Receptor desensitization of G protein-coupled receptors (GPCRs), which occurs during short-term (seconds to minutes) exposure of cells to agonists, is mediated by phosphorylation and receptor endocytosis. Recycling of the receptors is a requisite for resensitization of the response. The mechanisms that attenuate signaling by GPCRs are of considerable importance to regulation of intercellular signaling and maintenance of their ability to respond to agonists over time. This study evaluates the effect of nitric oxide (NO) on P2Y nucleotide receptor resensitization in cultured rat glomerular mesangial cells. The NO production in cultured mesangial cells was measured by using confocal microscopy and the fluorescence NO indicator 4,5-diaminofluorescein diacetate (DAF-2 DA). L-arginine increased and Nomega-nitro-L-arginine methyl ester (L-NAME) decreased NO production significantly (P < 0.05). Calcium responses to ATP were measured with fura-2 and imaging techniques. Repeated stimulation with ATP results in receptor desensitization that is characterized by lower calcium peak amplitude. Desensitization was induced by challenging mesangial cells with four consecutive 2-min pulses of ATP (0.1 mM) separated by 4.5-min control perfusions. Intracellular calcium concentration ([Ca2+]i) increase evoked by second, third, and fourth ATP challenges were about 40%, 26%, and 18% of the first one. The NO precursor, L-arginine (10 mM), and the NO donors, spermine-NONOate (500 microM) and sodium nitroprusside (SNP) (1 mM), were added before and during a fourth ATP challenge. Spermine-NONOate and L-arginine induced a recovery of the [Ca2+]i response to the fourth ATP challenge (P < 0.01 and 0.05, respectively). The NO synthase inhibitor, L-NAME (5 mM), applied along with ATP, was shown to enhance desensitization. 1H-(1,2,4)oxadiazolo(4,3-alpha)quinoxalin-1-one (ODQ, 30 microM), an inhibitor of guanylate cyclase, was used along with L-arginine, SNP, or spermine-NONOate. There was no significant difference with or without ODQ. Neither ODQ nor 8-Br-cGMP, an analog of cGMP, at different concentrations showed effects on ATP-stimulated [Ca2+]i. There was no elevation of [Ca2+]i when the cells were challenged by different concentrations (1 microM, 100 microM, 1 mM, 20 mM, and 30 mM) of caffeine, caffeine plus ATP (0.1 mM), and 4-chloro-3-ethylphenol (100 microM, 500 microM, and 1 mM), a new agonist of ryanodine receptors. The results indicate that NO can increase the P2Y receptor resensitization in rat glomerular mesangial cells by acting through a cGMP-independent pathway. No evidence was found for the existence of ryanodine-sensitive intracellular calcium stores in rat mesangial cells.