Second Messenger Of Atrial Natriuretic Factor Research Articles

Atrial natriuretic factor inhibits the phosphorylation of protein kinase C in plasma membrane preparations of cultured Leydig tumor cells.

The peptide hormone atrial natriuretic factor (ANF) exerts its effect in a receptor-mediated fashion and the membrane-bound form of guanylate cyclase represents a biologically active ANF receptor; thus, cGMP has been considered a second messenger of ANF. To understand the mechanisms of ANF action, we have studied its effect on protein phosphorylation in the plasma membrane preparations of murine Leydig tumor (MA-10) cells, which overexpress guanylate cyclase-coupled ANF receptor molecules in high density. After pretreatment of the plasma membranes with ANF (100 nM), a marked decrease in phosphorylation of the 78-kDa protein kinase C (PKC) and the 240-kDa protein was observed. Phosphorylation of the 78-kDa PKC was also inhibited by cGMP (0.1 mM); however, phosphorylation of the 240-kDa protein was not affected by cGMP. The quantitative analyses, as determined by densitometric scanning, revealed that both ANF and cGMP inhibited phosphorylation of the 78-kDa PKC by approximately 75% and 45%, respectively. The inhibitory effect of ANF on phosphorylation of the 240-kDa protein was almost 90%, but cGMP did not show any discernible effect on its phosphorylation in plasma membranes of MA-10 cells. Phosphorylation of the 78-kDa PKC was stimulated by Ca2+ and phospholipids, and it immunologically cross-reacted with antiserum against brain PKC. Furthermore, in these plasma membrane preparations, the 78-kDa PKC was immunoprecipitated and its phosphorylation was inhibited by ANF. These data provide evidence for a new signal transduction mechanism of ANF that negatively regulates phosphorylation of the 78-kDa PKC and the 240-kDa protein in a cGMP-dependent and -independent manner in Leydig cells.

Effects of atrial natriuretic factor on the renin-aldosterone system: In vivo and in vitro studies

We investigated the effect of high physiological plasma levels of human ∋atrial natriuretic factor (ANF) on renin and aldosterone secretion in normal sodium deplete men. In short term infusion studies (2 or 8 h duration), ANF plasma levels as observed after sodium loading (50–70 pg/ml) lowered basal renin (PRA) and aldosterone, but had only a marginal effect on angiotensin II-stimulated aldosterone secretion. Preliminary results of a study with long term infusion (6 days) of ANF during a period of dietary sodium depletion argue against a significant tonic inhibitory effect of ANF on the renin-aldosterone system in the preceding period of sodium repletion: the plasma aldosterone response to sodium depletion was similar with and without ANF infusion. The second messenger of ANF for the direct inhibition of aldosterone secretion from zona glomerulosa cells is still unknown. To test the hypothesis, that cGMP is the second messenger of ANF, we produced a rise in intracellular cGMP in rat and rabbit zona glomerulosa cells using the unspecific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the more cGMP specific phosphodiesterase specific inhibitor M + B2948 (Zaprinast). Both inhibitors simulated the action of ANF in suppressing steroid secretion and elevating cGMP levels. The results are compatible with the view that cGMP is of importance as a second messenger for ANF in adrenal zona glomerulosa cells. Selective inhibition of phosphodiesterases in combination with endopeptidase inhibition may be an interesting principle to enhance the action of endogenous and exogeneous ANF.

Renal and humoral responses to sustained cardiopulmonary baroreceptor deactivation in humans

The renal and neurohumoral effects of prolonged cardiopulmonary baroreflex unloading and the relationship of these changes to urinary sodium excretion have not been well documented in humans. In this study, 12 normal males underwent lower body negative pressure at -15 mmHg for 90 min, a maneuver that deactivates cardiopulmonary baroreceptors. Glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and filtration fraction (FF) were measured in eight of these subjects using inulin and p-aminohippuric acid clearance techniques. During reduction of central venous pressure, arterial blood pressure and heart rate did not change. Plasma concentrations of atrial natriuretic factor (ANF) decreased markedly (22 +/- 2 to 12 +/- 1 pg/ml, P = 0.0001) as did the second messenger of ANF's biological action, guanosine 3',5'-cyclic monophosphate, whereas renin and vasopressin were not significantly altered. There was a significant rise in plasma norepinephrine (1.6 +/- 0.2 to 2.4 +/- 0.4 nmol/l, P = 0.03). GFR (104 +/- 9 to 68 +/- 6 ml/min, P = 0.007) and FF (0.18 +/- 0.01 to 0.14 +/- 0.01, P = 0.007) decreased significantly, with maintenance of ERPF. There was a significant antinatriuresis without an antikaliuresis and a significant reduction in free water clearance. These changes in renal hemodynamics are unlike the known effects of renal vasoconstrictors, and the alterations in solute and free water clearance are consistent with the removal of the known actions of ANF from tubular target sites. Taken together, our findings suggest that a mechanism other than activation of vasoconstrictors, possibly the diminution of the influence of ANF on the kidney, may be operative in the renal adjustments to cardiopulmonary baroreflex deactivation in humans.

Stimulation of protein phosphorylation by atrial natriuretic factor in plasma membranes of bovine adrenal cortical cells

Atrial natriuretic factor (ANF) rapidly enhanced phosphorylation of plasma membrane proteins of bovine adrenal cortical cells. Pretreatment of the membranes with ANF (1 x 10(-8)M to 1 x 10(-7)M) resulted two- to four-fold in an incorporation of 32p-radioactivity from [gamma -32p]ATP as compared to the controls. The guanosine 3', 5' monophosphate (cGMP) which has been considered a second messenger of ANF also enhanced the phosphorylation of several proteins which were stimulated by ANF. However, the phosphorylation of certain proteins was stimulated differentially only by either ANF or cGMP. These results suggest that ANF-induced protein phosphorylation may play a role in transmembrane signalling pathway involving other second messenger(s) in addition to cGMP during the biological action of ANF.

ANF inhibits vasopressin-induced Ca2+ mobilization and contraction in glomerular mesangial cells.

It has been suggested that atrial natriuretic factor (ANF) may increase glomerular filtration rate (GFR) via alteration of the glomerular ultrafiltration area, which is at least in part regulated by the contractile state of mesangial cells. The present study therefore investigated the effect of ANF (atriopeptin III) on arginine vasopressin (AVP)-induced Ca2+ mobilization and contraction in cultured rat mesangial cells. AVP (10(-8) M) increased intracellular Ca2+ ([ Ca2+]i) as measured by the quin2 method (142.1 +/- 3.7 vs. 297.9 +/- 8.9 nM, P less than 0.001). This effect, however, was diminished by 10(-8) M ANF (297.9 +/- 8.9 vs. 233.1 +/- 9.3 nM, P less than 0.0025). ANF alone did not affect basal [Ca2+]i. AVP-stimulated 45Ca2+ efflux (cpm.mg prot-1.30 s-1) from mesangial cells within 30 s (1,432 +/- 70 basal vs. 2,751 +/- 287 AVP at 30 s, P less than 0.001). This effect of AVP on 45Ca2+ efflux was also inhibited by ANF (1,403 +/- 62 basal vs. 1,584 +/- 87 AVP and ANF, NS); ANF alone had no effect on spontaneous 45Ca2+ efflux. Furthermore, ANF inhibited AVP-induced 45Ca2+ uptake, which is purportedly one of the major determinants of the sustained phase of cell contraction. Cell contraction was assessed by digital imaging analysis. AVP (10(-7) M) caused contraction of 33.8 +/- 2.0% of the cells, but the contractile response decreased to 20.7 +/- 2.3% in the presence of 10(-9) M ANF and was virtually abolished in the presence of 10(-8) M ANF. Guanosine 3',5'-cyclic monophosphate (cGMP) has been proposed as the second messenger for ANF. ANF (10(-7) M) stimulated cGMP production significantly (0.54 +/- 0.12 vs. 22.30 +/- 5.91 pmol.mg prot-1.10 min-1, P less than 0.01), but it had no effect on cGMP at concentrations of 10(-8) M and below. 8-Bromo-cGMP (8-BrcGMP) attenuated the stimulatory effect of AVP on [Ca2+]i, 45Ca2+ efflux, and 45Ca2+ uptake. AVP-induced cell contraction was also decreased in the presence of 2 mM 8-BrcGMP (33.8 +/- 2.0% vs. 18.8 +/- 2.6%, P less than 0.005). The present results suggest that ANF may increase GFR in part by blocking the cellular action of AVP and possibly other vasoconstrictors on glomerular mesangial cells, thus affecting glomerular filtration area. A role of cGMP as second messenger for ANF is possible, but it appears unlikely to be the only mediator for the effects of ANF.

In vivo evidence that cGMP is the second messenger for atrial natriuretic factor.

cGMP generation has been associated with many of the vascular and endocrine actions of atrial natriuretic factor (ANF) in vitro. To examine the role of cGMP as a second messenger for the renal hemodynamic action of ANF in vivo, we measured glomerular filtration rate (GFR) and cGMP concentration in systemic artery, renal vein, and urine as well as in Bowman's space and end-proximal tubule (by free-flow micropuncture) after administration of ANF. ANF increased GFR by 45% and simultaneously induced a greater than 5-fold increase of cGMP concentration in glomerular ultrafiltrate (Bowman's space) when compared to controls. There was no significant increase in either systemic artery or renal vein cGMP concentration. Thus, the source of increased Bowman's space cGMP is not from the blood via filtration but rather from either glomerular mesangial or epithelial cells, which are not in direct contact with the circulation. Although a small amount of tubular handling of cGMP occurred along the length of the nephron, the augmented cGMP production from the glomerulus accounted for most of the 10- to 12-fold higher urinary cGMP excretion observed after ANF administration. Intrarenal arterial infusion of dibutyryl cGMP, but not dibutyryl cAMP, increased GFR in a dose-dependent fashion (from 10 to 1000 microM) by a mechanism similar to that of ANF--an increase in glomerular hydraulic pressure. Thus, ANF markedly stimulated glomerular production of cGMP, which coincided with a marked increase in GFR. Since dibutyryl cGMP itself was capable of increasing GFR, cGMP is the likely second messenger for ANF in vivo.