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 Mg/kg per day IP) in normal saline for 4 weeks followed by ouabain (27.8 figJkg 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 (6J9±1.17 vs 2.36±0. 52 fig/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. (Hypertension 1993^2:178-187)

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. {Hypertension 1991;18:316-324)

Endogenous Na + ,K + -ATPase inhibitors may have a role in the mechanism of low-renin hypertension. Two such compounds have been characterized: ouabain from human plasma and resibufogenin from toad plasma. Previously, we examined the acute effects of ouabain and bufalin (which has the same structure as resibufogenin except for one H + ) in normal rats. Bufalin raised blood pressure, but ouabain had little effect. In contrast, given chronically, ouabain substantially increased blood pressure in normal rats and 70% reduced renal mass rats on a salt-free diet. We have now examined the chronic effects of bufalin in rats. Normal rats received 14.8 ng/kg per day bufalin or an equimolar dose of ouabain intraperitoneally for 6 weeks; 70% reduced renal mass rats also received 14.8 fig/kg per day bufalin. Another group of normal rats received 29.6 jig/kg per day bufalin intraperitoneally for 6 weeks. Respective control animals received vehicle.In contrast to ouabain, blood pressure did not increase in normal rats receiving the 14.8 ^tg dose of bufalin. However, normal rats receiving 29.6 /ig bufalin and 70% reduced renal mass rats receiving 14.8 fig bufalin developed significant increases in blood pressure. Increases in blood pressure were associated with decreases in myocardial Na + ,K + -ATPase activity and correlated with increased plasma Na + ,K + -ATPase inhibitory activity. Thus, although bufalin is a more potent pressor agent than ouabain when both agents are given acutely, ouabain is at least as potent a vasopressor agent as bufalin when given chronically. Thus, both are pressor agents, more so in the presence of reduced renal mass, when given chronically in the rat. 13 Numerous investigators have attempted to isolate this endogenous NKA inhibitor. However, only two steroid NKA inhibitors have been purified and chemically characterized -resibufogenin, a bufodienolide derivative in toad skin and plasma, 14 and a substance with the same basic structure as ouabain from human plasma. 15 In an earlier study 16 designed to determine whether such endogenous inhibitors in fact produce the above cardiovascular changes, we compared the acute effects of equimolar doses of commercially available bufalin (aglycone, identical to resibufogenin except for one H + ) and ouabain (Fig 1) in normal rats. Bufalin infused intravenously into anesthetized rats increased BP, dP/dt, heart rate, urine volume, and urinary sodium excretion. When applied to tail arteries from normal rats in vitro, bufalin depolarized vascular smooth muscle cell membrane potentials. Although equimolar doses of ouabain caused the same directional changes in all parameters except heart rate and membrane potentials, the magnitudes of these changes were always less than those produced by bufalin (ouabain had no effect on heart rate or vascular smooth muscle cell membrane potentials). In fact, ouabain actually decreased BP at higher doses. In contrast, in a subsequent study 17 we found that ouabain given chronically raised BP in both normal and 70% reduced ...

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We have reported that streptozotocin-induced insulin-dependent diabetes mellitus in 25% reduced renal mass rats is associated with low-renin, volume-expanded hypertension and that the development of the hypertension can be prevented with insulin. In this study we examined the effect of insulin after the animals had developed sustained hypertension. Normotensive 25% reduced renal mass rats were treated with streptozotocin and, as expected, developed insulin-dependent diabetes mellitus and hypertension. After 4 weeks of sustained hypertension, neutral protamine Hagedorn insulin (6 to 8 IU/d) was administered subcutaneously for 4 weeks. As expected, insulin treatment decreased plasma glucose and increased body weight gain relative to untreated diabetic rats. On the other hand, insulin treatment did not reverse the hypertension and albuminuria. It also did not normalize extracellular fluid volume and plasma renin activity. Further-T he role of insulin in blood pressure regulation is complicated. Insulin increases renal sodium absorption and sympathetic nerve activity, both of which tend to elevate arterial pressure. On the other hand, insulin stimulates Na + -K + pump activity, thereby causing electrogenic hyperpolarization of the vascular muscle cell and vasodilation. Clinically, insulin treatment has no significant effect on insulin-dependent diabetes mellitus (IDDM) hypertension.1 Chronic administration of insulin in experimental IDDM animals has been shown to increase, 2 decrease, 3 and have no effect on 4 blood pressure. We have previously shown that hypertension regularly develops when rats with 25% reduced renal mass (RRM) are treated with streptozotocin 5 and that this hypertension is associated with albuminuria, increased extracellular fluid volume (ECFV), and the appearance in plasma of an Na + ,K + -ATPase inhibitor 6 and that the albuminuria, volume expansion, appearance of the Na + ,K + -ATPase inhibitor, and hypertension can be prevented by treatment with insulin at the time the IDDM is induced, ie, if the insulin treatment is started before the development of hypertension. 7 In the latter study, neutral protamine Hagedorn (NPH) insulin treatment (initially 4 IU/d for 2 to 3 days and then 6 IU/d) immediately after streptozotocin injection prevented the hypertension and all the other changes, even though blood glucose was not completely normalized. more, insulin treatment did not reverse the increase in plasma Na + ,K + -ATPase inhibitory activity (determined by both radioimmunoassay and bioassay) and the inhibition of myocardial microsomal Na + ,K + -ATPase activity observed in the untreated diabetic hypertensive rats. 5'-Nucleotidase, a membrane marker, was not different between insulin-treated and untreated diabetic rats. These results show that insulin, given as here described, does not reverse the insulin-dependent diabetes mellitus hypertension in 25% reduced renal mass rats once it is established, perhaps because it does not reverse the albuminuria, volume expansion, increase in endogeno...