This study aimed to investigate the influence of acute tissue hypoxygenation on the expression of NO synthase (NOS) genes in vivo. To this end, male Sprague-Dawley rats were exposed either to 9% oxygen or to 0.1% carbon monoxide for 6 h, and mRNA levels of NOS-I, -II, and -III in kidneys, livers, lungs, and left and right heart ventricles were assayed by ribonuclease protection. For comparison, mRNA levels of erythropoietin were also measured in these tissues. NOS-III mRNA was highly abundant in all organs investigated. NOS-II mRNA was detected in lungs and hearts but not in kidneys and livers. NOS-I mRNA was found in kidneys, lungs, and hearts but not in livers. NOS-III mRNA levels were upregulated by hypoxia in all tissues examined, with the least effect (1.2-fold) in the left ventricle and the greatest effect (2.6-fold) in the lung. NOS-II mRNA was substantially downregulated in the ventricles by both treatments but not changed in the lung. NOS-I mRNA was upregulated by carbon monoxide in kidneys and lungs and by 9% oxygen in the lung. These findings suggest that NOS-III and possibly also NOS-I gene expression behave like oxygen-regulated genes, whereas the general effect of tissue hypoxygenation on NOS-II gene expression is less clear. Because NOS-III is primarily expressed in endothelial cells, a general upregulation of NOS in these cells may be of relevance for the regulation and maintenance of blood flow through hypoxic tissues.

Utilizing a combination of mechanical and chemical unilateral denervation, we have examined the relevance of renal innervation for the expression of renin in kidneys of adult rats. Renal denervation led to a reduction by 57 +/- 4% of renin-containing areas in denervated kidneys as quantitated by morphometry of kidney sections immunoreactive against a polyclonal antirenin antibody. Preprorenin mRNA content in the denervated kidneys fell to 46 +/- 7% of the contralateral innervated kidneys. Treatment of rats with the beta 1-adrenoreceptor antagonist metoprolol (100 mg.kg-1.day-1) for 2 days decreased renal renin mRNA levels to 71% of control levels. Unilateral renal denervation led to a further decrease of renin mRNA levels also in metoprolol-treated animals to 60% of the values found in the contralateral kidneys. Hypotensive hemorrhage led to a 1.4-fold increase of renin mRNA in the kidneys of sham-treated animals. In unilaterally denervated rats renin mRNA increased to levels similar to those in sham-operated animals in both denervated and in contralateral innervated kidneys in response to bleeding. As a consequence, the ratio of abundance of renin mRNA in the denervated to the innervated kidneys rose to 86 +/- 7%. Pretreatment of the animals with metoprolol, on the other hand, prevented the rise of renin mRNA in response to hypotensive hemorrhage. Our findings suggest that in the adult organism renal neural input significantly contributes to the expression of renin under basal conditions, while it appears to be of less importance for stimulation of renin gene expression by severe blood loss.

This study sought to examine the involvement of prostaglandins and of nitric oxide (NO) in the macula densa-dependent activation of the renin system in vivo. For this purpose, male Sprague-Dawley rats were chronically infused with furosemide (12 mg/day) for 6 days to inhibit macula densa salt transport. To inhibit the synthesis of prostaglandins and of NO, animals were injected with indomethacin (2 mg/kg twice daily) and with nitro-L-arginine methyl ester (L-NAME; 40 mg/kg twice daily) for the last 2 days of the experiment, respectively. Furosemide infusion increased plasma renin activity (PRA) from 8.8 +/- 1.4 to 41 +/- 5.2 ng angiotensin I (ANG I).h-1.ml-1 and renin mRNA levels from 112 +/- 8 of standard to 249 +/- 18% of standard. After treatment with indomethacin, the furosemide-induced increases in renin mRNA levels was attenuated to 190 +/- 11% of standard. After injections of L-NAME, both the furosemide-induced increases of renin mRNA levels and of PRA were reduced to 126 +/- 14% of standard and 22 +/- 5 ng ANG I.h-1.ml-1, respectively. These findings suggest that activation of renin gene expression by blockade of the macula densa function is dependent on intact NO and prostaglandin formation, whereas for stimulation of renin secretion mainly intact NO formation appears to be necessary.

In our study we have examined the mRNA levels of nitric-oxide-(NO-)synthases in rat kidneys during states of stimulated and reduced renin gene expression, to find out whether renal mRNA levels of NO-synthases are correlated with the activity of the renin system. Stimulation of the renin system was achieved by unilateral renal artery clipping (2-kidney/1-clip rats), treatment with the angiotensin II (ANG II) antagonist losartan (40 mg/kg), application of furosemide (12 mg x kg-1 x day-1) and a low-sodium diet (0.02% w/w Na+), which increased renin mRNA levels to 464%, 495%, 309% and 219% of those of control animals, respectively. Inhibition of the renin system was achieved in the nonclipped (contralateral) kidneys of 2-kidney/1-clip rats and in the kidneys of rats which were fed a high-sodium diet (4% w/w Na+); in both cases renin mRNA levels decreased to about 50% of the control values. First screening of the gene expression of brain-type NO-synthase (b-NOS), endothelial NOS (e-NOS) and inducible NOS (i-NOS) during all these alterations of the renin system was done using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Results from such noncompetitive PCR experiments indicated that only b-NOS mRNA levels change concordantly with the levels of renin. These changes in b-NOS mRNA levels were checked by the more reliable method of RNase protection assay. Results of the RNase protection assay proved that the renal levels of b-NOS mRNA were significantly increased by about 50% after a low-sodium diet and hypoperfusion of the kidney. Given a stimulatory role of endothelium-derived relaxing factor (EDRF)/NO on the renin system our findings may provide the first evidence that increases of renal levels of b-NOS mRNA and, as a consequence, of renal EDRF/NO formation could be important mediators of the well-known effect of salt intake and hypoperfusion on the renin system.

This study was designed to examine the possible involvement of prostaglandins and nitric oxide (NO) in the renin stimulatory effect ofangiotensin 11 (AngII) antagonists. To this end, plasma renin activities (PRAs) and renal renin mRNA levels were assayed in rats that were treated with the Angconverting enzyme inhibitor ramipril or with the AnglI AT1-receptor antagonist losartan. Ramipril and losartan increased PRA values from 7.5 + 1.6 to 86 ± 6 and 78 ± 22 ng of AngI per h per ml and renin mRNA levels from 112 ± 9% to 391 + 20%o and 317 ± 10%o, respectively. Inhibition of prostaglandin formation with indomethacin did not influence basal or ramiprilaffected PRA. Basal renin mRNA levels also were unchanged by indomethacin, while increases in renin mRNA levels after ramipril treatment were slightly reduced by indomethacin. Inhibition of NO synthase by nitro-L-arginine methyl ester (L-NAME) reduced PRA values to 3.2 ± 0.9, 34 ± 13, and 12.1 ± 2.7 ng of AngI per h per ml in control, ramipril-treated, and losartan-treated animals, respectively. Renin mRNA levels were reduced to 77 + 14% under basal conditions and ramipril-and losartan-induced increases in renin mRNA levels were completely blunted after addition of L-NAME. The AngIH antagonists, furthermore, induced an upstream recruitment of reninexpressing cells in the renal afferent arterioles, which was also blunted by L-NAME. These findings suggest that renin mRNA levels are tonically increased by NO and that the action of NO is counteracted by AngII.One of the most prominent in vivo effects of angiotensin (Ang)-converting enzyme (ACE) inhibitors is a marked stimulation of renin secretion and renin gene expression in the kidneys (1-5). Since ANGII is known as a negative feedback regulator of renin secretion and of renin gene expression (6, 7), it is thought that the renin stimulatory effects of ACE inhibitors are related to the inhibition of AnglI formation and consequently to the lowering of circulating and tissue AnglI levels (8).There is evidence that several in vivo effects of ACE inhibitors are not, as originally thought, due to the inhibition of AnglI formation but to the inhibition of kinin degradation (9-12). Inhibition of kinin degradation results in elevated tissue levels of autacoids such as prostaglandins and nitric oxide (NO), the formation of which is stimulated by kinins (11)(12)(13)(14). Although NO is reported to inhibit the renin system (15-17), contradictory findings suggest that NO stimulates the renin system (18-25) and also that prostaglandins stimulate the renin system (for review, see ref. 26). With such a stimulatory effect of NO and prostaglandins on the renin system, it is possible that the renin stimulatory effect of ACE inhibitors could be related to an enhanced formation of prostaglandins and NO.To investigate the mode of action of ACE inhibitors on the renin system, we studied renin release and renin mRNA levels in the kidneys of conscious rats that were treated with the ACE inhibitor ramipril (27) or with the Angll AT1-rec...

Liberators of NO exert a dual effect on renin secretion from isolated mouse renal juxtaglomerular cells. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F180-F186, 1993.-This study aimed to examine the role of nitric oxide (NO) in the regulation of renin secretion from renal juxtaglomerular (JG) cells. Using primary cultures of mouse renal JG cells, we found that sodium nitroprusside (SNP) and 3-morpholino-sydnonimin-hydrochloride (SIN-l), two structurally different liberators of NO, led to a transient inhibition during the first hour followed by a marked dose-dependent stimulation of renin secretion lasting for an additional 20 h. This stimulatory effect was blunted by methylene blue (50 PM) and was reversible within minutes after removal of the NO liberators. SNP and SIN-l also stimulated guanylate cyclase activity in the cultures with a maximum within the first hour of incubation. Increasing intracellular guanosine 3',5'-cyclic monophosphate levels by 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (100 PM) or by atria1 natriuretic peptide (10 nM) decreased basal renin secretion but did not inhibit the effect of SNP. The stimulatory effect of SNP was not related to adenosine 3',5'-cyclic monophosphate levels in the JG cells and was blunted after chelation of extracellular calcium by 2 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Taken together, our findings suggest that liberators of NO have two effects on renin secretion from isolated JG cells: an inhibitory effect mediated by stimulation of soluble guanylate cyclase activity and a stimulatory effect mediated by an as yet unknown pathway that requires extracellular calcium. endothelium-derived relaxing factor; exocytosis THERE IS ACCUMULATING evidence that the endothehum and endothelium-derived factors may exert physiologically important functions for the regulation of renin secretion from renal juxtaglomerular (JG) cells (4). In this context, good evidence has been elaborated suggesting a stimulatory effect of prostacyclin (10) and an inhibitory effect of endothelin (19, 20,23,27) on renin secretion. Recent findings, moreover, suggest that the endothelium-derived relaxing factor (EDRF), which is considered to be nitric oxide (NO) (22), is also capable of modulating renin secretion (1, 7, 16,25,31). Indirect evidence for a relevant role of EDRF in the control of renin secretion may also arise from the observation that the enzyme NO synthase, which generates EDRF, is not only located in renal endothelial cells (18) but also in the macula densa cells that are neighbors to JG cells (24). The particular effect of EDRF/NO on renin secretion, however, is so far less clear. Studies on renal cortical slices have produced results suggesting an inhibitory effect of EDRF/NO on renin secretion (1,31). Because EDRF/NO is known to stimulate guanylate cyclase activity in various tissues (13,30), an inhibitory effect of EDRF/NO would be compatible with the concept that guanosine 3',5'-cyclic monophosphate (cGMP) is an inhibit...

This study aimed to characterize the influence of endogenous angiotensin II on renal renin gene expression during different states of a stimulated and of a suppressed renin system. To this end the renin system in male Sprague Dawley rats was stimulated by unilateral renal artery clipping (0.2 mm clip), by furosemide (60 mg/kg per diem) or isoproterenol (160 microg/kg per diem), and by ingestion of a low-salt diet (0.02%), or was suppressed by setting a contralateral renal artery clip (0.2-mm clip) or by ingestion of a high-salt diet (4%). During the last 2 days of these different treatment regimens, the animals were treated with the angiotensin II AT1 receptor antagonist losartan (40 mg/kg per diem) and renal renin mRNA levels were assayed. Renin gene expression was stimulated four- to fivefold by renal artery clipping and isoproterenol infusion, two- to three-fold by furosemide and a low-salt diet, and about four-fold by losartan. Additional treatment with losartan potentiated the stimulatory effects of a low-salt diet, of furosemide and of isoproterenol infusion on renin gene expression, whilst there was no significant additional effect of losartan on renin gene expression in clipped kidneys. Both contralateral renal artery clipping and a high-salt diet decreased renin mRNA levels to about 50% of the control value. In rts with a unilateral clip, additional losartan treatment caused renin mRNA to increase to about 350% of the control value in the contralateral kidney but to only 100% of the control value in animals on a high-salt diet. These findings suggest that the enhanced formation of angiotensin II during a low-salt intake, during tubular inhibition of salt reabsorption or during beta-adrenoreceptor activation plays a relevant negative feedback role in the activation of the renin gene. Moreover, in rats with one hypoperfused kidney, angiotensin II could be involved in the inhibition of renin gene expression in the contralateral kidney. In hypoperfused kidneys, however, and in animals on a high-salt diet, angiotensin II appears to play a only a minor feedback role in the regulation of the renin gene.

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