Cholera toxin increases the rate of antigen-stimulated calcium influx in rat basophilic leukemia cells

Cholera toxin pretreatment has been found to cause a 3-fold increase in the initial rate of antigen-stimulated secretion of serotonin from rat basophilic leukemia (RBL) cells. Under similar conditions, cholera toxin enhances the antigen-stimulated rise in cytoplasmic free ionized calcium levels and causes a 2-3-fold increase in the rate of antigen-stimulated influx of 45Ca. In intact RBL cells cholera toxin pretreatment potentiates the antigen-stimulated production of inositol phosphates, but in permeabilized cells, with strongly buffered free calcium levels, no effect of cholera toxin pretreatment on the antigen-stimulated activation of cellular phospholipase activities is observed. In addition, pretreatment of cells with tetradecanoylphorbol acetate inhibits antigen-stimulated production of inositol phosphates by greater than 95%, while the stimulated influx of 45Ca remains unaffected. These data indicate that the antigen-stimulated influx of calcium into RBL cells can be dissociated from the production of inositol phosphates in these cells. The observed effects of cholera toxin on exocytosis and Ca2+ influx in RBL cells are not due to the elevation of cellular cyclic AMP levels since a variety of agents capable of elevating cellular cyclic AMP levels do not mimic these effects. Together, these data suggest that a cholera toxin-sensitive guanine nucleotide-binding protein is involved in the pathway responsible for the antigen-stimulated influx of calcium into RBL cells.     

Serotonin stimulates calcium influx in isolated rat adrenal zona glomerulosa cells.

To investigate the role of calcium as a second messenger in serotonin-stimulated aldosterone secretion, radiolabelled calcium influx studies were carried out in purified rat adrenal zona glomerulosa cells using 45CaCl2. The results show that serotonin caused calcium influx within 45 seconds of addition and this continued for up to 105 seconds. Angiotensin II also caused calcium influx; however, the effect was significantly smaller than that of serotonin. Serotonin-stimulated calcium influx could be inhibited by the calcium antagonist verapamil and by methysergide, a selective serotonin receptor type-1/2 antagonist. The data indicate that serotonin directly stimulates calcium uptake in zona glomerulosa cells via calcium channels which are coupled to specific serotonin receptors.     

Role of increase in intracellular calcium in PTH-induced homologous desensitization in UMR-106 cells.

Effects of increase in intracellular calcium on PTH-induced homologous desensitization were investigated using calcium ionophores. Pretreatment of UMR-106 cells (rat osteoblast like osteosarcoma cell line) with calcium ionophores (A23187 or ionomycin) for 6h resulted in approximately 50% decrease of PTH-stimulated cAMP production. PTH receptor binding, assessed with 125I-[Nle8,Nle18,Tyr34]PTH-(1-34) as radioligand, was significantly decreased in 10(-6) M calcium ionophore-pretreated (for 6h) cells without affecting the dissociation constant (Kd) for PTH. Minimal effective treatment period was 2h and similar inhibitory effect was observed in 12h-treated cells. These data suggest that increase in intracellular calcium might also act on PTH receptor in the similar manner as protein kinase C activation to induce desensitization.     

Exogenous ATP-stimulated calcium uptake in isolated rat intestinal epithelial cells

ATP in the extracellular medium is known to stimulate Ca uptake into avian intestinal epithelial cells. We have now demonstrated a similar effect of ATP in mammalian intestinal epithelial cells and have further characterized this effect. Exogenous ATP increased 45Ca uptake 2-6 fold in isolated rat small intestinal epithelial cells, with a maximal effect at 1 mM and an ED50 of 290 microM. A strict structural requirement for nucleotide-stimulated 45Ca uptake was observed. ADP was much less effective than ATP and gamma-thio-ATP, and 5'-AMP, cyclic AMP, adenosine, non-adenine nucleotides, non-hydrolyzable ATP analogs and ATP analogs with ring substitutions at the 8 position were inactive. Prenylamine (100 microM) completely inhibited ATP-stimulated 45Ca uptake, while verapamil (100 microM) had only a small effect. In the intact intestine, ATP increased short-circuit current (Isc) when added to the mucosal side of the tissue. This effect was reduced by 10 microM and abolished by 100 microM prenylamine. The effect of ATP on Isc was markedly reduced in Cl-free solutions and in reduced-Ca solutions. Serosal and mucosal addition of the nonhydrolyzable ATP analog, beta, gamma-methylene-ATP, and serosal addition of ATP had little or no effect on Isc. The similarities between the effects of ATP in isolated cells and in the intact intestine suggest that the effect of ATP on Isc may be at least partially mediated through stimulation of Ca uptake into the epithelial cells.     

Role of capacitative calcium entry on glutamate-induced calcium influx in type-I rat cortical astrocytes

Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross-talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL-2H3), a model where calcium release-activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL-2H3 cells and (ii) glutamate-induced Ca2+ influx in astrocytes plays a primary role in glutamate-dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL-2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.     

Inhibition of transmembrane calcium influx induces decrease in proteoglycan synthesis in immature rat sertoli cells

Beyond increased cAMP synthesis, calcium influx has been involved in signal transduction triggered by the gonadotropin follicle‐stimulating hormone (FSH), the main regulator of Sertoli cells functions. In order to delineate a possible involvement of calcium in the regulation of proteoglycan synthesis, we have examined the effect of low‐voltage‐activated calcium channel blocker verapamil on both [³⁵S]‐sulfate and [³H]‐glucosamine incorporation into proteoglycan molecules neosynthesized by cultured Sertoli cells from 20‐day‐old rats. Verapamil induced a dose‐ and time‐dependent decrease in labeling of both secreted and cell‐associated proteoglycans, as determined by quantitative solid‐phase assay. This effect was mimicked by the addition of the calcium chelator EGTA, suggesting that verapamil effect resulted from the inhibition of transmembrane calcium influx. The decrease in apparent proteoglycan synthesis appeared to be attributable primarily to a lowering of the glycanation process, as shown by experiments using an exogenous acceptor for glycosaminoglycan synthesis. Moreover, verapamil induced a decrease in relative proportion of heparan sulfate proteoglycans in the cell layer. Pulse‐chase kinetics demonstrated that verapamil also altered proteoglycan catabolism, leading to glycosaminoglycan retention in the cell layer and inhibiting the proteoglycan desulfation step. We conclude that intracellular calcium is essential to maintain Sertoli cell proteoglycan expression and could thus be involved in the repression of Sertoli cell cAMP‐dependent syntheses such as estradiol production. J. Cell. Biochem. 76:322–331, 1999. © 1999 Wiley‐Liss, Inc.     

Inhibition of transmembrane calcium influx induces decrease in proteoglycan synthesis in immature rat Sertoli cells

Beyond increased cAMP synthesis, calcium influx has been involved in signal transduction triggered by the gonadotropin follicle-stimulating hormone (FSH), the main regulator of Sertoli cells functions. In order to delineate a possible involvement of calcium in the regulation of proteoglycan synthesis, we have examined the effect of low-voltage-activated calcium channel blocker verapamil on both [(35)S]-sulfate and [(3)H]-glucosamine incorporation into proteoglycan molecules neosynthesized by cultured Sertoli cells from 20-day-old rats. Verapamil induced a dose- and time-dependent decrease in labeling of both secreted and cell-associated proteoglycans, as determined by quantitative solid-phase assay. This effect was mimicked by the addition of the calcium chelator EGTA, suggesting that verapamil effect resulted from the inhibition of transmembrane calcium influx. The decrease in apparent proteoglycan synthesis appeared to be attributable primarily to a lowering of the glycanation process, as shown by experiments using an exogenous acceptor for glycosaminoglycan synthesis. Moreover, verapamil induced a decrease in relative proportion of heparan sulfate proteoglycans in the cell layer. Pulse-chase kinetics demonstrated that verapamil also altered proteoglycan catabolism, leading to glycosaminoglycan retention in the cell layer and inhibiting the proteoglycan desulfation step. We conclude that intracellular calcium is essential to maintain Sertoli cell proteoglycan expression and could thus be involved in the repression of Sertoli cell cAMP-dependent syntheses such as estradiol production.     

IgE receptor-activated calcium permeability pathway in rat basophilic leukemia cells: measurement of the unidirectional influx of calcium using quin2-buffered cells

The intracellular calcium indicator and buffer quin2 has been used to generate a large calcium buffering capacity in the cytoplasm of rat basophilic leukemia cells. Above 3 mM intracellular quin2, there is no further increase in the initial rate of antigen-induced 45Ca uptake, suggesting that 45Ca buffering by quin2 is now sufficient to prevent the immediate efflux of 45Ca from the cells. Thus, the initial rate of 45Ca uptake should reflect the true unidirectional influx of calcium that occurs when immunoglobulin E (IgE) receptors are aggregated by antigen. The antigen-induced calcium permeability pathway appears to be saturable, with a Km of about 0.7 mM and a Vmax of 0.9 nmol of calcium (10(6) cells)-1 min-1. Although net 45Ca uptake reaches a plateau a few minutes after antigen stimulation, the increase in plasma membrane permeability is maintained for at least an hour, provided that receptors for IgE remain aggregated. The initial rate of 45Ca influx correlates well with the subsequent secretion of [3H]serotonin in response to different concentrations of antigen. Both 45Ca uptake and [3H]serotonin secretion are maximal when only 10% of the receptors are occupied with antigen-specific IgE. Thus, 45Ca influx correlates more closely with secretion than with the number of IgE receptors aggregated by antigen.     

Effect of aging on populations of estrogen receptor-containing cells in the rat uterus

Estrogen receptor-containing cells were identified in uteri of mature and senescent rats by means of quantitative autoradiography. Cell numbers and density were assessed in the various uterine layers. It was determined that despite decreases in receptor concentrations in endometrium, endometrial stroma, myometrium, and epithelium, no significant cell loss occurred in any region. Receptor content per cell clearly decreased with age in the endometrial stroma, while only in the myometrium was there any suggestion of cell loss in the absence of receptor loss from individual cells. Thus, age-related reductions in uterine estradiol receptor levels may be due to different mechanisms in different tissue regions.     

Effect of calcium on rat intestinal alkaline phosphatase activity and molecular aggregation

Two fractions of rat intestinal alkaline phosphatase (IAP) were detected by Western blot: 168 ± 6 and 475 ± 45 kDa. The low molecular weight fraction constitutes 43% of the isolated proteins exhibiting 82% of the enzymatic activity, and a heavier fraction constitutes 57% of the isolated proteins and has 18% of the enzymatic activity. Calcium produced an increase of the 475-kDa form to the detriment of the 168-kDa form. This work also describes the kinetic and structural changes of IAP as a function of calcium concentration. With [Ca²⁺] < 10 mmole/L, the Ca²⁺-IAP interaction fitted a binding model with 7.8 ± 4.4 moles of Ca²⁺ /mole of protein, affinity constant = 19.1 ± 8.4 L/mmole, and enzymatic activity increased as a linear function of [Ca²⁺] (r = 0.946 p < 0.01). On the other hand, with [Ca²⁺] >10 mmole/L the data did not fit this model and, the enzymatic activity decreased as a function of [Ca²⁺] (r = ? 0.703 p < 0.05).     

Effect of calcium on rat intestinal alkaline phosphatase activity and molecular aggregation

Two fractions of rat intestinal alkaline phosphatase (IAP) were detected by Western blot: 168 +/- 6 and 475 +/- 45 kDa. The low molecular weight fraction constitutes 43% of the isolated proteins exhibiting 82% of the enzymatic activity, and a heavier fraction constitutes 57% of the isolated proteins and has 18% of the enzymatic activity. Calcium produced an increase of the 475-kDa form to the detriment of the 168-kDa form. This work also describes the kinetic and structural changes of IAP as a function of calcium concentration. With [Ca2+] < 10 mmole/L, the Ca(2+)-IAP interaction fitted a binding model with 7.8 +/- 4.4 moles of Ca2+ /mole of protein, affinity constant = 19.1 +/- 8.4 L/mmole, and enzymatic activity increased as a linear function of [Ca2+] (r = 0.946 p < 0.01). On the other hand, with [Ca2+] > 10 mmole/L the data did not fit this model and, the enzymatic activity decreased as a function of [Ca2+] (r = - 0.703 p < 0.05).     

Lanthanum increases the rat thymocyte cytoplasmic free calcium concentration by enhancing calcium influx

In the present study, I have examined the effect of lanthanum (La3+) on cytoplasmic free calcium concentration in isolated rat thymocytes employing the quin2 technique. As with its effect on 15Ca accumulation in rat thymocytes (Segal, J. and Ingbar, S.H. (1984) Endocrinology, 115, 160-166), La3+ produced a concentration-related increase in thymocyte cytoplasmic free calcium concentration. This effect of La3+ was very prompt in onset, evident within about 30 s from the time of addition of La3+. The lowest effective concentration of La3+ was 6 microM (+22.7% above control), and it increased progressively to reach maximal values at 25 microM (+100% above control). La3+ added to quin2-loaded thymocytes suspended in a calcium-free medium was without effect. In addition, La3+ had no significant effect on 45Ca efflux, and La3+ did not inhibit calcium-ATPase activity in the rat thymocytes. These results demonstrate that in rat thymocytes La3+ increases cytoplasmic free calcium concentration by increasing the extracellular calcium influx into the cell rather than the release of calcium from an intracellular pool.     

Properties of the basal calcium influx in human red blood cells

The basal ⁴⁵Ca²⁺ influx in human red blood cells (RBC) into intact RBC was measured. ⁴⁵Ca²⁺ was equilibrated with cells with t_1/2=15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5±0.2 μmol/l_{packed cells} (n=6) at 37 °C. The average value of the Ca²⁺ influx rate was 43.2±8.9 μmol/l_{packed cells} hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 °C. The basal Ca²⁺ influx was saturable with Ca²⁺ up to 5 mmol/l but at higher extracellular Ca²⁺ concentrations caused further increase of basal Ca²⁺ influx. The ⁴⁵Ca²⁺ influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3′,4′,5-tetrachloro-salicylanilide (TCS) 10⁻⁶-10⁻⁵ mol/l) inhibited in part the Ca²⁺ influx. The results show that the basal Ca²⁺ influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca²⁺ influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca²⁺ are coupled via the RBC H⁺ homeostasis.     

Properties of the basal calcium influx in human red blood cells

The basal (45)Ca(2+) influx in human red blood cells (RBC) into intact RBC was measured. (45)Ca(2+) was equilibrated with cells with t(1/2)=15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5+/-0.2 micromol/l(packed cells) (n=6) at 37 degrees C. The average value of the Ca(2+) influx rate was 43.2+/-8.9 micromol/l(packed cells) hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 degrees C. The basal Ca(2+) influx was saturable with Ca(2+) up to 5 mmol/l but at higher extracellular Ca(2+) concentrations caused further increase of basal Ca(2+) influx. The (45)Ca(2+) influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3',4',5-tetrachloro-salicylanilide (TCS) 10(-6)-10(-5) mol/l) inhibited in part the Ca(2+) influx. The results show that the basal Ca(2+) influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca(2+) influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca(2+) are coupled via the RBC H(+) homeostasis.     

The kinetics of calcium influx into rat liver slices

The unidirectional influx of calcium across rat liver slices is a carrier-mediated process which displays saturation kinetics. The presence of Mg2+ in the incubation medium competitively inhibits calcium influx into rat liver slices. Metabolic inhibitors such as ouabain and 2,4-dinitrophenol at a concentration of 1 X 10(-4) and 2.5 X 10(-4) M respectively, inhibited significantly (P less than 0.001) calcium influx across the liver slices. Calcium influx is dependent on the presence of sodium in the extracellular medium, and is significantly reduced (P less than 0.01) when sodium concentration in the preincubation solution is reduced to zero.     

Effect of intracellular pH on depolarization-evoked calcium influx in human sperm

Human sperm are endowed with putative voltage-dependent calcium channels (VDCC) that produce measurable increases in intracellular calcium concentration ([Ca²⁺]_i) in response to membrane depolarization with potassium. These channels are blocked by nickel, inactivate in 1–2 min in calcium-deprived medium, and are remarkably stimulated by NH₄Cl, suggesting a role for intracellular pH (pH_i). In a previous work, we showed that calcium permeability through these channels increases approximately onefold during in vitro "capacitation," a calcium-dependent process that sperm require to fertilize eggs. In this work, we have determined the pH_i dependence of sperm VDCC. Simultaneous depolarization and pH_i alkalinization with NH₄Cl induced an [Ca²⁺]_i increase that depended on the amount of NH₄Cl added. VDCC stimulation as a function of pH_i showed a sigmoid curve in the 6.6–7.2 pH_i range, with a half-maximum stimulation at pH 7.00. At higher pH_i (7.3), a further stimulation occurred. Calcium release from internal stores did not contribute to the stimulating effect of pH_i because the [Ca²⁺]_i increase induced by progesterone, which opens a calcium permeability pathway that does not involve gating of VDCC, was unaffected by ammonium. The ratio of pH_i-stimulated-to-nonstimulated calcium influx was nearly constant at different test depolarization values. Likewise, depolarization-induced calcium influx in pH_i-stimulated and nonstimulated cells was equally blocked by nickel. In our capacitating conditions pH_i increased 0.11 pH units, suggesting that the calcium influx stimulation observed during sperm capacitation might be partially caused by pH_i alkalinization. Additionally, a calcium permeability pathway triggered exclusively by pH_i alkalinization was detected. mammalian sperm; capacitation; intracellular calcium     

Effect of age, vitamin D, and calcium on the regulation of rat intestinal epithelial calcium channels

Transepithelial transport of calcium involves uptake at the apical membrane, movement across the cell, and extrusion at the basolateral membrane. Active vitamin D metabolites regulate the latter two processes by induction of calbindin D and the plasma membrane ATPase (calcium pump), respectively. The expression of calbindin D and the calcium pump declines with age in parallel with transepithelial calcium transport. The apical uptake of calcium is thought to be mediated by the recently cloned calcium channels-CaT1 (or ECaC2, TRPV6) and CaT2 (or ECaC1, TRPV5). The purpose of these studies was to determine whether there were age-related changes in intestinal calcium channel regulation and to identify the dietary factors responsible for their regulation. Young (2 months) and adult (12 months) rats were fed either a high calcium or low calcium diet for 4 weeks. The low calcium diet significantly increased duodenal CaT1 and CaT2 mRNA levels in both age groups, but the levels in the adult were less than half that of the young. The changes in calcium channel expression with age and diet were significantly correlated with duodenal calcium transport and with calbindin D levels. To elucidate the relative roles of serum 1,25(OH)2D3 and calcium in the regulation of calcium channel expression, young rats were fed diets containing varying amounts of calcium and vitamin D. Dietary vitamin D or exogenous 1,25(OH)2D3 more than doubled CaT1 mRNA levels, and this regulation was independent of dietary or serum calcium. These findings suggest that the apical calcium channels, along with calbindin and the calcium pump, may play a role in intestinal calcium transport and its modulation by age, dietary calcium, and 1,25(OH)2D3.     

Somatostatin and intestinal calcium transport in the rat

In intact rats we studied the influence of low doses of intravenously (i.v.) administered somatostatin (SRIF) on the net absorption and the bidirectional fluxes (lumen-to-plasma, LP; plasma-to-lumen, PL) of calcium in the duodenum, jejunum, ileum and caecum. In the duodenum SRIF inhibited the LP-flux and the net absorption of Ca significantly at infusion rates of 0.75 and 1.0 microgram SRIF . kg-1 . h-1. The PL-flux was not altered by any of the SRIF doses administered. In the other gut segments studied (jejunum, ileum, caecum) neither the net absorption nor the bidirectional Ca fluxes were changed by i.v. SRIF. It is concluded that SRIF in the plasma levels achieved in this study has an influence on the duodenal calcium absorption (CaA) of the rat; questions regarding the mechanisms of this action as well as the physiological significance of our findings are as yet unresolved.