Roberto Bruna - Academia.edu (original) (raw)

Papers by Roberto Bruna

Research paper thumbnail of A native, affinity-based protein blot for the analysis of streptavidin heterogeneity: Consequences for the specificity of streptavidin mediated binding assays

Research paper thumbnail of Zonal expression of angiotensin II-AT1-receptor genes in rat kidneys

Experimental nephrology

This study aimed to examine the relative distribution of ANGII-AT1-receptor gene expression in di... more This study aimed to examine the relative distribution of ANGII-AT1-receptor gene expression in different zones of the rat kidney, namely the glomeruli, cortex (CO), outer and inner stripes of outer medulla (OSOM and ISOM, respectively) and inner medulla (IM). By RNAse protection we found a ratio for the overall abundance of AT1 mRNA of 1, 0.68, 0.52, 0.86 and 0.46 for glomeruli, CO, OSOM, ISOM and IM, respectively. The intrazonal proportion of AT1a and AT1b mRNA was determined by RT-PCR, which yielded values for the ratio AT1a/AT1b of 1.1, 4.7, 4.4, 3.7 and 1.9 for glomeruli, CO, OSOM, ISOM, and IM, respectively. These findings suggest a zonal heterogeneity in the expression of AT1-receptor genes such that the highest expression is found in glomeruli and ISOM, whilst the papilla shows the lowest level of expression. Moreover, there appears to exist a corticopapillary gradient for the relative expression of AT1a and AT1b-receptor genes such that the preponderance of AT1a-receptor gen...

Research paper thumbnail of Juxtaglomerular cells in culture

Research paper thumbnail of Effect of synthetic atrial natriuretic peptide on rat renal juxtaglomerular cells

Journal of hypertension. Supplement : official journal of the International Society of Hypertension, 1986

We examined the effect of synthetic atrial natriuretic peptide (ANP) on unstimulated renin releas... more We examined the effect of synthetic atrial natriuretic peptide (ANP) on unstimulated renin release from rat renal juxtaglomerular cells. Using renal cortical cell cultures containing 80-90% juxtaglomerular cells, we found that ANP strongly inhibited renin release from the cells in a dose-dependent fashion. Half-maximal inhibition was observed at 10(-11) mol/l ANP. Furthermore, ANP produced a rise in the intracellular concentration of cyclic guanosine monophosphate (cGMP) and a fall in the concentration of cyclic adenosine monophosphate (cAMP). Data indicated that the inhibition of renin release by ANP was related to the rise in cGMP and not to the fall of cAMP. Atrial natriuretic peptide (10(-10) mol/l) had no influence on the transmembrane calcium influx nor did it alter the intracellular calcium concentration of the juxtaglomerular cell. We found, however, that the inhibitory effect of ANP on renin release could be attenuated by the calcium channel blocker verapamil. Our results s...

Research paper thumbnail of Determinants of renin secretion and renin synthesis in isolated mouse juxtaglomerular cells

Kidney international. Supplement, 1991

In this study we examined the effects of classic second messenger molecules on renin secretion an... more In this study we examined the effects of classic second messenger molecules on renin secretion and renin synthesis in primary cultures of mouse renal juxtaglomerular (JG) cells. Stimulation of cAMP formation by forskolin, inhibition of calmodulin by calmidazolium, and inhibition of Na+/H+ exchange by ethylisopropylamiloride enhanced renin secretion. Raising of intracellular cGMP by 8-bromo-cGMP and activation of protein kinase C by phorbol ester led to an inhibition of secretion. Renin synthesis was stimulated by forskolin. Calmidazolium, EIPA, 8-bromo-cGMP, and phorbol ester were without effect on basal renin synthesis. The data suggest that renin secretion is influenced by a number of transmembrane transduction systems which in their majority exert a negative control on renin secretion. Activation of adenylate cyclase appears to be a stimulatory control mechanism for both the secretion and the synthesis of renin. The findings suggest, moreover, that the second messenger controls o...

Research paper thumbnail of Calmodulin antagonists stimulate renin secretion and inhibit renin synthesis in vitro

The American journal of physiology, 1992

To find out whether calmodulin activity could be a common denominator for the cellular control of... more To find out whether calmodulin activity could be a common denominator for the cellular control of renin secretion and synthesis, we have examined the effects of calmodulin antagonists on the secretion and the synthesis of renin in primary cultures of mouse juxtaglomerular (JG) cells. We found that the calmodulin antagonists calmidazolium (CMDZ), N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), and trifluoperazine (TFP) strongly stimulated renin release from isolated JG cells with a rank order of potency CMDZ greater than TFP greater than W-7. With the same order of potency as on renin secretion, CMDZ, TFP, and W-7 also inhibited de novo synthesis of renin. This decrease of renin synthesis went in parallel with a general inhibition of protein synthesis in the cultured JG cells. A comparable inhibition of total protein synthesis and renin synthesis as with CMDZ was achieved with cycloheximide. Cycloheximide, however, did not alter renin secretion within 20 h of incubation. T...

Research paper thumbnail of Atrial natriuretic peptide inhibits renin release from juxtaglomerular cells by a cGMP-mediated process

Proceedings of the National Academy of Sciences, 1986

We have exmined the effect of a synthetic analogue of human et-atrial natriuretic peptide (ANP), ... more We have exmined the effect of a synthetic analogue of human et-atrial natriuretic peptide (ANP), APH, on renin release in cultured renal juxtaglomerular cells (JGA cells). Using cell cultures containing 80-90% renal juxtaglomerular cells, we found that ANP (10-13-10-9 M) strongly

Research paper thumbnail of Amiloride enhances the secretion but not the synthesis of renin in renal juxtaglomerular cells

Pfl�gers Archiv European Journal of Physiology, 1991

In this study we have examined a potential role of the sodium/proton exchange system in the regul... more In this study we have examined a potential role of the sodium/proton exchange system in the regulation of renin secretion. We found that the inhibitors of the Na+/H+ antiport, amiloride (1 mM) and ethylisopropylamiloride (EIPA, 50 microM), led to a 125% increase of renin secretion from cultured mouse juxtaglomerular cells. The stimulatory effect of EIPA on renin secretion was dependent on the extracellular concentrations of sodium and hydrogen ions. While lowering the extracellular pH from 7.3 to 7.0, and lowering [Na+]e from 130 mM to 5 mM had no effect on basal renin release, it markedly attenuated or even blunted the effect of EIPA on renin secretion. The stimulatory effect of forskolin on renin secretion, however, was not altered by decreases of extracellular pH and of sodium. Inhibition of basal renin release was achieved with angiotensin II (1 microM). In the presence of EIPA the inhibitory effect angiotensin II was markedly attenuated. Although effective on renin secretion, neither amiloride nor EIPA exerted a significant effect on the denovo synthesis of renin in cultured mouse JG cells. These findings are compatible with the idea that an amiloride-sensitive transport process, presumably the Na+/H+ exchanger, acts indirectly as an inhibitory signal transduction system for renin secretion from renal juxtaglomerular cells.

Research paper thumbnail of Endothelium derived relaxing factor is involved in the pressure control of renin gene expression in the kidney

Pfl�gers Archiv European Journal of Physiology, 1994

To study the influence of endothelium derived relaxing factor/nitric oxide (EDNO) on renin gene e... more To study the influence of endothelium derived relaxing factor/nitric oxide (EDNO) on renin gene expression, the effects of a 2-day treatment with the NO-synthase inhibitor nitro-L-arginine-methylester (L-NAME, 40 mg/kg twice a day) on plasma renin activity (PRA) and renal and adrenal renin m-RNA levels were examined in conscious rats with and without unilateral renal clips (0.2 mm). In sham-clipped animals L-NAME led to a decrease of PRA from 7.5 to 2.5 ng angiotensin (ANGI).h-1.ml-1 and to a 35% decrease of renal renin m-RNA levels. Unilateral renal artery clipping increased PRA to 35 and to 13 ng ANGI.h-1.ml-1 in vehicle and in L-NAME-treated rats, respectively. In the clipped kidneys renin m-RNA levels increased to 450% of control values in vehicle-treated animals and to 220% of control values in L-NAME-treated animals. In the contralaterals as opposed to clipped kidneys, renin m-RNA levels decreased to 16% and 50% of the control values in vehicle- and in L-NAME-treated animals, respectively. In the adrenal glands renin m-RNA levels were not significantly changed either by clipping of one renal artery or by treatment of animals with L-NAME. The NO-donor sodium nitroprusside (100 microM) was found to increase renin secretion and renin m-RNA levels in primary cultures of renal juxtaglomerular cells. These findings suggest that EDNO is involved in the control of the renin gene by the renal perfusion pressure.

Research paper thumbnail of Renin gene and angiotensin II AT1 receptor gene expression in the kidneys of normal and of two-kidney/one-clip rats

Pfl�gers Archiv European Journal of Physiology, 1995

This study aimed to investigate the inter-relation between the angiotensin II (ANG II) AT~ recept... more This study aimed to investigate the inter-relation between the angiotensin II (ANG II) AT~ receptor and renin gene expression in rat kidneys. To this end, renin mRNA levels and mRNA levels for A%~and ATtb were assayed by RNase protection in the kidneys of normal rats, in animals treated with the AT~ antagonist losartan and in rats bearing 0.2-mm left renal artery clips for 2 days. In normal rats, we found a negative correlation between renin mRNA levels and ATla receptor mRNA levels. Losartan led to a fourfold increase in renin mRNA levels without changing AT1 receptor mRNA levels. Unilateral renal artery clipping increased renin mRNA levels fourfold in the clipped kidney and suppressed renin mRNA levels in the contralateral kidneys. AT~ receptor mRNA levels were not changed in the contralateral intact kidneys, but were significantly decreased by 15-25% in the clipped kidneys. Renin mRNA levels were inversely correlated to AT~a mRNA levels in the clipped, but not in the contralateral, kidneys. Our findings suggest that the systemic activity of the renin angiotensin system has no regulatory influence on renal AT1 receptor gene expression. Renin mRNA levels in normal and in clipped kidneys appear to be negatively determined by the level of AT~a receptor gene expression. Thus modulation of ATla receptor gene expression could be a pathway for indirect modulation of renin gene expression by ANG II. This conclusion is in agreement with the observation that AT~ receptor antagonists are powerful stimulators of the renin system.

Research paper thumbnail of Extracellular calcium exerts a dual effect on renin secretion from isolated mouse juxtaglomerular cells

Pfl�gers Archiv European Journal of Physiology, 1993

Utilizing primary cultures of mouse renal juxtaglomerular cells we found that renin secretion dur... more Utilizing primary cultures of mouse renal juxtaglomerular cells we found that renin secretion during 20 h of incubation was stimulated by 100% when extracellular calcium was lowered from a basal level of 0.5 mM to below I gM, and was enhanced in a concentration-dependent fashion by 600 % when extracellular calcium was increased up to 10 mM. The stimulatory effect of low calcium on renin secretion was apparent after the first hour of incubation, whereas the stimulatory effect of increased calcium occurred with a delay of at least 1 h. During the first hour of incubation increased extracellular calcium blunted the stimulation of renin secretion induced by forskolin (10 pM). The stimutatory effect of increased calcium was attenuated in the presence of 8-pCPT-cGMP, a membrane-permeable cGMP analogue, and in the presence of 100 mM sucrose. The stimulatory effect of increased calcium was blunted in the presence of 0.5 mM cobalt, which itself stimulated renin secretion at normal (0.5 mM) calcium concentrations. Renin synthesis by the cultured cells at low calcium was markedly attenuated in proportion with total protein synthesis, whereas renin synthesis was not altered at increased calcium concentrations. Our findings suggest that a rise of intracellular calcium, induced by an increase of extracellular calcium, is associated with a transient inhibition followed by a marked and regulatable stimulation of renin secretion. Neither calcium depletion nor calcium elevation appears to exert specific effects on renin synthesis in juxtaglomerular cells.

Research paper thumbnail of Renin gene expression in the aging kidney: effect of sodium restriction

Mechanisms of Ageing and Development, 1995

... [6]. H. Huang, T. Baussant, R. Reader, JB Michel and P. Corvol ... [17]. A. Brecher, D. Shier... more ... [6]. H. Huang, T. Baussant, R. Reader, JB Michel and P. Corvol ... [17]. A. Brecher, D. Shier, H. Dene, S. Wang, J. Rapp, R. Franco-Saenz and P. Mulrow, Regulation of adrenal renin messenger ribonucleic acid by dietary sodium chloride. Endocrinology 124 (1989), pp. 2907–2913. ...

Research paper thumbnail of Opposite regulation of renin gene expression by cyclic AMP and calcium in isolated mouse juxtaglomerular cells

Kidney International, 1995

Opposite regulation of renin gene expression by cyclic AMP and calcium in isolated mouse juxtaglo... more Opposite regulation of renin gene expression by cyclic AMP and calcium in isolated mouse juxtaglomerular cells. A quantitative reverse transcriptase-polymerase chain reaction for mouse renin mRNA was utilized to study the influence of classic second messenger molecules on renin mRNA levels in primary cultures of juxtaglomerular (JG) cells isolated from the kidneys of C57/B16 mice. We found that forskolin (3 .LM), an activator of adenylate cyclase led to proportional increases of renin secretion and renin mRNA levels. The nitric oxide (NO) donor, sodium nitroprusside (100 MM), stimulated both renin secretion and renin gene expression, the effect on secretion being stronger than that on renin mRNA levels. An increase of the extracellular concentration of calcium from 0.5 to 3 ms led to a transient inhibition of renin secretion, followed by a marked stimulation of secretion and to a continuous suppression of renin mRNA levels. These were also decreased by the calcium ionophore A 23187 (1 zM). The membrane permeable 8-bromo-cyclic GMP (100 LM) inhibited basal renin secretion without an effect on renin mRNA levels. The phorbol ester phorbol-12-myristate-13-acetate (1 to 100 nM), which was used to stimulate protein kinase C activity, had no significant effects on renin secretion and renin mRNA levels, neither alone nor in combination with forskolin. These findings suggest that cAMP, NO and calcium are effective regulators of renin gene expression in renal JG cells, in a way that cAMP and NO are stimulators and calcium acts as an inhibitor.

Research paper thumbnail of Cyclosporine A enhances renin secretion and production in isolated juxtaglomerular cells

Kidney International, 1988

Cyclosporine A enhances renin secretion and production in isolated juxtaglomerular cells. Stimula... more Cyclosporine A enhances renin secretion and production in isolated juxtaglomerular cells. Stimulation of the renin-angiotensin system is a major side effect of the fungoid immunosuppressant cyclosporine A (CyA). The aim of this study was to find out whether or not this effect of CyA results from a direct interaction with renal juxtaglomerular (JG) cells, which are the site of renal renin synthesis and release. Using primary cell cultures from rat renal cortex containing more than 80% JG cells, we found that CyA (0.01 to 10 zg/ml) stimulated renin secretion threefold. This stimulation was paralleled by a dose-dependent twofold increase of inactive renin within the cells, while the active intracellular renin remained the same. In order to identify a possible second messenger which could mediate the effects of CyA on JG cells, we examined the simultaneous effects of a single concentration of CyA (I jsg/ml) on renin secretion, prostaglandin formation and intracellular cAMP concentration. However, prostaglandin formation and cAMP were not detectably altered by CyA in experiments where renin secretion was significantly enhanced. Our results indicate that cyclosporine A stimulates renin secretion and renin synthesis by a direct effect on renal juxtaglomerular cells. This action of CyA is not mediated by changes in cellular prostaglandin or intracellular cAMP.

Research paper thumbnail of Endothelins inhibit cyclic-AMP induced renin gene expression in cultured mouse juxtaglomerular cells

Kidney International, 1996

Research paper thumbnail of Role of cGMP as second messenger of adenosine in the inhibition of renin release

Kidney International, 1988

Role of cGMP as second messenger of adenosine in the inhibition of renin release. Adenosine is kn... more Role of cGMP as second messenger of adenosine in the inhibition of renin release. Adenosine is known to be a potent inhibitor of renin

Research paper thumbnail of Angiotensin II receptor gene expression in hypertrophied left ventricles of rat hearts

Journal of Hypertension, 1996

To examine the expression of angiotensin II AT1a, AT1b and AT2 receptor genes in the left ventric... more To examine the expression of angiotensin II AT1a, AT1b and AT2 receptor genes in the left ventricles of rats subjected to ventricular pressure overloading induced by aortic banding for 6 weeks and then 6 weeks medical treatment. Aortic banding was related to an increase in relative weight of the left ventricle from 1.73 +/- 0.06 (sham-operated) to 2.81 +/- 0.25 g/kg, an increase in beta-myosin: alpha-myosin messenger RNA (mRNA) ratio from 0.30 +/- 0.02 to 1.94 +/- 0.55 and an 18-fold increase in left ventricular atrial natriuretic peptide mRNA levels. In contrast, left ventricular pressure overload hypertrophy was not related to a significant change in the abundance of AT1a and AT1b mRNA, which were expressed in a relative ratio of 5:1. Similarly, the abundance of AT2 mRNA was not significantly changed in hypertrophied ventricles. In rats receiving the angiotensin II AT, receptor antagonist losartan (40 mg/kg) for 6 weeks after banding, relative heart weights were 2.39 +/- 0.14 g/kg, the beta-myosin: alpha-myosin ratio was 1.04 +/- 0.20 and atrial natriuretic peptide mRNA levels displayed a blunted increase (11-fold over sham-treated controls), documenting a significant amelioration of left ventricular hypertrophy by blockade of the AT1 receptor. Losartan treatment in parallel did not affect AT1a, AT1b and AT2 receptor mRNA levels, which were not different from those in vehicle-treated or sham-treated controls. These findings confirm that left ventricular hypertrophy in the rat is associated with increased ventricular expression of beta-myosin and of atrial natriuretic peptide and with reduced expression of alpha-myosin. Despite these significant changes in cardiac gene expression no alteration was observed in AT1a, AT1b and AT2 receptor mRNA levels.

Research paper thumbnail of Expression of cardiac angiotensin II AT1 receptor genes in rat hearts is regulated by steroids but not by angiotensin II

Journal of Hypertension, 1995

To examine the regulation by angiotensin II and by steroids of the expression of the angiotensin ... more To examine the regulation by angiotensin II and by steroids of the expression of the angiotensin II AT1a and AT1b receptor genes in rat hearts. Endogenous levels of angiotensin II in the rats were increased either by unilateral 0.2-mm renal artery clips or by subcutaneous infusions of frusemide (12 mg/day) and by low-sodium diet. To inhibit endogenous angiotensin II actions the rats received the AT1 receptor antagonist losartan (40 mg/kg per day) or the angiotensin converting enzyme inhibitor ramipril (8 mg/kg per day). Circulating levels of glucocorticoids were elevated by subcutaneous injections of dexamethasone (400 micrograms/kg per day) and levels of mineralocorticoids were increased by subcutaneous injections of deoxycorticosterone acetate (2 mg/kg per day). AT1a and AT1b messenger RNA (mRNA) levels were semiquantified by reverse-transcriptase polymerase chain reaction and related to actin mRNA. The AT1a mRNA:AT1b mRNA ratio in the hearts of untreated rats was 10:1. Unilateral renal artery clipping led to a 30% decrease in AT1a mRNA, whereas treatment with frusemide, losartan or ramipril had no effect on the AT1a or AT1b mRNA levels. Rats fed a low-sodium diet showed a 37% increase in AT1a gene expression. Dexamethasone increased AT1a mRNA by 100% and AT1b mRNA by 300%, whereas deoxycorticosterone acetate treatment decreased AT1a mRNA levels to 30% of the control values. The present results suggest that the expression of the predominant cardiac AT1a receptor gene is not feedback-regulated by endogenous angiotensin II, whereas steroid hormones appear to be effective regulators, because glucocorticoids stimulate AT1 receptor gene expression and mineralocorticoids inhibit it.

Research paper thumbnail of Molecular Mechanisms of Renin Release

Journal of Cardiovascular Pharmacology, 1990

Renin secretion from renal juxtaglomerular cells is controlled by the intrarenal blood pressure, ... more Renin secretion from renal juxtaglomerular cells is controlled by the intrarenal blood pressure, sodium chloride load of the organism, sympathetic nerve activity, and a number of hormones. The mechanisms by which these parameters exert their effects are not well understood. However, it is reasonable to assume that they act via signal transduction systems, implying the role of second messenger molecules in the control of renin secretion. This article summarizes present knowledge about the cellular mechanisms of renin secretion. Moreover, current concepts about the mechanisms by which the blood pressure and the sodium chloride load could influence renin secretion are discussed.

Research paper thumbnail of Rat Juxtaglomerular Cells are Endowed with DA-1 Dopamine Receptors Mediating Renin Release

Journal of Cardiovascular Pharmacology, 1988

Under control conditions a primary culture containing about 80-90% of granular juxtaglomerular (J... more Under control conditions a primary culture containing about 80-90% of granular juxtaglomerular (JG) cells prepared from rat kidneys continuously released renin into the culture medium at a rate of 17.9 +/- 1.4 ng angiotensin I/h per mg of cell proteins per 30 min (n = 14). Dopamine (1.0 microM), the DA-1 dopamine receptor agonist fenoldopam (0.5 microM), and isoproterenol (1.0 microM) increased renin secretion markedly (130-200%). Propranolol (0.1 microM) reduced the effects of isoproterenol significantly (80%), but not those of dopamine or fenoldopam. In contrast, SCH 23390 (0.01 microM), a DA-1 dopamine receptor antagonist, inhibited markedly only the renin release evoked by the latter two agonists, whereas S-sulpiride (10 microM), a DA-2 dopamine receptor antagonist, and phentolamine (10 microM), a nonselective alpha-adrenoceptor antagonist, did not modify the effects of either dopamine or fenoldopam. In rats, pithed to eliminate reflexogenic mechanisms regulating renin release, at the end of a 15 min i.v. infusion of fenoldopam (20 micrograms/kg per min) there was a significant increase in plasma renin activity. This effect was completely prevented by SCH 23390 (0.1 mg/kg i.v.) but not significantly changed by S-sulpiride (0.3 mg/kg i.v.) or phentolamine (3.0 mg/kg i.v.) plus propranolol (0.75 mg/kg i.v.). In conclusion, these results indicate that DA-1 dopamine receptors are present in rat kidney JG cells and that pharmacological stimulation of these receptors with dopamine or fenoldopam leads to renin secretion.

Research paper thumbnail of A native, affinity-based protein blot for the analysis of streptavidin heterogeneity: Consequences for the specificity of streptavidin mediated binding assays

Research paper thumbnail of Zonal expression of angiotensin II-AT1-receptor genes in rat kidneys

Experimental nephrology

This study aimed to examine the relative distribution of ANGII-AT1-receptor gene expression in di... more This study aimed to examine the relative distribution of ANGII-AT1-receptor gene expression in different zones of the rat kidney, namely the glomeruli, cortex (CO), outer and inner stripes of outer medulla (OSOM and ISOM, respectively) and inner medulla (IM). By RNAse protection we found a ratio for the overall abundance of AT1 mRNA of 1, 0.68, 0.52, 0.86 and 0.46 for glomeruli, CO, OSOM, ISOM and IM, respectively. The intrazonal proportion of AT1a and AT1b mRNA was determined by RT-PCR, which yielded values for the ratio AT1a/AT1b of 1.1, 4.7, 4.4, 3.7 and 1.9 for glomeruli, CO, OSOM, ISOM, and IM, respectively. These findings suggest a zonal heterogeneity in the expression of AT1-receptor genes such that the highest expression is found in glomeruli and ISOM, whilst the papilla shows the lowest level of expression. Moreover, there appears to exist a corticopapillary gradient for the relative expression of AT1a and AT1b-receptor genes such that the preponderance of AT1a-receptor gen...

Research paper thumbnail of Juxtaglomerular cells in culture

Research paper thumbnail of Effect of synthetic atrial natriuretic peptide on rat renal juxtaglomerular cells

Journal of hypertension. Supplement : official journal of the International Society of Hypertension, 1986

We examined the effect of synthetic atrial natriuretic peptide (ANP) on unstimulated renin releas... more We examined the effect of synthetic atrial natriuretic peptide (ANP) on unstimulated renin release from rat renal juxtaglomerular cells. Using renal cortical cell cultures containing 80-90% juxtaglomerular cells, we found that ANP strongly inhibited renin release from the cells in a dose-dependent fashion. Half-maximal inhibition was observed at 10(-11) mol/l ANP. Furthermore, ANP produced a rise in the intracellular concentration of cyclic guanosine monophosphate (cGMP) and a fall in the concentration of cyclic adenosine monophosphate (cAMP). Data indicated that the inhibition of renin release by ANP was related to the rise in cGMP and not to the fall of cAMP. Atrial natriuretic peptide (10(-10) mol/l) had no influence on the transmembrane calcium influx nor did it alter the intracellular calcium concentration of the juxtaglomerular cell. We found, however, that the inhibitory effect of ANP on renin release could be attenuated by the calcium channel blocker verapamil. Our results s...

Research paper thumbnail of Determinants of renin secretion and renin synthesis in isolated mouse juxtaglomerular cells

Kidney international. Supplement, 1991

In this study we examined the effects of classic second messenger molecules on renin secretion an... more In this study we examined the effects of classic second messenger molecules on renin secretion and renin synthesis in primary cultures of mouse renal juxtaglomerular (JG) cells. Stimulation of cAMP formation by forskolin, inhibition of calmodulin by calmidazolium, and inhibition of Na+/H+ exchange by ethylisopropylamiloride enhanced renin secretion. Raising of intracellular cGMP by 8-bromo-cGMP and activation of protein kinase C by phorbol ester led to an inhibition of secretion. Renin synthesis was stimulated by forskolin. Calmidazolium, EIPA, 8-bromo-cGMP, and phorbol ester were without effect on basal renin synthesis. The data suggest that renin secretion is influenced by a number of transmembrane transduction systems which in their majority exert a negative control on renin secretion. Activation of adenylate cyclase appears to be a stimulatory control mechanism for both the secretion and the synthesis of renin. The findings suggest, moreover, that the second messenger controls o...

Research paper thumbnail of Calmodulin antagonists stimulate renin secretion and inhibit renin synthesis in vitro

The American journal of physiology, 1992

To find out whether calmodulin activity could be a common denominator for the cellular control of... more To find out whether calmodulin activity could be a common denominator for the cellular control of renin secretion and synthesis, we have examined the effects of calmodulin antagonists on the secretion and the synthesis of renin in primary cultures of mouse juxtaglomerular (JG) cells. We found that the calmodulin antagonists calmidazolium (CMDZ), N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), and trifluoperazine (TFP) strongly stimulated renin release from isolated JG cells with a rank order of potency CMDZ greater than TFP greater than W-7. With the same order of potency as on renin secretion, CMDZ, TFP, and W-7 also inhibited de novo synthesis of renin. This decrease of renin synthesis went in parallel with a general inhibition of protein synthesis in the cultured JG cells. A comparable inhibition of total protein synthesis and renin synthesis as with CMDZ was achieved with cycloheximide. Cycloheximide, however, did not alter renin secretion within 20 h of incubation. T...

Research paper thumbnail of Atrial natriuretic peptide inhibits renin release from juxtaglomerular cells by a cGMP-mediated process

Proceedings of the National Academy of Sciences, 1986

We have exmined the effect of a synthetic analogue of human et-atrial natriuretic peptide (ANP), ... more We have exmined the effect of a synthetic analogue of human et-atrial natriuretic peptide (ANP), APH, on renin release in cultured renal juxtaglomerular cells (JGA cells). Using cell cultures containing 80-90% renal juxtaglomerular cells, we found that ANP (10-13-10-9 M) strongly

Research paper thumbnail of Amiloride enhances the secretion but not the synthesis of renin in renal juxtaglomerular cells

Pfl�gers Archiv European Journal of Physiology, 1991

In this study we have examined a potential role of the sodium/proton exchange system in the regul... more In this study we have examined a potential role of the sodium/proton exchange system in the regulation of renin secretion. We found that the inhibitors of the Na+/H+ antiport, amiloride (1 mM) and ethylisopropylamiloride (EIPA, 50 microM), led to a 125% increase of renin secretion from cultured mouse juxtaglomerular cells. The stimulatory effect of EIPA on renin secretion was dependent on the extracellular concentrations of sodium and hydrogen ions. While lowering the extracellular pH from 7.3 to 7.0, and lowering [Na+]e from 130 mM to 5 mM had no effect on basal renin release, it markedly attenuated or even blunted the effect of EIPA on renin secretion. The stimulatory effect of forskolin on renin secretion, however, was not altered by decreases of extracellular pH and of sodium. Inhibition of basal renin release was achieved with angiotensin II (1 microM). In the presence of EIPA the inhibitory effect angiotensin II was markedly attenuated. Although effective on renin secretion, neither amiloride nor EIPA exerted a significant effect on the denovo synthesis of renin in cultured mouse JG cells. These findings are compatible with the idea that an amiloride-sensitive transport process, presumably the Na+/H+ exchanger, acts indirectly as an inhibitory signal transduction system for renin secretion from renal juxtaglomerular cells.

Research paper thumbnail of Endothelium derived relaxing factor is involved in the pressure control of renin gene expression in the kidney

Pfl�gers Archiv European Journal of Physiology, 1994

To study the influence of endothelium derived relaxing factor/nitric oxide (EDNO) on renin gene e... more To study the influence of endothelium derived relaxing factor/nitric oxide (EDNO) on renin gene expression, the effects of a 2-day treatment with the NO-synthase inhibitor nitro-L-arginine-methylester (L-NAME, 40 mg/kg twice a day) on plasma renin activity (PRA) and renal and adrenal renin m-RNA levels were examined in conscious rats with and without unilateral renal clips (0.2 mm). In sham-clipped animals L-NAME led to a decrease of PRA from 7.5 to 2.5 ng angiotensin (ANGI).h-1.ml-1 and to a 35% decrease of renal renin m-RNA levels. Unilateral renal artery clipping increased PRA to 35 and to 13 ng ANGI.h-1.ml-1 in vehicle and in L-NAME-treated rats, respectively. In the clipped kidneys renin m-RNA levels increased to 450% of control values in vehicle-treated animals and to 220% of control values in L-NAME-treated animals. In the contralaterals as opposed to clipped kidneys, renin m-RNA levels decreased to 16% and 50% of the control values in vehicle- and in L-NAME-treated animals, respectively. In the adrenal glands renin m-RNA levels were not significantly changed either by clipping of one renal artery or by treatment of animals with L-NAME. The NO-donor sodium nitroprusside (100 microM) was found to increase renin secretion and renin m-RNA levels in primary cultures of renal juxtaglomerular cells. These findings suggest that EDNO is involved in the control of the renin gene by the renal perfusion pressure.

Research paper thumbnail of Renin gene and angiotensin II AT1 receptor gene expression in the kidneys of normal and of two-kidney/one-clip rats

Pfl�gers Archiv European Journal of Physiology, 1995

This study aimed to investigate the inter-relation between the angiotensin II (ANG II) AT~ recept... more This study aimed to investigate the inter-relation between the angiotensin II (ANG II) AT~ receptor and renin gene expression in rat kidneys. To this end, renin mRNA levels and mRNA levels for A%~and ATtb were assayed by RNase protection in the kidneys of normal rats, in animals treated with the AT~ antagonist losartan and in rats bearing 0.2-mm left renal artery clips for 2 days. In normal rats, we found a negative correlation between renin mRNA levels and ATla receptor mRNA levels. Losartan led to a fourfold increase in renin mRNA levels without changing AT1 receptor mRNA levels. Unilateral renal artery clipping increased renin mRNA levels fourfold in the clipped kidney and suppressed renin mRNA levels in the contralateral kidneys. AT~ receptor mRNA levels were not changed in the contralateral intact kidneys, but were significantly decreased by 15-25% in the clipped kidneys. Renin mRNA levels were inversely correlated to AT~a mRNA levels in the clipped, but not in the contralateral, kidneys. Our findings suggest that the systemic activity of the renin angiotensin system has no regulatory influence on renal AT1 receptor gene expression. Renin mRNA levels in normal and in clipped kidneys appear to be negatively determined by the level of AT~a receptor gene expression. Thus modulation of ATla receptor gene expression could be a pathway for indirect modulation of renin gene expression by ANG II. This conclusion is in agreement with the observation that AT~ receptor antagonists are powerful stimulators of the renin system.

Research paper thumbnail of Extracellular calcium exerts a dual effect on renin secretion from isolated mouse juxtaglomerular cells

Pfl�gers Archiv European Journal of Physiology, 1993

Utilizing primary cultures of mouse renal juxtaglomerular cells we found that renin secretion dur... more Utilizing primary cultures of mouse renal juxtaglomerular cells we found that renin secretion during 20 h of incubation was stimulated by 100% when extracellular calcium was lowered from a basal level of 0.5 mM to below I gM, and was enhanced in a concentration-dependent fashion by 600 % when extracellular calcium was increased up to 10 mM. The stimulatory effect of low calcium on renin secretion was apparent after the first hour of incubation, whereas the stimulatory effect of increased calcium occurred with a delay of at least 1 h. During the first hour of incubation increased extracellular calcium blunted the stimulation of renin secretion induced by forskolin (10 pM). The stimutatory effect of increased calcium was attenuated in the presence of 8-pCPT-cGMP, a membrane-permeable cGMP analogue, and in the presence of 100 mM sucrose. The stimulatory effect of increased calcium was blunted in the presence of 0.5 mM cobalt, which itself stimulated renin secretion at normal (0.5 mM) calcium concentrations. Renin synthesis by the cultured cells at low calcium was markedly attenuated in proportion with total protein synthesis, whereas renin synthesis was not altered at increased calcium concentrations. Our findings suggest that a rise of intracellular calcium, induced by an increase of extracellular calcium, is associated with a transient inhibition followed by a marked and regulatable stimulation of renin secretion. Neither calcium depletion nor calcium elevation appears to exert specific effects on renin synthesis in juxtaglomerular cells.

Research paper thumbnail of Renin gene expression in the aging kidney: effect of sodium restriction

Mechanisms of Ageing and Development, 1995

... [6]. H. Huang, T. Baussant, R. Reader, JB Michel and P. Corvol ... [17]. A. Brecher, D. Shier... more ... [6]. H. Huang, T. Baussant, R. Reader, JB Michel and P. Corvol ... [17]. A. Brecher, D. Shier, H. Dene, S. Wang, J. Rapp, R. Franco-Saenz and P. Mulrow, Regulation of adrenal renin messenger ribonucleic acid by dietary sodium chloride. Endocrinology 124 (1989), pp. 2907–2913. ...

Research paper thumbnail of Opposite regulation of renin gene expression by cyclic AMP and calcium in isolated mouse juxtaglomerular cells

Kidney International, 1995

Opposite regulation of renin gene expression by cyclic AMP and calcium in isolated mouse juxtaglo... more Opposite regulation of renin gene expression by cyclic AMP and calcium in isolated mouse juxtaglomerular cells. A quantitative reverse transcriptase-polymerase chain reaction for mouse renin mRNA was utilized to study the influence of classic second messenger molecules on renin mRNA levels in primary cultures of juxtaglomerular (JG) cells isolated from the kidneys of C57/B16 mice. We found that forskolin (3 .LM), an activator of adenylate cyclase led to proportional increases of renin secretion and renin mRNA levels. The nitric oxide (NO) donor, sodium nitroprusside (100 MM), stimulated both renin secretion and renin gene expression, the effect on secretion being stronger than that on renin mRNA levels. An increase of the extracellular concentration of calcium from 0.5 to 3 ms led to a transient inhibition of renin secretion, followed by a marked stimulation of secretion and to a continuous suppression of renin mRNA levels. These were also decreased by the calcium ionophore A 23187 (1 zM). The membrane permeable 8-bromo-cyclic GMP (100 LM) inhibited basal renin secretion without an effect on renin mRNA levels. The phorbol ester phorbol-12-myristate-13-acetate (1 to 100 nM), which was used to stimulate protein kinase C activity, had no significant effects on renin secretion and renin mRNA levels, neither alone nor in combination with forskolin. These findings suggest that cAMP, NO and calcium are effective regulators of renin gene expression in renal JG cells, in a way that cAMP and NO are stimulators and calcium acts as an inhibitor.

Research paper thumbnail of Cyclosporine A enhances renin secretion and production in isolated juxtaglomerular cells

Kidney International, 1988

Cyclosporine A enhances renin secretion and production in isolated juxtaglomerular cells. Stimula... more Cyclosporine A enhances renin secretion and production in isolated juxtaglomerular cells. Stimulation of the renin-angiotensin system is a major side effect of the fungoid immunosuppressant cyclosporine A (CyA). The aim of this study was to find out whether or not this effect of CyA results from a direct interaction with renal juxtaglomerular (JG) cells, which are the site of renal renin synthesis and release. Using primary cell cultures from rat renal cortex containing more than 80% JG cells, we found that CyA (0.01 to 10 zg/ml) stimulated renin secretion threefold. This stimulation was paralleled by a dose-dependent twofold increase of inactive renin within the cells, while the active intracellular renin remained the same. In order to identify a possible second messenger which could mediate the effects of CyA on JG cells, we examined the simultaneous effects of a single concentration of CyA (I jsg/ml) on renin secretion, prostaglandin formation and intracellular cAMP concentration. However, prostaglandin formation and cAMP were not detectably altered by CyA in experiments where renin secretion was significantly enhanced. Our results indicate that cyclosporine A stimulates renin secretion and renin synthesis by a direct effect on renal juxtaglomerular cells. This action of CyA is not mediated by changes in cellular prostaglandin or intracellular cAMP.

Research paper thumbnail of Endothelins inhibit cyclic-AMP induced renin gene expression in cultured mouse juxtaglomerular cells

Kidney International, 1996

Research paper thumbnail of Role of cGMP as second messenger of adenosine in the inhibition of renin release

Kidney International, 1988

Role of cGMP as second messenger of adenosine in the inhibition of renin release. Adenosine is kn... more Role of cGMP as second messenger of adenosine in the inhibition of renin release. Adenosine is known to be a potent inhibitor of renin

Research paper thumbnail of Angiotensin II receptor gene expression in hypertrophied left ventricles of rat hearts

Journal of Hypertension, 1996

To examine the expression of angiotensin II AT1a, AT1b and AT2 receptor genes in the left ventric... more To examine the expression of angiotensin II AT1a, AT1b and AT2 receptor genes in the left ventricles of rats subjected to ventricular pressure overloading induced by aortic banding for 6 weeks and then 6 weeks medical treatment. Aortic banding was related to an increase in relative weight of the left ventricle from 1.73 +/- 0.06 (sham-operated) to 2.81 +/- 0.25 g/kg, an increase in beta-myosin: alpha-myosin messenger RNA (mRNA) ratio from 0.30 +/- 0.02 to 1.94 +/- 0.55 and an 18-fold increase in left ventricular atrial natriuretic peptide mRNA levels. In contrast, left ventricular pressure overload hypertrophy was not related to a significant change in the abundance of AT1a and AT1b mRNA, which were expressed in a relative ratio of 5:1. Similarly, the abundance of AT2 mRNA was not significantly changed in hypertrophied ventricles. In rats receiving the angiotensin II AT, receptor antagonist losartan (40 mg/kg) for 6 weeks after banding, relative heart weights were 2.39 +/- 0.14 g/kg, the beta-myosin: alpha-myosin ratio was 1.04 +/- 0.20 and atrial natriuretic peptide mRNA levels displayed a blunted increase (11-fold over sham-treated controls), documenting a significant amelioration of left ventricular hypertrophy by blockade of the AT1 receptor. Losartan treatment in parallel did not affect AT1a, AT1b and AT2 receptor mRNA levels, which were not different from those in vehicle-treated or sham-treated controls. These findings confirm that left ventricular hypertrophy in the rat is associated with increased ventricular expression of beta-myosin and of atrial natriuretic peptide and with reduced expression of alpha-myosin. Despite these significant changes in cardiac gene expression no alteration was observed in AT1a, AT1b and AT2 receptor mRNA levels.

Research paper thumbnail of Expression of cardiac angiotensin II AT1 receptor genes in rat hearts is regulated by steroids but not by angiotensin II

Journal of Hypertension, 1995

To examine the regulation by angiotensin II and by steroids of the expression of the angiotensin ... more To examine the regulation by angiotensin II and by steroids of the expression of the angiotensin II AT1a and AT1b receptor genes in rat hearts. Endogenous levels of angiotensin II in the rats were increased either by unilateral 0.2-mm renal artery clips or by subcutaneous infusions of frusemide (12 mg/day) and by low-sodium diet. To inhibit endogenous angiotensin II actions the rats received the AT1 receptor antagonist losartan (40 mg/kg per day) or the angiotensin converting enzyme inhibitor ramipril (8 mg/kg per day). Circulating levels of glucocorticoids were elevated by subcutaneous injections of dexamethasone (400 micrograms/kg per day) and levels of mineralocorticoids were increased by subcutaneous injections of deoxycorticosterone acetate (2 mg/kg per day). AT1a and AT1b messenger RNA (mRNA) levels were semiquantified by reverse-transcriptase polymerase chain reaction and related to actin mRNA. The AT1a mRNA:AT1b mRNA ratio in the hearts of untreated rats was 10:1. Unilateral renal artery clipping led to a 30% decrease in AT1a mRNA, whereas treatment with frusemide, losartan or ramipril had no effect on the AT1a or AT1b mRNA levels. Rats fed a low-sodium diet showed a 37% increase in AT1a gene expression. Dexamethasone increased AT1a mRNA by 100% and AT1b mRNA by 300%, whereas deoxycorticosterone acetate treatment decreased AT1a mRNA levels to 30% of the control values. The present results suggest that the expression of the predominant cardiac AT1a receptor gene is not feedback-regulated by endogenous angiotensin II, whereas steroid hormones appear to be effective regulators, because glucocorticoids stimulate AT1 receptor gene expression and mineralocorticoids inhibit it.

Research paper thumbnail of Molecular Mechanisms of Renin Release

Journal of Cardiovascular Pharmacology, 1990

Renin secretion from renal juxtaglomerular cells is controlled by the intrarenal blood pressure, ... more Renin secretion from renal juxtaglomerular cells is controlled by the intrarenal blood pressure, sodium chloride load of the organism, sympathetic nerve activity, and a number of hormones. The mechanisms by which these parameters exert their effects are not well understood. However, it is reasonable to assume that they act via signal transduction systems, implying the role of second messenger molecules in the control of renin secretion. This article summarizes present knowledge about the cellular mechanisms of renin secretion. Moreover, current concepts about the mechanisms by which the blood pressure and the sodium chloride load could influence renin secretion are discussed.

Research paper thumbnail of Rat Juxtaglomerular Cells are Endowed with DA-1 Dopamine Receptors Mediating Renin Release

Journal of Cardiovascular Pharmacology, 1988

Under control conditions a primary culture containing about 80-90% of granular juxtaglomerular (J... more Under control conditions a primary culture containing about 80-90% of granular juxtaglomerular (JG) cells prepared from rat kidneys continuously released renin into the culture medium at a rate of 17.9 +/- 1.4 ng angiotensin I/h per mg of cell proteins per 30 min (n = 14). Dopamine (1.0 microM), the DA-1 dopamine receptor agonist fenoldopam (0.5 microM), and isoproterenol (1.0 microM) increased renin secretion markedly (130-200%). Propranolol (0.1 microM) reduced the effects of isoproterenol significantly (80%), but not those of dopamine or fenoldopam. In contrast, SCH 23390 (0.01 microM), a DA-1 dopamine receptor antagonist, inhibited markedly only the renin release evoked by the latter two agonists, whereas S-sulpiride (10 microM), a DA-2 dopamine receptor antagonist, and phentolamine (10 microM), a nonselective alpha-adrenoceptor antagonist, did not modify the effects of either dopamine or fenoldopam. In rats, pithed to eliminate reflexogenic mechanisms regulating renin release, at the end of a 15 min i.v. infusion of fenoldopam (20 micrograms/kg per min) there was a significant increase in plasma renin activity. This effect was completely prevented by SCH 23390 (0.1 mg/kg i.v.) but not significantly changed by S-sulpiride (0.3 mg/kg i.v.) or phentolamine (3.0 mg/kg i.v.) plus propranolol (0.75 mg/kg i.v.). In conclusion, these results indicate that DA-1 dopamine receptors are present in rat kidney JG cells and that pharmacological stimulation of these receptors with dopamine or fenoldopam leads to renin secretion.