Tricking the parathyroid gland with novel calcimimetic agents.
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Aniline Compounds↗
Biomedical subjects
E F Nemeth
Publications and source records attributed to E F Nemeth.
Allosteric activation of the Ca2+ receptor expressed in Xenopus laevis oocytes by NPS 467 or NPS 568.
The Ca2+ receptor is a G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to changes in the concentration of extracellular Ca2+. In this study, two novel phenylalkylamine compounds, NPS 467 and NPS 568, were examined for effects on Xenopus laevis oocytes expressing the bovine or human parathyroid Ca2+ receptors. Increases in chloride current (ICl) were elicited in oocytes expressing the bovine Ca2+ receptor when the extracellular Ca2+ concentration was raised above 1.5 mM, whereas Ca2+ concentrations > 3 mM were generally necessary to elicit responses in oocytes expressing the human Ca2+ receptor. NPS 467 and NPS 568 potentiated the activation of ICl by extracellular Ca2+ in oocytes expressing either Ca2+ receptor homolog, and this resulted in a leftward shift of the Ca2+ concentration-response curve. Neither compound was active in the absence of extracellular Ca2+. Certain inorganic and organic cations known to activate the Ca2+ receptor were substituted for elevated levels of extracellular Ca2+ to increase ICl and the effects of these agonists were also potentiated by NPS 568 or NPS 467. The effects of NPS 568 were stereoselective and the R-enantiomer was about 10-fold more potent than the corresponding S-enantiomer. Neither NPS 467 nor 568 affected ICl in water-injected oocytes or in oocytes expressing the substance K receptor or the metabotropic glutamate receptor 1a. These results provide compelling evidence that NPS 467 and NPS 568 act directly upon the parathyroid Ca2+ receptor to increase its sensitivity to activation by extracellular Ca2+. This activity suggests that these compounds are positive allosteric modulators of the Ca2+ receptor. As such, these compounds define a new class of pharmacological agents with potent and selective actions on the Ca2+ receptor.
Allosteric Regulation↗
Plasma levels of parathyroid hormone that induce anabolic effects in bone of ovariectomized rats can be achieved by stimulation of endogenous hormone secretion.
Parathyroid hormone (PTH) administration increases bone mass in normal and osteopenic animals. However, this treatment currently requires the daily injection of large amounts of PTH, and the relationship of these doses to plasma levels of PTH that are achievable physiologically is unknown. We determined in ovariectomized (ovx) rats: 1) the plasma PTH levels that occur after the subcutaneous injection of graded doses of rat PTH, 2) whether similar PTH levels can be achieved by stimulation of endogenous PTH secretion, and 3) whether a plasma PTH profile that is achievable physiologically is anabolic on bone. Injection of 1, 5, or 25 micrograms/kg rat PTH-(1-34) increased plasma PTH by 46, 164, or 520 pg/mL, respectively, above basal levels within 60 min. Infusion of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid for 2 h reduced plasma Ca2+ by 0.36 mmol/L and produced a total plasma PTH response (area under the plasma PTH curve) similar to that with the 5 micrograms/kg rat PTH injection. Then, 1, 5, or 25 micrograms/kg doses of rat PTH-(1-34) were injected subcutaneously daily for 28 days in 19-week-old rats that were ovx 7 weeks earlier. The 5 and 25 micrograms/kg doses significantly increased bone mineral density in the distal femur and trabecular bone area and average trabecular thickness in the proximal tibia. All doses of PTH significantly increased indices of trabecular connectivity and cancellous bone formation, including double-labeled surface, mineralizing surface, and surface-referent bone formation rate. In conclusion, anabolic effects on bone can be achieved with a plasma PTH profile similar to that attained following stimulation of the parathyroid gland by induced hypocalcemia. These data suggest that agents that transiently increase endogenous PTH secretion may represent a novel means to promote anabolic effects in skeletal tissues.
Absorptiometry, Photon↗
The parathyroid calcium receptor: a novel therapeutic target for treating hyperparathyroidism.
Parathyroid cells, C-cells, and certain cells in the kidney express a cell surface calcium (Ca2+) receptor which enables these cells to detect and respond to changes in the concentration of extracellular Ca2+. This receptor protein is a member of the G protein-coupled receptor superfamily and shares limited sequence homology only with metabotropic glutamate receptors. The Ca2+ receptor is the primary physiological mechanism regulating the secretion of parathyroid hormone (PTH) and plays a pivotal role in maintaining systemic Ca2+ homeostasis. Compounds that act as Ca2+ receptor agonists are called calcimimetics because they mimic or potentiate the effects of extracellular Ca2+ on parathyroid cell function. NPS R-568 is a small organic calcimimetic compound that acts as a positive allosteric modulator to increase the sensitivity of the Ca2+ receptor to activation by extracellular Ca2+. In normal rats, orally administered NPS R-568 decreases plasma levels of PTH and Ca2+ and, at higher doses, increases plasma levels of calcitonin. The changes in the circulating levels of these two hormones explain the hypocalcemia caused by this compound. NPS R-568 also effectively lowers plasma PTH levels in normal humans and in rat models of secondary hyperparathyroidism. Calcimimetic compounds that target the Ca2+ receptor provide a novel therapeutic approach for treating primary and secondary hyperparathyroidism.
Animals↗
Molecular cloning and functional expression of human parathyroid calcium receptor cDNAs.
Parathyroid cells express a cell surface receptor, coupled to the mobilization of intracellular Ca2+, that is activated by increases in the concentration of extracellular Ca2+ and by a variety of other cations. This "Ca2+ receptor" (CaR) serves as the primary physiological regulator of parathyroid hormone secretion. Alterations in the CaR have been proposed to underlie the increases in Ca2+ set-point seen in primary hyperparathyroidism due to parathyroid adenoma. We have isolated human CaR cDNAs from an adenomatous parathyroid gland. The cloned receptor, expressed in Xenopus oocytes, responds to extracellular application of physiologically relevant concentrations of Ca2+ and other CaR agonists. The rank order of potency of CaR agonists displayed by the native receptor (Gd3+ > neomycin B > Ca2+ > Mg2+) is maintained by the expressed receptor. The nucleotide sequence of the human CaR cDNA predicts a protein of 1078 amino acids with high sequence similarity to a bovine CaR, and displays seven putative membrane-spanning regions common to G protein-coupled receptors. The deduced protein sequence shows potential sites for N-linked glycosylation and phosphorylation by protein kinase C and has a low level of sequence similarity to the metabotropic glutamate receptors. Comparison of the cDNA sequence to that of the normal human CaR gene showed no alteration in the coding region sequence of the CaR in this particular instance of parathyroid adenoma. Human cDNA clones with differing 5'-untranslated regions were isolated, suggesting alternative splicing of the parathyroid CaR mRNA. A rare variant cDNA clone representing a 10 amino acid insertion into the extracellular domain was also isolated. Northern blot analysis of normal and adenomatous parathyroid gland mRNA identified a predominant transcript of approximately 5.4 kilobases, and less abundant transcripts of approximately 10, 4.8 and 4.2 kilobases in RNA from the adenoma. While there is no evidence for alteration of the primary amino acid sequence of the CaR in this adenoma, modulation of CaR biosynthesis through alternative RNA processing may play a role in set-point alterations.
Adenoma↗
Calcium receptor messenger ribonucleic acid levels in the parathyroid glands and kidney of vitamin D-deficient rats are not regulated by plasma calcium or 1,25-dihydroxyvitamin D3.
The level of extracellular ionized calcium ([Ca2+]o) is the primary physiological regulator of PTH secretion. Complementary DNAs encoding the calcium receptor (CaR) protein that mediates this response have been cloned from bovine and human parathyroid glands. This protein is a seven-transmembrane, G-protein-coupled receptor linked to the mobilization of intracellular Ca2+ in response to increases in [Ca2+]o. More recently, a rat kidney CaR has been cloned and shown to be 92% identical at the amino acid level to the bovine parathyroid CaR. Homologous or heterologous regulation of the expression and/or function of a variety of G-protein-coupled receptors has been documented in numerous cell types. Therefore, we determined whether [Ca2+]o and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], major regulators of PTH synthesis and secretion, affect CaR gene expression in parathyroid gland and kidney in rats. CaR messenger RNA (mRNA) levels were quantified in pairs of parathyroid glands and single kidneys from individual animals using a solution hybridization assay. The effects of Ca2+ and 1,25-(OH)2D3 on CaR gene expression were assessed independently in vitamin D-deficient (-D) rats. A wide range of plasma Ca2+ levels (0.7-1.9 mM) was produced by supplementing -D diets with varying amounts of calcium and by infusing CaCl2 i.v. for 7 days using osmotic minipumps. There was no correlation between plasma Ca2+ levels and steady state CaR mRNA levels in parathyroid gland (r = -0.18) or kidney (r = 0.25). In another group of -D rats, 1,25-(OH)2D3 was infused sc at 25 and 275 ng/kg.day for 10-12 days. Dietary calcium was adjusted to maintain normocalcemia in some of the groups. No effect of 1,25-(OH)2D3 administration on CaR mRNA levels occurred in parathyroid glands or kidney regardless of the resultant plasma Ca2+ or 1,25-(OH)2D3 levels. In conclusion, neither parathyroid gland nor kidney CaR mRNA levels are regulated by plasma Ca2+ and 1,25-(OH)2D3 levels in the experimental models examined here.
Animals↗
Voltage-activated potassium currents of rabbit osteoclasts: effects of extracellular calcium.
The effects of increased extracellular Ca2+ concentration ([Ca2+]e) were examined on a delayed-rectifier K+ current (IK) and an inward-rectifier K+ current (IK1) in rabbit osteoclasts. Elevation of [Ca2+]e from 1.8 to 18 mM shifted the half point for IK activation by +11.5 mV and the voltage dependence of inactivation by +9.7 mV and slowed the rate of IK activation and deactivation. These effects of elevated [Ca2+]e on IK are consistent with screening of cell surface negative charge. However, elevation of [Ca2+]e increased the voltage-dependent kinetics of IK inactivation at all potentials tested, inconsistent with that predicted by simple surface charge theory. This finding suggests an additional, regulatory role for [Ca2+]e in the gating of IK channels. Some osteoclasts had an IK1, which was decreased when [Ca2+]e was raised from 1.8 to 18 mM. The physiological function of both types of K+ currents remains to be determined, and it is not clear whether these currents are involved with the coupling of cytosolic [Ca2+] to [Ca2+]e.
Animals↗
Stimulus-secretion coupling in parathyroid cells deficient in protein kinase C activity.
The role of protein kinase C (PKC) in regulating cytosolic Ca2+ concentrations ([Ca2+]i) and parathyroid hormone (PTH) secretion was studied in bovine parathyroid cells rendered deficient in PKC activity by incubation with phorbol 12-myristate 13-acetate (PMA). Pretreatment with PMA caused a time- and concentration-dependent loss of functional PKC activity as assessed by the failure of [Ca2+]i and PTH secretion to respond to the subsequent addition of PKC activators or the inhibitor staurosporine. Pretreatment for 24 h with 1 microM PMA caused a loss of 85% of the total and 98% of the cytosolic PKC activity. Cells so pretreated were considered to be "PKC downregulated." Increasing the concentration of extracellular Ca2+ or Mg2+ caused corresponding increases in [Ca2+]i that were similar in control and in PKC-downregulated cells. PTH secretion regulated by extracellular Ca2+ or Mg2+ was likewise similar in control and PKC-downregulated cells. Stimulus-secretion coupling is thus unimpaired in parathyroid cells deficient in PKC activity. Cytosolic Ca2+ responses remained depressed in cells incubated for 24 h with low concentrations of PMA (30 or 100 nM). However, under these conditions, extracellular Ca2+ still suppressed PTH secretion similarly to control cells. These results reveal a dissociation between cytosolic Ca2+ and PTH secretion and suggest that signals other than cytosolic Ca2+ are involved in the regulation of PTH secretion.
Animals↗
Functional expression of the parathyroid cell calcium receptor in Xenopus oocytes.
Various studies suggest the existence of a plasma membrane receptor on parathyroid cells that senses changes in the concentration of extracellular Ca2+. To test this hypothesis, Xenopus laevis oocytes were injected with poly(A)(+)-enriched mRNA from bovine parathyroid cells and examined for their ability to respond to increases in the concentration of extracellular Ca2+ or other polycations. Cytosolic Ca2+ concentrations were measured indirectly by recording Cl- currents through the endogenous, cytosolic Ca(2+)-activated Cl- channel. Increasing the concentration of extracellular Ca2+ (from 0.7 to 5 mM) or Mg2+ (from 0.8 to 10 mM) elicited oscillatory increases in the Cl- current. Responses to either divalent cation were not observed in oocytes injected with water or with mRNA prepared from HL-60 cells or rat liver. Responses elicited by extracellular Mg2+ persisted when extracellular Ca2+ was reduced to low micromolar levels. La3+, Gd3+, or neomycin B also evoked oscillatory increases in the Cl- current in oocytes under conditions of low extracellular Ca2+ levels. These extracellular polycations all cause the mobilization of intracellular Ca2+ in oocytes injected with parathyroid cell mRNA like they do in intact parathyroid cells. The injection of parathyroid cell mRNA thus confers on oocytes the ability to detect and respond to changes in the concentration of extracellular polycations. The data provide compelling evidence for the existence of a cell surface Ca2+ receptor protein(s) on parathyroid cells that regulates cellular function.
Animals↗
Cytosolic calcium homeostasis in bovine parathyroid cells and its modulation by protein kinase C.
1. The effects of protein kinase C (PKC) activators and inhibitors on the mechanisms regulating cytosolic Ca2+ homeostasis in dissociated bovine parathyroid cells loaded with fura-2 were examined. 2. Stepwise increases in the concentration of extracellular Ca2+ (from 0.5 to 2 or 3 mM) elicited transient followed by sustained increases in the concentration of intracellular free Ca2+ ([Ca2+]i). Cytosolic Ca2+ transients reflected the mobilization of intracellular Ca2+ and influx of extracellular Ca2+ whereas sustained increases in [Ca2+]i resulted from the influx of extracellular Ca2+. Brief (1-2 min) pretreatment with phorbol myristate acetate (PMA) shifted the concentration-response curve for extracellular Ca(2+)-induced cytosolic Ca2+ transients to the right without affecting the maximal response. Cytosolic Ca2+ transients elicited by extracellular Mg2+ were similarly affected by PMA. 3. These effects of PMA were mimicked by various other activators of PKC with the rank order of potency PMA > phorbol dibutyrate > bryostatin , > (-)indolactam V > mezerein. Isomers or analogues of these compounds that do not alter PKC activity (4 alpha-phorbols and (+)indolactam V) did not alter [Ca2+]i. 4. PKC activators depressed evoked increases in [Ca2+]i when influx of extracellular Ca2+ was blocked with Gd3+. Cytosolic Ca2+ transients elicited by extracellular Mg2+ in the absence of extracellular Ca2+ were similarly inhibited by PKC activators. Activation of PKC thus inhibits the mobilization of intracellular Ca2+ elicited by extracellular divalent cations. 5. Increases in the concentration of extracellular Ca2+ caused corresponding increases in the formation of [3H]inositol 1,4,5-trisphosphate ([3H]InsP3). Pretreatment with PMA shifted the concentration-response curve for extracellular Ca(2+)-induced [3H]InsP3 formation to the right without affecting the maximal response. 6. PKC activators also caused some depression of steady-state increases in [Ca2+]i elicited by extracellular Ca2+. In contrast, PMA did not affect increases in [Ca2+]i elicited by ionomycin or thapsigargin. 7. Ba2+ was used to monitor divalent cation influx. PMA decreased the rate of rise of the fluorescent signal elicited by extracellular Ba2+. 8. All these effects of PKC activators on [Ca2+]i were blocked or reversed by staurosporine at concentrations (30-100 nM) that inhibited PKC activity in parathyroid cells. Staurosporine alone potentiated cytosolic Ca2+ responses evoked by submaximal concentrations of extracellular divalent cations. 9. PKC thus depresses both the mobilization of intracellular Ca2+ and the influx of extracellular Ca2+ in parathyroid cells. The effects on [Ca2+]i provide evidence for a Ca2+ receptor on the surface of parathyroid cells that uses transmembrane signalling mechanisms common to some other Ca(2+)-mobilizing receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
Alkaloids↗
Protein kinase C modulates hormone secretion regulated by extracellular polycations in bovine parathyroid cells.
1. The role of protein kinase C (PKC) in the regulation of parathyroid hormone (PTH) secretion was examined in dissociated bovine parathyroid cells. 2. Increasing the concentration of extracellular Ca2+ from 0.5 to 2 mM inhibited PTH secretion by 60-80%. Similar depressive effects on secretion were obtained by increasing the concentration of extracellular Mg2+ from 1 to 7 mM or by adding La3+ (to 40 microM). The PKC activator phorbol myristate acetate (PMA) depressed PTH secretion at the lower and potentiated secretion at the higher concentrations of extracellular Ca2+, Mg2+ or La3+. The inhibitory effect of PKC on secretion correlated positively with the magnitude of the inhibitory effect elicited by elevated extracellular Ca2+. 3. The stimulatory effects of PKC activators on PTH secretion were reversed completely and the inhibitory effects were reversed partially by the PKC inhibitor staurosporine. Staurosporine alone did not affect secretion at low (0.5 mM) or high (2 mM) concentrations of extracellular Ca2+ but it did depress secretion at intermediate concentrations (around 1 mM) of extracellular Ca2+. 4. The stimulatory effects of PKC activators on secretion were overcome by increases in the concentration of extracellular Ca2+ (to 5 or 10 mM) or La3+ (to 100 microM). In contrast, increasing the concentration of extracellular Mg2+ to 11 or 19 mM did not alleviate the potentiating effects of PKC activators. The different results obtained with Ca2+ and Mg2+ could not be explained by their different effects on cytosolic Ca2+ and suggests that different cations can have varying degrees of efficacy to activate functional responses linked to the Ca2+ receptor on bovine parathyroid cells. 5. PTH secretion stimulated by isoprenaline was not affected by PKC activators or staurosporine. Similarly, the inhibitory effects of extracellular ATP gamma S on secretion were unaffected by PKC activators. These results show that PKC activators affect specifically PTH secretion regulated by extracellular polycations. 6. The stimulatory effect of PKC activators on secretion parallels its inhibitory effects on [Ca2+]i and inositol trisphosphate formation, showing that PKC blunts the mechanisms associated with extracellular Ca(2+)-induced inhibition of secretion. The specificity of these actions suggests that PKC acts at a very early step of stimulus-secretion coupling in parathyroid cells, specific to that used by extracellular polycations and perhaps involving the Ca2+ receptor.
Adenosine Triphosphate↗
Arylamine toxins from funnel-web spider (Agelenopsis aperta) venom antagonize N-methyl-D-aspartate receptor function in mammalian brain.
The venom of the North American funnel-web spider Agelenopsis aperta contains a variety of arylamine toxins (the alpha-agatoxins) that paralyze insects by blocking glutamatergic neuromuscular transmission. We have tested six synthetic alpha-agatoxins for their ability to antagonize glutamate receptor function in mammalian brain. These compounds produce, at submicromolar concentrations, noncompetitive inhibition of N-methyl-D-aspartate (NMDA) receptor-mediated elevations in the concentration of cytosolic free calcium in cultured rat cerebellar granule neurons. In contrast, the alpha-agatoxins are relatively weak antagonists of elevations in the cytosolic free calcium concentration induced by non-NMDA receptor agonists. The alpha-agatoxins also produce reversible suppression of the NMDA receptor-mediated excitatory postsynaptic potential in rat hippocampal slices at concentrations that have little effect on the non-NMDA receptor-mediated population spike. We conclude that the alpha-agatoxins are selective and reversible noncompetitive antagonists at NMDA receptors in mammalian brain.
Animals↗
Modulation of N-methyl-D-aspartate receptor-mediated increases in cytosolic calcium in cultured rat cerebellar granule cells.
The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in rat cerebellar granule cells using the fluorescent indicator fura-2. Culturing the cells as monolayers on plastic squares which could be placed into cuvettes allowed measurements of [Ca2+]i to be performed on large and homogeneous populations of CNS neurons. Granule cells so cultured maintained low levels of [Ca2+]i (around 90 nM) which increased promptly upon the addition of various excitatory amino acids including N-methyl-D-aspartate (NMDA). Increases in [Ca2+]i elicited by NMDA were inhibited by Mg2+ (1 mM) and often potentiated by glycine (1 microM). The addition of TTX or strychnine (5 microM each) did not alter responses to NMDA or NMDA plus glycine. Cytosolic Ca2+ responses to NMDA/glycine were dependent on the presence of extracellular Ca2+ and were unaffected by concentrations of nifedipine or verapamil that blocked increases in [Ca2+]i elicited by K+ depolarization. Responses elicited by NMDA/glycine were inhibited competitively by 2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid and non-competitively by MK-801 or Mg2+. HA-966 and 7-chlorokynurenate inhibited responses to NMDA alone and blocked competitively the potentiating effects of glycine. The results demonstrate NMDA-mediated increases in [Ca2+]i in cerebellar granule cells that arise solely from influx of extracellular Ca2+ through dihydropyridine-insensitive channels. The strict dependence of the NMDA-evoked response on extracellular Ca2+ provides little evidence for a coupling of NMDA receptors to inositol phosphate metabolism and mobilization of intracellular Ca2+. The effect of various agents on NMDA/glycine-induced increases in [Ca2+]i parallels their effects on ligand binding to or current flow through the NMDA receptor-channel complex. The measurement of cytosolic Ca2+ in this preparation of neuronal cells thus appears especially well suited for assessing, on a functional level, the regulation of NMDA receptors in the CNS.
Animals↗
Lithium effects on dispersed bovine parathyroid cells grown in tissue culture.
It is not clear whether hypercalcemia and hyperparathyroidism associated with lithium therapy are the result of an unmasking of preexisting disease or a direct effect of lithium on the parathyroid glands. To investigate this phenomenon, parathyroid hormone (PTH) secretion and cytosolic calcium concentrations [( Ca]i) were measured in normal and lithium-treated dispersed bovine parathyroid cells grown in tissue culture and incubated with varying concentrations of extracellular calcium [( Ca]e) (0.5 to 2.5 mmol/L). Results indicate that lithium has two effects on parathyroid secretory response: (1) a decrease in low calcium-stimulated PTH release and (2) a potentiation of PTH release at physiologic concentrations of extracellular calcium. [Ca]i was assessed by use of fura-2, a calcium-sensitive fluorescent indicator. Resting [Ca]i levels were unaffected by lithium (103 +/- 13 nmol/L in controls vs 101 +/- 5 nmol/l in lithium-treated cells, mean +/- SE). Subsequent increases in [Ca]i in response to increases in [Ca]e were significantly less in lithium-treated cells, with no difference at maximal [Ca]e. Increases in [Ca]i in response to a submaximal concentration of extracellular magnesium were also blunted in cells pretreated with lithium. In conclusion, our data suggest that, at physiologic calcium concentrations, lithium decreases parathyroid cell sensitivity to changes in [Ca]e, reducing [Ca]i levels and increasing PTH secretory response.
Animals↗
The role of extracellular calcium in the regulation of intracellular calcium and cell function.
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Animals↗
Regulation of cytosolic calcium by extracellular divalent cations in C-cells and parathyroid cells.
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Animals↗
Cytosolic Ca2+ in melanotrophs: pharmacological insights into regulatory influences of electrical activity and ion channels.
The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in melanotrophs, the characteristic endocrine cells of the pars intermedia of the rat pituitary gland, using the fluorescent Ca indicator fura-2. The resting [Ca2+]i was 211 +/- 8 nM and was little affected by tetrodotoxin (TTX; 5 or 10 microM), which inhibits the spontaneous action potentials that occur in these cells. Removal of extracellular Ca2+ (by chelation with EGTA) or addition of the Ca channel blocker nimodipine (1 microM) produced a rapid fall in [Ca2+]i, which occurred whether TTX was present or not. Excess K+ (60 mM), veratridine (10 or 100 microM) and BAY K 8644 (1 microM) each caused a rapid rise in [Ca2+]i, which was blocked or truncated by EGTA or nimodipine. TTX blocked or truncated the increases in [Ca2+]i induced by veratridine, but not those induced by either excess K+ or BAY K 8644. The results show that manipulations that increase or decrease hormone output increase or decrease [Ca2+]i. Furthermore, the resting [Ca2+]i appears to depend importantly on Ca influx, since it is rapidly and markedly reduced by removal of extracellular Ca2+ or addition of a Ca channel blocker.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Adenine nucleotides mobilize cellular Ca2+ and inhibit parathyroid hormone secretion.
Measurements of the concentration of intracellular free calcium [( Ca2+]i) were used to screen for the presence of Ca2+-mobilizing receptors on dissociated and purified bovine parathyroid cells loaded with fura-2. Among a wide variety of agents known to mobilize cellular Ca2+ in other cells, only ATP and certain other nucleotides were capable of altering [Ca2+]i in parathyroid cells. The addition of ATP or adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) (10-200 microM) to parathyroid cells evoked a rapid and transient increase that was followed by a small, steady-state increase in [Ca2+]i. Cytosolic Ca2+ transients elicited by ATP or ATP gamma S persisted in the absence of extracellular Ca2+ and presence of a mitochondrial uncoupler but were blocked by pretreatment with ionomycin or fluoride. Cytosolic Ca2+ transients elicited by ATP were inhibited by increased concentrations of extracellular Ca2+, Mg2+, or Sr2+. Conversely, ATP depressed increases in [Ca2+]i elicited by these extracellular divalent cations. Parathyroid hormone (PTH) secretion was inhibited by ATP gamma S but not by those nucleotides that were without effect on [Ca2+]i. Loading cells with 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and fura-2 blocked cytosolic Ca2+ transients elicited by ATP gamma S but did not block the inhibitory effects of ATP gamma S on PTH secretion. The results show that the activation of a calcium-mobilizing receptor, in this case by ATP gamma S, is sufficient to inhibit PTH secretion. This favors the view that extracellular Ca2+ acts via a Ca2+-mobilizing receptor to regulate PTH secretion.
Adenine Nucleotides↗