Nitric oxide (NO) 1 is now recognized as a signaling molecule in many mammalian tissues where it has diverse functions as a neurotransmitter as well as an agent mediating apoptosis (1-7). Although NO was first discovered as a mediator of vascular smooth muscle relaxation, where it leads to a decrease in intracellular free calcium [Ca 2ϩ ] i (8), recent reports in interstitial cells in the mammalian gut (9), a macrophage line (10), and pancreatic  cells (11) demonstrate that treatments with NO and NO donors elicit increases in [Ca 2ϩ ] i . These effects persist in the absence of extracellular calcium and can be blocked by pretreatment with ryanodine (9, 11), suggesting that NO may activate a signal transduction cascade, which activates ryanodine-sensitive calcium release channels (RyRs). We have studied this novel aspect of NO action in the sea urchin egg, since Ca 2ϩ release mechanisms have been extensively studied in this system (12) and where multiple calcium mobilization pathways have been shown and are amenable to detailed analysis. In the sea urchin egg one Ca 2ϩ release mechanism is gated by the established second messenger, inositol 1,4,5-trisphosphate (IP 3 ), which is produced in response to the interaction of many extracellular stimuli with cell surface receptors (13). Another involves the activation of ryanodine-sensitive calcium release channels (14). RyRs are present on intracellular calcium stores in a wide range of cell types including sea urchin eggs (15). Here ryanodine receptors have been shown to be regulated by cADPR (16), a novel calcium-mobilizing metabolite that is synthesized from -NAD ϩ by ADP-ribosyl cyclases (12). Accumulating evidence suggests that cADPR is a widespread modulator of ryanodine receptor-mediated calcium release in many different types of mammalian cells (17-26) as well as in plants (27).A second messenger role for cADPR requires that it mediates the intracellular actions of hormones or neurotransmitters. We show that NO mobilizes calcium from intracellular stores in the sea urchin egg via a pathway in part involving cGMP and leading to the activation of the cADPR-sensitive calcium release mechanism. EXPERIMENTAL PROCEDURESCollection of Sea Urchin Eggs-Eggs were obtained by stimulating ovulation of female Lytechinus pictus (Marinus, Inc., Long Beach, CA) with an intracoelomic injection of 0.5 M KCl solution. These were then washed twice in artificial seawater (435 mM NaCl, 40 mM MgCl 2 , 15 mM MgSO 4 , 11 mM CaCl 2 , 10 mM KCl, 2.5 mM NaHCO 3 , 1 mM EDTA at pH 8.0), and jelly was removed by filtration through 85-m Nitex mesh.Imaging of Intracellular [Ca 2ϩ ] i in Eggs-Eggs were transferred to poly-L-lysine (10 mg/ml)-coated glass coverslips, allowed to adhere, and microinjected with fura-2, pentapotassium salt (10 mM in the pipette), in buffer consisting of 0.5 M KCl, 20 mM Pipes at pH 6.7 to a final cellular concentration of approximately 10 M. Injection volumes did not exceed 1% of cell volume. All experiments were performed at 22°C. Free cytosolic Ca 2...
Many hormones or neurotransmitters act at cell surface receptors to increase the intracellular free calcium concentration, triggering a wide range of cellular responses. As the source of this Ca2+ is often internal stores, additional messengers are required to convey the hormonal message from the plasma membrane. Cyclic ADP-ribose (cADPR) has been proposed as the endogenous activator of Ca(2+)-induced Ca2+ release by the ryanodine receptor in sea urchin eggs and in several mammalian cell types. A second messenger role for cADPR requires that its intracellular levels be under the control of extracellular stimuli. Here we demonstrate a novel action of 3',5'-cyclic guanosine monophosphate (cGMP) in stimulating the synthesis of cADPR from beta-NAD+ by activating its synthetic enzyme ADP-ribosyl cyclase in sea urchin eggs and egg homogenates. We suggest that cADPR may transduce signals generated by cell surface receptors or gaseous transmitters linked to cGMP production.
We have investigated the effects of in vivo lithium treatment on cerebral inositol phospholipid metabolism. Twice-daily treatment of rats with LiCl (3 mEq/kg) for 3 or 16 days resulted in a 25-40% reduction in agonist-stimulated inositol phosphate production, compared with NaCl-treated controls, in cortical slices prelabelled with [3H]inositol. A small effect was also seen with 5-hydroxytryptamine (5-HT) 24 h after a single dose of LiCl (10 mEq/kg). Dose-response curves to carbachol and 5-HT showed that lithium treatment reduced the maximal agonist response without altering the EC50 value. This inhibition was not affected by the concentration of LiCl in the assay buffer. Stimulation of inositol phosphate formation by 10 mM NaF in membranes prepared from cortex of 3-day lithium-treated rats was also inhibited, by 35% compared with NaCl-treated controls. Lithium treatment did not alter the kinetic profile of inositol polyphosphate formation in cortical slices stimulated with carbachol. Muscarinic cholinergic and 5-HT2 bindings were unaltered by lithium, as was cortical phospholipase C activity and isoproterenol-stimulated cyclic AMP formation. [3H]Inositol labelling of phosphatidylinositol 4,5-bisphosphate was significantly enhanced by 3-day lithium treatment. The results, therefore, indicate that subacute or chronic in vivo lithium treatment reduces agonist-stimulated inositol phospholipid metabolism in cerebral cortex; this persistent inhibition appears to be at the level of G-protein-phospholipase C coupling.
Cyclic ADP-ribose (cADPR), an endogenous NAD+ metabolite in many mammalian and invertebrate tissues, is a potent mediator of calcium mobilization in sea urchin eggs. Our results show that cADPR also stimulates calcium release from rat brain microsomes, marked release occurring over the concentration range 10-250 nM. This is not inhibited by concentrations of heparin which completely abolish inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. Ryanodine (100 microM) inhibits the cADPR response. Our results are consistent with cADPR being an endogenous messenger mediating Ca2+ release from ryanodine-sensitive pools in brain.
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