Depletion Of Extracellular Calcium Research Articles

Mechanism of action of Brazilin on gluconeogenesis in isolated rat hepatocytes.

The present study was undertaken to investigate the mechanism of action of brazilin on gluconeogenesis and ketogenesis in isolated rat hepatocytes and to elucidate the hypoglycemic mechanism of brazilin. Brazilin decreased gluconeogenesis at 100 micro M in hepatocytes isolated from diabetic rats. Brazilin also decreased basal and glucagon-induced gluconeogenesis in hepatocytes from normal rats. Fatty acids (octanoate or oleate)-induced gluconeogenesis was significantly reduced by brazilin, but ketogenesis was not influenced. The depletion of extracellular or intracellular calcium decreased gluconeogenesis in calcium-depleted media. Brazilin lowered dibutyryl cAMP (Bt2cAMP)-induced gluconeogenesis and the intracellular adenosine 3',5'-cyclic monophosphate (cAMP) level in glucagon-treated hepatocytes. It was also found that brazilin does not require calcium for inhibition of gluconeogenesis, but may inhibit the down-stream of cAMP signaling pathways. These data suggest that a decreased gluconeogenic flux in hepatocytes might at least partly contribute to the hypoglycemic effects of brazilin.

Functional characterization and expression of PBR in rat gastric mucosa: stimulation of chloride secretion by PBR ligands

Previous studies have demonstrated that gastric mucosa contained high levels of the polypeptide diazepam binding inhibitor, the endogenous ligand of the peripheral-type benzodiazepine receptor (PBR). However, the expression and function of this receptor protein in these tissues have not been investigated. Immunohistochemistry identified an intense PBR immunoreactivity in the mucous and parietal cells of rat gastric fundus and in the mucous cells of antrum. Immunoelectron microscopy revealed the mitochondrial localization of PBR in these cells. Binding of isoquinoline PK 11195 and benzodiazepine Ro5-4864 to gastric membranes showed that fundus had more PBR-binding sites than antrum, displaying higher affinity for PK 11195 than Ro5-4864. In a Ussing chamber, PK 11195 and Ro5-4864 increased short-circuit current (I(sc)) in fundic and antral mucosa in a concentration-dependent manner in the presence of GABA(A) and central benzodiazepine receptor (CBR) blockers. This increase in I(sc) was abolished after external Cl(-) substitution and was sensitive to chloride channels or transporter inhibitors. PK 11195-induced chloride secretion was also 1) sensitive to verapamil and extracellular calcium depletion, 2) blocked by thapsigargin and intracellular calcium depletion, and 3) abolished by the mitochondrial pore transition complex inhibitor cyclosporine A. PK 11195 had no direct effect on H(+) secretion, indicating that it stimulates a component of Cl(-) secretion independent of acid secretion in fundic mucosa. These data demonstrate that mucous and parietal cells of the gastric mucosa express mitochondrial PBR functionally coupled to Ca(2+)-dependent Cl(-) secretion, possibly involved in the gastric mucosa protection.

Activation of deoxycytidine kinase in lymphocytes is calcium dependent and involves a conformational change detectable by native immunostaining

Deoxycytidine kinase (dCK), the principal deoxynucleoside salvage enzyme, plays a seminal role in the bioactivation of a wide array of cytotoxic nucleoside analogues. Recently, activation of dCK has been considered as a protective cellular response to a number of DNA-damaging agents in lymphocytes. Regarding the molecular mechanism of the enzyme activation, a post-translational modification by protein phosphorylation has been suggested. Here we provide evidence that both the activation process and the maintenance of the activated state require free cytosolic calcium. BAPTA-AM, a cell-permeable calcium chelator selectively inhibited the activation of dCK in a time- and concentration-dependent manner while extracellular calcium depletion had no effect. On the other hand, elevation of cytoplasmic calcium levels by thapsigargin did not potentiate the enzyme, referring to the permissive function of calcium in the activation process. Denaturing Western blots of extracts from lymphocytes incubated with 2-chlorodeoxyadenosine, aphidicolin and/or BAPTA-AM clearly demonstrated that dCK protein levels were unchanged during these treatments. However, a striking correlation was found between enzyme activity and the intensity of dCK-specific signals in native Western blots. Extracts from CdA-treated cells were much better recognized by the antibody raised against the C-terminal peptide of dCK than the BAPTA-AM-treated samples. These results indicate that the calcium-dependent activation of dCK is accompanied by a conformational change that renders the C-terminal epitope more accessible to the antibody.

A simple method for screening photoelectric dyes towards their use for retinal prostheses.

Photoelectric dyes absorb light and convert photon energy to electric potentials. To test whether these dyes could be used for retinal prostheses, a simple in vitro screening system was developed. Retinal neurons were cultured from the eyes of chick embryos at the 10-day embryonic stage, at which time no retinal photoreceptor cells have yet developed. Intracellular calcium elevation was observed with Fluo-4 in cultured retinal neurons before and after photoelectric dye was applied at varying concentrations to the culture medium. Five of 7 photoelectric dyes tested in this in vitro system induced intracellular calcium elevation in cultured chick retinal neurons. The intracellular calcium elevation generated by the 5 photoelectric dyes was blocked by extracellular calcium depletion in the case of all 5 dyes, and, except for one dye, by the presence of voltage-gated calcium channel blockers. The photoelectric dyes absorbed light under an inverted microscope and stimulated retinal neurons. This simple in vitro system allows the screening of photoelectric dyes which can be used for retinal prostheses.

Role of Phospholipase A2 Activation and Calcium in CYP2E1-dependent Toxicity in HepG2 Cells

Previous studies suggested a role for calcium in CYP2E1-dependent toxicity. The possible role of phospholipase A2 (PLA2) activation in this toxicity was investigated. HepG2 cells that overexpress CYP2E1 (E47 cells) exposed to arachidonic acid (AA) +Fe-NTA showed higher toxicity than control HepG2 cells not expressing CYP2E1 (C34 cells). This toxicity was inhibited by the PLA2 inhibitors aristolochic acid, quinacrine, and PTK. PLA2 activity assessed by release of preloaded [3H]AA after treatment with AA+Fe was higher in the CYP2E1 expressing HepG2 cells. This [3H]AA release was inhibited by PLA2 inhibitors, alpha-tocopherol, and by depleting Ca2+ from the cells (intracellular + extracellular sources), but not by removal of extracellular calcium alone. Toxicity was preceded by an increase in intracellular calcium caused by influx from the extracellular space, and this was prevented by PLA2 inhibitors. PLA2 inhibitors also blocked mitochondrial damage in the CYP2E1-expressing HepG2 cells exposed to AA+Fe. Ca2+ depletion and removal of extracellular calcium inhibited toxicity at early time periods, although a delayed toxicity was evident at later times in Ca2+-free medium. This later toxicity was also inhibited by PLA2 inhibitors. Analogous to PLA2 activity, Ca2+ depletion but not removal of extracellular calcium alone prevented the activation of calpain activity by AA+Fe. These results suggest that release of stored calcium by AA+Fe, induced by lipid peroxidation, can initially activate calpain and PLA2 activity, that PLA2 activation is critical for a subsequent increased influx of extracellular Ca2+, and that the combination of increased PLA2 and calpain activity, increased calcium and oxidative stress cause mitochondrial damage, that ultimately produces the rapid toxicity of AA+Fe in CYP2E1-expressing HepG2 cells.

Ethanol-induced cephalic apoptosis requires phospholipase C-dependent intracellular calcium signaling.

Although the ability of ethanol to elicit neural crest cell apoptosis is well documented, the initial target of ethanol in these cells, and the biochemical pathway leading to their apoptosis, have yet to be determined. Recent work in preimplantation mouse embryos demonstrates that ethanol induces a phospholipase-C (PLC)-dependent calcium transient that mediates ethanol's effects. We tested whether a similar effect on calcium and PLC is involved in ethanol-induced neural crest apoptosis. Chicken embryos were collected and loaded with Fluo-3-AM to assess the effects of ethanol on intracellular calcium levels. Pharmacological agents were used to determine the sources and mechanism of intracellular calcium increases. In separate experiments, embryos were treated in ovo with pharmacological modulators of calcium signaling prior to ethanol exposure, and resulting levels of cell death were assessed by using the vital dye acridine orange. Ethanol exposure caused a localized increase in intracellular calcium levels in embryonic neural folds within 15 sec of ethanol exposure. Ethanol-induced apoptosis was specifically blocked by chelation of intracellular calcium before ethanol exposure. Pretreatment with the PLC inhibitor U73122 blocked ethanol-induced apoptosis as well as the intracellular calcium transient. Depletion of extracellular calcium resulted in a partial block of ethanol-induced apoptosis. Ethanol exposure alters calcium signaling within the neurulation-stage chicken embryo in a PLC-dependent manner. Increases in intracellular calcium and PLC activity are necessary for ethanol's induction of apoptosis within cephalic populations. These effects likely represent an early and crucial event in the pathway leading to ethanol-induced cell death.

Calcium channel blockers inhibit galvanotaxis in human keratinocytes.

Directed migration of keratinocytes is essential for wound healing. The migration of human keratinocytes in vitro is strongly influenced by the presence of a physiological electric field and these cells migrate towards the negative pole of such a field (galvanotaxis). We have previously shown that the depletion of extracellular calcium blocks the directional migration of cultured human keratinocytes in an electric field (Fang et al., 1998; J Invest Dermatol 111:751-756). Here we further investigate the role of calcium influx on the directionality and migration speed of keratinocytes during electric field exposure with the use of Ca(2+) channel blockers. A constant, physiological electric field strength of 100 mV/mm was imposed on the cultured cells for 1 h. To determine the role of calcium influx during galvanotaxis we tested the effects of the voltage-dependent cation channel blockers, verapamil and amiloride, as well as the inorganic Ca(2+) channel blockers, Ni(2+) and Gd(3+) and the Ca(2+) substitute, Sr(2+), on the speed and directionality of keratinocyte migration during galvanotaxis. Neither amiloride (10 microM) nor verapamil (10 microM) had any effect on the galvanotaxis response. Therefore, calcium influx through amiloride-sensitive channels is not required for galvanotaxis, and membrane depolarization via K(+) channel activity is also not required. In contrast, Sr(2+) (5 mM), Ni(2+) (1-5 mM), and Gd(3+) (100 microM) all significantly inhibit the directional migratory response to some degree. While Sr(2+) strongly inhibits directed migration, the cells exhibit nearly normal migration speeds. These findings suggest that calcium influx through Ca(2+) channels is required for directed migration of keratinocytes during galvanotaxis and that directional migration and migration speed are probably controlled by separate mechanisms.

Vasopressin-induced cytoplasmic and nuclear calcium signaling in cultured cortical astrocytes

We sought to determine whether vasopressin V 1a receptor (V 1aR) mRNA detected in cortical astrocytes [Brain Res. Mol. Brain Res. 45 (1997) 138] was translated into functional receptors by investigating the effector calcium signaling cascade associated with the vasopressin V 1a receptor subtype. Analysis of intracellular calcium dynamics using the calcium-sensitive dye fura-2 AM indicated that exposure of cortical astrocytes to V 1 vasopressin receptor agonist, [Phe 2,Orn 8]-oxytocin, induced a marked dose-dependent increase in intracellular calcium which was abolished by depletion of extracellular calcium. V 1 agonist treatment induced a rapid increase in calcium signal in both the cytoplasm and nucleus, which was followed by an accumulation of the calcium signal in the nucleus, suggesting translocation of cytoplasmic calcium into the nucleus. The nuclear calcium signal was sustained for several seconds followed by translocation back to the cytoplasm. Following the nuclear-to-cytoplasmic calcium translocation, total free intracellular calcium concentration decreased. The dynamic calcium cytoplasmic and nuclear localization was confirmed by laser scanning confocal microscopy coupled with the calcium-sensitive dye fluo-3 AM. To determine the source of calcium, V 1 agonist-induced 45Ca 2+ uptake and [ 3H]IP 1 accumulation were investigated. V 1 agonist induced significant and rapid uptake of 45Ca 2+ and a significant dose-dependent increase in [ 3H]IP 1 accumulation in cortical astrocytes. To our knowledge, this is the first documentation of a vasopressin receptor-induced calcium signaling cascade in cortical astrocytes and the first documentation of vasopressin receptor induction of nuclear calcium signaling.

Transient disruption of intercellular junctions enables baculovirus entry into nondividing hepatocytes.

Baculovirus infection has extended the capabilities for transfection of exogenous genes into a variety of mammalian cell types. Because rat hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented chemically defined medium are an excellent model system for studying liver function in vitro, we investigated the ability of baculoviruses to infect and deliver exogenous genes to cells in this culture system. Efficient delivery to hepatocytes in short-term culture becomes restricted to peripheral cells, or "edge" cells, as the hepatocytes acquire intercellular junctions and form islands with time in culture. This barrier to baculovirus entry can be overcome, and the percentage of internal cells within the hepatocyte islands that are infected with the baculovirus can be increased more than 100-fold, when cells are subjected to transient calcium depletion before and during infection. These findings suggest that at least in some cell types, such as hepatocytes, baculovirus entry may require contact with the basolateral surface. We conclude from this study that recombinant baculovirus infection following transient depletion of extracellular calcium results in delivery of exogenous genes to at least 75% of hepatocytes in long-term DMSO culture, thereby making it possible for the first time to carry out gain-of-function and loss-of-function studies in this cell system.

Mechanical stimulation activates Galphaq signaling pathways and 5-hydroxytryptamine release from human carcinoid BON cells.

5-Hydroxytryptamine (5-HT) released from enterochromaffin cells activates secretory and peristaltic reflexes necessary for lubrication and propulsion of intestinal luminal contents. The aim of this study was to identify mechanosensitive intracellular signaling pathways that regulate 5-HT release. Human carcinoid BON cells displayed 5-HT immunoreactivity associated with granules dispersed throughout the cells or at the borders. Mechanical stimulation by rotational shaking released 5-HT from BON cells or from guinea pig jejunum during neural blockade with tetrodotoxin. In streptolysin O-permeabilized cells, guanosine 5'-O- (2-thiodiphosphate) (GDP-beta-S) and a synthetic peptide derived from the COOH terminus of Galphaq abolished mechanically evoked 5-HT release, while the NH(2)-terminal peptide did not. An antisense phosphorothioated oligonucleotide targeted to a unique sequence of Galphaq abolished mechanically evoked 5-HT release and reduced Galphaq protein levels without affecting the expression of Galpha(11). Depletion and chelation of extracellular calcium did not alter mechanically evoked 5-HT release, whereas depletion of intracellular calcium stores by thapsigargin and chelation of intracellular calcium by 1,2-bis (o-Aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM) reduced 5-HT release. Mechanically evoked 5-HT release was inhibited by somatostatin-14 in a concentration-dependent manner. The results suggest that mechanical stimulation of enterochromaffin-derived BON cells directly or indirectly stimulates a G protein-coupled receptor that activates Galphaq, mobilizes intracellular calcium, and causes 5-HT release.

Extracellular calcium depletion as a mechanism of short-term synaptic depression.

Recent experiments have demonstrated that normal neural activity can cause significant decrements in external calcium levels, and that these decrements mediate a form of short-term synaptic depression. These findings raise the possibility that certain forms of short-term synaptic depression at glutamatergic synapses throughout the mammalian CNS may be influenced by similar changes in external calcium. We use a computational model of the extracellular space, combined with experimental data on calcium consumption, to show that such short-term depression can be accounted for by changes in calcium just outside active synapses, provided that external calcium diffusion is restricted. Remarkably, the model suggests the novel possibility that synapses may possess private pools of external calcium that enforce some forms of short-term depression in a synapse-specific manner.

3-(15-Hydroxypentadecyl)-2,4,4-trimethyl-2-cyclohexen-1-one and its effect on neuropeptide secretion

The aim of the present study was to describe the synthesis of a t rimethyl c yclohexenonic long chain f atty a lcohol ( t-CFA), and analyze its biological activity. Specifically, 3-(15-hydroxypentadecyl)-2,4,4-trimethyl-2-cyclohexen-1-one, the t-CFA containing 15 carbon atoms on the side chain ( t-CFA n=15) stimulated arginine vasopressin secretion in nerve terminals of the neurohypophysis. This effect was inhibited by extracellular calcium depletion, which suggets that t-CFA n=15 stimulates neuropeptide secretion through a calcium-dependent exocytosis mechanism.

Cutaneous expression of CRH and CRH-R. Is there a "skin stress response system?".

The classical neuroendocrine pathway for response to systemic stress is by hypothalamic release of corticotropin releasing hormone (CRH), subsequent activation of pituitary CRH receptors (CRH-R), and production and release of proopiomelanocortin (POMC) derived peptides. It has been proposed that an equivalent to the hypothalamic-pituitary-adrenal axis functions in mammalian skin, in response to local stress (see Reference 1). To further define such system we used immunocytochemistry, RP-HPLC separation, and RIA techniques, in rodent and human skin, and in cultured normal and malignant melanocytes and keratinocytes. Production of mRNA for CRH-R1 was documented in mouse and human skin using RT-PCR and Northern blot techniques; CRH binding sites and CRH-R1 protein were also identified. Addition of CRH to immortalized human keratinocytes, and to rodent and human melanoma cells induced rapid, specific, and dose-dependent increases in intracellular Ca2+. The latter were inhibited by the CRH antagonist alpha-helical-CRH(9-41) and by the depletion of extracellular calcium with EGTA. CRH production was enhanced by ultraviolet light radiation and forskolin (a stimulator for intracellular cAMP production), and inhibited by dexamethasone. Thus, evidence that skin cells, both produce CRH and express functional CRH-R1, supports the existence of a local CRH/CRH-R neuroendocrine pathway that may be activated within the context of a skin stress response system.

Phosphatidic acid increases inositol-1,4,5,-trisphosphate and [Ca 2+] i levels in neonatal rat cardiomyocytes

Phosphatidic acid (PA), which can be synthesized de novo, or as a product of phosphatidylcholine hydrolysis and/or phosphorylation of 1,2-diacylglycerol (DAG), mediates diverse cellular functions in various cell types, including cardiomyocytes. We set out to characterize the effect of PA on intracellular free calcium ([Ca 2+] i) and inositol-1,4,5-trisphosphate (IP 3) levels in primary cultures of neonatal rat cardiomyocytes. Addition of PA led to rapid, concentration and time dependent increases in both IP 3 and [Ca 2+] i levels in adherent cells. There was strong correlation in the concentration-response relationships between IP 3 and [Ca 2+] i increases evoked by PA. Incubation with the sarcoplasmic reticulum (SR) Ca 2+ pump inhibitor, cyclopiazonic acid (CPA), significantly attenuated the PA evoked [Ca 2+] i increase but had no significant effect on IP 3 accumulation. The phospholipase C (PLC) inhibitor, D-609, attenuated both IP 3 and [Ca 2+] i elevations evoked by PA whereas staurosporine (STS), a potent and non-selective PKC inhibitor, had no significant effect on either. Another PLC inhibitor, U73122, but not its inactive analog, U73343, also inhibited PA evoked increases in [Ca 2+] i. Depletion of extracellular calcium attenuated both basal and PA evoked increases in [Ca 2+] i. The PLA 2 inhibitors, bromophenylacyl-bromide (BPB) and CDP-choline, had no effect on PA evoked [Ca 2+] i responses. Neither the DAG analog, dioctanoylglycerol, nor the DAG kinase inhibitor, R59949, affected PA evoked changes in [Ca 2+] i. Taken together, these data indicate that PA, in a manner independent of PKC, DAG, or PLA 2, may enhance Ca 2+ release from IP 3 sensitive SR Ca 2+ stores via activation of PLC in neonatal rat cardiomyocytes.

Lymphocyte intracellular free calcium concentration is increased in preeclampsia

Objectives: We tested 2 hypotheses: (1) Preeclampsia is characterized by an increase in intracellular free calcium concentration in lymphocytes. (2) Levels of intracellular free calcium are influenced by the calcium concentration in the extracellular milieu or by parathyroid hormone. Study Design: Intracellular free calcium concentrations were measured in 4 groups of women: nonpregnant women (n = 25), normotensive pregnant women (n = 30), pregnant women with chronic hypertension (n = 15), and women with preeclampsia (n = 15). Intracellular free calcium concentration was measured in the basal state, at varying extracellular calcium ion concentrations, and in the presence of exogenous parathyroid hormone. Results: Women with preeclampsia had the highest basal lymphocyte intracellular free calcium concentration (121 ± 7 nmol/L, mean ± SEM) compared with normotensive pregnant women during the third trimester (94 ± 3 nmol/L, P < .001) and pregnant women in the third trimester with chronic hypertension (100 ± 3 nmol/L, P < .01). During the third trimester normotensive women and women with chronic hypertension had significantly higher basal intracellular free calcium concentrations than were found in women during the first trimester. Exposure of lymphocytes to an extracellular milieu of low calcium concentration resulted in an increase in intracellular free calcium concentration. Incubation with parathyroid hormone had no effect on intracellular free calcium concentration. Conclusions: Lymphocyte intracellular free calcium concentration is increased in preeclampsia and not in chronic hypertensive pregnancy and is greater during the third trimester than during the first trimester. Extracellular calcium depletion increases lymphocyte intracellular free calcium concentration. These data support the idea that a calcium deficit leading to an increased intracellular free calcium concentration during late pregnancy contributes to the pathogenesis of preeclampsia. (Am J Obstet Gynecol 1999;180:1209-14.)

Relationship between active oxygen species and cardenolide production in cell cultures of Digitalis thapsi: effect of calcium restriction

Elimination of calcium ions from the medium of undifferentiated cell cultures of Digitalis thapsi increased cardenolide production and induced extracellular H2O2 accumulation, as measured by the quenching of pyranine fluorescence. The addition of catalase reduced the response and the inclusion of superoxide dismutase enhanced the loss of fluorescence. This suggested that, besides H2O2, the superoxide anion was also formed before dismutating to H2O2. Additionally, exogenous H2O2 or superoxide dismutase stimulated cardenolide production whereas the addition of catalase markedly reduced it. These results point to a connection between H2O2 and cardenolide formation. The absence of calcium did not alter the levels of lipid peroxidation products; however, changes in the antioxidant system of D. thapsi cells were observed. Catalase activity was extremely low in control cultures and remained unaltered upon calcium elimination. Ascorbate peroxidase activity was not modified in calcium‐free cultures. By contrast, calcium deprivation stimulated superoxide dismutase activity and strongly inhibited glutathione reductase activity. Also, a significant decrease in reduced glutathione was observed. These responses were emulated by treatment of the cultures with the glutathione biosynthesis inhibitor buthionine sulfoximine and by ethyleneglycol‐bis‐β‐aminoethyl ether and LaCl3. All these results indicate that the depletion of extracellular calcium induces changes in the redox state of cells and suggest that this alteration stimulates cardenolide formation in D. thapsi cultures.

The effect of calcium-related factors on the predominance of IFN-gamma or interleukin-4 in cultured mononuclear cells.

Certain factors have been studied that might influence whether interferon-gamma (IFN-gamma) or interleukin-4 (IL-4) will predominate in cultures of peripheral blood mononuclear cells (PBMC). ELISA was used to measure cytokine protein, and PCR was used to quantitate mRNA. It was found that induction with plant lectins produced greater yields of IFN-gamma than induction with ionophores, but ionophores alone produced at least equal yields of IL-4 as did lectins. Phorbol myristate acetate (PMA) greatly enhanced IFN yields in the presence of ionophores but had no significant influence on IL-4 production. Calcium-independent cytokine induction using anti-CD28 and PMA resulted in production of both cytokines, whereas depletion of extracellular calcium and magnesium adversely influenced the yield of both IL-4 and IFN-gamma. Finally, calmidazolium, an inhibitor of calmodulin, had an enhancing effect on IFN-gamma yields when phytohemagglutinin (PHA) was the inducer and an adverse effect when A23187 was the inducer. In contrast, calmidazolium reduced IL-4 yields with both PHA and A23187 induction.

Effect of CRF and related peptides on calcium signaling in human and rodent melanoma cells

Corticotropin releasing factor (CRF) induces a rapid, within seconds, and dose-dependent increase in the intracellular Ca 2+ in both human and hamster melanoma cells. This effect is inhibited by depletion of extracellular calcium using 3 mM EGTA and is attenuated by the CRF receptor antagonist, α-helical-CRF(9-41). Other peptides of the CRF superfamily, sauvagine and urocortin, also induce increases in cytoplasmic calcium concentration but at higher concentrations than CRF. We conclude that malignant melanocytes express CRF receptors, which are coupled to activation of plasma membrane calcium channels.

Mapping of a carboxyl-terminal active site of parathyroid hormone by calciummimaging

We recently showed that the C-terminal fragment PTH (52–84) effectively increases intracellular free calcium ([Ca 2+] i in a subset of growth plate chondrocytes not activated by the N-terminal PTH fragment (1–34). Here we characterize the active site on C-terminal PTH (52–84) with respect to calcium (Ca 2+)-signaling and the mechanism involved by using synthetic PTH-subfragments in digital CCD ratio-imaging experiments. Our results show amino acids 73–76 to be the core region for increasing [Ca 2+] i. Ryanodine (1 μM), caffeine (10 mM), lithium (2 mM), or cyclopiazonic acid (2–5 μMI), agents that interfere with intracellular Ca 2+ release, all failed to block PTH (52–84) induced [Ca 2+] i increases. Depletion of extracellular calcium ([Ca 2+] o) blocked PTH (52–84) induced [Ca 2+]; increases, indicating a transmembrane Ca 2+ influx. In contrast to voltage-gated and Ca 2+ release activated Ca 2+ influx, PTH (52–84) evoked Ca 2+ influx was not blocked by nickel (1 mM). We conclude that PTH amino acids 73–76 are essential for activation of a nickel-insensitive Ca 2+ influx pathway in growth plate chondrocytes that is likely to be of relevance for matrix calcification, a key step in endochondral bone formation.

Expression of the Receptor Tyrosine Kinase Ret on the Plasma Membrane Is Dependent on Calcium

Mutations in the Ret receptor tyrosine kinase are responsible for a variety of human syndromes, including multiple endocrine neoplasia 2 and Hirschsprung's disease. Ret is expressed as a 150-kDa precursor form in the endoplasmic reticulum and a 170-kDa mature form at the plasma membrane. Here we show that expression of p170(ret) is dependent on calcium. Depletion of extracellular calcium completely blocks p170(ret) expression, which is not caused by a decrease in half-life of p170(ret) at the plasma membrane but by a defect in processing of p150(ret) into p170(ret). This processing defect can be mimicked by treating the cells with thapsigargin, a drug that releases calcium from internal stores, indicating that reduction in luminal calcium is responsible for the processing defect. We propose that a relatively high concentration of luminal calcium is necessary for the proper folding of Ret in the endoplasmic reticulum.