dibutyryl-cyclic-gmp and 7-nitroindazole

We have previously shown that cyclooxygenase-2 (COX-2) is localized to the cortical thick ascending limb of the loop of Henle (cTALH)/macula densa of the rat kidney, and expression increases in response to low-salt diet and/or angiotensin-converting enzyme (ACE) inhibition. Because of the localization of neuronal nitric oxide synthase (nNOS) to macula densa and surrounding cTALH, the present study investigated the role of nitric oxide (NO) in the regulation of COX-2 expression. For in vivo studies, rats were fed a normal diet, low-salt diet or low-salt diet combined with the ACE inhibitor captopril. In each group, one-half of them were treated with the nNOS inhibitors 7-nitroinidazole (7-NI) or S-methyl-thiocitrulline. Both of these NOS inhibitors inhibited increases in COX-2 mRNA and immunoreactive protein in response to low salt and low salt+captopril. For in vitro studies, COX-2 expression was studied in primary cultures of rabbit cTALH cells immunodisssected with Tamm-Horsfall antibody. Basal COX-2 immunoreactivity expression was stimulated by S-nitroso-N-acetyl-penicillamine (SNAP), an NO donor, and intracellular cGMP concentration. The cultured cells expressed immunoreactive nNOS, and 7-NI inhibited basal COX-2 immunoreactivity expression, which could be partially overcome by cGMP. In summary, these studies indicate that NO is a mediator of increased renal cortical COX-2 expression seen in volume depletion and suggest important interactions between the NO and COX-2 systems in the regulation of arteriolar tone and the renin-angiotensin system by the macula densa.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Captopril; Cells, Cultured; Citrulline; Cyclooxygenase 2; Dibutyryl Cyclic GMP; Gene Expression Regulation, Enzymologic; Immunohistochemistry; Indazoles; Isoenzymes; Kidney Cortex; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Penicillamine; Prostaglandin-Endoperoxide Synthases; Rabbits; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium Chloride, Dietary; Thiourea

Recently published data have provided evidence that nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) are signaling intermediates in the pathway through which muscle contraction stimulates glucose transport. As exercise promotes both NO production and calcium flux, we examined the relationships between NO-stimulated glucose uptake and calcium-, contraction-, and phosphatidylinositol-3-kinase (PI-3-K)-mediated glucose transport in the isolated incubated rat epitrochlearis muscle preparation. The NO donor sodium nitroprusside (SNP; 10 mmol/l) and dibutyryl cGMP (100 micromol/l) accelerated epitrochlearis glucose transport four- to fivefold above basal levels (P < 0.001) in a manner similar to in vitro contractile activity and the calcium releasing agent N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7; 100 micromol/l). In the case of SNP, this effect could be completely attributed to an increase in cell surface GLUT4. The effect of SNP on glucose transport was not inhibitable by either wortmannin (1.5 micromol/l) or dantrolene (12.5 micromol/l). Similarly, neither calcium nor contraction stimulation of glucose transport was affected by the NO synthase inhibitors NG-monomethyl-L-arginine (L-NMMA; 100 micromol/l) or 7-nitroindazole (1 mmol/l). Furthermore, whereas SNP raised epitrochlearis cGMP levels tenfold (P < 0.001), neither in vitro contractile activity nor W7 significantly elevated cGMP. These results indicate that NO/cGMP can markedly stimulate skeletal muscle glucose transport by increasing GLUT4 levels at the cell surface by a mechanism that does not depend on activation of PI-3-K. In addition, since calcium/contraction-stimulated glucose transport is not blocked by NO synthase inhibition and did not elevate cGMP, NO/cGMP may be part of a novel pathway that is distinct from both the insulin- and contraction-activated mechanisms.

Topics: 3-O-Methylglucose; Androstadienes; Animals; Calcium; Dantrolene; Dibutyryl Cyclic GMP; Enzyme Inhibitors; Female; Glucose; Indazoles; Muscle Contraction; Muscle, Skeletal; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; omega-N-Methylarginine; Phosphatidylinositol 3-Kinases; Rats; Rats, Sprague-Dawley; Sulfonamides; Wortmannin