RESUMEN
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.
Asunto(s)
Androstanoles/uso terapéutico , Antihipertensivos/uso terapéutico , Hipertensión/tratamiento farmacológico , Ouabaína/antagonistas & inhibidores , Androstanoles/farmacología , Androstanoles/toxicidad , Animales , Antihipertensivos/farmacología , Antihipertensivos/toxicidad , Humanos , Ratas , Ratas Endogámicas SHR , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacosRESUMEN
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 and/or increased plasma levels of endogenous ouabain (EO). Ouabain (OU) itself induces hypertension in rats and both OU and mutated adducin activate the renal Na/K-ATPase function both in vivo and 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) and/or increased EO levels (OS) at oral doses of 0.1-10 micro g/kg. In NRK cells either transfected with mutated adducin or incubated with 10(-9) M 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 micro g/kg 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.
Asunto(s)
Androstanoles/farmacología , Antihipertensivos/farmacología , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacos , Animales , Presión Sanguínea/efectos de los fármacos , Células Cultivadas , Humanos , Riñón/enzimología , Microsomas/enzimología , Ratas , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , TransfecciónRESUMEN
The synthesis and binding affinities to the digitalis Na(+),K(+)-ATPase receptor of a series of 3 beta,14 beta-dihydroxy-5 beta-androstane and 3 beta-hydroxy-14-oxoseco-D-5 beta-androstane derivatives bearing a 17 alpha-(aminoalkoxy)imino chain are reported; some derivatives were also studied for their inotropic activity. Our recently proposed model of interaction of molecules with the digitalis receptor was used to design these compounds. On that basis, the possibility to design novel potent inhibitors of Na(+),K(+)-ATPase without being constrained by the stereochemistry of the classical digitalis skeleton in the D-ring region was predicted. The binding affinities of the most potent compounds in the two series, (EZ)-17 alpha-[2-[(2-aminoethoxy)imino]ethyl]-5 beta-androstane-3 beta,14 beta-diol (6f) and (EZ)-3 beta-hydroxy-17 alpha-[2-[(2-aminoethoxy)imino]ethyl]-14,15-seco-5 beta-androstan-14-one (24c) are higher than that of the potent natural compound digitoxigenin, despite the unusual alpha-exit of the substituent in position 17 of 6f or the disruption of the D-ring in 24c. These results further support the validity of our recently proposed model of binding at the digitalis receptor. Results of the inotropic tests on guinea pig atrium deserve further investigation on the pharmacological profile of these derivatives.
Asunto(s)
Androstanos/síntesis química , Androstanoles/síntesis química , Inhibidores Enzimáticos/síntesis química , Oximas/síntesis química , Secoesteroides/síntesis química , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Androstanos/química , Androstanos/farmacología , Androstanoles/química , Androstanoles/farmacología , Animales , Función Atrial , Unión Competitiva , Digitoxigenina/química , Perros , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Cobayas , Atrios Cardíacos/efectos de los fármacos , Técnicas In Vitro , Riñón/química , Masculino , Modelos Moleculares , Contracción Miocárdica/efectos de los fármacos , Oximas/química , Oximas/farmacología , Ensayo de Unión Radioligante , Secoesteroides/química , Secoesteroides/farmacologíaRESUMEN
A series of digitalis-like compounds with a 17-aminoalkoxyiminoalkyl or -alkenyl substituent was synthesized and evaluated for inhibition of Na(+),K(+)-ATPase and for inotropic activity. The highest inhibition was found with compounds having the substituent in configuration 17beta and the amino group at a distance of 6 or 7 bonds from C(17) of the digitoxigenin skeleton. The presence of the oxime function strengthens the interaction with the receptor, more if alpha,beta-unsaturated, thus mimicking the electronic situation of the unsaturated lactone in natural digitalis compounds. The most active compounds showed Na(+),K(+)-ATPase inhibitory potencies (IC(50)) 17-25 times higher than the standards digitoxigenin and digoxin and 3-11 times higher inotropic potencies (EC(50)) in isolated guinea pig left atria. These features are supported by a molecular model suggesting the possible interactions of the groups described above with particular amino acid residues in the H1-H2 domains of Na(+),K(+)-ATPase. Some interactions are the classical ones already described in the literature; a new, very strong interaction of the basic group with the Cys138 was found and adds new possibilities to design compounds interacting with this region of the receptor. The most interesting compounds were also studied in vivo in the anesthetized guinea pig for evaluating their inotropic effect versus the lethal dose. Compounds 9 and 12 showed a slightly higher safety ratio than digoxin and deserve further evaluation.
Asunto(s)
Androstanos/síntesis química , Androstanoles/síntesis química , Cardiotónicos/síntesis química , Glicósidos Digitálicos/farmacología , Inhibidores Enzimáticos/síntesis química , Oximas/síntesis química , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/química , Androstanos/química , Androstanos/farmacología , Androstanoles/química , Androstanoles/farmacología , Animales , Sitios de Unión , Cardiotónicos/química , Cardiotónicos/farmacología , Perros , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Cobayas , Atrios Cardíacos/efectos de los fármacos , Técnicas In Vitro , Riñón/enzimología , Masculino , Modelos Moleculares , Contracción Miocárdica/efectos de los fármacos , Ouabaína/química , Ouabaína/metabolismo , Oximas/química , Oximas/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Relación Estructura-ActividadRESUMEN
Adducin point mutations are associated with genetic hypertension in Milan hypertensive strain (MHS) rats and in humans. In transfected cells, adducin affects actin cytoskeleton organization and increases the Na(+)-K(+)-pump rate. The present study has investigated whether rat and human adducin polymorphisms differently modulate rat renal Na(+)-K(+)-ATPase in vitro. We report the following. 1) Both rat and human adducins stimulate Na(+)-K(+)-ATPase activity, with apparent affinity in tens of nanomolar concentrations. 2) MHS and Milan normotensive strain (MNS) adducins raise the apparent ATP affinity for Na(+)-K(+)-ATPase. 3) The mechanism of action of adducin appears to involve a selective acceleration of the rate of the conformational change E(2) (K) --> E(1) (Na) or E(2)(K). ATP --> E(1)Na. ATP. 4) Apparent affinities for mutant rat and human adducins are significantly higher than those for wild types. 5) Recombinant human alpha- and beta-adducins stimulate Na(+)-K(+)-ATPase activity, as do the COOH-terminal tails, and the mutant proteins display higher affinities than the wild types. 6) The cytoskeletal protein ankyrin, which is known to bind to Na(+)-K(+)-ATPase, also stimulates enzyme activity, whereas BSA is without effect; the effects of adducin and ankyrin when acting together are not additive. 7) Pig kidney medulla microsomes appear to contain endogenous adducin; in contrast with purified pig kidney Na(+)-K(+)-ATPase, which does not contain adducin, added adducin stimulates the Na(+)-K(+)-ATPase activity of microsomes only about one-half as much as that of purified Na(+)-K(+)-ATPase. Our findings strongly imply the existence of a direct and specific interaction between adducin and Na(+)-K(+)-ATPase in vitro and also suggest the possibility of such an interaction in intact renal membranes.
Asunto(s)
Proteínas de Unión a Calmodulina/fisiología , Hipertensión/genética , Hipertensión/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Ancirinas/farmacología , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/aislamiento & purificación , Proteínas de Unión a Calmodulina/farmacología , Eritrocitos/enzimología , Eritrocitos/metabolismo , Humanos , Riñón/enzimología , Riñón/metabolismo , Mutación/fisiología , Ratas , Ratas Endogámicas , Proteínas Recombinantes/farmacología , Albúmina Sérica Bovina/farmacología , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificación , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
A new three-dimensional model for the relative binding mode of cassaine 1 and digitoxigenin 2 at the digitalis receptor site is proposed on the basis of the structural and conformational similarities among 1, 2 and its 14,15-seco analogues 3 and 4. Accordingly, the speculation that also 17alpha-substituted derivatives of the digitalis 5beta,14beta-androstane skeleton could efficiently bind to the Na+,K+-ATPase receptor is put forward and verified through the synthesis of some related compounds. The binding affinity shown by 2-(N,N-dimethylamino)ethyl 3beta, 14-dihydroxy-5beta,14beta-androstane-17alpha-acrylate 6 (IC50 = 5.89 microM) and, much more significantly, by the corresponding 14, 15-seco-14-oxo derivative 9 (IC50 = 0.12 microM) substantiates the new hypothesis and opens new prospects to the design of novel inhibitors of Na+,K+-ATPase as potential positive inotropic compounds.
Asunto(s)
Alcaloides/metabolismo , Androstanos/química , Androstanos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Abietanos , Alcaloides/química , Androstanos/síntesis química , Androstanos/metabolismo , Animales , Unión Competitiva , Digitalis/química , Perros , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Técnicas In Vitro , Riñón/efectos de los fármacos , Riñón/metabolismo , Modelos Moleculares , Conformación Molecular , Imitación Molecular , Plantas Medicinales , Plantas Tóxicas , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , EstereoisomerismoRESUMEN
Milan hypertensive rats (MHS) develop hypertension because of a primary renal alteration. Both apical and basolateral sodium transport are faster in membrane vesicles derived from renal tubules of MHS than in those of Milan normotensive control rats (MNS). These findings suggest that the increased renal sodium retention and concomitant development of hypertension in MHS may be linked to an altered transepithelial sodium transport. Since this transport is mainly under the control of the Na-K pump, we investigated whether an alteration of the enzymatic activity and/or protein expression of the renal Na,K-ATPase is detectable in prehypertensive MHS. We measured the Na,K-ATPase activity, Rb+ occlusion, turnover number, alpha 1- and beta 1-subunit protein abundance, and alpha 1 and beta 1 mRNA levels in microsomes from renal outer medulla of young (prehypertensive) and adult (hypertensive) MHS and in age-matched MNS. In both young and adult MHS, the Na,K-ATPase activity was significantly higher because of an enhanced number of active pump sites, as determined by Rb+ occlusion maximal binding. The higher number of pump sites was associated with a significant pretranslational increase of alpha 1 and beta 1 mRNA levels that preceded the development of hypertension in MHS. Since a molecular alteration of the cytoskeletal protein adducin is genetically associated with hypertension in MHS and is able to affect the actin-cytoskeleton and Na-K pump activity in transfected renal cells, we propose that the in vivo upregulation of Na-K pump in MHS is primary and linked to a genetic alteration of adducin.