Two endogenous digitalis-like cardiotonic steroids (CTS), endogenous ouabain (EO) and marinobufagenin (MBG), coexist in mammalian tissues. MBG acts as a selective inhibitor of ouabain-resistant alpha-1 isoform of Na/K-ATPase (NKA), the main isoform in the kidney, vascular smooth muscle (VSM) and adult cardiomyocytes. In Dahl salt-sensitive rats (DS) on a high NaCl intake, brain EO triggers peripheral MBG, which raises the blood pressure (BP). In a model of preeclampsia (PE), pregnant Sprague-Dawley rats (S-D) on a high NaCl intake raise BP in parallel with an increase in MBG levels. In vivo, administration of polyclonal and monoclonal (Mab) anti-MBG antibodies to hypertensive DS and pregnant rats with PE, lowered the BP. During the last year our research efforts concentrated (i) on the study of the relationship between central and peripheral CTS in salt-sensitive hypertension, (ii) on the development of therapeutic anti-MBG Mab, and (iii) on the studies of mechanisms of CTS-induced NKA-mediated cell signaling.? ? (i) Pathogenesis of NaCl-sensitive hypertension.? Following acute NaCl loading of DS, an initial transient rise of hippocampal EO, followed by an increase in EO in the supraoptic nucleus of the hypothalamus and pituitary, stimulates pituitary angiotensin II (AngII), and, via activation of sympathetic nervous system activates the adrenocortical renin-angiotensin system (RAS). Adrenocortical AngII acting through AT1 receptors stimulates production of MBG. An increase in MBG production induces inhibition of the Na pump in renal tubules and in VSM. The homeostatic response to high NaCl is not limited to CTS and RAS, and includes endothelin, vasopressin, natriuretic peptides, renal dopamine system, etc. Our results demonstrate that in response to acute NaCl loading MBG may stimulate Arg-vasopressin (AVP), since the administration of anti-MBG antibody to NaCl-loaded DS prevented NaCl-induced elevation of plasma AVP levels. Previously, we demonstrated that MBG is implicated in the mechanisms of ethanol dependence. Administration of MBG to rats suppressed voluntary alcohol intake, while immunization against MBG induced alcohol-seeking behavior. In humans alcohol withdrawal is associated with elevation of BP and renal Na retention. We hypothesized that MBG may be implicated in the pathogenesis of hypertension associated with alcohol withdrawal. In S-D rats, ethanol withdrawal was associated with a pressor response, fluid retention with a drop in hematocrit, and increased MBG and AVP levels. We hypothesized that AVP, which exhibits anti-addictive effects may be a factor linking MBG to central mechanisms regulating dependence and water-electrolyte metabolism. Indeed, administration of anti-MBG antibody to NaCl-loaded DS prevented elevation of plasma AVP levels, indicating a causative relationship between two hormones. These data suggest that AVP may limit the fluid excretory action of MBG without affecting its Na excretory effects. An important question is to what extent data on the relationship between brain EO and MBG obtained in DS, may be relevant to normotensive species including humans. We studied renal excretion of CTS in 8 healthy volunteers on a high NaCl intake (280 mmol/day for 6 days). The time courses of renal excretion of both CTS resembled those observed in NaCl-loaded DS, i.e., EO excretion rose two-fold in 3 days of a high NaCl intake and decreased to baseline levels in 5 days, while MBG excretion doubled in three days and remained elevated.? ? (ii) Development of therapeutic anti-MBG antibodies.? Heightened levels of MBG are associated with elevations of BP in patients with PE, in hypertensive subjects with end stage renal disease (ESRD), in hypertensive DS, and in experimental PE in rats. In hypertensive DS, in rats with pregnancy-induced hypertension and in volume expanded hypertension associated with ESRD, blockade of heightened levels of MBG with anti-MBG anntibody resulted in the reduction of BP and in an increase in the activity of VSM Na pump. In 20 patients with PE, plasma MBG levels exhibited substantial increase, and the activity of erythrocyte NKA was inhibited by 50% vs. that in 10 normotensive pregnant women. In vitro treatment of erythrocytes from PE patients with anti-MBG Mab restored the NKA activity, while DIGIBIND (Fab fragments of anti-digoxin antibody, that bind CTS) was less effective. An application for a patent has been filed to US Patent and Trademark Office in June 2006. Therapeutic effects of anti-MBG Mabs are not limited to depressor action. In rats with experimental ESRD, blockade of MBG markedly reduced cardiac fibrosis, reduced plasma levels of markers of oxidative stress, and was associated with increased levels of Fli-1, transcription factor, which inhibits collagen synthesis, in left ventricular (LV) myocardium. ? ? (iii) MBG-induced cell signaling and mechanisms of its modulation.? ANP, via a cGMP-dependent mechanism, dephosphoprylates VSM NKA, and markedly reduces its sensitivity to MBG. In renal medulla, ANP exhibits an opposite effect, i.e., induces NKA phosphorylation and sensitizes the Na pump to the inhibitory effect of MBG. Since VSM and renal medulla express PKG1 and PKG2 isoforms, respectively, we hypothesize that these two PKG isoforms mediate the opposing effects of ANP on NKA phosphorylation and MBG sensitivity. Thus, concurrent production of a vasorelaxant, ANP, and a vasoconstrictor, MBG, potentiate each other's natriuretic effects, but ANP may offset the vasoconstrictor effect of MBG. Aging, hypertension, diabetes mellitus and congestive heart failure are all associated with heightened levels of CTS and with a decline in cGMP-PKG signaling in the kidney and VSM. Such a decline may shift the balance between effects of MBG and ANP on renal and cardiovascular NKA towards lesser inhibition of renal Na pump and greater inhibition of VSM NKA and thus contribute to the pathogenesis of hypertension. We compared effects of 1 nmol/L ANP on MBG-induced NKA inhibition in VSM and renal medulla from young (8 weeks old) and aged (1 year old) male S-D rats. In both tissues in animals of both ages, 1 nmol/L MBG inhibited NKA by 20%. While in young rats ANP potentiated MBG-induced inhibition of renal NKA, and reversed inhibition of VSM NKA, the effect in aged animals was opposite: ANP did not alter MBG-induced inhibition of renal NKA, and in VSM, ANP potentiated MBG-induced NKA inhibition. Thus, in aged rats, ANP is not capable of potentiation of the physiological effect of MBG, natriuresis, and, likewise, aging is associated with a loss in the ability of ANP to offset MBG-induced vasoconstriction.? ? MBG is implicated in the pathogenesis of uremic cardiomyopathy. Rats subjected to chronic MBG infusion (10 ug/kg/day) or partial nephrectomy (PNX) develop substantial cardiac fibrosis. Immunization of PNX rats against MBG protects them from development of fibrosis in LV myocardium. The expression of Fli-1 was markedly decreased in the hearts of both PNX and MBG-treated rats, but was not affected in PNX rats immunized against MBG. In primary culture of rat fibroblasts, nanomolar concentrations of MBG caused a two-fold increase in reactive oxygen species production, a 150% increase in procollagen expression, and a decrease in Fli-1 expression. These data suggest that MBG produces cardiac fibrosis in a process involving oxidative stress and decreased Fli-1 expression.? ? Taken together, these findings demonstrate that CTS are important factors in pathogenesis of hypertension, PE, and ESRD, and open new pharmacological possibilities in the treatment of hypertension, including blockade of circulating MBG with a specific anti-MBG Mab and attenuation of MBG-induced inhibition of NKA. Pharmacological antagonism of CTS-induced cell signaling may give a new dimension to the treatment of uremic cardiomyopathy.
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