12-O-tetradecanoyl-phorbol-13-acetate decreases influx of extracellular Ca2+ induced by depolarization in GH4C1 cells: effects of pretreatment with 1,25-dihydroxycholecalciferol.

In GH4C1 rat pituitary cells, 1,25-dihydroxycholecalciferol (1,25(OH)2D3) causes amplification of both the TRH-induced spike phase in cytosolic free calcium [( Ca2+]i) and the increase in [Ca2+]i induced by depolarization with K+. In the present study we investigated the actions of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on Ca2(+)-homeostasis in GH4C1 cells pretreated with 1,25(OH)2D3 for 24 h. In control and 1,25(OH)2D3-pretreated cells, incubation with TPA (0.1-300 nM) for 15 min in the presence of 45Ca2+ did not affect the basal uptake of 45Ca2+. However, if the cells were treated with 50 mM K+, TPA induced a time- and concentration-dependent decrease in depolarization-induced net 45Ca2+ uptake. A maximal decrease of 30-50% was observed with 100-300 nM TPA, 1,25(OH)2D3 pretreated cells being more responsive to the action of TPA than control cells. sn-1-Oleoyl-2-acetyl-glycerol, which mimics the action of TPA on protein kinase C (PKC), did not alter depolarization-induced uptake of 45Ca2+. Two agents which inhibit PKC activity, polymyxin B and K252A, did not prevent the effect of TPA on depolarization-induced uptake of 45Ca2+, whereas staurosporin totally inhibited the action of TPA. In Fura-2 loaded cells pretreated with 1,25(OH)2D3, incubation with 200 nM TPA for 9 min decreased the depolarization-induced spike and plateau phases of change in [Ca2+]i; only the spike phase was decreased in control cells. TPA did not affect basal [Ca2+]i in either group. Treatment with TPA for only 3 min decreased the TRH-induced spike in [Ca2+]i only in 1,25(OH)2D3 pretreated cells; however, after a 5-min treatment with TPA, the TRH-induced spike in [Ca2+]i was decreased in both control and 1,25(OH)2D3 pretreated cells. TPA did not affect the spike in [Ca2+] induced by 50 nM ionomycin. Na+/Ca2+ exchange was not altered by TPA, nor did TPA enhance efflux of 45Ca2+ from cells preloaded with 45Ca2+ for 2.5 h. We conclude that, in GH4C1 cells, TPA modulates plasma membrane calcium flux, probably via an inhibitory action on voltage-operated Ca2+ channels. This inhibitory action may be independent of activation of PKC, and 1,25(OH)2D3 pretreated cells are more responsive to the actions of TPA than are control cells. These results are consistent with our previous findings that 1,25(OH)2D3 enhances voltage-dependent Ca2+ channel activity in GH4C1 cells.     

Influx of extracellular calcium mediates 1,25-dihydroxyvitamin D3-dependent transcaltachia (the rapid stimulation of duodenal Ca2+ transport)

We investigated the role of extracellular Ca2+ in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] rapid stimulation of intestinal Ca2+ transport (termed transcaltachia) in the perfused duodenal of vitamin D-replete chicks. The carboxylic ionophore ionomycin (2 microM) was found to stimulate 45Ca2+ transport from the lumen to the vascular effluent to the same extent as physiological levels of 1,25-(OH)2D3. The increase in duodenal 45Ca2+ transport caused by 1,25-(OH)2D3 was dependent on the presence of medium Ca2+, since it was abolished by prior addition of EGTA and was restored upon the addition of Ca2+. Depolarization of the basal lateral membrane of intestinal epithelial cells with 70 mM K+ caused a rapid increase in 45Ca2+ transport (30% above control values within 2 min and 250% after 20 min of vascular perfusion). The rise was also abolished by prior addition of EGTA. Intracellular calcium concentrations ([Ca2+]i) were measured in isolated duodenal cells from vitamin D-replete chicks using the fluorescent dye fura 2. A 1-min incubation with physiological concentrations of 1,25-(OH)2D3 (130 pM) caused an increase in [Ca2+]i from a basal level of 168 +/- 23 nM to 363 +/- 44 nM. Pretreatment of intestinal epithelial cells with the protein kinase-C activator tetradeconyl-phorbol acetate (100 nM) or the adenylate cyclase activator forskolin (10 microM), both shown to induce acute stimulation of intestinal 45Ca2+ transport in the perfused duodenum, also mimicked the stimulatory effect of 1,25-(OH)2D3 on [Ca2+]i. The increase in [Ca2+]i elicited by the 1,25-(OH)2D3 was due to Ca2+ influx from the extracellular medium, since it was blocked by the Ca2+ chelator EGTA (5 mM) and the Ca2+ channel antagonist nifedipine (1 microM). These results suggest that the acute effects of 1,25-(OH)2D3 on duodenal 45Ca2+ transport are triggered by the influx of Ca2+ through voltage-operated Ca2+ channels and that both protein kinase-C and protein kinase-A play an important role in mediating or modulating 1,25-(OH)2D3 effects on transcaltachia.     

1,25-dihydroxyvitamin D3 inhibits Na(+)-H+ exchange by stimulating membrane phosphoinositide turnover and increasing cytosolic calcium in CaCo-2 cells.

We have examined the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the phosphoinositol signal transduction pathway in the human colon cancer-derived cell line CaCo-2 and have studied the regulation of intracellular calcium ([Ca2+]i) and pH (pHi) by this secosteroid. CaCo-2 cells were prelabeled with [3H]myoinositol and treated with 10(-8) M 1,25-(OH)2D3 or vehicle for 90 sec. 1,25-(OH)2D3 caused a decrease in labeled phosphatidylinositol-4-5-bis-phosphate and an increase in labeled inositol 1,4,5-trisphosphate. Treatment with 10(-8) M 1,25-(OH)2D3 for 90 sec also raised the cellular content of diacylglycerol. In a dose-dependent manner, 1,25-(OH)2D3 caused the translocation of protein kinase-C activity from the cytosolic to the membrane fraction, which occurred after as little as 15 sec of exposure to the secosteroid, peaked at about 1-5 min, and then returned toward baseline values. In these CaCo-2 cells, baseline [Ca2+]i was 258 +/- 2 nM (mean +/- SE), as assessed using the fluorescent dye fura-2. After exposure to 10(-8) M 1,25-(OH)2D3, [Ca2+]i rapidly increased to 392 +/- 14 nM after 100 sec, fell, and then subsequently rose to a plateau of 350 +/- 3 nM after 400 sec. In Ca(2+)-free buffer, 1,25-(OH)2D3 caused only a transient rise in [Ca2+]i, indicating that 1,25-(OH)2D3 stimulated both the release of intracellular calcium stores and calcium influx. 1,25-(OH)2D3 caused a dose-dependent decrease in pHi in CaCo-2 cells, as assessed by the fluorescent dye BCECF, which was not observed in cells suspended in Na(+)-free buffer or pretreated with amiloride, indicating that the secosteroid inhibited Na(+)-H+ exchange. No effect of 1,25-(OH)2D3 on pHi was observed in cells in a Ca(2+)-free buffer or pretreated with the phospholipase-C inhibitor U-73,122, which also blocked the rise in [Ca2+]i, or in cells pretreated with the Ca2+/calmodulin inhibitor calmidazolium. Taken together, these studies indicate that 1,25-(OH)2D3 rapidly stimulates membrane phosphoinositide breakdown in CaCo-2 cells, generating the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, causing translocation of protein kinase-C to the membrane, and increasing [Ca2+]i by both releasing calcium stores and promoting calcium influx. Secondary to the rise in [Ca2+]i, Na(+)-H+ exchange is inhibited by a calcium/calmodulin-dependent pathway.     

1,25-Dihydroxycholecalciferol enhances both the bombesin-induced transient in intracellular free Ca2+ and the bombesin-induced secretion of prolactin in GH4C1 pituitary cells

In GH4C1 rat pituitary cells, 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] enhances both the synthesis of PRL and the TRH-induced transient increase in cytosolic free calcium ( [Ca2+]i). In the present report we investigated whether 1,25-(OH)2D3 could enhance the effect of the tetradecapeptide bombesin (BBS) in GH4C1 cells. Pretreatment of the cells with 1 nM 1,25-(OH)2D3 for 24 h enhanced the BBS-induced transient increase in [Ca2+]i compared to that in control cells, while having no significant effect on the plateau phase of [Ca2+]i. Addition of the Ca2+ channel blocker nimodipine or chelating extracellular Ca2+ with EGTA did not abolish the enhancement of the BBS response in 1,25-(OH)2D3-pretreated cells. Furthermore, the BBS-induced efflux of 45Ca2+ from cells preequilibrated with 45Ca2+ was larger in cells treated with 1,25-(OH)2D3. Incubating GH4C1 cells with 1,25-(OH)2D3 alone or in combination with BBS for up to 72 h did not stimulate synthesis of PRL. However, the BBS-induced secretion of PRL was enhanced in cells pretreated with 1,25-(OH)2D3 for 24 h compared with that in vehicle-treated control cells. The effect of 1,25-(OH)2D3 on BBS-induced secretion was dose dependent, with 10(-11) M 1,25-(OH)2D3 enhancing the stimulated secretion of PRL. We conclude that in GH4C1 cells, pretreatment with 1,25-(OH)2D3 enhances the BBS-induced transient increase in [Ca2+]i. This effect may be due to a modulation of the availability of sequestered intracellular Ca2+ and/or membrane Ca2+ conductance. Furthermore, pretreatment with 1,25-(OH)2D3 enhanced secretion of PRL stimulated by BBS. The enhanced transient increase in [Ca2+]i may be the factor inducing the enhanced BBS-induced secretion of PRL.     

The involvement of calcium ions in the effect of 1,25-dihydroxyvitamin D3 on HL-60 cells

1,25-Dihydroxyvitamin D3 (1,25[OH]2D3) was found to suppress growth of human leukemic cells (HL-60), and to induce the differentiation of these cells to monocyte-like cells. The purpose of the present study was to examine the role of calcium ions in the effects of 1,25(OH)2D3 on HL-60 cells. Incubation of the HL-60 cells with 1,25(OH)2D3 (10(-7) M) for 4 days caused a significant inhibition of 50% of cell growth. The number of differentiated cells increased simultaneously from 24 x 10(3) +/- 2 x 10(3) in the controls to 658 x 10(3) +/- 32 x 10(3) in the 1,25(OH)2D3 (10(-7) M)-treated cells. The role of calcium ions in the effects of 1,25(OH)2D3 on HL-60 cells was first studied by changing the available calcium in the medium and by measuring the effect of 1,25(OH)2D3 on intracellular Ca2+ levels. Limitation of the available Ca2+ by means of ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or verapamil enhanced the inhibitory effect on proliferation and decreased the number of differentiated cells obtained by 1,25(OH)2D3 alone. These effects could be abolished by restoring the Ca2+ levels. The role of the intracellular free Ca2+ ions in the effect of 1,25(OH)2D3 was further illustrated by measuring the intracellular Ca2+ levels. The intracellular free Ca2+ concentration in 1,25(OH)2D3 (10(-7) M)-treated HL-60 cells rose significantly from 117.0 +/- 6.3 nM in the untreated HL-60 cells to 145.0 +/- 7.5 nM in the treated cells (p less than 0.02). Addition of verapamil moderated the increase in intracellular free Ca2+ (125.0 +/- 5.2 nM) obtained by 1,25(OH)2D3 alone. Thus the elevation of intracellular free Ca2+ caused by 1,25(OH)2D3 treatment may be involved in the effect of the hormone on the HL-60 cells.     

Pretreatment with 1,25-dihydroxycholecalciferol enhances thyrotropin-releasing hormone- and inositol 1,4,5-trisphosphate-induced release of sequestered Ca2+ in permeabilized GH4C1 pituitary cells.

In GH4C1 cells 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] has been shown to enhance the TRH- and bombesin-induced increase in intracellular Ca2+ ([Ca2+]i). The aim of the present study was to investigate whether this increase in [Ca2+]i could be due to enhanced release of sequestered Ca2+ in cells pretreated with 1,25-(OH)2D3. In digitonin-permeabilized cells, the addition of 10 microM inositol 1,4,5-trisphosphate (IP3) rapidly increased free Ca2+ ([Ca2+]) to 50 +/- 10 nM (mean +/- SE) in cells pretreated with 1 nM 1,25-(OH)2D3 for 24 h, compared with 25 +/- 5 in control cells (P < 0.05). Furthermore, stimulating permeabilized cells with TRH increased [Ca2+]. The increase in control cells was 20 +/- 2, compared with 55 +/- 11 in cells pretreated with 1,25-(OH)2D3 (P < 0.05). Repeated additions of IP3 resulted in an attenuation of the response of [Ca2+] in both control cells and cells pretreated with 1,25-(OH)2D3. However, only the first addition of IP3 resulted in an enhanced increase in [Ca2+] in cells pretreated with 1,25-(OH)2D3 compared with control cells. If the cells were stimulated first with TRH and then with IP3, no difference in the [Ca2+] response was observed between control cells and cells pretreated with 1,25-(OH)2D3. Furthermore, if cells were stimulated with IP3 and then with TRH, no difference in the [Ca2+] response was observed between control cells and cells pretreated with 1,25-(OH)2D3. Stimulating the permeabilized cells with thapsigargin resulted in an increase in [Ca2+]. However, no difference in the response was observed between control cells and cells pretreated with 1,25-(OH)2D3. Addition of GTP or the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) had no effect on [Ca2+]. The results suggest that 1,25-(OH)2D3 has a modulatory effect on an IP3-sensitive intracellular Ca2+ pool in GH4C1 cells.     

Antagonistic effects of 24R,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 on L-type Ca2+ channels and Na+/Ca2+ exchange in enterocytes from Atlantic cod (Gadus morhua)

There is mounting evidence that vitamin D and its metabolites play important roles in regulating plasma calcium concentrations in teleost fish as in other vertebrates. The aims of the present study were to elucidate the possible cellular target mechanisms for the rapid actions of 24R,25(OH)(2)D(3), 25(OH)D(3) and 1,25(OH)(2)D(3) in Atlantic cod enterocytes at physiological doses, and to establish the concentration and thus the physiological range of circulating 24R,25(OH)(2)D(3), 25(OH)D(3) and 1,25(OH)(2)D(3) in the Atlantic cod. The plasma concentrations of 25(OH)D(3), 1,25(OH)(2)D(3) and 24R,25(OH)(2)D(3) were 15.3 +/- 2.7nM, 125.1 +/- 12.3pM and 10.1 +/- 23.5nM respectively. Exposure of enterocytes to 10mM calcium (Ca(2+)) evoked an increase in intracellular Ca(2+) concentrations ([Ca(2+)](i)). This increase was suppressed by 24R,25(OH)(2)D(3) dose-dependently, with an EC(50) of 4.9nM and a maximal inhibition of 60%. 24R,25(OH)(2)D(3) (20nM) abolished an increase in [Ca(2+)](i) (approximately 252%) in the control enterocytes exposed to 10microM S(-)-BAYK-8644, suggesting that the hormone acts by inhibiting Ca(2+) entry through L-type voltage-gated Ca(2+) channels. Administration of 20nM 24R,25(OH)(2)D(3) to enterocytes in the absence of extracellular Ca(2+) increased [Ca(2+)](i) by approximately 20%, indicating a release of Ca(2+) from intracellular stores. Administration of 25(OH)D(3) (20nM) resulted in a biphasic change in the enterocyte [Ca(2+)](i): within 1--5s, it decreased to 87 +/- 12nM below its mean basal [Ca(2+)](i) (334 +/- 13nM), followed by a rapid recovery of [Ca(2+)](i) to a new level, 10% lower than the initial [Ca(2+)](i). The rapid decrease, the recovery rate and the final [Ca(2+)](i) were all affected dose-dependently by 25(OH)D(3), with EC(50) values of 8.5, 17.0 and 18.9nM respectively. Furthermore, the effects of 25(OH)D(3) were sensitive to sodium (Na(+)), bepridil (10microM) and nifedipine (5 microM), suggesting that 25(OH)D(3) regulates the activity of both basolateral membrane-associated Na(+)/Ca(2+) exchangers and brush border membrane-associated L-type Ca(2+) channels. Administration of 25(OH)D(3) (10nM) to enterocytes in the absence of extracellular Ca(2+) increased [Ca(2+)](i) by approximately 18%, indicating a release of Ca(2+) from intracellular stores. 1,25(OH)(2)D(3) also affected enterocyte [Ca(2+)](i) in a biphasic manner: the rapid decrease, the recovery rate, and the mean final [Ca(2+)](i) were all affected dose-dependently, with EC(50) values of 8.3, 24.5 and 7.7nM respectively. The high EC(50) values for 1,25(OH)(2)D(3) compared with circulating concentrations of 1,25(OH)(2)D(3) (130pM) suggest that this effect is pharmacological, rather than of physiological relevance in enterocyte Ca(2+) homeostasis of the Atlantic cod. It is concluded that 24R,25(OH)(2)D(3) has a physiological role in decreasing intestinal Ca(2+) uptake via inactivation of L-type Ca(2+) channels, whereas the physiological role of 25(OH)D(3) is to increase enterocyte Ca(2+) transport via activation of Na(+)/Ca(2+) exchangers, concurrent with activation of L-type Ca(2+) channels.     

A new role for follicle-stimulating hormone in the regulation of calcium flux in Sertoli cells: inhibition of Na+/Ca++ exchange

Elucidation of mechanisms regulating intracellular calcium levels in steroidogenic tissues is important for understanding control of cellular function. We have previously described FSH receptor-mediated flux of 45Ca++ into cultured rat Sertoli cells and receptor-enriched proteoliposomes via voltage-sensitive and voltage-independent calcium channels. In the present study, we report heretofore unrecognized inhibitory effects of FSH on Na+/Ca++ exchange in these two systems. An outwardly directed Na+ gradient, developed by preincubating Sertoli cell monolayers in buffer made hypertonic with NaCl, resulted in uptake of 45Ca++ that was unaffected by calcium channel blocking agents, ruthenium red or methoxyverapamil, but was enhanced by ouabain, a specific inhibitor of Na+/K(+)-ATPase. Sodium-dependent 45Ca++ flux into Sertoli cells was inhibited in a concentration-related manner by increased extracellular Na+ (up to 135 mM). FSH consistently and reproducibly (28.9 +/- 3.8%, 10 separate assays) reduced sodium-dependent 45Ca++ influx in the absence or presence of ouabain. A lesser effect on Na+/Ca++ exchange was seen when Li+ replaced Na+ in the preincubation buffer, and a marked reduction occurred when Sertoli cells were incubated in buffer containing KCl, presumably due to membrane depolarization. FSH-sensitive Na+/45Ca++ exchange was also observed when using FSH receptor-enriched proteoliposomes. Our earlier calcium channel studies indicated that FSH affects Ca++ entry into Sertoli cells via a receptor-mediated process. The results reported here demonstrate that the interaction of FSH with its receptor is associated with changes in Na+/Ca++ exchange as well, and suggest that this activity may also be involved in regulating intracellular free Ca++ levels in the Sertoli cell.     

Regulatory effect of 1,25-dihydroxycholecalciferol on calcium fluxes in thyroid FRTL-5 cells.

The aim of the present study was to investigate the effect of 1,25-dihydroxycholecalciferol (1,25(OH)2-D3) on the regulation of calcium fluxes in rat thyroid FRTL-5 cells. The ATP-induced uptake of 45Ca2+ was decreased in cells pretreated with 1,25(OH)2D3 for 48 h. No effect was seen on basal uptake of 45Ca2+. At least a 24 h incubation period was required for the effect of 1,25(OH)2D3 to be expressed. Pretreatment with 1,25(OH)2D3 for 48 h did not change resting intracellular Ca2+ ([Ca2+]i) in fura-2-loaded FRTL-5 cells. However, the ATP-induced increase in [Ca2+]i was significantly enhanced in cells preincubated with 1,25(OH)2D3. The effect of 1,25(OH)2D3 was abolished in Ca(2+)-free buffer. No difference in the ionomycin-induced increase in [Ca2+]i was observed between control cells and cells pretreated with 1,25(OH)2D3. However, in Ca(2+)-free buffer the ionomycin response was decreased in cells incubated with 1,25(OH)2D3. The ATP-induced change in [Ca2+]i was decreased when ATP was added after ionomycin to cells treated with 1,25(OH)2D3. The results suggest that 1,25(OH)2D3 has a regulatory effect on Ca2+ fluxes in FRTL-5 cells, possibly by acting on Ca2+ sequestration.     

Na+/Ca2+ exchanger: target for oxidative stress in salt-sensitive hypertension

The Na+/Ca2+ exchanger regulates intracellular calcium ([Ca2+]i), and attenuation of Na+/Ca2+ exchange by oxidative stress might lead to dysregulation of [Ca2+]i. We have shown that the Na+/Ca2+ exchanger differs functionally and at the amino acid level between salt-sensitive and salt-resistant rats. Therefore, the purpose of these studies was to determine how oxidative stress affects the activities of the 2 Na+/Ca2+ exchangers that we cloned from mesangial cells of salt-resistant (RNCX) and salt-sensitive (SNCX) Dahl/Rapp rats. The effects of oxidative stress on exchanger activity were examined in cells expressing RNCX or SNCX by assessing 45Ca2+ uptake (reverse mode) and [Ca2+]i elevation (forward mode) in the presence and absence of H2O2 and peroxynitrite. Our results showed that 45Ca2+ uptake in SNCX cells was attenuated at 500 and 750 micromol/L H2O2 (63+/-12% and 25+/-7%, respectively; n=16) and at 50 and 100 micromol/L peroxynitrite (47+/-9% and 22+/-9%, respectively; n=16). In RNCX cells, 45Ca2+ uptake was attenuated at only 750 and 100 micromol/L H2O2 and peroxynitrite (61+/-9% and 63+/-6%, respectively; n=16). In addition, the elevation in [Ca2+]i was greater in SNCX cells than in RNCX cells in response to 750 micromol/L H2O2 (58+/-5.5 vs 17+/-4.1 nmol/L; n=13) and 100 micromol/L peroxynitrite (33+/-5 vs 11+/-6 nmol/L; n=19). The enhanced impairment of SNCX activity by oxidative stress might contribute to the dysregulation of [Ca2+]i that is found in this model of salt-sensitive hypertension.     

Vitamin D-induction of secretory responses in rat pituitary tumour (GH4C1) cells

1,25-Dihydroxyvitamin D3(1,25-(OH)2D3) selectively enhances prolactin gene expression in GH4C1 clonal rat pituitary tumour cells. Because this effect requires extracellular Ca2+, we studied the effect of 1,25-(OH)2D3 on another Ca2+-dependent process, agonist-induced hormone secretion. Pretreatment with 1,25-(OH)2D3 (1 nmol/l) caused at least 25-fold sensitization of GH4C1 cells to the voltage-sensitive Ca2+ channel agonist BAY K 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyr idine -5-carboxylate) as a prolactin secretagogue. This inductive effect of 1,25-(OH)2D3 followed a similar time-course to the enhancement of prolactin production. 1,25-(OH)2D3 had no effect on basal or BAY K 8644-induced 45Ca2+ uptake. The Ca2+-selective divalent cation ionophore 11,19,21-trihydroxy-4,6,8,12,14,18,20- heptamethyl-9-oxo-22-(tetrahydro-5 methyl-5-tetra hydro-5-(1-hydroxyethyl)-5-methyl-2-furanyl)-10,16-docosadienoic acid (ionomycin; 12 nmol/l-1.2 mumol/l) caused no significant increase in prolactin secretion in the absence of 1,25-(OH)2D3, but in cells treated with 1,25-(OH)2D3-(1 nmol/l), it increased prolactin secretion by 73% at 12 nmol/l and by a maximum of 98% at 0.12 mumol/l. These data demonstrate that vitamin D markedly enhances the responsiveness of GH4C1 functional pituitary tumour cells to two secretagogues which acts primarily through Ca2+-dependent mechanisms. They support the proposal that 1,25-(OH)2D3 acts in this cultured cell model either by effecting a redistribution of intracellular Ca2+ or by increasing the response of a Ca2+-sensitive effector system, but not by enhancing agonist-induced Ca2+ uptake.     

Activation of a beta-adrenergic-sensitive signal transduction pathway by the secosteroid hormone 1,25-(OH)2-vitamin D3 in chick heart.

In recent studies we have established that 1 alpha, 25-dihydroxy-vitamin D3[1,25(OH)2D3] rapidly stimulates dihydropyridine-sensitive calcium channel-mediated Ca2+influx in chick cardiac muscle by a non-genomic action which is accompanied by PKA-dependent phosphorylation of a 45 kDa microsomal membrane protein. To investigate the signal transduction pathway activated by 1,25(OH)2D3 in heart, we have compared the effects of the secosteroid hormone with those of the beta-adrenergic agonist isoproterenol (IPT) by employing cultured chick embryonic cardiac cells (myocytes) and thin-slice preparations of differentiated adult heart muscle. The increases in 45Ca2+ uptake and intracellular calcium ([Ca2+]i), cyclic AMP accumulation and changes in microsomal protein phosphorylation evoked by 1,25(OH)2D3 could be reproduced by IPT. When combined treatments with the sterol and the beta-adrenergic agonist were performed, no additive stimulation of these parameters was observed, suggesting that a common signal transduction pathway mediates the effects of 1,25(OH)2D3 and IPT. The participation of a guanine nucleotide binding protein (G protein) in the 1, 25(OH)2D3-induced changes in heart was investigated. AlF4(-), an activator of G proteins, and cholera and pertussis toxins, like 1, 25(OH)2D3 increased 45Ca2+ uptake by myocytes. AlF4(-) did not further stimulate the effects of 1,25(OH)2D3 thereby showing that a G protein is involved in the hormone action. Moreover, 1,25(OH)2D3 potentiated pertussis toxin but was unable to modify choleric toxin-dependent myocyte Ca2+ influx. Altogether, these results provide evidence indicating that the non-genomic action of 1,25(OH)2D3 on cardiac muscle calcium influx involves modulation of the beta-adrenergic-sensitive adenylyl cyclase/cAMP/PKA pathway coupled to a Gs protein.     

Vitamin D-enhanced thyrotrophin release from rat pituitary cells: effects of Ca2+, dihydropyridines and ionomycin

Vitamin D may regulate pituitary function, as there are selective effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on gene expression in clonal pituitary tumour cells, and on TRH-induced TSH release in normal rat pituitary cells in vitro. The role of Ca2+ in 1,25-(OH)2D3-enhanced TSH release from primary rat pituitary cell cultures was investigated. Pretreatment with 10 nmol 1,25-(OH)2D3/l for 24 h augmented KCl (3-60 mmol/l)-induced TSH release over 1 h at all KCl concentrations greater than 7.5 mmol/l (P less than 0.001), with a 76% enhancement of TSH release induced by 30 mmol KCl/l (P less than 0.001). The Ca2+ channel antagonist nifedipine (10 nmol/l-10 mumol/l) caused a concentration-dependent inhibition of KCl (60 mmol/l)-induced TSH secretion. Pretreatment with 1,25-(OH)2D3 enhanced KCl-induced release at all concentrations of nifedipine (P less than 0.001). The Ca2+ selective divalent cation ionophore ionomycin (1 nmol/l-1 mumol/l), and the Ca2+ channel agonist BAY K 8644 (10 nmol/l-1 mumol/l) increased prolactin secretion but did not increase TSH release, and 1,25-(OH)2D3 had no effect. At an extracellular Ca2+ concentration of less than 500 nmol/l, TRH-induced TSH release was observed only after treatment with 1,25-(OH)2D3 (P less than 0.01). As the extracellular Ca2+ concentration was increased, greater increments of TRH-induced TSH release were observed following pretreatment with 1,25-(OH)2D3 (P less than 0.01). However, the effect of 1,25-(OH)2D3 in the thyrotroph was independent of the pretreatment extracellular Ca2+ concentration. We have shown that 1,25-(OH)2D3 acts selectively on the thyrotroph to enhance in-vitro responsiveness to TRH and KCl. These data suggest that the action of 1,25-(OH)2D3 in the thyrotroph is to enhance intracellular signal transduction. They further support a permissive or regulatory role of vitamin D in the normal pituitary gland.     

Effects of active oxygen generated by DTT/Fe2+ on cardiac Na+/Ca2+ exchange and membrane permeability to Ca2+

Sarcolemmal vesicles isolated from bovine heart were preincubated at 37 degrees C with an oxygen radical generating system consisting of 1 mM dithiothreitol (DTT) and 50 microM FeSO4. Exposure of the vesicles for 1 to 40 mins stimulated Na+/Ca2+ exchange about 2.5-fold. The DTT/Fe2+ treatment decreased the apparent Km for Ca2+ of Nai+-dependent Ca2+ uptake by 80% (from 63 to 13 microM). The effect on Vmax was much smaller however. The resulting stimulation of exchange activity was diminished by the presence of desferrioxamine (95%) or catalase (60%). In contrast, superoxide dismutase and sodium formate did not prevent the effects of DTT/Fe2+ on the exchanger. Neither Zn2+ nor Ga3+ could replace Fe2+ in the stimulation of Na+/Ca2+ exchange. Passive Ca2+ efflux was determined by first allowing Na+/Ca2+ exchange to continue to plateau values and then diluting the loaded vesicles in the presence of EGTA. Ca2+ leakage from the vesicles was slightly but significantly (P less than 0.05) increased by the action of DTT/Fe2+, the rate constants for the passive Ca2+ efflux being 0.22 and 0.26/min in control and treated groups, respectively. The calcium loading observed in myocytes in ischemia/reperfusion injury suggests that the stimulation of Na+/Ca2+ exchange by active oxygen may moderate the myocardial response to oxygen mediated injuries including ischemia/reperfusion injury. However, the clinical relevance of these phenomena is far from clear as the stimulation depends in part on the Km for Ca2+ prior to treatment.     

Na+-Ca2+ exchange in cultured vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) contract as intracellular free calcium ([Ca2+]i) rises. While Na+-Ca2+ exchange has been proposed to contribute to transmembrane Ca2+ flux, its role in cultured VSMC is unknown. Accordingly, we have investigated the role of Na+-Ca2+ exchange in unidirectional and net transmembrane Ca2+ fluxes in cultured rat aortic VSMC under basal conditions and following agonist-mediated stimulation. Transmembrane Ca2+ uptake was significantly increased in response to a low external Na+ concentration ([Na+]o) compared with 140 mM [Na+]o. Na+-dependent Ca2+ uptake in response to low [Na+]o was further increased by intracellular Na+ loading by preincubation of the VSMC with 1 mM ouabain. Under steady-state conditions, Ca2+ content varied inversely with [Na+]o, increasing from 1.0 nmol Ca2+/mg protein at 140 mM [Na+]o to 4.0 nmol Ca2+/mg protein at 20 mM [Na+]o. Increasing [K+]o to 55 mM also enhanced Na+-dependent Ca2+ influx. Augmentation of Ca2+ uptake with K+ depolarization was not significantly inhibited by the calcium channel antagonist verapamil. Transmembrane Ca2+ efflux was increased in response to 130 mM [Na+]o compared with zero [Na+]o (iso-osmotic substitution with choline+), and was further stimulated by the vasoconstrictor angiotensin II, which is known to elevate [Ca2+]i. These changes in [Ca2+]i were studied directly using fura-2 fluorescence measurements. Elevated [Ca2+]i levels returned to baseline more rapidly in the presence of normal (130 mM) [Na+]o compared with zero [Na+]o (iso-osmotic substitution with choline+). These findings suggest that a bidirectional Na+-Ca2+ exchange mechanism is present in cultured rat aortic VSMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of age on calcium uptake in isolated duodenum cells: role of 1,25-dihydroxyvitamin D3

Uptake of Ca2+ in cells isolated from rat duodenum declined in the senescent rats. This age-related change was not due to an alteration in the rate of Ca2+ efflux or in the size of the cell. The decrease appeared specific, as alpha-methyl glucoside uptake was not altered. Cell population, as monitored by sucrase activity for villus cells, was not different between duodenal cells isolated from 6- and 24-month-old rats. Kinetic analysis shows the Vmax, the apparent maximum uptake capacity, decreased in the cells from senescent rats whereas the Km, the apparent affinity to Ca2+, was unchanged. Serum levels of 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D [1,25-(OH)2D] were determined as a function of age; the levels of 25OHD were not significantly different in 3-, 6-, 12-, and 24-month-old rats. On the other hand, serum 1,25-(OH)2D decreased throughout the age range studied. Since duodenal Ca2+ uptake is closely regulated by 1,25-(OH)2D3, we tested the hypothesis that low serum 1,25-(OH)2D in the senescent rats may have contributed to the decline in duodenal Ca2+ uptake. In vivo administration of 1,25-(OH)2D3 to senescent rats significantly enhanced Ca2+ uptake activity in the isolated duodenal cells. After 1,25-(OH)2D3 treatment, Ca2+ uptake activity in cells isolated from senescent rats was only slightly less than that in cells from adult rats. We conclude that duodenal Ca2+ uptake declined in the senescent rats, and this age-related change was most likely due to the low serum level of 1,25-(OH)2D and not the result of a decrease in any duodenal response to 1,25-(OH)2D3.     

Effects of intracellular acidosis on [Ca2+]i transients, transsarcolemmal Ca2+ fluxes, and contraction in ventricular myocytes

We examined the effects of intracellular acidosis produced by washout of NH4Cl on [Ca2+]i transients (indo-1 fluorescence), cell contraction (video motion detector), and 45Ca and 24Na fluxes in cultured chick embryo ventricular myocytes. Exposure of cells to 10 mM NH4Cl produced intracellular alkalosis (pH 7.6), and subsequent washout resulted in a transient acidosis (pH 6.5). Exposure to 10 mM NH4Cl slightly decreased [Ca2+]i transients but increased the amplitude of cell contraction. Subsequent washout of NH4Cl initially increased diastolic [Ca2+]i and decreased the peak positive and negative d[Ca2+]i/dt, while the amplitude of cell contraction was markedly decreased. Subsequently, peak systolic [Ca2+]i increased with partial recovery of contraction. A similar increase in [Ca2+]i and decrease in contraction after washout of NH4Cl was observed in single paced adult guinea pig ventricular cells. Acidosis decreased 45Ca uptake by sarcoplasmic reticulum vesicles isolated from chick embryo ventricle. However, the [Ca2+]i increase caused by intracellular acidosis was also observed in the presence of 10 mM caffeine, suggesting that altered sarcoplasmic reticulum handling of calcium is not the only mechanism involved. Intracellular acidosis only slightly increased total 24Na uptake under these conditions, an effect resulting from the combination of a stimulation of amiloride-sensitive sodium influx (Na(+)-H+ exchange) and inhibition of sodium influx via Na(+)-Ca2+ exchange, manifested by a significant decrease in 45Ca efflux. Further support for a lack of involvement of an increased [Na+]i in the observed increase in [Ca2+]i during acidosis was low-sodium, nominal 0-calcium extracellular solution, an experimental condition that minimizes the possible effects of Na(+)-H+ exchange and Na(+)-Ca2+ exchange. We conclude that the [Ca2+]i increase caused by intracellular acidosis in cultured ventricular cells is primarily due to changes in [Ca2+]i buffering and [Ca2+]i extrusion, rather than to an increase in transsarcolemmal calcium influx. Intracellular acidosis also markedly decreases the sensitivity of the contractile elements to [Ca2+]i in cultured chick embryonic and adult guinea pig ventricular myocytes.     

Liponomic control of Ca2+ transport: relationship to mechanism of action of 1,25-dihydroxyvitamin D3.

Isolated vesicles prepared from the brush border membranes of chicken duodenal mucosal cells (enterocytes) take up calcium by a passive but saturable process. The rate of uptake (Vmax) is increased 2.5- to 3-fold, with no change in Km, in vesicles prepared from 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-treated chickens compared to vesicles from vitamin D-deficient controls. Preincubation of vesicles with either cis- or trans-vaccinic acid (cVA or tVA, respectively) or their methyl esters in vitro also alters the rates of calcium transport. Methyl cVA causes an increase in rate of calcium uptake into vesicles from vitamin D-deficient chickens but not in those from 1,25(OH)2D3-treated chickens. This increase is 80-90% of that seen after 1,25(OH)2D3 treatment. Higher concentrations of methyl cVA produce no further increases. Conversely, methyl tVA causes a decrease in rate of calcium uptake in vesicles from 1,25(OH)2D3-treated chickens but no change in vesicles from vitamin D-deficient controls. This decrease reduces the rate of calcium uptake to nearly the same value as seen in vesicles from vitamin D-deficient controls. Higher concentration of methyl tVA produce no further suppression of uptake rate. The changes seen were in the Vmax and not in the Km of the transport process. The fatty acids did not alter the process of Na+-dependent glucose uptake in the same membrane. These data demonstrate that a small alteration in the lipid structure of this membrane can specifically shift the activity of the calcium transport process.     

Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for Ca2+ entry after stimulation with ATP

The relationship between ATP-induced uptake of 45Ca2+ and the ATP-induced changes in [Ca2+]i was investigated in rat FRTL-5 thyroid cells. Addition of 1 microCi 45Ca2+/ml together with ATP induced a time- and dose-dependent increase in uptake of 45Ca2+, the uptake being still significantly above control after 30 min. Resting intracellular free Ca2+ levels ([Ca2+]i), measured using Fura-2, was determined to be 60 +/- 14.3 nM (mean +/- SE). ATP induced a rapid, transient increase in [Ca2+]i (785 +/- 56.2 nM) followed by a plateau phase (127 +/- 34.3 nM). In a Ca(2+)-free buffer, the ATP-induced transient was significantly decreased (357 +/- 57.4 nM, p less than 0.05), and the plateau phase was abolished. The results suggested that stimulating FRTL-5 cells with ATP induced an influx of Ca2+, possibly by a mechanism dependent on a transient increase in [Ca2+]i. To further test this possibility, the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was tested. In cells loaded with BAPTA, the ATP-induced uptake of 45Ca2+ was greatly enhanced, while the ATP-induced transient increase in [Ca2+]i was almost totally abolished. In cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ after termination of the ATP response induced a rapid increase in [Ca2+]i. Furthermore, addition of Mn2+ to cells stimulated with ATP induced a more rapid quenching of Fura-2, compared to that seen in control cells. The results indicate that stimulating FRTL-5 cells with ATP induces a rapid release of Ca2+ from intracellular stores, followed immediately by an increase in plasma membrane conductance and influx of extracellular Ca2+. The ATP-induced change in [Ca2+]i may function as a signal enhancing influx of extracellular Ca2+, although some other unknown mechanism(s) is also needed.     

Rapid changes in skeletal muscle calcium uptake induced in vitro by 1,25-dihydroxyvitamin D3 are suppressed by calcium channel blockers

Previous investigations have shown that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] stimulates muscle Ca uptake through a nuclear mechanism. The possibility that 1,25-(OH)2D3 would induce rapid changes in muscle Ca fluxes independent of de novo protein synthesis was investigated in the present work. In vitro preparations of soleus muscles obtained from vitamin D-deficient chicks were used. A significant increase in 45Ca labeling of the tissue was already observed after 3-min treatment with 2.4 X 10(-10) M 1,25-(OH)2D3. This early stimulation in muscle Ca uptake became maximal at 10-15 min. Cycloheximide (50 microM) did not block the effect of the metabolite at 15 and 30 min. However, the antibiotic effectively blocked the increase in Ca uptake induced by 1,25-(OH)2D3 after 1-h treatment. The rapid 1,25-(OH)2D3-dependent stimulation of 45Ca labeling of soleus muscle was not associated to changes in lipid synthesis as assessed by measurements of 3H-glycerol incorporation into the tissue lipids. However, the calcium antagonists verapamil and nifedipine (50 microM) abolished the stimulation in Ca uptake produced by 1,25-(OH)2D3 in 5 min. These results suggest that 1,25-(OH)2D3 can act directly at the muscle membrane level affecting Ca fluxes through Ca channels.