Motilal B. Pamnani

Long-term ouabain administration produces hypertension in rats.

Ouabain has recently been identified as an endogenous Na(+)-K+ pump inhibitor. We administered ouabain chronically to normotensive rats with varying degrees of reduced renal mass (RRM) and to normal two-kidney rats to see whether hypertension could be produced. Normal male Wistar rats and rats with 25%, 60%, and 70% RRM received ouabain (13.9 micrograms/kg per day IP) in normal saline for 4 weeks followed by ouabain (27.8 micrograms/kg per day IP) for 3 to 4 more weeks. Respective control animals received vehicle only. Blood pressure was recorded weekly by tail plethysmography. Animals received tap water and standard rat chow, except for 70% RRM rats, which received distilled water and sodium-free chow. After 6 to 8 weeks of treatment, with rats under thiobutabarbital anesthesia, direct blood pressure was determined. Plasma, tissue, and urinary ouabain levels were measured with a specific radioimmunoassay. Animals receiving ouabain developed significant increases in mean blood pressure compared with control animals (70% RRM, 147 +/- 4 vs 116 +/- 4 mm Hg; 60% RRM, 140 +/- 4 vs 107 +/- 3 mm Hg; 25% RRM, 131 +/- 5 vs 100 +/- 2 mm Hg; no RRM, 116 +/- 4 vs 98 +/- 5 mm Hg). Plasma ouabain levels measured 24 hours after the last ouabain dose were not different in animals receiving ouabain vs those receiving vehicle. However, kidney tissue ouabain levels were significantly greater (6.39 +/- 1.17 vs 2.36 +/- 0.52 micrograms/kg, P < .05) in animals receiving ouabain. In conclusion, ouabain, given chronically, is associated with the development of hypertension in RRM rats as well as in normal rats. Blood pressure was greater in animals with greater degrees of RRM for a given ouabain dose.

Humoral factors and the sodium-potassium pump in volume expanded hypertension

Abstract Bioassay studies in the old and recent literature suggest the presence of an unknown slowly acting pressor agent in the blood of animals and man with volume expanded (low renin) hypertension. Recent studies in our laboratories suggest that the sodium-potassium pump activity of blood vessels is suppressed in animals with one-kidney, one wrapped, one-kidney, one clip, and one-kidney, DOCA, salt hypertension. Similar reduction of Na + , K + -ATPase activity has been observed in the left ventricle of animals with one-kidney, one clip and one-kidney, DOCA salt hypertension. The changes do not appear to result from increased pressure since they have also been observed in veins and right ventricle. Acute volume expansion of the normal rat with saline suppresses pump activity in the tail artery and plasma from these animals suppresses pump activity when applied to a tail artery from another rat. Data in the literature indicate that the adrenergic nerve terminals are depleted of norepinephrine. Suppression of pump activity, with ouabain for example, is known to activate cardiovascular muscle and reduce norepinephrine uptake by nerve terminals. These observations suggest a role for a slowly acting ouabain-like humoral agent, which acts directly on cardiovascular muscle to increase contractility and on nerve endings to reduce reflex compensation, in the genesis of volume expanded hypertension.

Effects of three sodium-potassium adenosine triphosphatase inhibitors

Reports from several laboratories suggest the presence of an ouabainlike compound in plasma and various animal tissues, particularly during acute volume expansion and in low-renin hypertension. It has been hypothesized that this compound, through inhibition of the Na+-K+ pump, can constrict blood vessels, enhance vasoconstriction in response to agonists, increase cardiac contractility, raise blood pressure, and cause natriuresis/diuresis and therefore is implicated in the pathophysiology of the low-renin, volume-expanded type of hypertension. However, so far, only two steroid Na+-K+ pump inhibitors (namely, a bufodienolide derivative [resibufogenin], obtained from toad skin and plasma and a factor with the same carbon, oxygen, and hydrogen content as ouabain obtained from the plasma of volume-expanded humans) have been purified and structurally characterized. To determine whether such endogenous Na+-K+ pump inhibitors can in fact produce the above effects on the cardiovascular and renal systems, we infused commercially available bufalin (aglycone, identical to resibufogenin except for one H+), ouabain, and ouabagenin (aglycone) at equimolar doses in normotensive rats. Relative to ouabain, bufalin produced significantly greater dose-dependent increases in blood pressure, left ventricular rate of pressure change, heart rate, and excretion of urinary volume and sodium. Ouabagenin was without effect on any of these parameters. These data indicate that a Na+-K+ pump inhibitor can cause an increase in blood pressure despite potent diuretic and natriuretic effects and that, in rats, bufalin is much more potent in this respect than ouabain or ouabagenin.

Effects of rat atrial extract on sodium transport and blood pressure in the rat.

Abstract Atrial cardiocytes contain specific atrial granules (SAGs) which are the storage site of atrial natriuretic factor (ANF). The purpose of the present study was to determine whether ANF produces natriuresis by inhibiting Na+-K+ pump activity and whether this factor is similar to the humoral sodium transport inhibiting factor (HSTIF) previously demonstrated in acutely volume expanded animals and humans as well as in experimental and human essential hypertension. Our results indicate that, in contrast to the HSTIF, ANF does not inhibit membrane Na+, K+-ATPase, vascular smooth muscle cell Na+-K+ pump activity, or sodium transport in the toad bladder. Intravenous infusion of ANF in the bilaterally nephrectomized, hexamethoniumtreated rat produces only a small transient pressor response, probably due to potentiation of endogenous norepinephrine. These findings strongly suggest that the ANF is not the same as the HSTIF detected on acute volume expansion and in some forms of hypertension. They also suggest that the diuretic and natriuretic effects of ANF are due to mechanism(s) other than blood pressure elevation and inhibition of Na+-K+ pump activity.

Vascular Na+-K+ Pump Activity in Dahl S and R Rats

Abstract Ouabain-sensitive 86rubidium uptake was used to estimate sodium-potassium pump activity in the tail arteries of Dahl salt-sensitive and Dahl salt-resistant rats on normal and high oral intakes of salt. Uptake was increased in the salt-sensitive strain relative to the resistant strain at a given salt intake. It was also increased in a given strain when the salt intake was increased. In each case the increased ouabain-sensitive uptake was associated with increased ouabain-insensitive uptake which in part reflects the permeability of the cell membrane to rubidium. The results suggest that the increased pump activity is a secondary compensatory response to increased passive penetration of sodium. In this respect, the Dahl salt-sensitive rat is similar to SHR, another genetic model, but different from the other low-renin, presumably, volume-expanded models of hypertension we have studied.

Mechanism of antihypertensive effect of dietary potassium in experimental volume expanded hypertension in rats

Dietary potassium supplementation lowers blood pressure (BP) and attenuates complications in hypertensive subjects, particularly those with the low renin volume expanded (LRVE) variety. We and others have shown that the plasma level of a digitalis like substance (DLS) is elevated in this type of hypertension. We therefore, examined the effect of increases in dietary potassium on the plasma level of endogenous DLS, myocardial and renal Na+, K+-ATPase (NKA) activities, BP, and renal excretory function in reduced renal mass (RRM)-salt hypertension in the rat, a classical model of LRVE hypertension. 70% RRM rats were divided in 4 groups, namely those consuming: 1) a sodium free and normal potassium (1.3% as KCl) diet (RRM-0 Na), 2) a normal sodium and normal potassium diet (RRM-NaK), 3) a normal sodium and high potassium (2 X normal) diet (RRM-Na2K), and 4) a normal sodium and 4 times normal potassium diet (RRM-Na4K). At the end of 4 weeks of dietary treatment, direct BP was recorded, plasma level of DLS determined by bioassay and with a radioimmunoassay for digoxin (DIF) and myocardial and renal NKA activities were measured. As expected, compared to RRM-0Na rats, RRM-NaK rats developed hypertension. BP increased significantly less in RRM-Na2K, whereas BP did not increase in RRM-Na4K rats. Hypertension in RRM-NaK rats was associated with an increase in plasma DLS and DIF and decrease in renal and myocardial NKA activities. DLS was increased (DIF was not changed) and myocardial NKA also decreased in rats consuming double potassium. However, quadrupling potassium in the diet (RRM-Na4K) normalized DLS and DIF and increased myocardial and renal NKA activities, compared to RRM-0Na rats. Also compared to RRM-0Na, water consumption, urinary volume excretion, sodium, and potassium increased in the other 3 groups, more so in RRM-Na4K rats. These data show that quadrupling the potassium in the diet prevents the BP increase in RRM rats and this is associated with diuresis/natriuresis and normalization of DLS, perhaps because the diuresis/natriuresis normalizes blood volume.

Role of a humoral sodium-potassium pump inhibitor in experimental low renin hypertension

Recent evidence suggests that the vascular sodium-potassium pump suppression previously observed in animals with various models of low renin hypertension results from a circulating heat stable ouabain-like agent. It appears to come from or be influenced by the anteroventral third ventricle area of the brain and its action on blood vessels results in depolarization of the smooth muscle cell. Suppression of the vascular sodium-potassium pump, with ouabain for example, increases contractile activity and the contractile responses to vasoactive agents. Thus the humoral pump inhibitor may be involved in the genesis and maintenance of experimental low renin hypertension.

Role of ouabain-like factors and Na-K-ATPase inhibitors in hypertension--some old and recent findings.

Three lines of evidence led to our suggestion in 1976 that sodium pump inhibitors are involved in volume expanded hypertension. These were 1) pressor activity of low renin hypertensive blood 2) natriuretic and sodium pump inhibiting activities of volume expanded blood and 3) potassium vasoactivity which was blocked by ouabain and suppressed potassium vasodilatation, myocardial Na-K-ATPase, and artery, vein and WBC sodium pumps in low renin hypertension. This led to bioassay of plasma from acutely volume expanded dogs and from dogs with one-kidney, one wrapped hypertension for sodium pump inhibiting activity that acts on arteries. Positive results were reported in 1980. The assay was also positive in rats with one-kidney, one clip and reduced renal mass hypertension (but not in rats with spontaneous or salt sensitive hypertension) and in humans with acute volume expansion and low renin essential hypertension (but not in humans with normal renin hypertension). Thus the inhibitor which acts on the sodium pump in arteries appears to be present only in low renin hypertension.

Role of Atrial Natriuretic Factor in Regulation of Blood Pressure in Normotensive Rats Having Reduced Renal Mass

Abstract Experiments were carried out in normotensive, saline-drinking, 60% reduced renal mass rats to determine the effect of an in vivo blockade of endogenous atrial natriuretic factor (ANF) on blood pressure. We used a 60% reduction in renal mass because blood pressure in these normotensive animals is extremely sensitive to any slight further reduction of renal excretory function. Six weeks following the reduction of renal mass and documentation of normotension, rats were injected intraperitoneally twice daily for 12 days with ANF antibody prepared against the C-terminal heptapeptide of AP III conjugated to bovine thyroglobulin. Control rats similarly prepared, received normal rabbit serum (NRS). Blood pressure progressively increased in rats receiving the antibody, and its withdrawal returned blood pressure to control levels within 4–5 days. Serum from either normal rabbits or rabbits immunized with bovine thyroglobulin or peptides unrelated to ANF had no effect on blood pressure in the control animals. These experiments show that in the normotensive saline-drinking rat with reduced renal mass, an antibody to AP III raises blood pressure. This suggests that ANF here is acting to prevent the rise in blood pressure.

Pharmacologic agents for the in vivo detection of vascular sodium transport defects in hypertension

Anatagonists to angiotensin, catecholamines, aldosterone, and vasopressin have long been used to help determine agonist roles in hypertension. We here call attention to a possible extension of this approach to detect, evaluate, and treat vascular sodium transport defects in hypertension. Two basic types of transport defects have been identified in the blood vessels of hypertensive animals, increased sodium permeability and decreased sodium pump activity. Intravenous injection of 6-iodo-amiloride, a sodium channel blocker and vasodilator, produces an immediate and sustained decrease in blood pressure in two genetic models of hypertension characterized by increased permeability of the vascular smooth muscle cell membrane to sodium (Okamoto spontaneously hypertensive rat, Dahl salt sensitive rat), whereas it produces only a transient fall in arterial pressure in two renal models of hypertension having normal sodium permeability in vascular smooth muscle cells (reduced renal mass-saline rat, one-kidney, one clip rat). Canrenone, a metabolic product of spironolactone which can compete with oubain for binding to Na+,K+-ATPase at the digitalis receptor site, decreases blood pressure in a low renin, volume expanded model of hypertension which has been shown to have depressed sodium pump activity in arteries and increased sodium pump inhibitor in plasma (reduced renal mass-saline rat) but has no effect on blood pressure in a genetic model of hypertension which has been shown to have increased sodium pump activity secondary to increased sodium permeability (spontaneously hypertensive rat). Thus, a sodium channel blocker and a competitor to ouabain binding can detect and determine the functional significance of sodium transport defects in the blood vessels of intact hypertensive animals. Studies in red and white blood cells suggest that similar defects may exist in the blood vessels of hypertensive humans. Thus, this approach, probing for vascular transport defects in the intact animal, may ultimately also be useful in the clinical setting.