Activation of the natriuretic peptide system lowers blood pressure and causes the excretion of salt. Atrial natriuretic peptide and B-type natriuretic peptide are the humoral mediators of this effect; they act primarily by binding to membrane-bound natriuretic peptide receptor A (NPRA) and stimulating its intrinsic guanylate cyclase activity. To study whether genetically determined differences in NPRA expression affect blood pressure we have generated mice with one, two, three, or four copies of the gene encoding NPRA (Npr1 in the mouse). Atrial natriuretic peptide-dependent guanylate cyclase activity ranged progressively from approximately one-half normal in one-copy animals to twice normal in four-copy animals (P < 0.001). On different diets (0.05%, 2%, and 8% NaCl), the blood pressures of F 1 male mice having only one copy of Npr1 averaged 9.1 mmHg (1 mmHg ‫؍‬ 133 Pa) above those of wild-type two-copy males (P < 0.001), whereas males with three copies of the gene had blood pressures averaging 5.2 mmHg below normal (P < 0.01). The blood pressures of the one-copy F 1 animals were significantly higher (by 6.2 mmHg; P < 0.01) on the high-salt than on the low-salt diet. The blood pressures of four-copy F 3 males were significantly lower (by 7 mmHg; P < 0.05) on the high-salt than on the low-salt diet. These results demonstrate that below normal Npr1 expression leads to a salt-sensitive increase in blood pressure, whereas above normal Npr1 expression lowers blood pressures and protects against high dietary salt.

Vasodilator prostaglandin PGE(2) protects the kidney from excessive vasoconstriction during contraction of extracellular fluid volume and pathophysiological states. However, it is not yet clear which of the four known E-prostanoid (EP) receptors is localized to resistance vessels and mediates net vasodilation. In the present study, we assessed the presence, signal transduction, and actions of EP receptor subtypes in preglomerular arterioles of Sprague-Dawley rat kidneys. RNA encoding EP(1), an EP(1)-variant, and EP(4) receptors was identified by RT-PCR in freshly isolated preglomerular microvessels; cultured preglomerular vascular smooth muscle cells (VSMC) had EP(1)-variant and EP(4) RNA but lacked EP(1). EP(2) and EP(3) receptors were undetectable in both vascular preparations. In studies of cell signaling, stimulation of cAMP by various receptor agonists is consistent with primary actions of PGE(2) on the EP(4) receptor, with no inhibition of cAMP by EP(1) receptors. Studies of cytosolic calcium concentration in cultured renal VSMC support an inhibitory role of EP(4) during ANG II stimulation. In vivo renal blood flow (RBF) studies indicate that the EP(4) receptor is the primary receptor mediating sustained renal vasodilation produced by PGE(2), whereas the EP(1) receptor elicits transient vasoconstriction. The EP(1)-variant receptor does not appear to possess any cAMP or cytosolic calcium signaling capable of affecting RBF. Collectively, these studies demonstrate that the EP(4) receptor is the major receptor in preglomerular VSMC. EP(4) mediates PGE(2)-induced vasodilation in the rat kidney and signals through G(s) proteins to stimulate cAMP and inhibit cytosolic calcium concentration.

In order to exert an appropriate biological effect, the action of the vasoconstrictive hormone angiotensin II (ANG II) is modulated by vasoactive factors such as prostaglandins PGE2 and PGI2. The present study investigates whether prostaglandins alter ANG II-mediated increases in cytosolic calcium concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC) isolated from rat renal preglomerular arterioles. [Ca2+]i was assessed using the calcium-sensitive dye fura 2 and a microscope-based photometer system. ANG II (10(-7) M) caused a biphasic, time-dependent [Ca2+]i response: an initial peak increase from 52 +/- 7 to 264 +/- 25 nM, followed by a sustained plateau of 95 +/- 9 nM in cultured VSMC. Coadministration of PGE2 or PGI2 or synthetic mimetics caused dose-dependent decreases in the peak [Ca2+]i response to ANG II, with attenuation of 40-50%. This degree of inhibition was even more pronounced in individual freshly isolated preglomerular VSMC. Increasing cAMP levels in cultured VSMC, by using either a cell-permeable analog or inhibiting phosphodiesterase activity, mirrored the antagonistic effects of prostaglandins on ANG II-stimulated increases in [Ca2+]i. Radioimmunoassays demonstrate that ANG II (10(-7) M) stimulates production of PGI2 and PGE2; the stable prostacyclin metabolite 6-keto-PGF(1alpha) was released in 10-fold greater concentrations than PGE(2.) Indomethacin blockade of prostaglandin production potentiated both the peak (264 to 337 +/- 26 nM) and sustained [Ca2+]i responses (95 to 181 +/- 22 nM) to ANG II. When prostaglandin analogs were added during indomethacin treatment, the ANG II response was restored to the typical pattern. In conclusion, we demonstrate that modulation of intracellular calcium levels is one mechanism by which prostaglandins can buffer ANG II-mediated constriction in renal preglomerular VSMC. PGI2 is more potent than PGE2 in this regard.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) exert their physiological actions by binding to natriuretic peptide receptor A (NPRA), a receptor guanylate cyclase (rGC) that synthesizes cGMP in response to both ligands. The family of rGCs is rapidly expanding, and it is plausible that there might be additional, as yet undiscovered, rGCs whose function is to provide alternative signalling pathways for one or both of these peptides, particularly given the low affinity of NPRA for BNP. We have investigated this hypothesis, using a genetically modified (knockout) mouse in which the gene encoding NPRA has been disrupted. Enzyme assays and NPRA-specific Western blots performed on tissues from wild-type mice demonstrate that ANP-activated cGMP synthesis provides a good index of NPRA protein expression, which ranges from maximal in adrenal gland, lung, kidney, and testis to minimal in heart and colon. In contrast, immunoreactive NPRA is not detectable in tissues isolated from NPRA knockout animals and ANP- and BNP-stimulatable GC activities are markedly reduced in all mutant tissues. However, testis and adrenal gland retain statistically significant, high-affinity responses to BNP. This residual response to BNP cannot be accounted for by natriuretic peptide receptor B, or any other known mammalian rGC, suggesting the presence of a novel receptor in these tissues that prefers BNP over ANP.

Abstract. In a previous study of cultured preglomerular vascular smooth muscle cells, it was demonstrated that, although the stable prostacyclin analog iloprost alone had no effect on the intracellular calcium concentration ([Ca2+]i), it did significantly attenuate the increase in [Ca2+]i stimulated by angiotensin II (AngII). In this study, the mechanisms by which iloprost interacts with calcium signaling pathways stimulated by AngII were examined. [Ca2+]i was assessed using the calcium-sensitive fluorescent dye fura-2. Initial studies identified two major components of the [Ca2+]i response to AngII in this homogeneous preparation of vascular smooth muscle cells from renal resistance vessels. Mobilization of internal stores was evident as an immediate TMB-8-sensitive peak increase in [Ca2+]i (52 ± 6 to 297 ± 26 nM, P < 0.001) in a calcium-free medium. After [Ca2+]i had returned to baseline levels during continued AngII stimulation, a nifedipine-sensitive entry pathway was revealed by the sustained stimulatory effect of added external calcium, which increased [Ca2+]i to 112 ± 13 nM (P < 0.001). Coadministration of iloprost with AngII attenuated both the immediate peak (154 ± 14 nM) and sustained plateau (61 ± 9 nM) phases. Increases in endogenous levels of cAMP induced by the phosphodiesterase inhibitor milrinone mirrored the actions of iloprost, suggesting that the prostacyclin analog exerted its actions via cAMP activation. Blockade of cAMP-dependent protein kinase with KT 5720 reversed the effects of both iloprost and milrinone. When iloprost or milrinone was introduced after the initial mobilization peak had dissipated, the plateau phase of calcium entry was unchanged (92 ± 9 nM). The concept that iloprost does not directly modulate calcium entry was further supported by data showing that the activation of L-type calcium channels by BAY-K 8644 was unchanged during iloprost treatment. On the basis of the observation that iloprost did not alter thapsigargin stimulation of Ca2+-ATPase activity, it is concluded that the actions of cAMP are distinct from increasing calcium uptake into the sarcoplasmic reticulum. This study provides new information on the ability of iloprost to primarily attenuate inositol-1,4,5-triphosphate-mediated calcium mobilization via cAMP, with secondary inhibition of L-type calcium entry channels. These data clarify the mechanism by which prostaglandins buffer AngII constriction in resistance arterioles.