The central effects of a nitric oxide synthase inhibitor (N omega-nitro-L-arginine) on blood pressure and plasma renin.

An endothelium-derived relaxing factor has been identified as nitric oxide (NO). Peripheral and central administration of nitric oxide synthase inhibitors result in an increase in renal sympathetic nerve activity and an increase in blood pressure. The goal of our study was to determine if the increase in blood pressure following central NO synthase inhibition with N omega-nitro-L-arginine (L-NNA) is caused by the release of renin. Six groups of Sprague-Dawley rats were used. Group I (control) received intracerebroventricular (i.c.v.) artificial cerebrospinal fluid. Groups II & III received i.c.v. L-NNA, 5 & 15 micrograms/min. respectively. Group IV was treated with intravenous L-NNA, 15 micrograms/min. Group V, after bilateral nephrectomy, received i.c.v. L-NNA, 15 micrograms/min. Group VI received i.c.v. L-arginine, 60 micrograms/min. and i.c.v. L-NNA, 15 micrograms/min., simultaneously. Plasma renin concentration was measured in groups I, III, IV & V. Mean arterial blood pressure was significantly increased in groups II, III & V, i.e., following i.c.v. infusion of L-NNA. The increase in mean arterial blood pressure was significantly greater when the dose was increased from 5 to 15 micrograms/min. and it was eliminated when L-arginine was added to the infusion. The increase in blood pressure was attended by no change in heart rate. While the plasma renin concentration increased significantly in group III, this could not explain the increase in blood pressure since the nephrectomized group (V) showed no increase in renin concentration but an equivalent increase in blood pressure. The results show that acute central administration of a low dose of L-NNA increases blood pressure in rats and this increase can be prevented by central administration of L-arginine. However, intravenous infusion of the same dose (15 micrograms/min.) of L-NNA does not change blood pressure. We conclude that L-NNA acts directly within the central nervous system to increase blood pressure by a renin-independent mechanism. These results imply that central nitric oxide plays an important role in the regulation of blood pressure.

Time course of stimulation of renal renin messenger RNA by furosemide.

Renin secretion responds rapidly to a variety of stimuli; however, reported changes in renal renin messenger RNA (mRNA) levels in vivo have been observed only after prolonged stimulation. Studies were designed to test whether rapid changes in renin mRNA levels can be produced in vivo. In the first series, Sprague-Dawley rats received furosemide (10 mg/kg) intraperitoneally and a low sodium diet (0.05% sodium); renin secretion was significantly stimulated at 8 or 16 hours after treatment, but renin mRNA levels did not change. In a second series, rats were pretreated with deoxycorticosterone acetate (200 mg/kg) and saline drinking water for 3 days and then killed 0, 2, 4, 8, or 48 hours after furosemide administration. The renin mRNA level was unchanged at 2 hours but was stimulated twofold at 4 and 8 hours and threefold at 48 hours. In additional animals, the response of renin mRNA 4 hours after furosemide was found not to be potentiated by the converting enzyme inhibitor quinapril (5 mg/kg). The results demonstrate that with acute stimulation, renin mRNA levels lag 2-4 hours behind the change in plasma renin levels.

Analysis of active renin heterogeneity.

Active renin is a heterogeneous enzyme that can be separated into multiple forms with high-resolution isoelectric focusing. The isoelectric heterogeneity may result from differences in glycosylation between the different forms. In order to determine the relationship between active renin heterogeneity and differences in composition or attachment of oligosaccharides, two separate experiments were performed: (i) Tunicamycin, which interferes with normal glycosylation processing, increased the proportion of relatively basic renin forms secreted into the incubation media by rat renal cortical slices. (ii) Endoglycosidase F, which enzymatically removes carbohydrate from some classes of glycoprotein, similarly increased the proportion of relatively basic forms when incubated with active human recombinant renin. In addition, further studies with inhibitors of human renin activity revealed that the heterogeneous renin forms were similarly inhibited by two separate renin inhibitors. These results are consistent with the hypothesis that renin isoelectric heterogeneity is due in part to differences in carbohydrate moiety attachment and that the heterogeneity of renin does not influence access of direct renin inhibitors to the active site of renin.

Naloxone attenuates development of hypertension in DOCA-salt hypertensive rats.

The effects of naloxone on the development of hypertension were studied in unilaterally nephrectomized rats implanted with deoxycorticosterone acetate (DOCA; 200 mg/kg) and given saline to drink. Intraperitoneal (i.p.) infusion of naloxone at 150 micrograms/hr significantly lowered systolic blood pressure (SBP) compared to rats not receiving naloxone, (135 +/- 4.4 vs 158 +/- 5.9 mmHg on day 16). IP infusion of naloxone at 300 micrograms/hr produced the same reductions of SBP as that at 150 micrograms/hr in DOCA-salt treated rats. In other experiments intracerebroventricular (i.c.v.) infusion of naloxone at 7 micrograms/hr also significantly attenuated the DOCA-salt hypertension. The same dose given i.p. had no effect on the development of hypertension. Naloxone had no effect on plasma renin activity (PRA), plasma atrial natriuretic peptide (ANP), or concentrations of Na+ and K+ in plasma. The present data demonstrate that naloxone significantly attenuates the development of hypertension in rats given DOCA and fed a high salt diet. The attenuation of blood pressure could not be associated with the changes in PRA or plasma ANP. These results imply that the central opiate receptors play an important role in the pathogenesis of this model of hypertension.

Evidence for the functional significance of multiple renin forms.

Our laboratory demonstrated the existence of 6 forms of renin, (F1-F6) each with a unique isoelectric point (pI). We ascribe the heterogeneity to differences in glycosylation. This heterogeneity has been demonstrated to exist in all animals studied in our laboratory, across a wide evolutionary scale. Multiple forms have been found in fish, amphibians, birds and mammals, including humans. We have been able to demonstrate that hepatic inactivation of the renin forms in humans is different for each form. Isolation and purification of the forms allowed injection of a single form into rats. IV infusion of F4 resulted in a significant natriuresis and diuresis, while the other forms had no significant renal effects. However, if the same forms were infused into the cerebroventricles at a much lower dose, F6 caused a natriuresis. Pretreatment with converting enzyme inhibitor abolished that effect. We were able to show that Spontaneously Hypertensive Rats (SHR) exhibited a renin profile that was altered in a predictable way and was significantly correlated with their blood pressure. The hepatic clearance of each form was also different, so that the forms have different half lives. These data support the hypothesis that renin heterogeneity is dependent upon glycosylation and is functionally significant.

Science education: too much of too little.

By all measures attempted, scientific literacy of the American public is sadly wanting. The vast majority of our secondary school children and adults have no knowledge of most of the basic terms or concepts of science. The reasons for this shortcoming are many but prominent among them are sadly deficient texts, teachers untrained in the subject matter they teach, and college and university scientists who divorce themselves from the problem, although probably deploring it. Our institutions are no aid. They reward scientific productivity (read: number of papers published per year and research dollars), not teaching. Some suggested cures are production of better texts, training of science teachers in the field in which they teach, and, most importantly, involvement of scientists in the process. We must be willing to spend some of our time with secondary school pupils and their teachers. All will gain from the experience.

Central infusion of aldosterone increases blood pressure by mechanisms independent of Na retention.

Experiments were performed to test the hypothesis that Na retention and Na in the diet are not required to initiate central aldosterone induced hypertension. Rats were fed either standard rat chow or Na-deficient diet and infused intracerebroventricularly (i.c.v.) with aldosterone (28 ng/h) dissolved in artificial cerebrospinal fluid (vehicle) or vehicle alone. In Na-replete rats the central infusion of aldosterone did not promote Na or water retention, prior to increases in systolic blood pressure (SBP). Infusion of aldosterone in Na-deficient rats also initiated a rise in SBP, although the response was delayed. In neither group of rats did aldosterone infusion significantly change plasma Na, K, renin, norepinephrine (NE) or vasopressin (AVP) concentrations. There was no significant increase in plasma aldosterone concentration in Na replete rats centrally infused with aldosterone. Infusion of vehicle had no effect on SBP. We conclude that central aldosterone infusion initiates an increase in blood pressure by a mechanism independent of Na retention. Furthermore, increased concentrations of systemic renin, vasopressin, and activation of the sympathetic nervous system do not appear to be involved in maintaining hypertension.

Naloxone prevents increased vascular sensitivity in Goldblatt hypertensive rats.

These experiments were designed to determine if the opiate antagonist naloxone affects vascular sensitivity in 2K-1C hypertensive rats. Group 1 was 2K-1C hypertensive rats. Group 2 was 2K-1C rats given a naloxone infusion (100 micrograms/h) for 14 days. Group 3 received naloxone without clipping. Group 4 was untreated rats. At day 14 following clipping, systolic blood pressure was increased significantly in the 2K-1C rats. Those infused with naloxone showed a significant attenuation of the increase in blood pressure. Vascular responses to norepinephrine and KCl in the aortae from all groups were tested. Strips from untreated, 2K-1C rats were more sensitive to the contractile effects of norepinephrine than those from naloxone-treated, 2K-1C rats, and from both groups of normotensive rats. Contractile responsiveness to depolarizing concentrations of KCl were not different among the four groups. These data demonstrate that naloxone attenuates the development of renal hypertension and prevents the increase in vascular responsiveness to norepinephrine.

Blood pressure and acidic renin forms in stroke-prone hypertensive rats.

Six forms of renin, distinguishable by their isoelectric focusing characteristics, are found in rat kidney and plasma (forms 1-6, form 1 having the most basic pI). To test the hypothesis that high blood pressure in stroke-prone spontaneously hypertensive rats (SHRSP) is associated with a particular fraction of the renin forms, systolic blood pressure (SBP) and renin profile were measured in SHRSP, normotensive Wistar-Kyoto rats (WKY), and their genetic crosses (F1). Adult SHRSP showed an increase in the proportion of forms 4, 5, and 6 compared with WKY. F1 showed values between SHRSP and WKY. A strong positive correlation was noted between the fraction of the acidic forms of renin and SBP. The mean blood pressure and the fraction of the acidic renin forms in SHRSP and WKY pups also fitted the same regression line. However, no correlation existed between renal renin content (RRC) and SBP. SHRSP pups had a higher RRC than age-matched WKY or F1. RRC in normal adult SHRSP was lower than that in WKY or F1 of the same age. In old animals, SHRSP again showed the highest RRC, and microangiography of the kidney of old SHRSP revealed a loss of fine vascularity with few visible glomeruli. These results indicate that the established phase of hypertension in SHRSP is associated with an increased proportion of the acidic forms of renin, which may have a unique role in the establishment or maintenance of hypertension. It is also suggested that high RRC in the early developmental stage plays a role in the developing phase of hypertension.

Clonidine and morphine increase atrial natriuretic peptide secretion in anesthetized rats.

In order to determine whether the activity of central alpha 2-adrenergic and opioid receptors influence plasma atrial natriuretic peptide (ANP) levels, clonidine and morphine were infused into the lateral cerebral ventricle for 45 min in anesthetized Sprague-Dawley rats. The central administration of a low dose of clonidine (10 ng/min) caused a significant increase in plasma ANP without changing arterial blood pressure or central venous pressure. Pretreatment with yohimbine (5 micrograms/min) completely blocked the effect of clonidine. Central infusion of morphine (100 ng/min) also elevated plasma ANP levels and naloxone (5 micrograms/min) blunted this effect. Intravenous infusion of the same dose of clonidine or morphine did not affect plasma ANP levels. Moreover, the effect of clonidine on plasma ANP was partially blocked by pretreatment with naloxone (5 micrograms/min). These results suggest that central alpha 2-adrenergic and opioid receptors may be involved in ANP secretion.

Functional relevance of the microheterogeneity of active renin.

We previously demonstrated the presence of six active forms of renin in rats, based on their isoelectric points (pI). Intravenous or intraventricular injections yielded different renal effects with the different forms. Additional work is presented supporting the hypothesis that these forms are functionally different. Renin form 2 (pI = 5.7), form 4 (pI = 5.2) or form 6 (pI = 4.8) was infused intravenously (IV) in Spague-Dawley rats. The total renin infused over 30 minutes was the amount that could generate 1000 ng angiotensin I (Ang I)/h/100g body weight. Arterial blood pressure, urine volume, sodium, and potassium excretion were determined at 15-minute intervals. Plasma renin activity (PRA) and plasma aldosterone concentrations were measured 45 minutes after the start of infusion of renin forms. Infusion of renin form 4 increased urine volume and sodium excretion significantly. Renin forms 2 and 6 were without effect on renal excretion in spite of the fact that the same activity was infused. Both PRA and aldosterone concentrations after renin infusions were similar for all groups. Nonrenin renal protein with the same pI as form 4 was used as a control. This preparation was inactive and implies that our data with renin form 4 were not the result of contamination with a nonrenin protein having a pI of 5.2. In addition, the urinary responses could not be attributable to change in renal perfusion pressure as arterial blood pressure was not altered. These data support the hypothesis that there are functional differences among the different renin forms.

Central administration of aldosterone increases blood pressure in rats.

Experiments were designed to determine whether hypertension in rats caused by a central infusion of aldosterone requires supplemental sodium and uninephrectomy. Group 1 was uninephrectomized and received an intracerebroventricular (i.c.v.) infusion of aldosterone (9 ng/h) plus 1M NaCl, dissolved in 0.01% ethyl alcohol-artificial cerebrospinal fluid (vehicle). Group 2 received the same infusion but was not uninephrectomized. Group 3 received an i.c.v. infusion of aldosterone alone in vehicle. Group 4 received an i.c.v. infusion of vehicle with intravenous (i.v.) infusion of aldosterone plus NaCl. All rats received a diet of standard Purina rat chow and tap water ad libitum. Systolic blood pressure of groups 1 and 2 was significantly increased. Rats treated with i.c.v. aldosterone alone also showed a significant increase in blood pressure on day 21. However, i.v. infusion of the same dose of aldosterone did not change blood pressure. The results show that hypertension induced with chronic central infusion of aldosterone does not require uninephrectomy. We conclude that aldosterone may act directly within the central nervous system to increase blood pressure.

Naloxone attenuates development of hypertension in two-kidney one-clip Goldblatt rats.

The present experiments were designed to determine if an opiate antagonist affects blood pressure in two-kidney one-clip Goldblatt rats. Male Sprague-Dawley rats were divided into three groups. Group 1 received an infusion of saline intraperitoneally via an osmotic pump and left renal artery constriction (RAC). In group 2, rats were treated the same as group 1, except that they received an intraperitoneal infusion of naloxone (100 micrograms/h). Group 3 received the same infusion of naloxone without RAC. Naloxone-infused Goldblatt rats showed a significantly lower systolic blood pressure (SBP) than saline-infused Goldblatt rats (132 +/- 7 vs. 160 +/- 9 mmHg at day 14), but a higher SBP than control (132 +/- 7 vs. 106 +/- 1 mmHg). Infusion of naloxone did not significantly change SBP in normotensive rats. Renal renin activity in the clipped kidney was higher than in the nonclipped kidney in groups 1 and 2. Plasma renin activity (PRA) in both groups of Goldblatt rats was higher than in group 3, but no significant difference was found between the two groups of Goldblatt rats (groups 1 and 2). Naloxone (1.5 microM) did not affect the basal secretion of renin by isolated cortical slices from untreated rats. The present data demonstrate that naloxone significantly attenuates the development of hypertension in two-kidney one-clip rats. The attenuation of blood pressure was not associated with the changes in PRA, renal renin activity, or plasma aldosterone concentrations. The data support the hypothesis that the endogenous opioid system may be involved in the development of renovascular hypertension.

Multiple renin forms in the adrenal gland.

Renin heterogeneity has been described in rat kidney and plasma. In this study, we used the isoelectric focusing method to 1) characterize the adrenal renin forms in control rats, in rats on low- and high-Na diets, and in nephrectomized rats; and 2) examine their resemblance with plasma renin. Active renin (AR) and inactive trypsin-activatable renin (IR) were measured in adrenal homogenates and plasma. Aliquots were subjected to isoelectric focusing gels. Activation with trypsin (5 mg/ml) was performed before or after isoelectric focusing. Results showed that adrenal glands contained AR and IR. The content of adrenal AR increased significantly only in rats fed a low-Na diet. Following anesthesia, nephrectomy, or high-Na intake, the content of adrenal AR and IR was not significantly changed. In plasma, an inverse relationship between AR and IR was found. Adrenal glands contained six forms of AR focusing at the same pH as those of plasma AR but in different proportions. After activation of IR in adrenal glands, two additional renin forms focusing at pH 6.4 and 6.1 were found, whereas after activation of plasma IR, two peaks focusing at pH 5.9 and 4.8 were significantly enhanced. Adrenal AR forms were modified by alterations of salt and water balance differently than plasma AR. These results support the hypotheses of an endogenous production of renin forms by the adrenal gland.

Dopaminergic modulation of some central actions of angiotensin II in vivo.

Studies were performed in 12 conscious sheep of both sexes to determine if a brain dopaminergic pathway is involved in modulating the central actions of angiotensin II (Ang II) in regulating body temperature and plasma renin activity (PRA). Previous data showed that intracerebroventricular (ICV) infusion of Ang II significantly decreased PRA and body temperature. In contrast, converting enzyme inhibitor SQ 20881 (SQ) or dopamine (DA) significantly increased PRA and body temperature of sheep. In the present study, ICV infusion of the DA antagonist metoclopramide (MCP) (20 micrograms/min) significantly decreased PRA to 68 +/- 5% of the basal level. When sheep were pretreated with ICV MCP (20 micrograms/min) for 2 hr and then infused ICV with MCP (20 micrograms/min) plus DA (20 micrograms/min), Ang II (25 ng/min), or SQ (1 microgram/min), the PRA and temperature responses to DA, Ang II, or SQ were all abolished or attenuated significantly. The converse did not hold. Sheep pretreated with SQ (1 microgram/min) still showed a significant increase in body temperature (0.43 +/- 0.05 degree C) when infused with DA (20 micrograms/min). These results support the hypothesis that a central DA pathway is involved in the modulation of the actions of centrally administered Ang II on temperature and PRA.

Functional differences of six forms of renin in rats.

Six forms of renin with different isoelectric points (pIs) have been described in rats. This study was designed to determine if any of the renin forms have different biological activities. Each form of rat renin was semipurified and injected intravenously or intraventricularly in Sprague-Dawley rats anesthetized with pentobarbital sodium or Inactin. Changes in blood pressure (BP), renal function, sodium, and water excretion were observed, before and following equipressor doses; the peak response of BP was similar for all forms. However, the half-lives were significantly different. Form 4 (pI = 5.2) caused a significant increase in urine flow, Na, and K excretion, and urinary osmolality when given intravenously. The other forms were without significant effect. Infusion of converting enzyme inhibitor not only completely blocked the BP response, but also prevented the natriuresis and diuresis. This was observed in rats anesthetized with pentobarbital sodium or Inactin. Intraventricular infusions resulted in a diuresis and natriuresis when form 6 (pI = 4.8) was infused, but not with other forms. BP remained unchanged throughout. This study presents evidence that functional differences exist between renin forms.

Temperature sensitivity of renin-angiotensin system in the ground squirrel.

The alteration of renin release by alpha- and beta-adrenoceptors located on the juxtaglomerular cells has been shown to be temperature sensitive in nonhibernating mammals. These experiments investigate the effects of temperature on renin secretion by cortical slices of kidneys from the thirteen-lined ground squirrel Spermophilus tridecemlineatus. At 37 degrees C, beta-stimulation (isoproterenol 10(-7) M) increased the release of renin by slices taken from nonhibernating ground squirrels but had no effect on those taken from hibernating squirrels. The alpha-agonist phenylephrine (10(-5) M) had no effect on slices from nonhibernating squirrels but enhanced the release rate in those from hibernating ground squirrels, providing the first evidence of in vitro stimulation of renin release by an alpha-agonist. When incubated at 11 degrees C, kidney slices from both hibernating and nonhibernating animals were unresponsive to both alpha- and beta-agonists until incubation times were doubled. Under these prolonged conditions, phenylephrine again stimulated renin release. These results indicate that both in vitro and in vivo cooling alter the responses of alpha- and beta-adrenoceptors to renin-releasing stimuli.