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The evidence that high levels of endogenous ouabain (EO), a closely related isomer of ouabain, are implicated in human hypertension and cardiac hypertrophy and failure stimulated the pharmacological research for developing novel anti-hypertensive agents active as ouabain antagonists. The pathogenetic mechanisms through which increased EO levels affect cardiovascular system involve the modulation of Na-K ATPase, the key enzyme responsible for renal tubular sodium reabsorption and the activation of signalling transduction pathways implicated in growth-related gene transcription. By studying both genetic and experimental rat models of hypertension and comparing them with humans, our group has demonstrated that elevated levels of circulating EO and the genetic polymorphism of the cytoskeletal protein adducin associate with hypertension and high renal Na-K pump activity. Ouabain itself induces hypertension and up-regulates renal Na-K pump when chronically infused at low doses into rats (OS). In renal cultured cells, either incubated for several days with nanomolar concentrations of ouabain or transfected with the hypertensive adducin genetic variant, the Na-K pump results enhanced. Moreover, both EO and adducin polymorphism affect cardiac complications associated to hypertension, the former through the activation of a signalling transduction pathway. As a consequence, a compound able to interact with the cellular and molecular alterations, sustained by EO or mutated adducin, may represent the suitable treatment for those patients in whom these mechanisms are at work. A new antihypertensive compound, PST 2238, that selectively antagonises the pressor effect and the alteration of renal Na-K pump, sustained both by ouabain and adducin polymorphism, is described. A selective ability of PST 2238 to antagonise the ouabain-induced organ hypertrophy is also documented. The specificity of PST 2238 mechanism of action is supported by the absence of interactions with receptors or hormones involved in blood pressure regulation and by the lack of diuretic activity and diuretic-associated side effects. It is concluded that this compound could be useful for the treatment of those forms of essential hypertension in which renal Na handling alterations and cardiac complications are associated with either increased EO levels and/or adducin polymorphism.

A primary impairment of the kidney sodium excretion has been documented both in hypertensive patients (EH) and genetic animal models (Milan hypertensive rat [MHS]) carrying mutations of the cytoskeletal protein adducin andor increased plasma levels of endogenous ouabain (EO). Ouabain (OU) itself induces hypertension in rats and both OU and mutated adducin activate the renal NaK-ATPase function both in vivoand in cultured renal cells (NRK). A new antihypertensive agent, PST 2238, able to selectively interact with these alterations has been developed. PST lowers blood pressure (BP) by normalizing the expression and activity of the renal Na-K pump selectively in those rat models carrying the adducin mutation (MHS) andor increased EO levels (OS) at oral doses of 0.1-10 ?gkg. In NRK cells either transfected with mutated adducin or incubated with 10?9M OU, PST normalizes the Na-K pump activity. Recently, an association between EO and cardiac complications has been observed in both EH and rat models consistent with a prohypertrophic activity of OU. OS rats showed a 10 increase of left ventricle and kidney weights as compared with controls, and PST 2238 (1 ?gkg OS) prevented both ventricle and renal hypertrophy. This effect was associated with the ability of PST to antagonize the OU-dependent activation of growth-related genes, in the membrane subdomains of caveolae. In conclusion, PST is a new antihypertensive agent that may prevent cardiovascular complications associated with hypertension through the selective modulation of the Na-K pump function.

A genetic alteration in the adducin genes is associated with hypertension and up-regulation of the expression of renal Na,K-ATPase in Milan-hypertensive (MHS) rats, in which increased ouabain-like factor (OLF) levels are also observed. PST 2238, a new antihypertensive compound that antagonizes the pressor effect of ouabain in vivo and normalizes ouabain-dependent up-regulation of the renal Na-K pump, was evaluated for its ability to lower blood pressure and regulate renal Na,K-ATPase activity in MHS genetic hypertension. In this study, we show that PST 2238, given orally at very low doses (1 and 10 μg/kg for 5–6 weeks), reduced the development of hypertension in MHS rats and normalized the increased renal Na,K-ATPase activity and mRNA levels, whereas it did not affect either blood pressure or Na,K-ATPase in Milan-normotensive (MNS) rats. In addition, a similar antihypertensive effect was observed in adult MHS rats after a short-term treatment. In cultured rat renal cells with increased Na-K pump activity at Vmaxdue to overexpression of the hypertensive variant of adducin, 5 days of incubation with PST 2238 (10−10--10−9M) lowered the pump rate to the level of normal wild-type cells, which in turn were not affected by the drug. In conclusion, PST 2238 is a very potent compound that in MHS rats reduces blood pressure and normalizes Na-K pump alterations caused by a genetic alteration of the cytoskeletal adducin. Because adducin gene mutations have been associated with human essential hypertension, it is suggested that PST 2238 may display greater antihypertensive activity in those patients carrying such a genetic alteration.

The inhibition of the long-term pressor effect of ouabain may be useful for the therapy of essential hypertension. Here, for the first time, a selective inhibitor of the ouabain pressor effect is described. In vitro, 17β-(3-furyl)-5β-androstane-3β, 14β, 17α-triol (PST 2238) displaced ouabain from its binding sites on purified sodium, potassium ATPase enzyme (Na-K ATPase) (IC501.7 × 10−6M) without interacting with other receptors involved in blood pressure regulation or hormonal control. In cultured renal cells, incubation with ouabain (10−10to 10−8M) for 5 days stimulated the Na-K pump at Vmax, whereas PST 2238 showed the same effect at micromolar concentration. The ouabain-dependent increase in the Na-K pump rate was abolished by PST 2238 at concentrations from 10−14to 10−9M. In rats made hypertensive by chronic infusion of 50 μg/kg/day of ouabain, PST 2238 given p.o at very low doses (0.1–1 μg/kg/day for 4 weeks) abolished the increase in blood pressure and renal Na-K ATPase activity caused by ouabain. PST 2238 did not affect either blood pressure or renal Na-K ATPase activity in normotensive rats. In conclusion, PST 2238 is a very potent compound that normalizes both blood pressure and alterations in the Na-K pump caused by ouabain. Thus it represents the prototype of a new class of antihypertensive drugs that could be effective in forms of hypertension sustained by the concomitant increase of endogenous ouabain levels and alterations in the Na-K pump.

The maximal rate of activity of Na extrusion by the Na pump, Na-K outward cotransport, Na-Li countertransport and the rate constant for passive permeability to Na, K and Li were measured in the RBC of 24 normotensive subjects with both parents normotensive, 45 hypertensive subjects and 24 of their normotensive offspring. The Na extrusion by the Na pump and the passive permeability to Na, K and Li a.e equal. The hypertensives have significantly greater Na-Li countertransport and smaller Na-K cotransport when compared to the normotensives.Na-K cotransport and Na-Li countertransport are positively correlated, thus suggesting some relationship between the two systems. When arbitrary normal limits are set the hypertensives are divided in three groups: normal cotransport and countertransport (22.2%) high countertransport (31.1%) and low cotransport (44.4%). In nine hypertensive families studied if either alteration was observed in a hypertensive propositus, this was of the same kind as the one in case observed in any first degree relative, whether already hypertensive or young normotensive. The observed alterations are primitive to the development of hypertension and possibly related to its pathogenesis.

Renal and cardiac function were measured in 65 offspring of hypertensive parents (OHP) and in 56 offspring of normotensive parents (ONP). in two additional groups of 24 OHP and 42 ONP plasma renin activity (PRA) and plasma active (PRAC), inactive (PRIC) and total (PRTC) concentration were measured. OHP had significantly higher renal plasma flow (p < 0.01), glomerular filtration rate (p < 0.02) and 24-hour urinary output than ONP, while PRA was lower (p < 0.01). The measurements of the different forms of renin gave the following results:PRIC and PRTC were lower in OHP than in ONP, but the only statistically significant difference concerns PRIC (p < 0.025). A possible interpretation of these findings is that a primary increased tubular ion and water reabsorption might be the cause of the kidne function pattern seen in OHP.

1. The maximal rate of activity of sodium extrusion by the sodium pump, Na+—K+ outward cotransport, passive permeability to sodium and potassium, Na+—Li+ countertransport, and passive permeability to lithium were measured in 45 essential hypertensive patients, 24 young normotensive subjects with at least one hypertensive parent and 24 normotensive subjects with both parents normotensive. 2. The maximal rate of activity of the sodium pump and the rate constants for passive permeability to sodium, potassium and lithium were similar in the three groups. 3. The mean value for Na+—K+ outward cotransport was significantly lower and that for Na+—Li+ countertransport significantly higher in the hypertensive patients than in the normotensive subjects without a family history of hypertension, but there was a great overlap between individual values. The offspring of hypertensive parents had intermediate values. 4. A highly significant positive correlation existed between Na+—K+ cotransport and Na+—Li+ countertransport both in the normotensive and in the hypertensive subjects, indicating that in the latter there were some with high Na+—Li+ countertransport and others with low Na+—K+ cotransport. 5. In nine hypertensive families studied, if an alteration of the transport system was detected in a hypertensive propositus, the same type of alteration was also found in his still normotensive offspring, thus indicating a familial tendency for the alteration to occur.

Summary:

Endogenous ouabain-like factor (OLF) is present in mammai tissues and after standardized extraction procedure can be similarly quantified by two independent assays: RIA and Na-KATPase inhibition. OLF was quantified both from plasma and tissues obtained from MHS hypertensive and MNS normotensive rats, maintained under the same enviromental and dietary conditions, and from plasma of healthy volunteers and essential hypertensive patients. OLF biochemical characterization shows that it behaves like ouabain except for a 1000-fold higher affinity for the ouabain low-affinity Na-KATPase isoforms than ouabainTissue and plasma levels of OLF are higher in MHS than in MNS rats and are not influenced by exogenous OLF sources. Plasma OLF is also increased in a subgroup of hypertensive patients. Both in rats and humans a primary cell membrane alteration affecting ion transports seems to be linked to the increased levels of OLF. An antihypertensive compound which selectively antagonizes the pressor effect of OLF and corrects the ion transport defect is under development and can represent a new pharmacological approach to the treatment of hypertension

The Milan hypertensive strain of rats (MHS) develops a genetic form of renal hypertension that, when compared to its normotensive control (MNS), shows renal dysfunction similar to that of a subset of human patients with primary hypertension. MHS and MNS were shown to be homozygous by multilocus minisatellite analysis and monolocus microsatellite markers. We show here that one point mutation in each of two genes coding for the membrane skeleton protein adducin is associated with blood pressure in the Milan strain of rats. Adducin is a heterodimer formed by alpha and beta subunits that promotes the assembly of actin with spectrin. MHS and MNS differ, respectively, by the amino acids Y and F at position 316 of the alpha subunit. In the beta-adducin locus, MHS is always homozygous for R at position 529 while in MNS either R or Q occurs in that position. The R/Q heterozygotes showed lower blood pressure than any of the homozygotes. In vitro phosphorylation studies suggest that both of these amino acid substitutions occur within protein kinase recognition sites. Analysis of an F2 generation demonstrated that Y alleles segregated with a significant increment in blood pressure. This effect is modulated by the presence of the R allele of the beta subunit. Taken together, these findings strongly support a role for adducin polymorphisms in causing variation of blood pressure in the Milan strain of rats.

1. Erythrocyte Na+-K+ cotransport and Na+-Li+ countertransport were measured at the same time in 56 essential hypertensive patients, 24 of their normotensive offspring and 36 matched normotensive controls. 2. The mean value of Na+-K+ cotransport is slower and of Na+-Li+ countertransport is faster in the essential hypertensive group and in some of their normotensive offspring, but a great overlap with the value of the normotensive group was observed. 3. Na+-K+ cotransport values were bimodally distributed in the hypertensive group, with the first mode at 250 and the second at 600 µmol h-1 l-1 of erythrocytes. 4. The ratio Na+-K+ cotransport/Na+-Li+ countertransport was calculated. It was found that such a ratio distinguishes between normotensive and essential hypertensive subjects better than the individual transport systems alone.