Antony Galione

Pharmacological properties of the Ca2+‐release mechanism sensitive to NAADP in the sea urchin egg

The sea urchin egg homogenate is an ideal model to characterize Ca2+‐release mechanisms because of its reliability and high signal‐to‐noise‐ratio. Apart from the InsP3‐ and ryanodine‐sensitive Ca2+‐release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist. The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca2+‐releasing agent, NAADP, compared to inositol trisphosphate (InsP3) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors. NAADP‐induced Ca2+‐release was blocked by L‐type Ca2+‐channel blockers and by Bay K 8644, while InsP3‐ and cyclic ADPR‐induced Ca2+‐release were insensitive to these agents. L‐type Ca2+‐channel blockers did not displace [32P]‐NAADP binding, suggesting that their binding site was different. Moreover, stopped‐flow kinetic studies revealed that these agents blocked NAADP in a all‐or‐none fashion. Similarly, a number of K+‐channel antagonists blocked NAADP‐induced Ca2+‐release selectively over InsP3‐ and cyclic ADPR‐induced Ca2+‐release. Radioligand studies showed that these agents were not competitive antagonists. As has been shown for InsP3 and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca2+‐release mechanisms. The presence of K+ was not essential for NAADP‐induced Ca2+‐release, since substitution of K+ with other monovalent cations in the experimental media did not significantly alter Ca2+ release by NAADP. On the contrary, cyclic ADPR and InsP3‐sensitive mechanisms were affected profoundly, although to a different extent depending on the monovalent cation which substituted for K+. Similarly, modifications of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly affected NAADP‐induced Ca2+‐release. While the alkaline condition permitted InsP3 and cyclic ADPR‐induced Ca2+‐release, the acidic condition completely hampered both Ca2+‐release mechanisms. The present results characterize pharmacologically and biochemically the novel Ca2+‐release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.

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

BACKGROUNDnCyclic 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).nnnRESULTSnIn 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.nnnCONCLUSIONSnThese 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.

Nitric oxide induces intracellular Ca2+ mobilization and increases secretion of incorporated 5‐hydroxytryptamine in rat pancreatic β‐cells

This study is the first to demonstrate that low concentrations of aqueous NO induce intracellular Ca2+ mobilization and an increase in secretory activity of rat pancreatic β‐cells. Application of NO solution (2 μM) resulted in a transient increase in the free intracellular Ca2+ concentration ([Ca2+]i) of isolated cells, as assessed by video ratio imaging and single wavelength microfluorimetry. Amperometry revealed a simultaneous increase in the release of preloaded 5‐hydroxytryptamine from the isolated cells. The NO‐induced Ca2+ response primarily involves mobilization of endoplasmic reticulum Ca2+ stores, since the response was retained when cells were transferred to low Ca2+ medium, and completely inhibited when cells were pretreated with 10 μM thapsigargin. The Ca2+ response was also inhibited when cells were incubated with a high concentration of ryanodine (200 μM), suggesting that Ca2+ mobilization is via a ryanodine‐sensitive store.

Human lymphocyte antigen CD38 catalyzes the production of cyclic ADP-ribose

The human lymphocyte antigen CD38 has been shown to share sequence homology with ADP‐ribosyl cyclase, the enzyme that catalyzes the conversion of NAD+ to cyclic ADP‐ribose (cADPR), a potent Ca2+‐mobilizing agent. In this study COS1 cells from African Green Monkey kidney were transiently transfected with CD38 cDNA, inducing expression of authentic CD38 on the cell surface. We demonstrate that CD38 expressed in this manner can convert NAD+ to cADPR in the extracellular medium as assessed by Ca2+ release from sea‐urchin egg microsomes.

Calcium store depletion potentiates a phosphodiesterase inhibitor‐ and dibutyryl cGMP‐evoked calcium influx in rat pituitary GH3 cells

A role for cGMP in the control of capacitative Ca2+ influx was identified in rat pituitary GH3 cells. Application of 50 μM‐1 mM of the non‐specific phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine (IBMX), or the specific cGMP‐phosphodiesterase inhibitor, zaprinast, induced a dose‐dependent increase in the intracellular free Ca2+ concentration [Ca2+]i of the pituitary cell line, as assessed by video ratio imaging using fura‐2. Response onset times were identical and response profiles were similar in all cells analysed. Application of 50 μM dibutyryl cGMP to GH3 cells resulted in heterogeneous Ca2+ responses, consisting of single or multiple transients with varying onset times. In all cases, increases in [Ca2+]i were predominantly due to Ca2+ influx, since no responses were detected in low Ca2+ medium, or following pre‐incubation of cells with 1 μM verapamil, or nicardipine. Depleting intracellular Ca2+ stores by prior treatment of cells with 1 μM thapsigargin resulted in a dramatic potentiation in the Ca2+ influx mediated by both phosphodiesterase inhibitors and dibutyryl cGMP, suggesting that cGMP modulates a dihydropyridine‐sensitive Ca2+ entry mechanism in GH3 cells which is possibly regulated by the state of filling of Ca2+ stores.

Microinjection of cyclic ADP-ribose triggers a regenerative wave of Ca2+ release and exocytosis of cortical alveoli in medaka eggs.

Medaka (Oryzias latipes) eggs microinjected with the Ca(2+)-mobilising messenger cyclic adenosine diphosphate ribose (cADPR) underwent a wave of exocytosis of cortical alveoli and were thus activated. The number of eggs activated was sharply dependent on the concentration of cADPR in the pipette, the threshold concentration was approximately 60 nM. After injection, a pronounced latency preceded the onset of cortical alveoli exocytosis; this latency was independent of the concentration of cADPR but decreased markedly with increasing temperature. Heat-treated cADPR, which yields the inert non-cyclised product ADP-ribose, was ineffective in activating eggs. When cADPR was injected into aequorin-loaded eggs, a wave of luminescence arose at the site of cADPR injection and then swept out across the egg with a mean velocity of approximately 13 microns/s; the velocity was independent of the concentration of injected cADPR. In such a large cell (diameter of around 1 mm), this is considerably faster than that possible by simple diffusion of cADPR, which unambiguously demonstrates that cADPR must activate a regenerative process. cADPR has been demonstrated to modulate Ca(2+)-induced Ca2+ release (CICR) via ryanodine receptors (RyRs) in many cell types, and consistent with this was the finding that microinjection of the pharmacological RyR modulator, ryanodine, also activated medaka eggs. These results suggest that a cADPR-sensitive Ca2+ release mechanism is present in the medaka egg, that cADPR is the most potent activator of medaka eggs described to date, and that it activates eggs by triggering a wave of CICR from internal stores that in turn stimulates a wave of exocytosis.

Lithium-induced decrease in spontaneous Ca2+ oscillations in single GH3 rat pituitary cells.

1 Measurement of [Ca2+]i in single rat pituitary GH3 cells by dynamic single cell imaging techniques demonstrated that under basal conditions there is a large variation in the temporal pattern of [Ca2+]i signalling between individual cells ranging from high frequency asynchronous oscillations to quiescence. 2 We have reported previously that treatment of GH3 cells with 1 mm Li+ (a concentration used therapeutically in the treatment of manic depression) for 7 days reduces basal and thyrotrophin‐releasing hormone (TRH)‐stimulated levels of mass inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3]. In the present study, we show that this is associated with a reduction in the number of cells exhibiting basal Ca2+ oscillations over a sampling period of 60 s, whereas the maximum amplitude of oscillations is unaffected. 3 The pattern of [Ca2+]i responses to the agonist TRH varied considerably between individual cells, making quantitation of the responses difficult; however, data obtained from measurements made on a population of cells showed that increases in peak [Ca2+]i induced by high concentrations of TRH were reduced in cells treated with 1 mm Li+ for 7 days relative to control cells. 4 The sensitivity of the phosphoinositide pathway to [Ca2+]i was investigated by loading GH3 cells with BAPTA/AM at a concentration sufficient to lower ‘basal’ [Ca2+]i in a population of cells and to inhibit agonist‐stimulated increases in [Ca2+]i. Under these conditions, basal and TRH‐stimulated mass Ins(1,4,5)P3 levels were unaffected. 5 These results demonstrate that a 7‐day Li+ treatment leads to an alteration in Ca2+ signalling, in particular by reducing the number of cells exhibiting high frequency Ca2+ oscillations under basal conditions. The significance of these results to the clinical effectiveness of Li+ in the treatment of manic depression is discussed.