Characteristics of GTP-mediated microsomal Ca2+ release

Guanosine triphosphate (GTP) can release Ca2+ and enhance responses to D-myo-inositol 1,4,5-trisphosphate (IP3) in crude liver microsomes in the presence of polyethylene glycol (PEG) (Dawson et al. (1986) Biochem. J. 234, 311-315). The mechanism of these responses has been further investigated. GTP gamma S which antagonizes the actions of GTP on microsomes, does not promote Ca2+ re-uptake when added after the completion of GTP-mediated Ca2+ release. However, the effects of GTP could be reversed by washing or dilution of the microsomes. Addition of PEG to the incubation medium promoted the aggregation of microsomes. Electron microscopy provided no evidence for the fusion of microsomal vesicles in the presence or absence of GTP. In the presence of PEG, GTP produced an alteration of the permeability properties of the microsomal membrane as indicated by increased leakage of an intraluminal esterase, a reduction in the mean buoyant density of the vesicles, and a decrease in the latency of mannose 6-phosphate hydrolysis. All three effects developed relatively slowly, whereas the effects of GTP on Ca2+ fluxes occurred more rapidly (complete within 15 min). A low permeability to mannose 6-phosphate was restored upon washing away the GTP. These results suggest that non-specific permeability changes may underly the effects of GTP on Ca2+ release and that, under certain conditions, GTP can reversibly modulate the permeability of a transmembrane 'pore' in microsomal membranes that can pass ions and macromolecules. The possibility that such a pore serves to link IP3-sensitive vesicles with other Ca2+-containing compartments is discussed.     

Inositol 1,4,5-trisphosphate and guanine nucleotides activate calcium release from endoplasmic reticulum via distinct mechanisms

A sensitive and specific guanine nucleotide regulatory process has recently been shown to rapidly mediate a substantial release of Ca2+ from endoplasmic reticulum within the N1E-115 neuronal cell line (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464). The relationship between this mechanism and Ca2+ efflux mediated by the intracellular regulator inositol 1,4,5-trisphosphate (IP3) has been investigated. Using saponin-permeabilized N1E-115 cells, studies reveal a number of distinctions between the activation of Ca2+ release mediated by GTP and IP3. Thus, the GTP-mediated Ca2+ release process is specifically activated by polyethylene glycol which increases both GTP sensitivity and the extent of GTP-activated Ca2+ release; in contrast, IP3-dependent Ca2+ release is unaffected by polyethylene glycol. The non-hydrolyzable GTP analogue guanosine 5'-O-(3-thio)triphosphate, which completely inhibits GTP-mediated Ca2+ release, does not alter release mediated by IP3. Decreasing the release temperature from 37 to 4 degrees C decreases IP3-activated Ca2+ release by only 20%, whereas the action of GTP on Ca2+ release is abolished at 4 degrees C. Activation of Ca2+ release by IP3 is completely inhibited by increasing free Ca2+ from 0.1 to 10 microM, whereas the fraction of GTP-dependent Ca2+ release (approximately 50% of ionophore-releasable Ca2+) remains unaltered with increasing free Ca2+. These distinctions between IP3- and GTP-mediated Ca2+ release indicate that the two effectors function via distinct mechanisms to activate Ca2+ release; however, they do not preclude the possibility that coupling between the two mechanisms can occur or that a common Ca2+-translocating pathway activated by both effectors exists.     

Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the N1E-115 neuronal cell line

The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D. L., and Chueh, S. H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 microM IP3 effects rapid release (t1/2 less than 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 microM A23187. Half-maximal Ca2+ release occurs with 0.5 microM IP3, and maximal release with 3 microM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 microM IP3 rapidly releases (t1/2 less than 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 microM GTP, but not guanosine 5'-(beta, gamma-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t1/2 value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 microM GTP, with maximal release at 3-5 microM GTP; 20 microM GMPPNP has no effect on release and only slightly inhibits 5 microM GTP; 20 microM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 microM GTP. Using permeabilized N1E-115 cells, 5 microM GTP with 3% PEG releases greater than 50% of releasable Ca2+; without PEG, GTP still mediates approximately 30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.     

GTP- and inositol 1,4,5-trisphosphate-activated intracellular calcium movements in neuronal and smooth muscle cell lines

Recent evidence has revealed that a highly sensitive and specific guanine nucleotide regulatory process controls intracellular Ca2+ release within N1E-115 neuroblastoma cells (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464). The present report documents GTP-induced Ca2+ release within quite distinct cell types, including the DDT1MF-2 smooth muscle cell line. GTP-induced Ca2+ release has similar GTP sensitivity and specificity among cells and rapidly mobilizes up to 70% of Ca2+ specifically accumulated within a nonmitochondrial Ca2+-pumping organelle within permeabilized DDT2MF-2 cells. Maximal GTP-induced release of Ca2+ is observed to be greater than inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (the latter being approximately 30% of total releasable Ca2+). After maximal IP3-induced release, further IP3 addition is ineffective, whereas subsequent addition of GTP further releases Ca2+ to equal exactly the extent of Ca2+ release observed by addition of GTP in the absence of IP3. This suggests that IP3 releases Ca2+ from the same pool as GTP, whereas GTP also releases from an additional pool. The effects of GTP appear to be reversible since simple washing of GTP-treated cells restores their previous Ca2+ uptake properties. Electron microscopic analysis of GTP-treated membrane vesicles reveals their morphology to be unchanged, whereas treatment of vesicles with 3% polyethylene glycol, known to enhance GTP-mediated Ca2+ release, clearly induces close coalescence of membranes. In the presence of 4 mM oxalate, GTP induces a rapid and profound uptake, as opposed to release, of Ca2+. The findings suggest that GTP-activated Ca2+ movement is a widespread phenomenon among cells, which can function on the same Ca2+ pool mobilized by IP3, and although activating Ca2+ movement by a mechanism distinct from IP3, does so via a process that does not appear to involve fusion between membranes.     

The mechanism of action of GTP on Ca2+ efflux from rat liver microsomal vesicles.

1. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]), if added before GTP, blocks both Ca2+ efflux promoted by GTP and the effect of GTP on enhancement of inositol 1,4,5-triphosphate (IP3)-promoted Ca2+ release from preloaded microsomal vesicles. If, however, GTP[S] is added after GTP, it does not reverse the Ca2+ efflux promoted by GTP, nor does it inhibit IP3-promoted Ca2+ release. 2. The effect of GTP in enhancing IP3-promoted Ca2+ release is maintained after washing the microsomal vesicles free of added GTP. After this treatment, enhancement of IP3-promoted Ca2+ efflux can be observed in the absence of poly(ethylene glycol). 3. Electron microscopy shows that during GTP treatment of microsomal vesicles there is rapid production of very large vesicular structures, apparently produced by fusion of smaller vesicles. 4. Light-scattering changes are detectable during the fusion process. 5. Both Ca2+ efflux promoted by GTP and the enhancement of IP3-promoted Ca2+ release seen in the presence of GTP can probably be attributed to GTP-dependent vesicle fusion.     

Potentiation by glucose metabolites of inositol trisphosphate-induced calcium mobilization in permeabilized rat pancreatic islets

Saponin-permeabilized rat pancreatic islets degraded exogenously added inositol 1,4,5-trisphosphate (IP3), and degradation was inhibited in the presence of either fructose 1,6-bisphosphate or diphosphoglycerate. The addition of either fructose-1,6-P2 or diphosphoglycerate to 45Ca2+-labeled permeabilized islets potentiated 45Ca2+ release caused by IP3 (by either exogenously added IP3 or IP3 generated endogenously in the presence of carbachol or guanosine 5'-3-O-(thio)triphosphate (GTP gamma S). The effect of diphosphoglycerate and fructose-1,6-P2 on 45Ca2+ release correlated well with the effects of these agents on the recovery of radioactivity in IP3. These results further support our previous proposal that in pancreatic islets intracellular calcium mobilization may be sustained in part via the inhibition of IP3 degradation by metabolites produced during stimulation with insulinotropic concentrations of glucose (Rana, R.S., Sekar, M.C., Hokin, L.E., and MacDonald, M.J. (1986) J. Biol. Chem. 261, 5237-5240).     

GTP and Ca2+ Modulate the Inositol 1,4,5-Trisphosphate-Dependent Ca2+ Release in Streptolysin O-Permeabilized Bovine Adrenal Chromaffin Cells

The inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied using streptolysin O-permeabilized bovine adrenal chromaffin cells. The IP3-induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3-induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 microM GTP fully restored the sensitivity to IP3. Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) could not replace GTP but prevented the action of GTP. The effects of GTP and GTP gamma S were reversible. Neither GTP nor GTP gamma S induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 microM free Ca2+, a half-maximal Ca2+ no Ca2+ release was observed with 0.1 microM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 microM) were required to evoke Ca2+ release. At 8 microM free Ca2+, even 0.25 microM IP3 failed to induce release of Ca2+ from the store. The IP3-induced Ca2+ release at constant low (0.2 microM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+.     

Effect of guanosine triphosphate on the release of Ca2+ from intracellular store sites of saponin-treated human peripheral lymphocytes

The effects of guanosine triphosphate (GTP) on the release and uptake of Ca2+ in nonmitochondrial intracellular store sites of human peripheral lymphocytes were examined. GTP in the presence of 3% polyethylene glycol released Ca2+ from the intracellular store sites of lymphocytes in a dose-dependent manner, and the maximal release was obtained at 10 microM GTP. GDP and 5'-GMP also enhanced the release of Ca2+. On the other hand, Ca2+ uptake in the presence of oxalate by saponin-treated lymphocytes was stimulated by GTP and this stimulation was abolished when polyethylene glycol was concomitantly present. The dose dependence of the stimulated Ca2+ uptake by GTP was much the same as that of the Ca2+ released by GTP. These results indicate that GTP has an inherent activity to release Ca2+ as well as to stimulate the uptake of Ca2+ in nonmitochondrial intracellular store sites of saponin-treated lymphocytes. The stimulatory effect of polyethylene glycol on GTP-mediated Ca2+ release may occur by inhibiting functions of the Ca2+ pump.     

GTP gamma S effects on phosphatidylinositol phospholipase C alpha isoenzyme activity isolated from guinea pig uterine smooth muscle at different stages of pregnancy.

The ability of GTP gamma S to activate release of inositol polyphosphates from isolated permeabilised guinea pig uterine smooth muscle cells and from partially purified PI-PLC alpha has been studied. Streptolysin O permeabilised and [3H]inositol prelabelled cells show a time dependent release of inositol polyphosphates, predominantly inositol 4-phosphate. Ca2+ stimulated IP release with a Ka of 161 +/- 1.1 nM and this was further enhanced in an additive manner by GTP gamma S between 1-100 microM; the Ka for Ca2+ in the presence of 0.1 mM GTP gamma S was 117 +/- 0.7 nM. GTP gamma S activation of IP production did not require Ca2+ in the medium. Permeabilisation of the uterine smooth muscle cells with Streptolysin O readily released PI-PLC activity into the medium. However, unlike studies with isolated membranes 63.4 +/- 6.4% of the enzyme activity remained associated with membranes and/or particulate fractions of the cell. Studies were undertaken with PI-PLC alpha, the predominant isoenzyme form, partially purified from uterine smooth muscle at different stages of pregnancy by Q-Sepharose and Heparin-Agarose chromatography. The enzyme co-purifies with firmly associated GTP-binding activity. Enzyme prepared from near-term uterus is activated by 0.1 mM GTP gamma S, up to 100% when Ca2+ is between 0.1-1 microM, while 10 microM AlF4- under those conditions caused complete inhibition of the enzymes. Responses for enzymes prepared from non-pregnant uteri were broadly similar. In contrast enzyme preparations from guinea pig uteri at 20-60 days of pregnancy show an inhibition of activity in response to 0.1 mM GTP gamma S addition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inositol 1,4,5-trisphosphate-induced calcium release from platelet plasma membrane vesicles

A platelet membrane preparation, enriched in plasma membrane markers, took up 45Ca2+ in exchange for intravesicular Na+ and released it after the addition of inositol 1,4,5-trisphosphate (IP3). The possibility that contaminating dense tubular membrane (DTS) vesicles contributed the Ca2+ released by IP3 was eliminated by the addition of vanadate to inhibit Ca+-ATPase-mediated DTS Ca2+ sequestration and by the finding that only plasma membrane vesicles exhibit Na+-dependent Ca2+ uptake. Ca2+ released by IP3 was dependent on low extravesicular Ca2+ concentrations. IP3-induced Ca2+ release was additive to that released by Na+ addition while GTP or polyethylene glycol (PEG) had no effect. These results strongly suggest that IP3 facilitates extracellular Ca2+ influx in addition to release from DTS membranes.     

Guanosine 5'-triphosphate releases calcium from rat liver and guinea pig parotid gland endoplasmic reticulum independently of inositol 1,4,5-trisphosphate

GTP releases calcium from rat liver microsomes and guinea pig parotid gland microsomal subfractions independently of the presence of inositol 1,4,5-trisphosphate (IP3). Non-hydrolyzable guanine nucleotide analogues have no effect and inhibit the effect of GTP. The mechanism of GTP-mediated calcium release differs from IP3-mediated calcium release as indicated by the following findings: GTP-induced calcium release depends on the presence of compounds which increase the viscosity of the medium (polyethylene glycol, polyvinylpyrrolidone, or bovine serum albumin); GTP-mediated calcium release is much slower; GTP-mediated calcium release is strongly temperature-dependent, whereas IP3-mediated calcium release is not; GTP-mediated calcium release is much more sensitive to a decrease of intravesicular free calcium than IP3-mediated calcium release.     

Effect of GTP and Ca2+ on inositol 1,4,5-trisphosphate induced Ca2+ release from permeabilized rat exocrine pancreatic acinar cells

The effects of Ca2+ and GTP on the release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3) sensitive Ca2+ compartment were investigated with digitonin permeabilized rat pancreatic acinar cells. The amount of Ca2+ released due to IP3 directly correlated with the amount of stored Ca2+ and was found to be inversely proportional to the medium free Ca2+ concentration. Ca2+ release induced by 0.18 microM IP3 was half maximally inhibited at 0.5 microM free Ca2+, i.e. at concentrations observed in the cytosol of pancreatic acinar cells. GTP did not cause Ca2+ release on its own, but a single addition of GTP (20 microM) abolished the apparent desensitization of the Ca2+ release which was observed during repeated IP3 applications. This effect of GTP was reversible. GTP gamma S could not replace GTP. Desensitization still occurred when GTP gamma S was added prior to GTP. The reported data indicate that GTP, stored Ca2+ and cytosolic free Ca2+ modulate the IP3 induced Ca2+ release.     

Inositol 1,4,5-trisphosphate induced calcium release from corn coleoptile microsomes

The effect of inositol 1,4,5-trisphosphate (IP3) on Ca2+ release from microsomes of corn coleoptiles was investigated. Addition of micromolar concentrations of IP3 to Ca2+ loaded microsomes resulted in rapid release of 20-30% of sequestered Ca2+. Maximal and half maximal Ca2+ release occurred at 20 and 8 microM of IP3 respectively. Part of the Ca2+ released by IP3 was reaccumulated into microsomes within 4 min. The amount of Ca2+ released by IP3 was found to be dependent on free Ca2+ concentration in the incubation medium at the time of release. Maximum Ca2+ release was observed around 0.1 microM free Ca2+ concentration in the assay medium. These data suggest that IP3 might act as a second messenger in plants in a manner similar to animal systems by altering cytosolic levels of calcium.     

Effects of GTP on Ca2+ movements across endoplasmic reticulum membranes

Our initial observation that GTP could, under some experimental conditions, have profound effects on Ca2+ movements across endoplasmic reticulum membranes arose from attempts to increase the sensitivity of rat liver microsomes to inositol 1,4,5 trisphosphate (IP3). Most preparations of microsomal fractions from rat liver release only a very small percentage of accumulated Ca2+ on addition of IP3. We found, rather empirically, that the addition of microM concentrations of GTP greatly enhanced the amount of Ca2+ releasable by IP3. The initial, very appealing, hypothesis was to postulate a direct effect of GTP on the IP3-sensitive Ca2+ channel. This idea is no longer tenable, as will be described below. The more likely explanation, that GTP has its effect by either fusing small microsomal vesicles together or by allowing some form of communication between adjacent membranes is considerably more complex mechanistically and also possibly has far reaching implications for the mechanisms by which cells organise and maintain their reticular structures.     

GTP requirement for inositol-1,4,5-trisphosphate-induced Ca2+ release from sarcoplasmic reticulum in smooth muscle

We have examined inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release from the sarcoplasmic reticulum (SR) in the skinned vascular smooth muscle. The amount of Ca2+ in the SR was estimated indirectly by caffeine-induced contraction of the skinned preparation. The Ca2+ release from the SR by IP3 required GTP. A non-hydrolyzable analogue of GTP, guanosine 5'-(beta gamma-imido) triphosphate (GppNHp) could substitute for GTP in the IP3-induced Ca2+ release. These results suggest an involvement of GTP-binding protein in the mechanism of Ca2+ release from the SR by IP3 in smooth muscle.     

Effect of inositol 1,4,5-trisphosphate and GTP on calcium release from pituitary microsomes

Microsomal vesicles from bovine anterior pituitary accumulate Ca2+ and maintain a steady-state ambient Ca2+ level of 200 nM. IP3 and GTP both induce calcium release from the microsomal vesicles. The effect of IP3 is inhibited by polyethylene glycol (PEG), and the effect of GTP is absolutely dependent on PEG. Half-maximal effect of IP3 (without PEG) is 0.26 micron, the maximal calcium release attaining 7% of the A23187-releasable pool. The same values for GTP (in the presence of PEG) are 80 microM and 10%, respectively. GTP potentiates the effect of IP3. This potentiation is not mediated by protein phosphorylation.     

Liver glucose-6-phosphatase activity is modulated by physiological intracellular Ca2+ concentrations

The effect of varying concentrations of free Ca2+ on the formation of Pi from mannose-6-P or of Pi and [U-14C]glucose from [U-14C]glucose-6-P was investigated in isolated fasted rat hepatocytes made permeable by freezing and in liver microsomes. Free Ca2+ concentration was adjusted by the use of Ca-EGTA buffers. In permeabilized cells, glucose-6-phosphatase (EC 3.1.3.9) activity was inhibited up to 50% and in intact microsomes up to 70% by increasing free Ca2+ concentrations from 0.01 to 10 microM. The inhibition was reversible and competitive with respect to glucose-6-P. Treatment of microsomes with 0.4% deoxycholate exposed 90% of latent mannose-6-phosphatase activity which was insensitive to Ca2+. The results indicate that Ca2+ affects the glucose-6-P translocase rather than the phosphohydrolase component. It is concluded that the glucose-6-phosphatase system is modulated by changes in Ca2+ concentrations in the range of those occurring in the liver cell upon hormonal stimulation.     

Ca2+ release triggered by NAADP in hepatocyte microsomes

NAADP (nicotinic acid-adenine dinucleotide phosphate) is fast emerging as a new intracellular Ca2+-mobilizing messenger. NAADP induces Ca2+ release by a mechanism that is distinct from IP3 (inositol 1,4,5-trisphosphate)- and cADPR (cADP-ribose)-induced Ca2+ release. In the present study, we demonstrated that micromolar concentrations of NAADP trigger Ca2+ release from rat hepatocyte microsomes. Cross-desensitization to IP3 and cADPR by NAADP did not occur in liver microsomes. We report that non-activating concentrations of NAADP can fully inactivate the NAADP-sensitive Ca2+-release mechanism in hepatocyte microsomes. The ability of thapsigargin to block the NAADP-sensitive Ca2+ release is not observed in sea-urchin eggs or in intact mammalian cells. In contrast with the Ca2+ release induced by IP3 and cADPR, the Ca2+ release induced by NAADP was completely independent of the free extravesicular Ca2+ concentration and pH (in the range 6.4-7.8). The NAADP-elicited Ca2+ release cannot be blocked by the inhibitors of the IP3 receptors and the ryanodine receptor. On the other hand, verapamil and diltiazem do inhibit the NAADP- (but not IP3- or cADPR-) induced Ca2+ release.     

Stimulatory effect of glucose 6-phosphate on the non-mitochondrial Ca2+ uptake in permeabilized hepatocytes and Ca2+ release by inositol trisphosphate

The relationships between Ca2+ transport and glucose-6-phosphatase activity, previously studied in isolated liver microsomes, were investigated in permeabilized hepatocytes in the presence of mitochondrial inhibitors. It was found that the addition of glucose 6-phosphate to the cells markedly stimulates the MgATP-dependent Ca2+ uptake. A progressive increase in the stimulation of Ca2+ uptake was seen with increasing amounts of glucose 6-phosphate up to 5 mM concentrations. Vanadate, when added in adequate concentrations (20-40 microM) to the hepatocytes inhibits both the glucose-6-phosphatase activity and the stimulation of Ca2+ uptake by glucose 6-phosphate, while not affecting the MgATP-dependent Ca2+ uptake. The addition of inositol 1,4,5-trisphosphate to permeabilized hepatocytes in which Ca2+ had been accumulated in the presence of MgATP and glucose 6-phosphate, results in a rapid release of Ca2+.     

High extracellular Ca2+ and Mg2+ stimulate accumulation of inositol phosphates in bovine parathyroid cells

We examined the effects of the divalent cations Ca2+ and Mg2+ on inositol phosphate accumulation in bovine parathyroid cells prelabelled with [3H]inositol to determine whether the high extracellular Ca2+ and Mg2+-evoked transients in cytosolic Ca2+ in these cells might result from increases in cellular IP3 levels. In the presence of Li+, both Ca2+ and Mg2+ produced rapid, 2-6-fold increases in IP3 and IP2 and a linear increase in IP of 6-8-fold at 30 min. Smaller (1.5-2-fold) increases in IP2 and IP3 were evident within 7.5-15 s upon exposure to high (3 mM) Ca2+ in the absence of Li+. The relative potencies of Ca2+ and Mg2+ (Ca2+ 3-fold more potent than Mg2+) in elevating inositol phosphates were similar to those for their effects in inhibiting PTH release. Fluoride (5 and 10 mM) also produced similar increases in inositol phosphate accumulation, presumably through activation of phospholipase C by a guanine nucleotide (G) protein-dependent process. Thus, high extracellular Ca2+ and Mg2+-induced spikes in cytosolic Ca2+ in bovine parathyroid cells may be mediated by increases in IP3, perhaps through a receptor-mediated process linked to phospholipase C by a G-protein.