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.     

Cyclic ADP-ribose induces Ca2+ release from caffeine-insensitive Ca2+ pools in canine salivary gland cells

Cyclic ADP-ribose (cADPR), a novel putative messenger of the ryanodine receptor, was examined regarding its ability to mobilize Ca2+ from intracellular Ca2+ stores in isolated cells of parotid and submandibular glands of the dog. cADPR induced a rapid and transient Ca2+ release in the digitonin-permeabilized cells of salivary glands. cADPR-induced Ca2+ release was inhibited by ryanodine receptor antagonists ruthenium red, ryanodine, benzocaine, and imperatoxin inhibitor but not by the inositol 1,4,5-trisphosphate (IP3)-receptor antagonist heparin. Thapsigargin, at a concentration of 3 to 30 microM, inhibited IP3-induced Ca2+ release, while higher concentrations were required to inhibit cADPR-induced Ca2+ release. Cross-potentiation was observed between cADPR and ryanodine or SrCl2, suggesting that cADPR sensitizes the Ca2+-induced Ca2+ release mechanism. Cyclic AMP plays a stimulatory role on cADPR- and IP3-induced Ca2+ release in digitonin-permeabilized cells. Calmodulin also potentiated cADPR-induced Ca2+ release, but inhibited IP3-induced Ca2+ release. Acetylcholine and ryanodine caused the rise in intracellular free Ca2+ concentration ([Ca2+]i) in intact submandibular and parotid cells. Caffeine did not produce any increase in Ca2+ release or [Ca2+]i rise in any preparation. ADP-ribosyl cyclase activity was found in the centrifuged particulate fractions of the salivary glands. These results suggest that cADPR serves as an endogenous modulator of Ca2+ release from Ca2+ pools through a caffeine-insensitive ryanodine receptor channel, which are different from IP3-sensitive pools in canine salivary gland cells. This system is positively regulated by cyclic AMP and calmodulin.     

The transmembrane glycoprotein CD38 is a catalytically active transporter responsible for generation and influx of the second messenger cyclic ADP-ribose across membranes

CD38 is a type II transmembrane glycoprotein expressed in many vertebrate cells. It is a bifunctional ectoenzyme that catalyzes both the synthesis of Cyclic ADP-ribose (cADPR) from NAD+ and the degradation of cADPR to ADP-ribose by means of its ADP-ribosyl cyclase and cADPR-hydrolase activities, respectively. The cyclase also converts NGD+ to cyclic GDP-ribose (cGDPR), which is refractory to cADPR-hydrolase. cADPR, but not cGDPR, is a potent calcium mobilizer from intracellular stores. It has been demonstrated to be a new second messenger involved in the regulation of calcium homeostasis in many cell types, from plants to mammals. The number of physiological processes shown to be regulated by cADPR is steadily increasing. A topological paradox exists because ectocellularly generated cADPR acts intracellularly. Here we demonstrate that the catalytic functioning of CD38 is accompanied by a cADPR (cGDPR) -transporting activity across natural and artificial membranes. In resealed membranes from CD38(+) human erythrocytes, transport of catalytically generated cADPR or cGDPR was saturation dependent and occurred against a concentration gradient. Likewise, CD38-reconstituted proteoliposomes were active in concentrating NAD+ (NGD+) -derived cADPR (cGDPR) inside the vesicle compartment. Moreover, the cADPR-transporting activity in CD38 proteoliposomes prevented the hydrolase-catalyzed degradation to ADPR that occurs conversely with detergent-solubilized CD38, resulting in selective influx of cADPR. In the CD38 proteoliposomes, catalytically active CD38 exhibited monomeric, dimeric, and tetrameric structures. In CD38 sense- but not in antisense-transfected HeLa cells, externally added NAD+ resulted in significant, transient increases in cytosolic calcium. These data suggest that transmembrane juxtaposition of two or four CD38 monomers can generate a catalytically active channel for selective formation and influx of cADPR (cGDPR) to reach cADPR-responsive intracellular calcium stores.     

Signal transduction in gastrointestinal smooth muscle

Signal transduction in gastric and intestinal smooth muscle is mediated by receptors coupled via distinct G proteins to various effector enzymes, including PI-specific PLC-beta 1 and PLC-beta 3, and phosphatidylcholine (PC)-specific PLC, PLD and PLA2. Activation of these enzymes is different in circular and longitudinal muscle cells, generating Ca(2+)-mobilizing (IP3, AA, cADPR) and other (DAG) messengers responsible for the initial and sustained phases of contraction, respectively. IP3-dependent Ca2+ release occurs only in circular muscle. Ca2+ mobilization in longitudinal muscle involves a cascade initiated by agonist-induced transient activation of PLA2 and formation of AA, AA-dependent depolarization of the plasma membrane and opening of voltage-sensitive Ca2+ channels. The influx of Ca2+ induces Ca2+ release by activating sarcoplasmic ryanodine receptor/Ca2+ channel and stimulates cADPR formation which enhances Ca(2+)-induced Ca2+ release. The initial [Ca2+]i transient in both muscle cell types results in Ca2+/calmodulin-dependent activation of MLC kinase, phosphorylation of MLC20 and interaction of actin and myosin. The sustained phase is mediated by a Ca(2+)-independent isoform of PKC, PKC-epsilon DAG for this process is generated by PLC- and PLD-mediated hydrolysis of PC. Relaxation is mediated by cAMP-and/or cGMP-dependent protein kinase which inhibit the initial [Ca2+]i transient and reduce the sensitivity of MLC kinase to [Ca2+]i. Relaxation induced by the main neurotransmitters, VIP and PACAP, involves two cascades, one of which reflects activation of adenylyl cyclase. A distinct cascade involves G-protein-dependent stimulation of Ca2+ influx leading to Ca2+/calmodulin-dependent activation of a constitutive eNOS in muscle cells; the generation of NO activates soluble guanylyl cyclase. The resultant activation of PKA and PKG is jointly responsible for muscle relaxation.     

Ca2+-induced Ca2+ release in chromaffin cells seen from inside the ER with targeted aequorin

The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4, 5-trisphosphate (InsP3)- producing agonists released only 60-80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.     

CD38 and ADP-ribosyl cyclase catalyze the synthesis of a dimeric ADP-ribose that potentiates the calcium-mobilizing activity of cyclic ADP-ribose

CD38, a lymphocyte differentiation antigen, is also a bifunctional enzyme catalyzing the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and its hydrolysis to ADP-ribose (ADPR). An additional enzymatic activity of CD38 shared by monofunctional ADP-ribosyl cyclase from Aplysia californica is the exchange of the base group of NAD+ (nicotinamide) with various nucleophiles. Both human CD38 (either recombinant or purified from erythrocyte membranes) and Aplysia cyclase were found to catalyze the exchange of ADPR with the nicotinamide group of NAD+ leading to the formation of a dimeric ADPR ((ADPR)2). The dimeric structure of the enzymatic product, which was generated by recombinant CD38 and by CD38(+) Namalwa cells from as low as 10 microM NAD+, was demonstrated using specific enzyme treatments (dinucleotide pyrophosphatase and 5'-nucleotidase) and mass spectrometry analyses of the resulting products. The linkage between the two ADPR units of (ADPR)2 was identified as that between the N1 of the adenine nucleus of one ADPR unit and the anomeric carbon of the terminal ribose of the second ADPR molecule by enzymatic analyses and by comparison with patterns of cADPR cleavage with Me2SO:tert-butoxide. Although (ADPR)2 itself did not release Ca2+ from sea urchin egg microsomal vesicles, it specifically potentiated the Ca2+-releasing activity of subthreshold concentrations of cADPR. Therefore, (ADPR)2 is a new product of CD38 that amplifies the Ca2+-mobilizing activity of cADPR.     

Calcium signaling by cyclic ADP-ribose, NAADP, and inositol trisphosphate are involved in distinct functions in ascidian oocytes

ADP-ribosyl cyclase catalyzes the synthesis of two structurally and functionally different Ca2+ releasing molecules, cyclic ADP-ribose (cADPR) from beta-NAD and nicotinic acid-adenine dinucleotide phosphate (NAADP) from beta-NADP. Their Ca2+-mobilizing effects in ascidian oocytes were characterized in connection with that induced by inositol 1,4,5-trisphosphate (InsP3). Fertilization of the oocyte is accompanied by a decrease in the oocyte Ca2+ current and an increase in membrane capacitance due to the addition of membrane to the cell surface. Both of these electrical changes could be induced by perfusion, through a patch pipette, of nanomolar concentrations of cADPR or its precursor, beta-NAD, into unfertilized oocytes. The changes induced by beta-NAD showed a distinctive delay consistent with its enzymatic conversion to cADPR. The cADPR-induced changes were inhibited by preloading the oocytes with a Ca2+ chelator, indicating the effects were due to Ca2+ release induced by cADPR. Consistently, ryanodine (at high concentration) or 8-amino-cADPR, a specific antagonist of cADPR, but not heparin, inhibited the cADPR-induced changes. Both inhibitors likewise blocked the membrane insertion that normally occurred at fertilization consistent with it being mediated by a ryanodine receptor. The effects of NAADP were different from those of cADPR. Although NAADP induced a similar decrease in the Ca2+ current, no membrane insertion occurred. Moreover, pretreatment of the oocytes with NAADP inhibited the post-fertilization Ca2+ oscillation while cADPR did not. A similar Ca2+ oscillation could be artificially induced by perfusing into the oocytes a high concentration of InsP3 and NAADP could likewise inhibit such an InsP3-induced oscillation. This work shows that three independent Ca2+ signaling pathways are present in the oocytes and that each is involved in mediating distinct changes associated with fertilization. The results are consistent with a hierarchical organization of Ca2+ stores in the oocyte.     

A specific cyclic ADP-ribose antagonist inhibits cardiac excitation-contraction coupling

BACKGROUND: Cyclic ADP-ribose (cADPR) has been shown to act as a potent cytosolic mediator in a variety of tissues, regulating the release of Ca2+ from intracellular stores by a mechanism that involves ryanodine receptors. There is controversy over the effects of cADPR in cardiac muscle, although one possibility is that endogenous cADPR increases the Ca2+ sensitivity of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum. We investigated this possibility using 8-amino-cADPR, which has been found to antagonize the Ca2+-releasing effects of cADPR on sea urchin egg microsomes and in mammalian cells (Purkinje neurons, Jurkat T cells, smooth muscle and PC12 cells). RESULTS: In intact cardiac myocytes isolated from guinea-pig ventricle, cytosolic injection of 8-amino-cADPR substantially reduced contractions and Ca2+ transients accompanying action potentials (stimulated at 1Hertz). These reductions were not seen with injection of HEPES buffer, with heat-inactivated 8-amino-cADPR, or in cells pretreated with ryanodine (2 microM) to suppress sarcoplasmic reticulum function before injection of the 8-amino-cADPR. L-type Ca2+ currents and the extent of Ca2+ loading of the sarcoplasmic reticulum were not reduced by 8-amino-cADPR. CONCLUSIONS: These observations are consistent with the hypothesis that endogenous cADPR plays an important role during normal contraction of cardiac myocytes. One possibility is that cADPR sensitizes the CICR mechanism to Ca2+, an action antagonized by 8-amino-cADPR (leading to reduced Ca2+ transients and contractions). A direct effect of 8-amino-cADPR on CICR cannot be excluded, but observations with caffeine are not consistent with a non-selective block of release channels.     

A novel MADS-box gene of potato (Solanum tuberosum L.) expressed during the early stages of tuberization

Cyclic ADP-ribose (cADPR) is a Ca(2+)-mobilizing cyclic nucleotide derived from NAD+. Accumulating evidence indicates that it is an endogenous modulator of the Ca(2+)-induced Ca2+ release mechanism in cells. In this study, we show that ADP-ribosyl cyclase catalyzes the cyclization of not only NAD+ but also several of its analogs with various purine bases (guanine, hypoxanthine, or xanthine) substituting for adenine. Unlike cADPR, the resulting cyclic products are fluorescent. Comparisons with various model compounds indicate that only 7-methyl substituted purine nucleosides and nucleotides are fluorescent, and the pH-dependence of their UV spectra is most similar to that of the fluorescent cADPR analogs, indicating that the site of cyclization of these analogs is at the N7-position of the purine ring. This finding is novel since the site of cyclization is at the N1-position for cADPR as determined by X-ray crystallography. That a single enzyme can cyclize a variety of substrates at two different sites has important implications mechanistically, and a model is proposed to account for these novel catalytic properties. Among the analogs synthesized, cyclic GDP-ribose is highly resistant to hydrolysis, while cyclic IDP-ribose can be readily hydrolyzed by CD38, a bifunctional enzyme involved in the metabolism of cADPR. These unique properties of the analogs can be used to develop fluorimetric assays for monitoring separately the cyclization and hydrolytic reactions catalyzed by the metabolic enzymes of cADPR. The convenience of the method in measuring kinetic parameters, pH-dependence, and modulator activity of the metabolic enzymes of cADPR is illustrated.     

Autoantibodies against CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) that impair glucose-induced insulin secretion in noninsulin- dependent diabetes patients

Cyclic ADP-ribose (cADPR) has been shown to be a mediator for intracellular Ca2+ mobilization for insulin secretion by glucose in pancreatic beta cells, and CD38 shows both ADP-ribosyl cyclase to synthesize cADPR from NAD+ and cADPR hydrolase to hydrolyze cADPR to ADP-ribose. We show here that 13.8% of Japanese non-insulin-dependent diabetes (NIDDM) patients examined have autoantibodies against CD38 and that the sera containing anti-CD38 autoantibodies inhibit the ADP-ribosyl cyclase activity of CD38 (P 相似文献   

Cyclic-AMP-dependent Ca2+ influx elicited by prostaglandin D2 in freshly isolated nonchromaffin cells from bovine adrenal medulla

We previously reported that prostaglandin D2 (PGD2) specifically elevates intracellular cyclic AMP in nonchromaffin cells isolated from bovine adrenal medulla (Biochim. Biophys. Acta (1989) 1011, 75-80). Here we again found that PGD2 increased intracellular Ca2+ concentration ([Ca2+]i) in freshly isolated nonchromaffin cells and investigated the cellular mechanisms of PGD2-induced [Ca2+]i increase using the Ca2+ indicator fura-2 and a fluorescence microscopic imaging system. Treatment of the cells with PGD2 receptor agonists BW245C and ZK110841 resulted in both marked stimulation of cyclic AMP formation and an increase in [Ca2+]i. The [Ca2+]i response was also induced by bypassing of the receptor with forskolin, a direct activator of adenylate cyclase, but not by PGE2 or PGF2 alpha both of which are devoid of the ability to generate cyclic AMP in the cells. These cyclic AMP and [Ca2+]i responses induced by PGD2 were completely blocked by the PGD2 receptor antagonist BWA868C. The time-course of cyclic AMP production stimulated by PGD2 coincided with that of the [Ca2+]i increase. While the Ca(2+)-mobilizing hormone bradykinin stimulated a rapid inositol phosphate accumulation in nonchromaffin cells, PGD2 did not stimulate it significantly. Removal of extracellular Ca2+ markedly reduced the Ca2+ response to PGD2 in magnitude and duration, but did not alter the peak [Ca2+]i response to bradykinin. These results demonstrate that PGD2 receptor activation induces the increase in [Ca2+]i via cyclic AMP mainly by increasing the Ca2+ influx from the outside, unlike inositol trisphosphate which causes release of Ca2+ from internal stores.     

The CD38/cyclic ADP-ribose system: a topological paradox

CD38 was first identified as a lymphocyte differentiation antigen that showed typical properties of an orphan receptor involved in many programs of cell proliferation and activation. However, CD38 proved also to be a bifunctional ectoenzyme that catalyzes the transient formation of cyclic ADP-ribose (cADPR) in a variety of cell types. This property raises many intriguing and so far unanswered questions, since cADPR is a new second messenger molecule directly involved in the control of calcium homeostasis by means of receptor-mediated release of calcium from ryanodine-sensitive intracellular stores. The relationship between receptor-like and enzymatic properties of CD38 is still unknown. The apparent topological paradox of ectocellular synthesis and intracellular activity of cADPR might be explained by: (a) influx of cADPR across the plasma membrane to reach its target stores, as suggested by experiments on cerebellar granule cells; and (b) NAD(+)-induced internalization, following membrane oligomerization, of CD38 with consequent partial import of cADPR metabolism to an intracellular compartment, as recently observed in lymphoid B cells. These two distinct mechanisms and other potential ones (e.g. binding of ectocellularly formed cADPR to cell surface receptors and initiation of signal-transducing pathways across the plasmamembrane) seem to be paradigmatic of processes affecting different types of cells. Although in some biological systems, such as Aplysia and sea urchin egg, cADPR metabolism is restricted to the intracellular environment, in mammalian cells the CD38/cADPR system provides new challenges in terms of subcellular compartmentation and qualifies as an unusual example of "ectobiochemistry" with potential, still unrecognized, properties of cellular regulation.     

Pituitary adenylate cyclase-activating polypeptide causes Ca2+ release from ryanodine/caffeine stores through a novel pathway independent of both inositol trisphosphates and cyclic AMP in bovine adrenal medullary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca2+ release and Ca2+ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca2+ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP3), cyclic AMP, and the intracellular Ca2+ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP3 and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP3, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP3 production, the Ca2+ release induced by PA-CAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP3 channels. The potencies of the peptides to cause Ca2+ release in the presence of cinnarizine were similar. The Ca2+ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca2+ release was unaffected by Rp-adenosine 3',5'-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca2+ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP3 production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca2+ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca2+ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP3 and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca2+ in adrenal chromaffin cells.     

Multiple mechanisms of activating Ca2+ entry in freshly isolated rabbit aortic endothelial cells

In Fura-2-loaded, freshly isolated rabbit aortic endothelial cells the Ca2+ entry pathway was investigated using the Mn2(+)-quenching technique. Acetylcholine (ACh) interaction with muscarinic receptors activated Mn2+ influx through the plasma membrane. Sarcoplasmic-endoplasmic reticulum Ca2+ ATPase blockers such as cyclopiazonic acid (CPA), thapsigargin and BHQ, which block the endoplasmic reticulum Ca2+ pump and do not interact with receptors, also activated Mn2+ influx. Mn2+ influx activated by either ACh or CPA was blocked by the following agents: SKF96365, a receptor-operated Ca2+ channel (ROC) blocker; NCDC, a PLC and ROC blocker, and genistein, a tyrosine kinase inhibitor. D600, the L-type Ca2+ channel blocker, had no significant effect on Mn2+ influx. Caffeine blocked the ACh-induced Ca2+ release but had no effect on the ACh-induced Mn2+ influx. Similarly dantrolene, which blocked intracellular Ca2+ release induced by ACh, did not affect the ACh-activated Mn2+ influx. These data suggest that ACh can activate Ca2+ influx without depletion of the ACh-sensitive intracellular Ca2+ store. It is concluded (1) that in freshly isolated endothelial cells depletion of the intracellular Ca2+ store is not necessary for ACh-activated Ca2+ influx, and (2) that receptor activation and intracellular Ca2+ store depletion may activate the same Ca2+ entry pathway through parallel mechanisms.     

CD38 disruption impairs glucose-induced increases in cyclic ADP-ribose, [Ca2+]i, and insulin secretion

Increases in [Ca2+]i in pancreatic beta cells, resulting from Ca2+ mobilization from intracellular stores as well as Ca2+ influx from extracellular sources, are important in insulin secretion by glucose. Cyclic ADP-ribose (cADPR), accumulated in beta cells by glucose stimulation, has been postulated to serve as a second messenger for intracellular Ca2+ mobilization for insulin secretion, and CD38 is thought to be involved in the cADPR accumulation (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). Here we created "knockout" (CD38(-/-)) mice by homologous recombination. CD38(-/-) mice developed normally but showed no increase in their glucose-induced production of cADPR in pancreatic islets. The glucose-induced [Ca2+]i rise and insulin secretion were both severely impaired in CD38(-/-) islets, whereas CD38(-/-) islets responded normally to the extracellular Ca2+ influx stimulants tolbutamide and KCl. CD38(-/-) mice showed impaired glucose tolerance, and the serum insulin level was lower than control, and these impaired phenotypes were rescued by beta cell-specific expression of CD38 cDNA. These results indicate that CD38 plays an essential role in intracellular Ca2+ mobilization by cADPR for insulin secretion.     

Pituitary adenylate cyclase-activating polypeptide causes rapid Ca2+ release from intracellular stores and long lasting Ca2+ influx mediated by Na+ influx-dependent membrane depolarization in bovine adrenal chromaffin cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to increase intracellular Ca2+ concentrations ([Ca2+]i) and catecholamine release in adrenal chromaffin cells. We measured [Ca2+]i with fura-2 and recorded ion currents and membrane potentials with the whole cell configuration of the patch-clamp technique to elucidate the mechanism of PACAP-induced [Ca2+]i increase in bovine adrenal chromaffin cells. PACAP caused [Ca2+]i to increase due to Ca2+ release and Ca2+ influx, and this was accompanied by membrane depolarization and inward currents. The Ca2+ release was suppressed by ryanodine, an inhibitor of caffeine-sensitive Ca2+ stores, but was unaffected by cinnarizine, an inhibitor of inositol trisphosphate-induced Ca2+ release. Ca2+ influx and inward currents were both inhibited by replacement of extracellular Na+, and Ca2+ influx was inhibited by nicardipine, an L-type Ca2+ channel blocker, or by staurosporine, a protein kinase C (PKC) inhibitor, but was unaffected by a combination of omega- conotoxin-GVIA, omega-agatoxin-IVA, and omega-conotoxin- MVIIC, blockers of N-, P-, and Q-type Ca2+ channels. Moreover, 1-oleoyl-2-acetyl-sn-glycerol, a PKC activator, induced inward currents and Ca2+ influx. These results indicate that PACAP causes both Ca2+ release, mainly from caffeine-sensitive Ca2+ stores, and Ca2+ influx via L-type Ca2+ channels activated by membrane depolarization that depends on PKC-mediated Na+ influx.     

Co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in the cortical thick ascending limb cell of the rat kidney. Inhibition of hormone-dependent cAMP accumulation by extracellular Ca2+

The Ca2+-sensing receptor protein and the Ca2+-inhibitable type 6 adenylyl cyclase mRNA are present in a defined segment of the rat renal tubule leading to the hypothesis of their possible functional co-expression in a same cell and thus to a possible inhibition of cAMP content by extracellular Ca2+. By using microdissected segments, we compared the properties of regulation of extracellular Ca2+-mediated activation of Ca2+ receptor to those elicited by prostaglandin E2 and angiotensin II. The three agents inhibited a common pool of hormone-stimulated cAMP content by different mechanisms as follows. (i) Extracellular Ca2+, coupled to phospholipase C activation via a pertussis toxin-insensitive G protein, induced a dose-dependent inhibition of cAMP content (1.25 mM Ca2+ eliciting 50% inhibition) resulting from both stimulation of cAMP hydrolysis and inhibition of cAMP synthesis; this latter effect was mediated by capacitive Ca2+ influx as well as release of intracellular Ca2+. (ii) Angiotensin II, coupled to the same transduction pathway, also decreased cAMP content; however, its inhibitory effect on cAMP was mainly accounted for by an increase of cAMP hydrolysis, although angiotensin II and extracellular Ca2+ can induce comparable release of intracellular Ca2+. (iii) Prostaglandin E2, coupled to pertussis toxin-sensitive G protein, inhibited the same pool of adenylyl cyclase units as extracellular Ca2+ but by a different mechanism. The functional properties of the adenylyl cyclase were similar to those described for type 6. The results establish that the co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in a same cell allows an inhibition of cAMP accumulation by physiological concentrations of extracellular Ca2+.     

Inositol trisphosphate and cyclic ADP ribose as long range messengers generating local subcellular calcium signals

The process of messenger-mediated release of Ca2+ from intracellular stores, which is of great importance in virtually all cell types including neurons, can best be studied in cells lacking voltage-gated Ca2+ channels in the plasma membrane. In pancreatic acinar cells agonist-evoked repetitive cytosolic Ca2+ spikes are due to release of Ca2+ via inositoltrisphosphate (IP3) and ryanodine receptors and reuptake into the stores via thapsigargin-sensitive Ca2+ pumps. At low acetylcholine (ACh) or cholecystokinin concentrations the cytosolic Ca2+ spikes are mostly confined to the secretory granule area of the polarized pancreatic acinar cells. Similar results can be obtained by intracellular infusion of IP3 (or one of its non-metabolizable analogues) or cyclic ADP ribose. This suggests that high affinity IP3 and ryanodine receptors are concentrated in the secretory granule area. We have generated an 'artificial synapse' on isolated acinar cells by having a cell-attached patch pipette filled with ACh on the basal membrane. Initially, ACh is prevented from making contact with the receptors by the negative potential applied to the pipette. When the pipette polarity is switched to positive ACh can bind to its receptors. Using digital Ca2+ imaging it could be seen that the first cytosolic rise often occurred in the secretory granule area, a considerable distance away from the site of the agonist-receptor interaction. This shows the long-range action of the messenger(s) IP3 and or cyclic ADP ribose generated by the ACh-receptor interaction. The local Ca2+ spikes in the secretory granule area are sufficient for exocytotic secretory responses as seen in capacitance measurements.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dural invasion of meningiomas adjacent to the tumor margin on Gd-DTPA-enhanced MR images: histopathologic correlation

In order to investigate the possible involvement of cyclic ADP ribose as an intracellular messenger for hormone-evoked cytosolic Ca2+ signalling, we performed experiments on intracellularly perfused mouse pancreatic acinar cells. Both a stable inositol 1,4,5 trisphosphate analogue (IP3) and cyclic ADP ribose (cADPR) evoked regular spikes of Ca2+ dependent ion current, reflecting cytosolic Ca2+ spiking. The effect of cADPR, but not IP3, was abolished by the presence intracellularly of the cADPR antagonist 8-NH2-cADPR. External application of cholecystokinin (CCK) in a physiological concentration (2.5-5 pM) evoked a mixture of short-lasting and longer-lasting spikes of Ca2+-dependent ion current. These effects were abolished by the presence intracellularly of 8-NH2-cADPR (18 muM). Increasing the CCK concentration to 15 pM could overcome the inhibition by 18 muM of the antagonist. These experiments provide fresh evidence for the involvement of cADPR receptors in the hormone-evoked cytosolic Ca2+ signalling process in pancreatic acinar cells.