Production and Role of Extracellular Guanosine Cyclic 3′, 5′ Monophosphate in Sodium Uptake in Human Proximal Tubule Cells

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Abstract
The present study was designed to determine the capability of human renal proximal tubule (RPT) to generate and export guanosine cyclic 3′, 5′ monophosphate (cGMP) in response to direct stimulation of soluble guanylyl cyclase by nitric oxide (NO) donors. In addition, we investigated whether cGMP extrusion from human RPT cells is required for inhibition of cellular sodium uptake. RPT cells were cultured from fresh human kidneys (normotensive subjects, n=4, mean age 65±4.7 years, 3 men, 1 woman; hypertensive patients, n=6, mean age 64±6.1 years, 4 men, 2 women) after unilateral nephrectomy. The fluorescence dye Sodium Green was employed to determine cytoplasmic Na + concentration. In the presence of the Na + /K + ATPase inhibitor ouabain, fluorescence was monitored at the appropriate wavelength (excitation 485 nm, emission 535 nm). Nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP, 10 −4 M), increased both intracellular and extracellular cGMP (from 1.26±0.21 to 88.7±12.6 pmol/mg protein and from 0.58±0.10 to 9.24±1.9 pmol/mL, respectively, P + uptake by 37.4±6.8% ( P −6 M)-induced increase in intracellular cGMP accumulation (from 4.9±0.9 to 9.8±1.5 pmol/mg protein, P P + uptake. Neither intracellular nor extracellular cGMP were influenced by l -arginine, the metabolic precursor of NO, or N G -nitro- l -arginine methyl ester, an inhibitor of NO synthase. After exogenous administration of cGMP (10 −5 M) or its membrane-permeable analogue 8-Br-cGMP (10 −5 M), only 8-Br-cGMP crossed the cell membrane to increase intracellular cGMP (from 1.36±0.19 to 289.7±29.4 pmol/mg protein, P + uptake. In conclusion, human RPT cells contain soluble guanylyl cyclase and are able to generate and export cGMP in response to NO. Because human RPT cells do not themselves contain constitutive NO synthase, the NO-generating cGMP must be derived from sources outside the human RPT. The cGMP cellular export system is critical in the regulation of RPT cellular Na + absorption in humans.