Calcium-mobilizing Agent Research Articles

SARM1 is a multi-functional NAD(P)ase with prominent base exchange activity, all regulated bymultiple physiologically relevant NAD metabolites

SummarySARM1 is an NAD(P) glycohydrolase and TLR adapter with an essential, prodegenerative role in programmed axon death (Wallerian degeneration). Like other NAD(P)ases, it catalyzes multiple reactions that need to be fully investigated. Here, we compare these multiple activities for recombinant human SARM1, human CD38, and Aplysia californica ADP ribosyl cyclase. SARM1 has the highest transglycosidation (base exchange) activity at neutral pH and with some bases this dominates NAD(P) hydrolysis and cyclization. All SARM1 activities, including base exchange at neutral pH, are activated by an increased NMN:NAD ratio, at physiological levels of both metabolites. SARM1 base exchange occurs also in DRG neurons and is thus a very likely physiological source of calcium-mobilizing agent NaADP. Finally, we identify regulation by free pyridines, NADP, and nicotinic acid riboside (NaR) on SARM1, all of therapeutic interest. Understanding which specific SARM1 function(s) is responsible for axon degeneration is essential for its targeting in disease.

Genetic variants of two-pore calcium channel 2 rs1551305 and its association with type 2 diabetes risk

ContextThe prevalence of diabetes is highest in the Eastern Mediterranean Region, with Egypt leading the region (11% for both sexes) and lowest in the European Region (7% for both sexes). Genome-wide association studies of single-nucleotide polymorphisms (SNPs) have identified a number of variants that are associated with β-cell function and insulin resistance. Two-pore calcium channel 2 (TPCN2) localizes to the lysosome and is a likely receptor for the calcium-mobilizing agent nicotinic acid adenine dinucleotide phosphate. Several studies have indicated that nicotinic acid adenine dinucleotide phosphate may play a role in the insulin signaling of β-cells.Aimof the study The aim of this study was to investigate the association between TPCN2 rs1551305 SNPs and the development of type 2 diabetes.Patients and methodsA sample of 158 Egyptian participants was divided into two groups. Group one included 79 type 2 diabetic patients and group two included 79 healthy controls. TPCN2 rs1551305 SNPs were determined by the real-time PCR technique.ResultsA significant increase in the frequency of G/G genotype in diabetic patients was found. A/A genotype was significantly more frequent in the control group (P=0.001). G allele was also significantly higher in diabetic patients (P=0.008). The G/G genotype showed a 21.37-fold increase in the risk of developing diabetes mellitus.ConclusionThe previous findings suggest that TPCN2 rs1551305 SNP is associated with the risk of type 2 diabetes development.

Zinc ions cause the thimerosal-induced signal of fluorescent calcium probes in lymphocytes

Most fluorescent probes for the investigation of calcium signaling also detect zinc ions. Consequently, changes in the intracellular zinc concentration could be mistaken for calcium signals. Thimerosal (TMS) is used as a calcium-mobilizing agent and we analyzed the contribution of zinc ions to the signal observed with fluorescent calcium probes after TMS stimulation. Our findings show that the fluorescent signal in lymphocytes is entirely due to zinc release. Experiments in the T lymphocyte cell line Jurkat and primary human lymphocytes show that TMS and its active metabolite, ethyl mercury, cause an increase in signal intensity with probes designed for the detection of either calcium or zinc ions. The TMS/ethyl mercury-induced signal of the calcium probes Fluo-4 and FURA-2 was completely absent when the zinc chelator TPEN [N,N,N′,N′-tetrakis-(2-pyridyl-methyl)ethylenediamine] was added. In contrast, the signal caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was unaffected by TPEN. In light of these observations, zinc may also contribute to calcium signals caused by mercury-containing compounds other than TMS, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered.

The Involvement of Cyclic ADPR in Photoperiodic Flower Induction of Pharbitis nil

Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium-mobilizing agent synthesized from NAD+ by ADP-ribosyl cyclases described for several animal cells. Pharmacological studies suggest that cADPR is an endogenous modulator of Ca2+-induced Ca2+ release channels. There is also information about the sub-micromolar concentration of cADPR in plant cells. Whether cADPR can act as a Ca2+-mobilizing intracellular messenger in plant tissue is an unresolved question. Despite the obvious importance of monitoring cADPR cellular levels under various physiological conditions in plants, its measurement has been technically difficult and requires specialized reagents. In the present study a widely applicable sensitivity assay for cADPR is described. We show that Pharbitis nil tissue from cotyledons contains a certain cADPR level. To explain the possible roles of this second messenger in photoperiodic flower induction, some physiological experiments were also performed. The exogenous applications of cADPR to Pharbitis nil plants, which were exposed to a 12-h-long subinductive night, significantly increased flowering response. Nevertheless 8-Br-cADPR inhibited flowering when these compounds were applied during a 16-h-long inductive night. The effect of ruthenium red, a calcium channel blocker and ryanodine, a calcium channel stimulator, on the photoperiodic induction of flowering was also studied. Ruthenium red, when applied before and during an inductive 16-h dark period, slightly inhibited flowering, whereas ryanodine, when applied before and during a 12-h long subinductive night, stimulated flower bud formation. We also confirmed evidence that Ca2+ ions are involved in the photoperiodic induction of flowering. Thus, the obtained results may suggest the involvement of cyclic ADPR-activated Ca2+ mobilization in the photoperiodic flower induction process in Pharbitis nil.

Dissociation of endothelial nitric oxide synthase phosphorylation and activity in uterine artery endothelial cells

Pregnancy enhanced nitric oxide production by uterine artery endothelial cells (UAEC) is the result of reprogramming of both Ca(2+) and kinase signaling pathways. Using UAEC derived from pregnant ewes (P-UAEC), as well as COS-7 cells transiently expressing ovine endothelial nitric oxide synthase (eNOS), we investigated the role of phosphorylation of five known amino acids following treatment with physiological calcium-mobilizing agent ATP and compared with the effects of PMA (also known as TPA) alone or in combination with ATP. In P-UAEC, ATP stimulated eNOS activity and phosphorylation of eNOS S617, S635, and S1179. PMA promoted eNOS phosphorylation but without activation. PMA and ATP cotreatment attenuated ATP-stimulated activity despite no increase in phospho (p)-T497 and potentiation of p-S1179. In COS-7 cells, PMA inhibition of ATP-stimulated eNOS activity was associated with p-T497 phosphorylation. Although T497D eNOS activity was reduced to 19% of wild-type eNOS with ATP and 44% with A23187, we nonetheless observed more p-S1179 with ATP than with A23187 (3.4-fold and 1.8-fold of control, respectively). Furthermore, the S1179A eNOS mutation partly attenuated ATP- but not A23187-stimulated activity, but when combined with T497D, no further reduction of eNOS activity was observed. In conclusion, although phosphorylation of eNOS is associated with activation in P-UAEC, no single or combination of phosphorylation events predict activity changes. In COS-7 cells, phosphorylation of T497 can attenuate activity but also influences S1179 phosphorylation. We conclude that in both cell types, observed changes in phosphorylation of key residues may influence eNOS activation but are not sufficient alone to describe eNOS activation.

Arachidonic acid induces calcium-dependent mitochondrial formation of species promoting strand scission of genomic DNA

Both the phospholipase A 2 activator melittin and reagent arachidonic acid (AA) are poor inducers of DNA single strand breaks in U937 cells. These responses, however, were dramatically increased by the calcium-mobilizing agent caffeine (Cf) or by the respiratory substrate pyruvate via a mechanism that involved enforced mitochondrial Ca 2+ accumulation and that was sensitive to lipoxygenase inhibitors. In permeabilized cells, the DNA damage generated by AA in combination with either Cf, L-malate or CaCl 2 was blunted by catalase. AA generated DNA strand scission also in HeLa cells supplemented with pyruvate via a mechanism identical to that observed in U937 cells. This response was associated with an enforced formation of free radical species. These results demonstrate that mitochondria play a pivotal role in the DNA-damaging response evoked by AA and provide the bases for a calcium-dependent mechanism whereby the AA produced during inflammatory processes may affect various pathologic conditions, including carcinogenesis.

Distinct Nuclear Localization and Activity of Tissue Transglutaminase

Tissue transglutaminase is a calcium-dependent transamidating enzyme that has been postulated to play a role in the pathology of expanded CAG repeat disorders with polyglutamine expansions expressed within the affected proteins. Because intranuclear inclusions have recently been shown to be a common feature of many of these codon reiteration diseases, the nuclear localization and activity of tissue transglutaminase was examined. Subcellular fractionation of human neuroblastoma SH-SY5Y cells demonstrated that 93% of tissue transglutaminase is localized to the cytosol. Of the 7% found in the nucleus, 6% copurified with the chromatin-associated proteins, and the remaining 1% was in the nuclear matrix fraction. In situ transglutaminase activity was measured in the cytosolic and nuclear compartments of control cells, as well as cells treated with the calcium-mobilizing agent maitotoxin to increase endogenous tissue transglutaminase activity. These studies revealed that tissue transglutaminase was activated in the nucleus, a finding that was further supported by cytochemical analysis. Immunofluorescence studies revealed that nuclear proteins modified by transglutaminase exhibited a discrete punctate, as well as a diffuse staining pattern. Furthermore, different proteins were modified by transglutaminase in the nucleus compared with the cytosol. The results of these experiments clearly demonstrate localization of tissue transglutaminase in the nucleus that can be activated. These findings may have important implications in the formation of the insoluble nuclear inclusions, which are characteristic of codon reiteration diseases such as Huntington's disease and the spinocerebellar ataxias.

CADP-ribose formation by blood platelets is not responsible for intracellular calcium mobilization.

Human platelet CD38 is a multifunctional ectoenzyme catalysing the synthesis and hydrolysis of cADP-ribose (cADPR), a recently identified calcium-mobilizing agent that acts independently of D-myo-inositol 1,4,5-trisphosphate and is known to be expressed by human platelets. The present work shows that ADP-ribosyl cyclase activity is exclusively a membrane activity, of which the major part is located in plasma membranes and a small part in internal membranes. In broken cells, cyclase activity was insensitive to the presence of calcium and was not modulated by agonists such as thrombin or ADP, whereas in intact cells thrombin increased cADPR formation by 30%, an effect due to fusion of granules with the plasma membrane. In order to assess the role of cADPR as a calcium-mobilizing agent, vesicles were prepared from internal membranes and loaded with 45CaCl2. These vesicles were efficiently discharged by IP3 in a dose-dependent manner, but were not responsive to cADPR or ryanodine in the presence or absence of calmodulin. Thus cADPR is unlikely to play a role in intracellular calcium release in human blood platelets.

Identification of bovine liver mitochondrial NAD+ glycohydrolase as ADP-ribosyl cyclase.

The present investigation identifies bovine liver mitochondrial NADase (NAD+ glycohydrolase) as a member of the class of bifunctional ADP-ribosyl cyclases/cyclic ADP-ribose hydrolases, known to be potential second messenger enzymes. These enzymes catalyse the synthesis and degradation of cyclic ADP-ribose, a potent intracellular calcium-mobilizing agent. The mitochondrial enzyme utilized the NAD+ analogues nicotinamide guanine dinucleotide (NGD+) and nicotinamide hypoxanthine dinucleotide (NHD+) to form fluorescent cyclic purine nucleoside diphosphoriboses. ADP-ribosyl cyclase activity was also demonstrated using 32P-labelled NAD+ as substrate. The identity of NADase and ADP-ribosyl cyclase was supported by their co-migration in SDS/polyacrylamide gels. Cyclase activity was visualized directly within the gel by detecting the formation of fluorescent cyclic IDP-ribose from NHD+. The enzyme catalysed the hydrolysis of cyclic ADP-ribose to ADP-ribose. Moreover, in the presence of nicotinamide and cyclic ADP-ribose the enzyme synthesized NAD+. Both the ADP-ribosyl cyclase and NADase activities of the enzyme were strongly inhibited by reducing agents. Treatment of the NADase with dithiothreitol caused the apparent inactivation of the enzyme. Subsequent removal of the reducing agent and addition of oxidized glutathione led to a partial recovery of enzymic activity. The results support a model for pro-oxidant-induced calcium release from mitochondria involving cyclic ADP-ribose as a specific messenger, rather than the non-enzymic modification of proteins by ADP-ribose.

Activation of the mitogen-activated protein kinase cascade is suppressed by low concentrations of dexamethasone in mast cells.

Antigen stimulation of mast cells via the IgE receptor, Fc epsilon RI, results in recruitment of the cytosolic tyrosine kinases, Lyn and Syk, and the phosphorylation of proteins. We examined the effects of the glucocorticoid dexamethasone on these events in a cultured (RBL-2H3) mast cell line. Nanomolar concentrations of dexamethasone suppressed phosphorylation of proteins that were associated with the activation of the mitogen-activated protein (MAP) kinase/phospholipase A2 pathway without inhibiting initial events. For example, tyrosine phosphorylation of the subunits of Fc epsilon RI, Lyn, or Syk or of the Ras-guanine nucleotide exchange factor, Vav, was not suppressed in cells treated with up to 1 microM dexamethasone. In contrast, phosphorylation of Raf1, MEK1, p42mapk, and cytosolic phospholipase A2, as well as the associated increase in MAP kinase activity and release of arachidonic acid, were markedly inhibited in cells treated with as little as 10 nM dexamethasone--a concentration that only partially inhibited hydrolysis of inositol phospholipids or release of secretory granules. Prolonged exposure to dexamethasone also resulted in a partial decrease in expression of MEK1, p42mapk, and cytosolic phospholipase A2, which may contribute further to the effects of dexamethasone on this pathway. Activation of the MAP kinase/phospholipase A2 pathway by the calcium-mobilizing agent thapsigargin was similarly suppressed in dexamethasone-treated cells. These findings suggested that an early step in the pathway, possibly a step immediately before the activation of Raf1, was suppressed by low concentrations of dexamethasone.

Extracellular Synthesis of cADP-Ribose from Nicotinamide-Adenine Dinucleotide by Rat Cortical Astrocytes in Culture

cADPR is an endogenous calcium-mobilizing agent that in vertebrates is synthesized from nicotinamide-adenine dinucleotide (NAD) by bifunctional enzymes with ADP-ribosyl cyclase and cADPR hydrolase activity. ADP-ribosyl cyclase and cADPR hydrolase activity have been reported in the brain, but the cellular localization of these activities has not been determined previously. In the present study, selective culturing techniques were employed to localize ADP-ribosyl cyclase activity and cADPR hydrolase activity to astrocytes or neurons in cultures derived from rat embryonic cerebral cortex. ADP-ribosyl cyclase activity was determined by incubating cultures with 1 mM NAD in the extracellular medium for 60 min at 37 degrees C and measuring formation of cADPR by bioassay and by HPLC. Astrocyte cultures and mixed cultures of astrocytes and neurons had mean specific activities of 0.84 +/- 0.06 and 0.9 +/- 0.18 nmol cADPR produced/mg protein/hr, respectively. No detectable ADP-ribosyl cyclase activity was found in neuron-enriched/ astrocyte-poor cultures. cADPR hydrolase activity was detectable by incubating cultures with 300 microM cADPR for 60 min at 37 degrees C and assaying loss of cADPR or accumulation of ADPR. The demonstration of extracellular ADP-ribosyl cyclase and cADPR hydrolase activities associated with astrocytes may have important implications for the role of extracellular cADPR in signal transduction and in intercellular communication in the nervous system.

Expression of Cyclic ADP-Ribose–Synthetizing CD38 Molecule on Human Platelet Membrane

CD38 is a cell surface molecule widely used as a marker for immature and activated lymphocytes. It has been recently shown that CD38 displays three enzymatic activities: hydrolysis of NAD+ to ADP-ribose, synthesis of cyclic ADP-ribose from NAD+, and hydrolysis of cyclic ADP-ribose to ADP-ribose. Thus, CD38 plays a key role in the synthesis of cyclic ADP-ribose, a calcium-mobilizing compound. We investigate here the expression and cellular localization of CD38 in human platelets using a specific monoclonal antibody. Results showed that CD38 is expressed by human platelet membranes. Moreover, we show that platelet CD38 possesses NAD glycohydrolase, ADP-ribose cyclase, and cyclic ADP-ribose hydrolase activities. This finding indicates that the calcium-mobilizing agent cyclic ADP-ribose can be synthetized by human platelets and raises the question about the possible role of CD38 expression and enzymatic activities in the signal transduction pathways leading to platelet activation.

2′-Phospho-Cyclic ADP-ribose, a Calcium-mobilizing Agent Derived from NADP

Cyclic adenosine diphosphoribose (cADPR), a metabolite of NAD, appears to modulate changes in intracellular free Ca2+ levels by activation of ryanodine-sensitive Ca2+ channels. We report here that an ADPR cyclase purified from Aplysia californica readily catalyzes the conversion of NADP to 2'-phospho-cyclic adenosine diphosphoribose (2'-P-cADPR), cyclized at N-1 of the adenine moiety. An enzyme from canine spleen previously shown to contain NAD glycohydrolase, ADPR cyclase, and cADPR hydrolase activities also utilized NADP and 2'-P-cADPR as substrates. The apparent Km value for NADP was 1.6 microM compared with 9.9 microM for NAD, and the Vmax with NADP was twice that with NAD, indicating that 2'-P-cADPR is a likely metabolite in mammalian cells. 2'-P-cADPR was as active as cADPR in eliciting Ca2+ release from rat brain microsomes, but was unable to elicit Ca2+ release following conversion to 2'-P-ADPR by the action of canine spleen NAD glycohydrolase. 2'-P-cADPR and 1-D-myo-inositol 1,4,5-trisphosphate (IP3) appear to act by distinct mechanisms as microsomes desensitized to IP3 still released Ca2+ in response to 2'-P-cADPR and vice versa. Also, inhibition of IP3-induced Ca2+ release by heparin had no effect on release by 2'-P-cADPR. Both 2'-P-cADPR and cADPR appear to act by a similar mechanism based on similar kinetics of Ca2+ release, similar dose-response curves, cross-desensitization, and partial inhibition of release by procaine. The results of this study suggest that 2'-P-cADPR may function as a new component of Ca2+ signaling and a possible link between NADP metabolism and Ca2+ homeostasis.

Nitric Oxide-induced Mobilization of Intracellular Calcium via the Cyclic ADP-ribose Signaling Pathway

Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium-mobilizing agent synthesized from beta-NAD+ by ADP-ribosyl cyclases in sea urchin eggs and in several mammalian cells (Galione, A., and White, A. (1994) Trends Cell Biol. 4, 431 436). Pharmacological studies suggest that cADPR is an endogenous modulator of Ca2+-induced Ca2+ release mediated by ryanodine-sensitive Ca2+ release channels. An unresolved question is whether cADPR can act as a Ca2+-mobilizing intracellular messenger. We show that exogenous application of nitric oxide (NO) mobilizes Ca2+ from intracellular stores in intact sea urchin eggs and that it releases Ca2+ and elevates cADPR levels in egg homogenates. 8-Amino-cADPR, a selective competitive antagonist of cADPR-mediated Ca2+ release, and nicotinamide, an inhibitor of ADP-ribosyl cyclase, inhibit the Ca2+-mobilizing actions of NO, while, heparin, a competitive antagonist of the inositol 1,4,5-trisphosphate receptor, did not affect NO-induced Ca2+ release. Since the Ca2+-mobilizing effects of NO can be mimicked by cGMP, are inhibited by the cGMP-dependent-protein kinase inhibitor, Rp-8-pCPT-cGMPS, and in egg homogenates show a requirement for the guanylyl cyclase substrate, GTP, we suggest a novel action of NO in mobilizing intracellular calcium from microsomal stores via a signaling pathway involving cGMP and cADPR. These results suggest that cADPR has the capacity to act as a Ca2+-mobilizing intracellular messenger.

A role for protein tyrosine kinase in the steroidogenic pathway of angiotensin II in bovine zona glomerulosa cells.

Stimulation of aldosterone synthesis in bovine adrenal zona glomerulosa (ZGB) cells by angiotensin II (AngII) is believed to be mediated by the phospholipase C (PLC) pathway that results in the increase of cytosolic free calcium concentration and in the activation of protein kinase C (PKC). However, the cell proliferation and contraction associated with AngII action are known to be mediated in part by protein tyrosine kinases (PTK). To assess the potential role of PTK in the stimulatory effect of AngII on adrenal steroidogenesis, the actions of a series of PTK inhibitors on this metabolic pathway were examined in isolated ZGB cells. Tyrphostin 23 (TP23) caused a dose-dependent inhibition of AngII-stimulated aldosterone production with an IC50 of 15 microM and reached complete inhibition at 100 microM. Genistein (GS) was more potent with an IC50 of 35 nM and complete inhibition at 10 microM. The stimulation of aldosterone production by the calcium-mobilizing agent thapsigargin (Thaps) was also dose-dependently inhibited by TP and GS with the same potency. A specific PKC inhibitor, calphostin C (0.1 microM) caused only a 51.7% inhibition of AngII-stimulated aldosterone production. In the same way, a specific Ca2+/calmodulin-dependent protein kinase inhibitor, KN-62 (1 microM), reduced aldosterone production stimulated by AngII by 64%. As expected, thapsigargin-stimulated aldosterone biosynthesis was not affected by calphostin C, but was completely inhibited by KN-62. These results demonstrate for the first time that protein tyrosine kinase activity is part of the angiotensin II signalling pathway in bovine zona glomerulosa cells. The activation of this PTK occurs subsequently to the mobilization of intracellular calcium. This calcium-dependent protein tyrosine kinase pathway is essential for the steroidogenic response to AngII in bovine zona glomerulosa cells.

The Calcium-Mobilizing Agent, Thapsigargin, Inhibits Progesterone Production in Rat Luteal Cells by a Calcium-Independent Mechanism*

In rat luteal cells, an increase in intracellular [Ca]i impairs luteal function similar to that of prostaglandin F2a (PGF2a). However, calcium per se is not the mediator of the antigonadotropic action of PGF2a. Thapsigargin, a plant sesquiterpene lactone, increases intracellular calcium concentration concentration ([Ca]i) in several cell types by a mechanism that involves specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. To further investigate the antigonadotropic role of [Ca]i and the mechanism of action of PGF2a in rat luteal cells, the action of thapsigargin on cellular functional responses was examined in the absence and presence of PGF2a. Thapsigargin dose dependently increased [Ca]i and inhibited cAMP accumulation and progesterone production in response to LH. The inhibitory effect of thapsigargin on cAMP accumulation was calcium dependent but in contrast, inhibition of LH-stimulated progesterone production was independent of calcium mobilization by thapsigargin. Steroidogenesis stimulated by (Bu)2cAMP was also inhibited by thapsigargin. Thus, thapsigargin mimicked some effects of PGF2a with inhibitory sites of action on both cAMP accumulation and progesterone production. Thapsigargin also blocked the mobilization of [Ca]i by PGF2a, but when coincubated with PGF2a an additive effect on inhibition of LH-stimulated progesterone production occurred. However, no additive effects of thapsigargin and PGF2a on gonadotropin-sensitive cAMP accumulation were evident. In conclusion, although thapsigargin and PGF2a may share some similar actions, their antigonadotropic effects are mediated differently.

Inositol 1,4,5-trisphosphate releases intracellular Ca2+ in permeabilized chick atria

Inositol 1,4,5-trisphosphate (IP3) and caffeine evoked transient, reversible, and concentration-dependent increases in tension in saponin-treated chick atrial muscle. Contractures evoked by IP3 and caffeine were detected in solutions with 70 microM EGTA at pCa 7.0. In the presence of 7 mM EGTA, neither IP3 nor caffeine was able to evoke a contracture. Maximally effective concentrations of IP3 (20 microM) and caffeine (20 mM) developed tensions to approximately 44 and 83% of that elicited by pCa 5.0 (maximum tension = 100%), respectively. The IP3- or caffeine-induced contractures were consistently reproduced when the sarcoplasmic reticulum (SR) had previously been loaded with calcium. Preexposure to caffeine suppressed the following IP3-induced response. When ryanodine (1-10 microM) was present throughout the SR-loading cycle, the responses to IP3 and caffeine were prevented. However, when ryanodine was added after the SR was loaded with calcium, neither the response to IP3 nor that to caffeine was affected. These results are consistent with the hypothesis that ryanodine inhibition requires prior activation of the SR calcium-release channel. It is concluded that both IP3 and caffeine increased tension in the SR by releasing calcium from it. The effect of IP3 is consistent with its messenger role as a calcium-mobilizing agent.

Regulation of relaxin release from monodispersed porcine luteal cells: effect of calcium ionophore A23187 and calcium channel blockers.

The role of calcium ion mobilization in modulation of secretion of the ovarian protein hormone relaxin by porcine luteal cells was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis, a plaque, developed around relaxin-releasing luteal cells. The rate of development of plaques in time-course experiments was used in this study as an index of the rate of relaxin release. Exposure of luteal cell-containing monolayers to the calcium-mobilizing agent A23187 (40 nM to 5 microM) resulted in a dose-related increase in the rate of relaxin-plaque formation. This effect [and the influence of a stimulatory secretagogue, prostaglandin E2 (PGE2; 1 microM)] was suppressed by coculture with Co2+ (5 mM), a calcium channel blocker. These results are consistent with the view that calcium ion redistribution within porcine luteal cells forms a pathway that subserves, at least in part, the rates of basal and stimulated relaxin release in vitro. However, A23187 was equally effective in enhancing the rate of plaque formation when the monolayers were bathed in a low calcium medium (mean +/- SEM, 6.61 +/- 0.92 microM Ca2+), rather than a calcium-replete medium (1.56 +/- 0.09 mM Ca2+). Likewise, neither basal nor PGE2-stimulated (1 microM) relaxin secretion was abrogated by culture of monolayers in low calcium medium. These data suggest that the stimulatory effect of A23187 (and perhaps PGE2) arose predominantly through redistribution of calcium stored within intracellular sites in luteal cells, rather than entrance of calcium into the cell from the extracellular medium. Yet, incompatible with this interpretation, we observed that TMB-8 [8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride; a putative inhibitor of intracellular calcium redistribution] enhanced rather than blocked A23187- or PGE2-stimulated relaxin release. This result is consistent with the possibility that TMB-8 mobilized (rather than blocked) calcium in this cell system or that it acted via calcium-independent mechanisms. We conclude that calcium mobilization has the potential to act as a molecular pathway that transduces secretion of an ovarian peptide hormone, relaxin. However, the exact nature and physiological role(s) of the Ca2+ pathway in the control of relaxin secretion and the interrelationships of this mechanism with other intracellular messengers that may also modulate ovarian peptide secretion remain to be more clearly defined.

Inhibition by glucagon of the calcium pump in liver plasma membranes.

The ATP-dependent calcium transport in plasma membrane vesicles prepared from rat liver was inhibited by 0.1 to 10 microM glucagon. Inhibition of the high affinity (Ca2+-Mg2+)-ATPase was observed concomitantly. This effect was neither mimicked by cyclic AMP nor by dibutyryl cyclic AMP. A study of the structure-activity relationships of six glucagon derivatives demonstrated the specificity of glucagon action since only one or two analogs markedly altered the (Ca2+-Mg2+)-ATPase activity. The study also demonstrated the total absence of correlation between adenylate cyclase activation and (Ca2+-Mg2+)-ATPase inhibition induced by these glucagon derivatives. The decrease in the maximal velocities induced by glucagon of both calcium transport and (Ca2+-Mg2+)-ATPase activity were related to a reduction in the rate of dephosphorylation of the Ca-dependent phosphorylated intermediate of the enzyme. This phosphorylated intermediate was characterized as a 32P-labeled 110,000-dalton protein which accumulated to 50 to 150% over the basal level in the presence of glucagon. The present results demonstrate a novel aspect of the role of glucagon as a calcium-mobilizing agent.