases elicited by BAY K8644. These results indicate that PRL synthesis by GH4C1 cells can be specifically regulated by agents that enhance or block the movement of Ca2+ through voltage-sensitive channels. They also suggest that hormone synthesis by a secretory cell may be coupled to electrical activity by the opening of Ca2+ channels.     

Action of Ca2+ agonists/antagonists in mammalian peripheral neurons

The action of several ligands on the low- (LVA,T) and high-threshold (HVA,L and N) Ca channels of adult rat sensory neurons and human neuroblastoma IMR32 cells has been investigated. In both cell types, 40 microM Cd2+ and 6.4 microM /omega-Conotoxin (omega-CgTx) selectively blocked the HVA channels, sparing the majority of LVA channels that were antagonized by amiloride and Ni2+. In 50% of the cells, however, /omega-CgTx spared also a 15% of HVA channels that proved to be sensitive to BAY K 8644. The agonistic action of BAY K 8644 on [omega-CgTx-resistant HVA channels caused a large Ba current increase, prolonged current deactivation and acceleration of HVA channels inactivation that was particularly evident in adult rat DRG.     

Calcium channel agonists and antagonists: effects of chronic treatment on pituitary prolactin synthesis and intracellular calcium

PRL synthesis by GH cells in culture has previously been shown to increase when calcium is added to cultures grown in calcium-depleted medium or when cultures are treated for 18 h or longer with the dihydropyridine calcium channel agonist BAY K8644, whereas the antagonist nimodipine inhibits PRL. The experiments described here were designed to test whether differences in PRL synthesis caused by the dihydropyridines are due to changes in PRL mRNA levels, whether structurally different classes of calcium channel blockers alter PRL production, and whether long term treatment with calcium channel agonists and antagonists alters intracellular free calcium, [Ca2+]i. PRL synthesis and PRL mRNA levels were increased similarly by BAY K8644 and decreased in parallel by the dihydropyridine antagonist nimodipine, while overall protein and RNA synthesis were not changed by either the agonist or antagonist. Two calcium channel blockers which act at different sites on L-type channels than the dihydropyridines also inhibited PRL synthesis without affecting GH; 5 microM verapamil reduced PRL by 64% and 15 microM diltiazem by 89%. Partial depolarization with 5-25 mM KCl increased PRL synthesis up to 2-fold. The intracellular free calcium ion concentration was estimated by Quin 2 and averaged 142 nM for control cultures in normal medium, and 128 and 168 nM for cultures treated 72 h with nimodipine or BAY K8644, respectively. Nimodipine totally prevented the calcium rise obtained upon depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of BAY K 8644, nifedipine, and low Ca2+ on halothane and caffeine potentiation.

The purpose of this investigation was to examine the effects of the Ca2+ agonist BAY K 8644 and the Ca2+ antagonist nifedipine on halothane- and caffeine-induced twitch potentiation of mammalian skeletal muscle. Muscle fiber bundles were taken from normal Landrace pigs and exposed to BAY K 8644 (10 microM), nifedipine (1 microM), and low Ca2+ media administered alone and in combination with halothane (3%) or with increasing concentrations of caffeine (0.5-8.0 mM). Both BAY K 8644 and halothane potentiated twitches by approximately 80%; when they were administered in combination, twitch potentiation was nearly double that caused by either drug alone. In the presence of nifedipine, halothane increased twitches by less than 30%. Low Ca2+ significantly depressed twitches by approximately 25% but also inhibited halothane's inotropic effect. BAY K 8644 augmented caffeine potentiation but only at low caffeine concentrations (0.5-2.0 mM). Nifedipine and low Ca2+ failed to inhibit caffeine's inotropic effects. These results suggest that halothane potentiates twitches via a mechanism that involves or is influenced by extracellular Ca2+.     

Voltage-Sensitive Calcium Channels in Differentiated Neuroblastoma × Glioma Hybrid (NG108–15) Cells: Characterization by Quin 2 Fluorescence

Depolarization of differentiated neuroblastoma X glioma (NG108-15) cells with KCl (50 mM) or veratridine (50 microM) stimulated Ca2+ accumulation, was detected by quin 2 fluorescence. Intracellular Ca2+ concentrations ([Ca2+]i) were elevated about threefold from 159 +/- 7 to 595 +/- 52 nM (n = 12). Ca2+ entry evoked by high extracellular K+ concentration ([K+]o) was voltage-dependent and enhanced by the dihydropyridine agonists, BAY K 8644 and CGP 28 392, in a dose-dependent manner. CGP 28 392 was less potent and less efficacious than BAY K 8644. The (+) and (-) stereoisomers of 202-791 showed agonist and antagonist properties, respectively. (+)-202-791 was less potent, but as efficacious as BAY K 8644. In the absence of KCl, BAY K 8644 had no effect on Ca2+ entry. Voltage-sensitive calcium channel (VSCC) activity was blocked by organic Ca2+ channel antagonists (nanomolar range) both before and after KCl treatment and also by divalent metal cations (micromolar range). High [K+]o-induced Ca2+ accumulation was dependent on external Ca2+, but not on external Na+ ions ([Na]o), and was insensitive to both tetrodotoxin (3 microM) and tetraethylammonium (10 microM). In contrast, veratridine-induced Ca2+ accumulation required [Na+]o, and was blocked by tetrodotoxin, but not by nimodipine (1 microM). Veratridine-induced Ca2+ accumulation was slower (approximately 45 s), smaller in magnitude (approximately 30% of [K+]o-induced Ca2+ entry), and also enhanced by BAY K 8644 (approximately 50%). VSCC were identified in neuronal hybrid (NG108-15 and NCB-20) cells, but not in glial (C6BU-1), renal epithelial (MDCK), and human astrocytoma (1321N1) cells. NG108-15 cells differentiated with 1.0 mM dibutyryl cyclic AMP showed greater VSCC activity than undifferentiated cultures. These results suggest that cultured neural cells provide a useful system to study Ca2+ regulation via ion channels.     

Cadmium-resistant mutant of Bacillus subtilis 168 with reduced cadmium transport.

Cd2+ and Mn2+ accumulation was studied with wild-type Bacillus subtilis 168 and a Cd2+-resistant mutant. After 5 min of incubation in the presence of 0.1 microM 109Cd2+ or 54Mn2+, both strains accumulated comparable amounts of 54Mn2+, while the sensitive cells accumulated three times more 109Cd2+ than the Cd2+-resistant cells did. Both 54Mn2+ and 109Cd2+ uptake, which apparently occur by the same transport system, demonstrated cation specificity; 20 microM Mn2+ or Cd2+ (but not Zn2+) inhibited the uptake of 0.1 microM 109Cd2+ or 54Mn2+. 54Mn2+ and 109Cd2+ uptake was energy dependent and temperature sensitive, but 109Cd2+ uptake in the Cd2+-resistant strain was only partially inhibited by an uncoupler or by a decrease in temperature. 109Cd2+ uptake in the sensitive strain followed Michaelis-Menten kinetics with a Km of 1.8 microM Cd2+ and a Vmax of 1.5 mumol/min X g (dry weight); 109Cd2+ uptake in the Cd2+-resistant strain was not saturable. The apparent Km value for the saturable component of 109Cd2+ uptake by the Cd2+-resistant strain was very similar to that of the sensitive strain, but the Vmax was 25 times lower than the Vmax for the sensitive strain. The Km and Vmax for 54Mn2+ uptake by both strains were very similar. Cd2+ inhibition of 54Mn2+ uptake had an apparent Ki of 3.4 and 21.5 microM Cd2+ for the sensitive and Cd2+-resistant strains, respectively. Mn2+ had an apparent Ki of 1.2 microM Mn2+ for inhibition of 109Cd2+ uptake by the sensitive strain, but the Cd2+-resistant strain had no defined Ki value for inhibition of Cd2+ uptake by Mn2+.     

Characteristics of angiotensin II-, K+- and ACTH-induced calcium influx in adrenal glomerulosa cells. Evidence that angiotensin II, K+, and ACTH may open a common calcium channel

The characteristics of angiotensin II-, K+-, and adrenocorticotropin (ACTH)-induced calcium influx were studied in isolated adrenal glomerulosa cells. Basal calcium influx rate is 0.64 +/- 0.09 nmol/min/mg of protein. Addition of angiotensin II (1 nM) causes a rapid 230% increase in calcium influx rate. This angiotensin II-induced calcium influx is sustained and is rapidly reversed by angiotensin II antagonist, [Sar1,Ala8]angiotensin II. Addition of either K+ or ACTH (1 nM) causes a 340 or 160% increase, respectively, in the rate of calcium influx. The effect of either angiotensin II, K+, or ACTH on calcium influx is dependent on extracellular calcium. The apparent Km for calcium is 0.46, 0.35, and 0.32 mM, respectively. When the extracellular concentration of K+ is 2 mM, neither angiotensin II nor ACTH stimulates calcium influx. Conversely, when extracellular K+ is increased to 6 mM, both angiotensin II and ACTH cause a greater stimulation of calcium influx than at 4 mM K+. When extracellular K+ is increased to 10 mM, calcium influx is 360% of the basal influx seen at 4 mM K+, and neither angiotensin II nor ACTH further stimulates the influx rate. Nitrendipine (1 microM) blocks both angiotensin II- and K+-induced calcium influx completely. In contrast, 10 microM nitrendipine does not completely block ACTH-induced calcium influx. The calcium channel agonist, BAY K 8644, also stimulates calcium influx; 10 nM BAY K 8644 leads to a rate of calcium influx which is 185% of basal. This BAY K 8644-induced increase in calcium influx and that caused by either angiotensin II or ACTH are additive. In contrast, BAY K 8644 has more than an additive effect on the calcium influx when paired with 6 mM K+. These results suggest that angiotensin II, K+, and ACTH stimulate calcium influx via a common calcium channel but act by different mechanisms to alter its function.     

Protein kinase C mediated regulation of calcium channels in PC-12 pheochromocytoma cells

Depolarization of PC-12 pheochromocytoma cells with K+ produces an immediate increase in catecholamine release. The stimulation of release is blocked by Co2+, removal of extracellular Ca2+ or by dihydropyridine drugs such as nitrendipine. Release is enhanced by other dihydropyridines such as BAY K8644. Release is accompanied by a voltage dependent uptake of 45Ca2+ which is also blocked by Co2+ or nitrendipine and enhanced by BAY K8644. The phorbol ester phorbol 12-myristate-13-acetate (TPA) in the range 10(-9)-10(-6) M produced little effect by itself but augmented the K+ evoked release of catecholamine. An analog of TPA which does not activate protein kinase C was ineffective. In contrast, TPA in the same concentration range blocked influx of 45Ca2+ induced by 70 mM K+ or 70 mM K+/BAY K8644. 45Ca2+ influx produced by A23187 was not blocked by TPA. The results suggest a system by which protein kinase C may regulate the output of transmitters from secretory cells.     

Contribution of external and internal Ca2+ to changes in intracellular free Ca2+ produced by mitogens in Swiss 3T3 fibroblasts: the role of dihydropyridine sensitive Ca2+ channels

Changes in intracellular free Ca2+ concentration [( Ca2+]i) produced by growth factors and mitogens have been studied using aequorin-loaded Swiss 3T3 cells. Decreasing free Ca2+ in the external medium by using EGTA had no significant effect on the increase in [Ca2+]i produced by vasopressin, bradykinin, bombesin or prostaglandin E2, but reduced the increase in [Ca2+]i produced by platelet derived growth factor (PDGF) by 58%, by prostaglandin E1 44% and by prostaglandin F2 alpha 47%. The dihydropyridine Ca2+-channel antagonist nifedipine at 10 microM inhibited the [Ca2+]i response to PDGF by 41% in both the presence of and in the absence of external Ca2+. Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate (BAY K8644), a Ca2+-channel agonist, at 10 microM produced an increase in [Ca2+]i and decreased the [Ca2+]i response to PDGF by 39%. Nifedipine did not block 45Ca2+ uptake or release by inositol 1,4,5-trisphosphate in saponin-permeabilized Swiss 3T3 fibroblasts but BAY K8644 inhibited 45Ca2+ release by inositol 1,4,5-trisphosphate. The results suggest that the increase in [Ca2+]i caused by PDGF in Swiss 3T3 fibroblasts is due to the influx of external Ca2+ through dihydropyridine sensitive Ca2+ channels, as well as release of internal Ca2+.     

Stimulation of Calcium Uptake in PC12 Cells by the Dihydropyridine Agonist BAY K 8644

Abstract: Methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (BAY K 8644), an analog of dihydropyridine calcium channel antagonists, stimulated ⁴⁵Ca uptake into PC12 pheochromocytoma cells. Half-maximal stimulation occurred at 80 n M BAY K 8644. Enhancement of uptake was inhibited by cationic and organic calcium channel blockers, but not by tetrodotoxin, which is consistent with an effect on voltage-dependent calcium channels. Stimulation of ⁴⁵Ca uptake by BAY K 8644 occurred only at elevated concentrations of extracellular K⁺, suggesting that BAY K 8644 may interact with calcium channels in the open (activated) state.     

Ca2+ channels in chick neural retina cells characterized by 1,4-dihydropyridine antagonists and activators

The voltage-sensitive calcium channel in cultured chick neural retina cells was characterized by the actions of the enantiomers of Bay K 8644 and 202-791 and other 1,4-dihydropyridines. These cells showed time- and voltage-dependent Ca2+ uptake that was stimulated by K+ depolarization and blocked by the inorganic calcium channel blockers Cd2+ and Co2+. A small fraction only (15% maximum) of the uptake was inactivated by predepolarization of the cells with 80 mM K+. Ca2+ uptake was sensitive to the 1,4-dihydropyridine calcium channel antagonists and activators. (S)-Bay K 8644 and (S)-202-791 stimulated the Ca2+ uptake, and (R)-Bay K 8644 and (R)-202-791 as well as nitrendipine and PN 200-110 inhibited Ca2+ uptake stimulated by K+ depolarization or channel activators. The K+ depolarization-stimulated uptake was inhibited by 90%, but the activator-stimulated uptake was completely blocked by the 1,4-dihydropyridine antagonists. The potencies of these agents as inhibitors of Ca2+ uptake were significantly lower than the binding affinities in membrane preparations from the same cells or their binding and pharmacologic affinities in vascular smooth muscle. K+ depolarization or (S)-Bay K 8644 induced 45Ca2+ uptake was not observed in a glial cell culture. [3H]Nitrendipine and [3H]PN 200-110 bound to membrane preparations of the cells consistent with the presence of a single type of high affinity binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accumulation of cadmium in pancreatic beta cells is similar to that of calcium in being stimulated by both glucose and high potassium

The transport of Cd2+ and the effects of this ion on secretory activity and metabolism were investigated in beta cell-rich pancreatic islets isolated from obese-hyperglycemic mice. The endogenous cadmium content was 2.5 mumol/kg dry wt. After 60 min of incubation in a Ca2+-deficient medium containing 2.5 microM Cd2+ the islet cadmium content increased to 0.18 mmol/kg dry wt. This uptake was reduced by approx. 50% in the presence of 1.28 mM Ca2+. The incorporation of Cd2+ was stimulated either by raising the concentration of glucose to 20 mM or K+ to 30.9 mM. Whereas D-600 suppressed the stimulatory effect of glucose by 75%, it completely abolished that obtained with high K+. Only about 40% of the incorporated cadmium was mobilized during 60 min of incubation in a Cd2+-free medium containing 0.5 mM EGTA. It was possible to demonstrate a glucose-induced suppression of Cd2+ efflux into a Ca2+-deficient medium. Concentrations of Cd2+ up to 2.5 microM did not affect glucose oxidation, whereas, there was a progressive inhibition when the Cd2+ concentration was above 10 microM. Basal insulin release was stimulated by 5 microM Cd2+. At a concentration of 160 microM, Cd2+ did not affect basal insulin release but significantly inhibited the secretory response to glucose. It is concluded that the beta cell uptake of Cd2+ is facilitated by the activation of voltage-dependent Ca2+ channels. Apparently, the accumulation of Cd2+ mimics that of Ca2+ also involving a component of intracellular sequestration promoted by glucose.     

Signal transduction and ligand-receptor dynamics in the neutrophil. Ca2+ modulation and restoration

Intracellular Ca2+ rises when neutrophils are stimulated with formyl peptide ligands. There is enough Ca2+ released to complex approximately 200 microM Quin 2, (220 +/- 90 microM, 7 donors). This result is interpreted in terms of a fixed storage pool of Ca2+ of 44 pmol/10(6) cells. When extracellular Ca2+ is removed from the medium with 5 mM EGTA (final pH 7.4) just prior to cell stimulation, neither the magnitude nor the early time course of the Quin 2 response to formyl peptide is dramatically influenced. This result supports the concept that neither Ca2+ influx nor efflux, which are elevated in stimulated cells, contributes in a major way to the free Ca2+ pool which is monitored by Quin 2 during the early activation phase of cell responses. We have used intracellular Quin 2, and extracellular Ca2+ without the use of EGTA or ionophores to manipulate the levels of intracellular Ca2+. This is accomplished by depleting cells of intracellular Ca2+ by loading with Quin 2 in the absence of Ca2+. Intracellular Ca2+ is modulated by adding back Ca2+ to the medium. Using simultaneous analyses of cell function and Quin 2 fluorescence, we find that at least two aspects of cellular responsiveness (degranulation and O2- production) depend upon the level of available Ca2+. In contrast, the first phase, at least, of a biphasic rapid light scattering response which is related to actin polymerization is independent of Ca2+. We find that the Ca2+- sensitive cell responses can be partially restored in Ca2+-depleted cells if Ca2+ is provided within 30 s, a period which may reflect the putative lifetime of the transiently active ligand-receptor complex.     

Lead Enters Bovine Adrenal Medullary Cells Through Calcium Channels

Agents that stimulate secretion also accelerate the rate of Pb uptake into adrenal medullary cells. For example, when cells are suspended in a medium containing 5 microM Pb2+, depolarization by 77 mM K increases the rate of Pb uptake from 12 +/- 1 to 47 +/- 5 mumol/(L cells X min). K-induced Pb uptake has an apparent Km for Pb2+ of 2.6 microM, and is antagonized by Ca2+ with a K0.5 of 1.4 mM. The Ca channel blocker D-600 inhibits Pb entry with a K0.5 of 0.4 microM. Pb uptake is also stimulated by the Ca channel agonist BAY K 8644. These observations suggest that Pb passes through Ca channels. The permeability of the channels to Pb appears to be at least 10 times the permeability to Ca.     

Ca2+ channel modulation and kinase-C activation in a pituitary cell line: induction of immediate early genes and inhibition of proliferation.

We have studied the interaction between dihydropyridine (DHP) Ca2+ modulators and the phorbol ester phorbol 12-myristate 13-acetate (PMA) on whole cell Ca2+ currents, 45Ca2+ uptake, immediate early gene (IEG) expression, and proliferation in the rat pituitary GH4C1 cell line. When short (3- to 5-msec) depolarizing voltage clamp steps were used to activate L-type Ca2+ channels, the DHP Ca2+ agonist (-)Bay K 8644 markedly enhanced Ca2+ entry by slowing channel closing upon repolarization. In contrast, the Ca2+ agonist induced only small and inconsistent increases in c-fos mRNA and did not measurably increase NGFI-A. Ca2+ channel activation by depolarization with 50 mM KCl in the presence of (-)Bay K 8644 induced large increases in 45Ca2+ uptake, but failed to markedly induce either of the IEGs. The phorbol ester PMA did not alter T- or L-type Ca2+ current or 45Ca2+ uptake by GH4C1 cells, but triggered large increases in both c-fos and NGFI-A mRNA. In combination, PMA and (-)Bay K 8644 acted synergistically to increase mRNAs for both IEGs. The effect of the DHPs was stereospecific; (+)Bay K 8644, a Ca2+ antagonist, inhibited PMA-induced increases in c-fos and NGFI-A mRNAs. Both PMA and (-)Bay K 8644 inhibited the proliferation of GH4C1 cells, measured by cell count or [3H]thymidine incorporation. The inhibition by the Ca2+ agonist was stereoselective and approximately additive to that of PMA. These results indicate that the expression of c-fos IEG and that of NGFI-A IEG are differentially regulated by separate second messenger pathways in GH4C1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cadmium uptake in Escherichia coli K-12.

109Cd2+ uptake by Escherichia coli occurred by means of an active transport system which has a Km of 2.1 microM Cd2+ and a Vmax of 0.83 mumol/min X g (dry weight) in uptake buffer. 109Cd2+ accumulation was both energy dependent and temperature sensitive. The addition of 20 microM Cd2+ or Zn2+ (but not Mn2+) to the cell suspensions preloaded with 109Cd2+ caused the exchange of Cd2+. 109Cd2+ (0.1 microM) uptake by cells was inhibited by the addition of 20 microM Zn2+ but not Mn2+. Zn2+ was a competitive inhibitor of 109Cd2+ uptake with an apparent Ki of 4.6 microM Zn2+. Although Mn2+ did not inhibit 109Cd2+ uptake, the addition of either 20 microM Cd2+ or Zn2+ prevented the uptake of 0.1 microM 54Mn2+, which apparently occurs by a separate transport system. The inhibition of 54Mn2+ accumulation by Cd2+ or Zn2+ did not follow Michaelis-Menten kinetics and had no defined Ki values. Co2+ was a competitive inhibitor of Mn2+ uptake with an apparent Ki of 34 microM Co2+. We were unable to demonstrate an active transport system for 65Zn2+ in E. coli.     

Characterization of Ca2+-stimulated secretion in permeable GH3 pituitary cells

In this report, the secretory response to Ca2+ in GH3 rat pituitary cells permeabilized by electric field discharge has been compared in both magnitude and Ca2+ sensitivity to prolactin (PRL) release from intact GH3 cells. The half-maximally effective [Ca2+] for stimulating PRL release in permeable cells was approximately 0.5 microM, and maximal stimulation was obtained at 3-10 microM Ca2+. The magnitude of Ca2+ stimulation in permeable cells was in the same range as that obtained from an equal number of intact cells stimulated by depolarizing K+. Moreover, the Ca2+ sensitivity of PRL release in intact GH3 cells (measured by Quin 2 fluorescence) closely resembled the Ca2+ sensitivity determined in permeable cells. Release of a sulfated proteoglycan whose release is stimulated by secretagogues in intact cells was stimulated by Ca2+ in permeable cells with the same Ca2+ sensitivity as for PRL release. Maximal Ca2+ stimulation of PRL release in permeable cells required the addition of MgATP. Other energy sources (ADP, GTP, and inorganic phosphate) also supported Ca2+-stimulated secretion but were less effective. The above results indicated that PRL release from permeable cells resembles the physiological process in intact cells. The permeable cell system should prove useful in investigating the mechanism mediating the effect of Ca2+ on secretion, although our studies with pharmacological agents have so far proved inconclusive. Among calmodulin antagonists tested, only trifluoroperazine inhibited Ca2+-stimulated secretion, whereas pimozide and calmidazolium did not.