Uptake of atrial natriuretic peptide and production of cGMP in cultured human glomerular endothelial cells
Article
Green, DF, Duruibe, VA, Blyden, G et al. (1994). Uptake of atrial natriuretic peptide and production of cGMP in cultured human glomerular endothelial cells
. JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY, 5(4), 1091-1098.
Green, DF, Duruibe, VA, Blyden, G et al. (1994). Uptake of atrial natriuretic peptide and production of cGMP in cultured human glomerular endothelial cells
. JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY, 5(4), 1091-1098.
Interactions between human glomerular endothelial cells and atrial natriuretic peptide (ANP) were studied with 125I-α-human-ANP binding and intracellular accumulation of cGMP. Uptake for α-hANP (1-28 or 5-28) by homogeneous cultures of human glomerular endothelial cells was dose and time dependent with optimal uptake occurring after 30 min of incubation at 37°C. Scatchard analysis of the specific binding data with a two-compartment model identified both high (Kd = 0.3 nM)- and low (Kd = 10 nM)-affinity receptors, with a binding site density of 12,000 and 18,060 receptors per cell, respectively. α-hANP markedly stimulated glomerular endothelial cell-associated cGMP. After a 2-min incubation, cGMP increased 1.3-fold (from 17.88 ± 1.29 to 23.33 ± 3 pmol/mg of protein), in the presence of 1 nM ANP, to more than threefold (from 21 ± .1 to 80.5 ± 14.5 pmol/mg of protein) with 1 μM ANP (P < 0.05). In contrast, a 10 μM concentration of the clearance receptor C-ANP4-23 increased cGMP by 1.6 ± 0.6 fold. ANP stimulation of intracellular cGMP was 100 times more sensitive in human glomerular endothelial than in mesangial cells. In comparison, higher doses of bradykinin were necessary to evoke similar responses in glomerular endothelial cells. In the presence of 10 μM bradykinin, cellular cGMP increased by 1.75 ± 0.6-fold versus control cells. However, unlike ANP, bradykinin-stimulated cGMP synthesis was significantly inhibited by prior treatment with oxyhemoglobin (10-5 M), an inhibitor of soluble guanylate cyclase, and NG-nitro-L-arginine (NO2Arg), a specific inhibitor of endothelial-derived relaxing factor (EDRF). ANP-stimulated cGMP synthesis was also attenuated in the presence of both hemoglobin and NO2Arg; however, the difference was not significant compared with ANP alone. In addition, α-hANP had no effect on intracellular free calcium or the accumulation of cAMP. In summary, human glomerular endothelial cells possess both high- and low-affinity binding sites for ANP, which respond by the activation of guanylate cyclase. The attenuation of ANP-stimulated cGMP synthesis in vitro by specific blockers of EDRF suggests that locally released EDRF may modulate the cellular effects of ANP.
