calcimycin and 9-(tetrahydro-2-furyl)-adenine

We tested whether dilation of outer medullary descending vasa recta (OMDVR) is mediated by cAMP, nitric oxide (NO), and cyclooxygenase (COX). Adenosine (A; 10(-6) M)-induced vasodilation of ANG II (10(-9) M)-preconstricted OMDVR was mimicked by the cAMP analog 8-bromoadenosine 3',5'-cyclic monophosphate (10(-10) to 10(-4) M) and reversed by the adenylate cyclase inhibitor SQ-22536. Adenosine (10(-4) M) stimulated OMDVR cAMP production greater than threefold. NO synthase blockade with N(G)-nitro-L-arginine methyl ester and N(G)-monomethyl-L-arginine (10(-4) M) did not affect adenosine vasodilation. Adenosine induced endothelial cytoplasmic calcium transients that were small. Indomethacin (10(-6) M) reversed adenonsine-induced dilation of OMDVR preconstricted with ANG II, endothelin, 4-bromo-calcium ionophore A23187, or carbocyclic thromboxane A(2). In contrast, selective A(2)-receptor activation dilated endothelin-preconstricted OMDVR even in the presence of indomethacin. We conclude that OMDVR vasodilation by adenosine involves cAMP and COX but not NO. COX blockade does not fully inhibit selective A(2) receptor-mediated OMDVR dilation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenine; Adenosine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Angiotensin II; Animals; Calcimycin; Calcium; Colforsin; Cyclic AMP; Cyclooxygenase Inhibitors; Endothelium, Vascular; Enzyme Inhibitors; Female; Ionophores; Kidney Medulla; Kinetics; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; omega-N-Methylarginine; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Signal Transduction; Thromboxane A2; Vasodilation

This study was conducted to determine the mechanism of arachidonic acid (AA) release elicited by phenylephrine (PHE) stimulation of alpha adrenergic receptor (AR), and its modulation by cyclic adenosine 3',5'-monophosphate (cAMP) in Rat-1 fibroblasts (R-1Fs) transfected with the alpha-1A, alpha-1B or alpha-1D AR. PHE increased AA release and also caused a marked accumulation of cAMP in R-1Fs expressing the alpha-1 AR subtypes, but not in those transfected with vector alone. PHE also enhanced phospholipase D (PLD), but not phospholipase A2 (PLA2) activity. The increase in PHE-induced AA release, PLD activity and cAMP accumulation differed among the various alpha AR subtypes with: alpha-1A > alpha-1B > alpha-1D AR. The effect of PHE to increase AA release was attenuated by C2-ceramide, an inhibitor of PLD; propranolol, a phosphatidate phosphohydrolase inhibitor; and RHC-80267, a diacylglycerol lipase inhibitor in R-1Fs expressing the alpha-1A AR. Forskolin, which activates adenylyl cyclase, increased cAMP accumulation and inhibited PHE-induced AA release and PLD activity in alpha-1A-AR-expressing R-1Fs. 8-(4-chlorophenyl-thio)-cAMP, a nonhydrolyzable analog of cAMP, also attenuated the rise in AA release and PLD activity elicited by PHE in these cells. In contrast, SQ 22536, an adenylyl cyclase inhibitor, and KT 5720, a protein kinase A inhibitor, increased PHE-induced AA release and PLD activity in R-1Fs expressing the alpha-1A AR. These data suggest that the alpha-1A, alpha-1B and alpha-1D ARs are coupled to PLD activation and cAMP accumulation. Moreover, PHE promotes AA release in R-1Fs expressing the alpha-1A AR through PLD activation. Furthermore, cAMP generated by alpha-1A AR stimulation acts as an inhibitory modulator of PLD activity and AA release via protein kinase A.

Topics: Adenine; Adenylate Cyclase Toxin; Animals; Arachidonic Acid; Calcimycin; Carbazoles; Cell Line; Cholera Toxin; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Glycerides; GTP-Binding Proteins; Indoles; Lipase; Phenylephrine; Phospholipase D; Phospholipases A; Phospholipases A2; Prazosin; Pyrroles; Radioligand Assay; Rats; Receptors, Adrenergic, alpha-1; Transfection; Virulence Factors, Bordetella

The effects of thyroid hormone on renin secretion, renin content, and renin mRNA levels in juxtaglomerular (JG) cells harvested from rat kidneys were determined by radioimmunoassays and reverse transcriptase-polymerase chain reaction. Despite a lack of immediate effect, incubation with triiodothyronine dose dependently increased renin secretion during the first 6 h and elevated renin content and renin mRNA levels during the subsequent period. Simultaneous incubation with triiodothyronine and the calcium ionophore A-23187 abolished the increase in renin secretion and attenuated the increase in renin content but did not affect the increase in renin mRNA levels. During simultaneous incubation with triiodothyronine and the adenylate cyclase inhibitor SQ-22536 or membrane-soluble guanosine 3',5'-cyclic monophosphate (cGMP), the increases in renin secretion, content, and mRNA were similar to those observed in the presence of triiodothyronine alone, except for a cGMP-induced attenuation of the increase in renin secretion. These findings suggest that thyroid hormone stimulates renin secretion by JG cells through the calcium-dependent mechanism, whereas the stimulation of renin gene expression by thyroid hormone does not involve intracellular calcium or cyclic nucleotides.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Animals; Calcimycin; Cells, Cultured; Dibutyryl Cyclic GMP; Enzyme Inhibitors; Ionophores; Juxtaglomerular Apparatus; Male; Polymerase Chain Reaction; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Renin; RNA, Messenger; Triiodothyronine

Exposure of human neutrophils to the tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP) leads to a transient, 2-3 fold elevation of adenosine-3',5'-cyclic monophosphate (cAMP) that peaks at 5-15 seconds. This cAMP transient has been hypothesized as constituting an early activation event that may be responsible for subsequent functional responses. In order to evaluate the dependence of several FMLP-stimulated functional responses on elevated cAMP levels, we utilized 9-(tetrahydro-2-furyl)adenine (SQ 22,536), a putative inhibitor of adenylate cyclase. Pretreatment of cells with SQ 22,536 (1-1000 microM) caused dose-dependent inhibition of the FMLP (0.1 microM)-induced cAMP elevation (ID50 approximately 5 microM). Similar results were observed when cells were activated by the divalent cation ionophore A23187 (20 microM). At 1000 microM, a drug concentration which completely abolished the cAMP transient, SQ 22,536 had no effect on FMLP-stimulated superoxide radical (O2-) generation, granule enzyme release, or chemotaxis and only a modest inhibitory effect on A23187-induced O2- production. These studies strongly suggest that these FMLP- and A23187-induced responses occur independently of a transient elevation of cAMP and that, in intact human neutrophils, SQ 22,536 is a non-toxic inhibitor of adenylate cyclase.

Topics: Adenine; Adenylyl Cyclases; Calcimycin; Chemotaxis; Cyclic AMP; Cytoplasmic Granules; Humans; In Vitro Techniques; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Superoxides; Time Factors