Damage Signaling by Extracellular Nucleotides: A Role for Cyclic Nucleotides in Elevating Cytosolic Free Calcium?

Extracellular ATP (eATP) is now held to be a constitutive damage-associated molecular pattern (DAMP) that is released by wounding, herbivory or pathogen attack. The concentration of eATP must be tightly regulated as either depletion or overload leads to cell death. In Arabidopsis thaliana, sensing of eATP is by two plasma membrane legume-like lectin serine–threonine receptor kinases (P2K1 and P2K2), although other receptors are postulated. The transcriptional response to eATP is dominated by wound- and defense-response genes. Wounding and pathogen attack can involve the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) which, in common with eATP, can increase cytosolic-free Ca2+ as a second messenger. This perspective on DAMP signaling by eATP considers the possibility that the eATP pathway involves production of cyclic nucleotides to promote opening of cyclic nucleotide-gated channels and so elevates cytosolic-free Ca2+. In silico analysis of P2K1 and P2K2 reveals putative adenylyl and guanylyl kinase sequences that are the hallmarks of “moonlighting” receptors capable of cAMP and cGMP production. Further, an Arabidopsis loss of function cngc mutant was found to have an impaired increase in cytosolic-free Ca2+ in response to eATP. A link between eATP, cyclic nucleotides, and Ca2+ signaling therefore appears credible.

Endothelial vasodilator production by uterine and systemic arteries. I. Effects of ANG II on PGI2 and NO in pregnancy

Uterine vasculature is less responsive than systemic vasculature to angiotensin II (ANG II)-induced vasoconstriction. We hypothesized that pregnancy augments basal and ANG II-stimulated endothelial prostacyclin (PGI2) and/or nitric oxide (NO) production, which locally increase vascular smooth muscle (VSM) adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP), respectively. Uterine (UA) and systemic arteries (SA) from pregnant (P) and nonpregnant (NP) sheep were incubated with isobutylmethylxanthine. Basal PGI2, cAMP, and cGMP production was 2.4-, 1.6-, and 5.9-fold greater (P < 0.01) in UA from P vs. NP sheep; endothelium removal lowered (P < 0.05) values 69, 44, and 88%. Basal SA PGI2 and cAMP, but not cGMP, also were elevated by pregnancy. Indomethacin (Indo; 100 microM) decreased PGI2 and cAMP, but not cGMP production; N omega-nitro-L-arginine methyl ester (L-NAME; 10 microM) and methylene blue (MB, 10 microM) only decreased cGMP. Basal UA, but not SA, NO synthase activity (conversion of [3H]arginine to [3H]citrulline), was 1.8-fold higher in pregnancy and decreased (P < 0.01) after endothelium removal and with L-NAME. ANG II (50 nM) increased PGI2 (86%) and cAMP (56%) production only in UA from P sheep (P < 0.05); this was abolished by endothelium removal or Indo. ANG II also increased (P < 0.01) cGMP production by UA from both groups but only by SA from P ewes; this was absent in denuded, L-NAME-, or MB-treated vessels. Stimulation of VSM cGMP production with sodium nitroprusside (50 microM) was inhibited by MB, but not L-NAME or endothelium removal. In pregnancy, endothelial PGI2 and NO production are enhanced and may contribute to attenuated ANG II vasoconstriction via VSM cAMP and cGMP.

Phosphatase inhibitors potentiate adrenergic-stimulated cAMP and cGMP production in rat pinealocytes

The role of phosphoprotein phosphatase in the regulation of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in rat pinealocytes was investigated using the three phosphatase inhibitors calyculin A, tautomycin, and okadaic acid. Calyculin A (0.1 microM) was found to enhance the isoproterenol- and norepinephrine-stimulated cAMP accumulation six- and threefold, respectively, whereas tautomycin and okadaic acid were less effective. The effect of calyculin A was rapid (within 5 min) and persisted in the presence of phosphodiesterase inhibition. However, in contrast to protein kinase C activation or intracellular calcium elevation, the phosphatase inhibitors were less effective in potentiating the cAMP response stimulated by forskolin or cholera toxin, and their effects were not blocked by calphostin C or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. The adrenergic-stimulated cGMP response was also less sensitive to the phosphatase inhibition. Therefore, our results suggest that 1) the adrenergic-stimulated cAMP signal is subjected to the tonic inhibition by phosphoprotein phosphatase; 2) phosphatase inhibitors enhance cAMP synthesis through their actions at the receptor level; and 3) the cAMP signal is more sensitive to the regulation by phosphorylation than cGMP in rat pinealocytes.

Intracellular pH on adrenergic-stimulated cAMP and cGMP production in rat pinealocytes

In rat pinealocytes, activation of alpha 1-adrenergic receptors leads to increases in intracellular pH (pHi). In this study, the role of pHi on adrenergic regulation of cyclic nucleotide accumulation was investigated using ammonium chloride, which increased pHi, and sodium propionate, which reduced pHi. Ammonium chloride significantly enhanced the norepinephrine-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) responses, while it selectively potentiated the isoproterenol (ISO)-stimulated cGMP response. Reduction of pHi by sodium propionate reduced the norepinephrine-stimulated cGMP accumulation by 70%, and its effect on the ISO-stimulated cGMP response was stimulatory. Treatment with sodium propionate effectively neutralized the enhancing effects of ammonium chloride on the adrenergic-stimulated cAMP and cGMP responses. These effects of sodium propionate and ammonium chloride on cyclic nucleotides appeared to reflect altered rate of synthesis, and they were also in part secondary to changes in intracellular Ca2+. Our findings indicate that the receptor-mediated changes in pHi may play an integral part in the adrenergic regulation of cAMP and cGMP production in rat pinealocytes.

Surface cAMP receptors mediate multiple responses during development in Dictyostelium: evidenced by antisense mutagenesis.

Cell surface cAMP receptors (cARs) have been implicated in multiple aspects of development in Dictyostelium. Antisense mutagenesis has recently provided strong evidence that cARs are necessary for aggregation (Klein et al., 1988. Science (Wash. DC). 241:1467-1472). We show here that the expression of cAR1 antisense mRNA which prevents the appearance of cAR1 antigen also prevents the expression of cAMP-binding activity and blocks multiple cAMP-mediated responses. Chemotactic sensitivity to cAMP was lost as were stimulus-induced cAMP and cGMP production. Furthermore, the expression of developmentally regulated marker genes, dependent on repeated cAMP stimulation, was altered. As a result, the developmental program was severely impaired; most of the cells failed to aggregate and undergo further differentiation.