Captopril inhibits ouabain-sensitive Na+/K+-ATPase

Captopril has been reported to inhibit ouabain-sensitive Na+/K+-ATPase activity in erythrocyte membrane fragments. We investigated the effect of captopril on two physiological measures of Na+/K+ pump activity: 22Na+ efflux from human erythrocytes and K+-induced relaxation of rat tail artery segments. Captopril inhibited 22Na+ efflux from erythrocytes in a concentration-dependent fashion, with 50% inhibition of total 22Na+ efflux at a concentration of 4.8 X 10(-3) M. The inhibition produced by captopril (5 X 10(-3) M) and ouabain (10(-4) M) was not greater than that produced by ouabain alone (65.3 vs. 66.9%, respectively), and captopril inhibited 50% of ouabain-sensitive 22Na+ efflux at a concentration of 2.0 X 10(-3) M. Inhibition by captopril of ouabain-sensitive 22Na efflux was not explained by changes in intracellular sodium concentration, inhibition of angiotensin-converting enzyme or a sulfhydryl effect. Utilizing rat tail arteries pre-contracted with norepinephrine (NE) or serotonin (5HT) in K+-free solutions, we demonstrated dose-related inhibition of K+-induced relaxation by captopril (10(-6) to 10(-4) M). Concentrations above 10(-4) M did not significantly inhibit K+-induced relaxation but did decrease contractile responses to NE, although not to 5HT. Inhibition of K+-induced relaxation by captopril was not affected by saralasin, teprotide or indomethacin. We conclude that captopril can inhibit membrane Na+/K+-ATPase in intact red blood cells and vascular smooth muscle cells. The mechanism of pump suppression is uncertain, but inhibition of ATPase should be considered when high concentrations of captopril are employed in physiological studies.     

Possible mechanisms of positive inotropic action of synthetic human calcitonin gene-related peptide in isolated rat atrium

The effect of synthetic human calcitonin gene-related peptide (hCGRP) on the isolated and electrically driven left atria of rats were investigated. The peptide at concentrations of 3 X 10(-9)-3 X 10(-7) M produced positive inotropic effects on the left atria in a dose-dependent manner. Verapamil (10(-5) M) and adenosine (10(-4) M) reduced the positive inotropic effect of hCGRP at concentrations of 3 X 10(-9) and 3 X 10(-8) M, but not at that of 3 X 10(-7) M. Ouabain (5 X 10(-5) M) inhibited the effect of hCGRP in concentrations of 3 X 10(-7) and 3 X 10(-8) M, but not in that of 3 X 10(-9) M. Simultaneous pretreatment with verapamil (10(-5) M) and ouabain (5 X 10(-5) M) suppressed the positive inotropy by hCGRP at all concentrations tested. On the other hand, tetrodotoxin (10(-6) M) potentiated only the positive inotropic effect of 3 X 10(-7) M hCGRP. Metoprolol (10(-7) M) and theophilline (10(-3) M) did not affect the inotropic effect of hCGRP. These results suggest that the positive inotropic effect of hCGRP is not mediated by beta-adrenoceptors but by two distinct mechanisms of action, which was inhibited by verapamil but not by ouabain (facilitation of Ca++ influx in lower concentrations of hCGRP) and which was blocked by ouabain but not by verapamil and potentiated by tetrodotoxin (inhibition of Na+/Ca++ exchange mechanism at higher concentrations of hCGRP).     

Enzymatic inhibition of lysine transport across the small intestine in vivo

1. Trypsin, at different concentrations, significantly inhibited lysine absorption (P less than 0.05) in a dose-dependent pattern. 2. Maximum inhibition equivalent to 35% below control value was reached with 10 micrograms/ml (100 BAEE units) trypsin with a non-reversible inhibitory effect. 3. Chymotrypsin at 10 micrograms/ml produced a significant decrease (P less than 0.05) of lysine absorption although it did not exceed 5%. Perfusion of both enzymes did not show an additive inhibitory effect. 4. Lysine absorption showed a 39% decrease with 10 micrograms/ml trypsin and 1 X 10(-4) M ouabain, whereas ouabain alone produced 34% inhibition. 5. Lysine absorption showed a 71% decrease with 10 micrograms/ml trypsin in a sodium-free medium, and 70% inhibition with Na-free medium alone. 6. The inhibition of lysine absorption after trypsin treatment could be due to inhibition of the active component of lysine transport.     

The alpha-2 isomer of the sodium pump is inhibited by calcium at physiological levels

The inhibition of the (Na,K)ATPase by calcium was investigated in plasma membrane preparations of rat axolemma, skeletal muscle and kidney outer medulla. Ouabain titration curves demonstrated that physiological calcium (0.08-5 microM) inhibited mainly the high affinity alpha 2 isomer. In axolemma all the (Na,K)ATPase had high ouabain affinity and calcium inhibited 40-50% of the activity with a Ki of 1.9 +/- 0.9 x 10(-7) M. In skeletal muscle high and low ouabain affinity components were present in equal amounts and calcium inhibited only the high affinity component with a Ki of 1.3 +/- 0.3 x 10(-7) M. Kidney enzyme had a low affinity for ouabain and showed very little sensitivity to calcium in the physiological range. It was demonstrated that high calcium levels inhibit the enzyme in a general sense, irrespective of the isomer, with a Ki of 6.5 +/- 6 x 10(-4) M for the kidney and 5.9 +/- 4 x 10(-4) M for the axolemma enzymes. In axolemma, enzyme activity was studied as a function of sodium concentration. Physiological calcium reduced Vmax while not significantly changing K 0.5 for sodium binding.     

Enhancement (by ATP, insulin, and lack of divalent cations) of ouabain inhibition of cation transport and ouabain binding in frog skeletal muscle; effect of insulin and ouabain on sarcolemmal (Na + K)MgATPase.

Using small, intact frog muscles, the basic properties of Na+ and K+ transport were shown to resemble those of the (Na+ + K+)Mg2+ATPase (EC 3.6.1.3) isolated from skeletal muscle. (a) External K+ is essential for Na+ exit and K+ entry after the muscles are Na+-loaded and K+-depleted; (b) the ouabain concentration causing maximum inhibition of recovery is the same for transport as for the inhibition of the isolated enzyme. Ouabain causes a decrease in the sorbitol space and causes muscle fibre swelling. Absence of Ca2+ and Mg2+ inhibits recovery of normal Na+ and K+ concentrations and increases the sorbitol space. Insulin stimulates K+ uptake and Na+ loss in intact muscles but has no effect on the isolated sarcolemmal (Na+ + K+)Mg2+ATPase. Absence of divalent cations, addition of external ATP and of insulin enhance the ouabain inhibition of recovery. Bound ouabain was measured using [3H]ouabain and [14C]sorbitol (to measure the extracellular space). The process of binding was slowly reversible and was saturable within a range of ouabain concentrations from 1.48 X 10(-7) to 5.96 X 10(-7) M. From the nonexchangeable ouabain bound, the density of glycoside receptors was estimated to be 650 molecules per square micrometre of membrane surface. The absence of divalent cations, addition of external ATP and of insulin significantly enhanced the amount of ouabain bound. Substitution of Na+ and K+ by choline greatly reduced the bound ouabain.     

Paradoxical production of mouse thymocyte activating factor by ouabain-treated human mononuclear cells

At concentrations as low as 10(-7) M, the cardiotonic glycosteroid ouabain, a specific inhibitor of the membrane Na+, K+-ATPase, is known to inhibit in vitro human lymphocyte proliferation produced in mixed lymphocyte cultures or induced by various stimulating agents (PHA, Con A, PWM, soluble antigens), while mouse lymphocyte proliferation is unaffected at this concentration. Ouabain inhibits most of proliferative response parameters at all stages of the transformation. This observation prompted us to suggest that ouabain could also act through inhibition of interleukin production which is known to occur during the first hours after T-cell stimulation in the presence of monocytes. In order to check the possible influence of ouabain on interleukin production, conditioned media from stimulated human mononuclear cells, prepared in the presence or in the absence of inhibitor, were tested for their ability to promote a mouse thymocyte response to PHA. Instead of the expected inhibition, we found that ouabain, even at high concentrations (2 X 10(-6) M) enhanced the stimulatory effect and/or the production of murine thymocyte activating factor(s). Moreover conditioned media from serum-free cultures of unstimulated human mononuclear cells exposed for 24 hr to low ouabain concentrations (10(-8) to 10(-7) M) showed a high activating effect on the response of murine thymocytes to PHA. This soluble factor produced upon ouabain treatment is produced by adherent cells and appears to be functionally similar to interleukin 1.     

Selective inhibition of human erythrocyte Na+/K+ ATPase by cardiac glycosides and by a mammalian digitalis like factor

Na+/K+ATPase is a transport membrane protein which contains the functional receptor for digitalis compounds. In this work we compare the inhibition curves of Na+/K+ATPase measured by the inhibition of 86Rb uptake in human red blood cells by cardiac glycosides and by an endogenous digitalis like factor (EDLF) extracted from human newborn cord blood. The curves of Na+/K+TPase inhibition show a monophasic shape for ouabain, strophantidin, digitoxin, proscillaridin and EDLF whereas a biphasic shape for ouabagenin, digoxin, digoxigenin and digitoxigenin. All the drugs are potent inhibitors of erythrocyte Na+/K+ATPase with an IC50 ranging from 1.8 x 10(-9) M to 1.4 x 10(-11) M for the higher affinity binding site and from 1.8 x 10(-6) M to 5.5 x 10(-9) M for the lower affinity site. Digitoxigenin is the most active showing the higher active site at 1.4 x 10(-11) M. Ouabain and digoxin have higher affinity compared with their corresponding genins, while digitoxigenin shows a binding site with higher affinity than the respective cardiac glycosides. The increased affinity of the drugs to Na+/K+ATPase may be related to a lipophilic region in correspondence of the carbons 10, 9, 11, 12, 13 of the steroid nucleus, situated in the opposite side with respect of the C-OH-14. The comparison of the inhibition curves and the HPLC profile of newborn EDLF and of the investigated cardenolides suggest that EDLF may be a compound identical or very similar to ouabain.     

Measurement of spontaneous mutation rates at the Na+/K+ ATPase locus (ouabain resistance) of human fibroblasts using improved growth conditions

The mutation rate for the Na+/K+ ATPase locus (ouabain resistance, OuaR) in mammalian cells in culture has been reported to be 10-100-fold lower than the mutation rate of other gene loci in culture, such as the hypoxanthine phosphoribosyl transferase (HPRT) locus. Determination of the mutation rate to ouabain resistance is sensitive to culture conditions and the concentration of ouabain used to select mutants. Our improved growth conditions for human cells have permitted absolute cloning efficiencies of 70-90% and population doubling times of 16-17 h with both normal human diploid fibroblasts, KD, and their chemically induced neoplastic derivative, Hut-11A. Ouabain at 10(-7) M was found to be adequate to select for resistant (OuaR) mutants with an absolute recovery efficiency of 54-102%. Under these conditions, the mutation rates to ouabain resistance for human cells were measured and found to be 1-8.5 X 10(-7)/cell/generation for KD cells and 6-13 X 10(-7)/cell/generation for Hut-11A cells. These rates are 5-25 times higher than previously reported for human cells. Improved growth and the use of a lower concentration of ouabain for selection may allow for the increased recovery of OuaR mutants and an improved estimate of the mutation rate at this locus, which is only 2-10-fold less than the mutation rate at the HPRT locus in the same cells.     

The static magnetic field effects on ouabain H3 binding by cancer tissue

Radioactive labeled ouabain was used for estimating the static magnetic field (SMF) induced cell volume changes. Ouabain is a specific inhibitor of Na+/K+ ATPase, and can be used for estimating its quantity--thus giving information about the cell volume changes. Ouabain binding by cancer and normal glandular tissues of breast cancer patients and normal glandular tissues of healthy women was measured after exposure of tissues to SMF 0.2T. SMF exposure led to a decrease of ouabain binding in both normal and cancer tissues when ouabain concentration in the external medium was 10(-9) M, while in the case of higher concentrations of ouabain (10(-7) M, 10(-6) M) an increase of ouabain binding was seen. The normal glandular tissues of healthy women were sensitive to SMF only at the highest concentrations of ouabain used in our experiments. The SMF-induced decrease of binding at low ouabain concentrations was considered as an evidence for the dehydration effect of SMF. It is suggested that the SMF could influence the cancer cell metabolism through cell hydration changes.     

Autoradiographic localisation of [3H]ouabain bound to cultured epithelial cell monolayers of MDCK cells

[3H]Ouabain binding to intact MDCK (cultured monolayers of dog kidney) cells of 60 serial passages is dependent upon ouabain concentration, time and medium K+. By utilising high K+ incubations to estimate non-specific [3H]ouabain-binding, the concentration of ouabain giving half maximal specific binding was estimated to be 1.0 . 10(-7) M and the total maximum binding to be 2.33 . 10(5) sites/cell. Ouabain inhibition of (Na+, K+)-pump function was monitored by the cellular uptake of 86Rb over 5 min. The larger fraction of 86Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 . 10(-7) M. The cellular distribution of the (Na+ + K+)-ATPase was investigated using [3H]ouabain autoradiography of intact freeze-dried epithelial monolayers of MDCK cells grown upon millipore filter supports. Binding of [3H]ouabain is localised over the lateral cellular membranes. Autoradiographic silver grain density is close to background levels over both the apical and basal (attachment) membranes.     

Inhibition of DNA synthesis in hamster cheek pouch tissue in organ culture by dibutyryl cyclic AMP and a homologous extract

Hamster cheek pouch tissue cultured with 10⁻⁴M dibutyryl cyclic AMP exhibits inhibition of the rate of DNA synthesis. When tissue is cultured with dibutyryl for different time periods throughout the 18 hours of incubation, the effect on the rate of DNA synthesis is very similar. A combination of dibutyryl cyclic AMP and an epithelial cell extract from hamster cheek pouch tissue containing a natural epithelial chalone, appears to have an additive effect on the inhibition of the DNA synthesis rate.     

Effects of autonomic blockers on linear and nonlinear indexes of blood pressure and heart rate in SHR

Endothelium-derived hyperpolarizing factor (EDHF) is released in response to agonists such as ACh and bradykinin and regulates vascular smooth muscle tone. Several studies have indicated that ouabain blocks agonist-induced, endothelium-dependent hyperpolarization of smooth muscle. We have demonstrated that epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of arachidonic acid, function as EDHFs. To further test the hypothesis that EETs represent EDHFs, we have examined the effects of ouabain on the electrical and mechanical effects of 14,15- and 11,12-EET in bovine coronary arteries. These arteries are relaxed in a concentration-dependent manner to 14,15- and 11,12-EET (EC(50) = 6 x 10(-7) M), bradykinin (EC(50) = 1 x 10(-9) M), sodium nitroprusside (SNP; EC(50) = 2 x 10(-7) M), and bimakalim (BMK; EC(50) = 1 x 10(-7) M). 11,12-EET-induced relaxations were identical in vessels with and without an endothelium. Potassium chloride (1-15 x 10(-3) M) inhibited [(3)H]ouabain binding to smooth muscle cells but failed to relax the arteries. Ouabain (10(-5) to 10(-4) M) increased basal tone and inhibited the relaxations to bradykinin, 11,12-EET, and 14,15-EET, but not to SNP or BMK. Barium (3 x 10(-5) M) did not alter EET-induced relaxations and ouabain plus barium was similar to ouabain alone. Resting membrane potential (E(m)) of isolated smooth muscle cells was -50.2 +/- 0.5 mV. Ouabain (3 x 10(-5) and 1 x 10(-4) M) decreased E(m) (-48.4 +/- 0.2 mV), whereas 11,12-EET (10(-7) M) increased E(m) (-59.2 +/- 2.2 mV). Ouabain inhibited the 11,12-EET-induced increase in E(m). In cell-attached patch clamp studies, 11,12-EET significantly increased the open-state probability (NP(o)) of a calcium-activated potassium channel compared with control cells (0.26 +/- 0.06 vs. 0.02 +/- 0.01). Ouabain did not change NP(o) but blocked the 14,15-EET-induced increase in NP(o). These results indicate that: 1) EETs relax coronary arteries in an endothelium-independent manner, 2) unlike EETs, potassium chloride does not relax the coronary artery, and 3) ouabain inhibits bradykinin- and EET-induced relaxations as has been reported for EDHF. These findings provide further evidence that EETs are EDHFs.     

Cerebral intraventricular 6-hydroxydopamine prevents vascular changes in the mineralocorticoid hypertensive rat

The effect of cerebral intraventricular administration of 6-hydroxydopamine (6-OHDA) on blood pressure and vascular smooth muscle responsiveness in deoxycorticosterone acetate (DOCA)-treated rats was assessed. Rats treated with 6-OHDA and DOCA had significantly lower systolic blood pressures (142 +/- 8 mm Hg) than rats treated with DOCA alone (185 +/- 5 mm Hg). After 5 weeks of DOCA treatment, femoral arteries and aortae were excised from these rats, cut helically into strips, and placed in a muscle bath to record isometric force. Dose-response curves to serotonin were shifted to the left in femoral arteries from DOCA-treated rats compared to both control and 6-OHDA-DOCA-treated rats (ED50: DOCA = 6.8 X 10(-8) M, control = 27.9 X 10(-8) M, 6-OHDA-DOCA = 13.4 X 10(-8) M). Arachidonic acid, the prostaglandin precursor, produced greater maximal contractions in femoral artery strips of DOCA-treated rats (358 +/- 56 mg) than in those from controls (115 +/- 31 mg). The maximal response to arachidonic acid in arteries from 6-OHDA-DOCA rats (203 +/- 78 mg) was not different from control values. Ouabain produced a greater maximal response in aortic strips from DOCA rats (658 +/- 165 mg) compared to those from control (196 +/- 72 mg) or 6-OHDA-DOCA (309 +/- 87 mg) rats. We conclude that increased vascular responsiveness to serotonin, arachidonic acid, and ouabain in DOCA hypertensive rats is secondary to a central action of the mineralocorticoid.     

Ouabain-insensitive Na+ stimulation of a microsomal Mg2+ -ATPase in gills of sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested.     

Therapeutic success of boron neutron capture therapy (BNCT) mediated by a chemically non-selective boron agent in an experimental model of oral cancer: a new paradigm in BNCT radiobiology

The hypothesis of boron neutron capture therapy (BNCT) research has been that the short-range, high-linear energy transfer radiation produced by the capture of thermal neutrons by (10)B will potentially control tumor and spare normal tissue only if the boron compound selectively targets tumor tissue within the treatment volume. In a previous in vivo study of low-dose BNCT mediated by GB-10 (Na(2)(10)B(10)H(10)) alone or combined with boronophenylalanine (BPA) in the hamster cheek pouch oral cancer model that was primarily designed to evaluate safety and feasibility, we showed therapeutic effects but no associated normal tissue radiotoxicity. In the present study, we evaluated the response of tumor, precancerous and normal tissue to high-dose BNCT mediated by GB-10 alone or combined with BPA. Despite the fact that GB-10 does not target hamster cheek pouch tumors selectively, GB-10-BNCT induced a 70% overall tumor response with no damage to normal tissue. (GB-10+BPA)-BNCT induced a 93% overall tumor response with no normal tissue radiotoxicity. Light microscope analysis showed that GB-10-BNCT selectively damages tumor blood vessels, sparing precancerous and normal tissue vessels. In this case, selective tumor lethality would thus result from selective blood vessel damage rather than from selective uptake of the boron compound.     

Association of the positive inotropic action of ouabain with a second species of digitalis receptors

Studies were conducted to determine whether Na-K ATPase or a second species of digitalis receptors in canine cardiac sarcolemma membrane preparations is associated with the positive inotropic action of nontoxic concentrations of ouabain. [3H]ouabain association and dissociation experiments using highly enriched sarcolemma preparations from canine ventricle indicate the presence of two species of ouabain binding receptors. Ouabain binding to Na-K ATPase of the sarcolemma preparation requires supporting ligands and is characterized by fast association and very slow dissociation in vitro. The second species of digitalis receptor does not require supporting ligands for ouabain binding and is characterized by slow association and fast dissociation. To determine which species of digitalis receptor is associated with the positive inotropic action of digitalis, ouabain washout experiments were conducted using various isolated canine myocardial preparations. Washout of the positive inotropic effects of 1.2-2.4 X 10(-7) M ouabain gave half-life values of 1.5-2.0 h for the various myocardial preparations. [3H]ouabain dissociation from the second species of digitalis receptors gave half-life values of 1.7-1.8 h, whereas dissociation from the sarcolemma Na-K ATPase gave half-life values of 8.9-9.3 h for the various sarcolemma preparations utilized. Therefore, based on similarities in half-life values between ouabain inotropy and [3H]ouabain dissociation from the second class of digitalis receptors, it is postulated that the positive inotropic action of digitalis glycosides is associated with the second species of digitalis receptors in the sarcolemma and not with the digitalis inhibitory receptor of Na-K ATPase for nontoxic concentrations of digitalis.     

Calcium Enhances In Vitro Free Radical-Induced Damage to Brain Synaptosomes, Mitochondria, and Cultured Spinal Cord Neurons

Preincubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) in Ca2+-free Krebs buffer resulted in a 27% inhibition of synaptosomal gamma-aminobutyric acid (GABA) uptake. Addition of 1.5 mM CaCl2 increased the inhibition with X/XO to 46%, and inhibition was essentially complete when the calcium ionophore A23187 also was included. In other studies, preincubation of purified rat brain mitochondria with the combination of X/XO and 4 microM CaCl2 produced a significant (38%) decrease in state 3 respiration with glutamate/malate as substrate that was not seen with either X/XO or Ca2+ alone. Similar results were obtained using cultured mouse spinal cord neurons in which incubation with X/XO/ADP/FeCl2 and A23187 produced membrane damage as assessed by a 32% reduction of neuronal Na+, K+-ATPase activity. Neither X/XO/ADP/FeCl2 nor A23187 alone caused detectable inhibition. These results demonstrate the synergistic damaging effect of free radicals and Ca2+ on membrane function. In addition, they suggest that free radical-induced peroxidation of membrane lipid, occurring focally during complete or nearly complete ischemia in vivo, could result in intense cellular perturbation when coupled with increased intracellular Ca2+.     

Purification of cardiac (Na+,K+)-activated adenosine triphosphatase from rat

A procedure is described for preparation of highly active (Na+,K+)-ATPase from rat heart which has a specific activity of 200-600 mumol Pi/mg/h. The procedure is simple and can be applied to small amounts of heart muscle (approximately 1 g). The ATPase activity was more than 90% sensitive to ouabain (at concentrations up to 1 mM). The ouabain sensitivity is biphasic with about 20% of the ATPase activity being inhibited at approximately 3 X 10(-7) M ouabain.     

Effect of ouabain on mechanical and electrical properties of rat and guinea pig hearts at 180 C.

The effects of ouabain 10(-6) M on rat and guinea pig hearts have been studied at 18 degrees C, in order to reduce almost fully both the Na+, K+-dependent ATPase activity and the ouabain induced inhibition of this enzyme. In isolated guinea pig hearts the positive inotropic response to ouabain obtained at 32 degrees C disappeared at 18 degrees C. On the contrary, the contractile strength of rat hearts was slightly reduced by ouabain and in the same manner at both temperatures. Current and voltage clamp experiments carried out at 18 degrees C in ventricular fibres revealed that ouabain 10(-6) M decreased both the action potential overshoot and the fast sodium current in rat and guinea pig, by reduction of the membrane sodium conductance. Ouabain did not change the calcium current in guinea pig preparations, whereas in rat heart muscle this current was reduced. The effects of ouabain on both the action potential plateau and outward repolarizing current indicated some inconsistencies from preparation to preparation and cannot therefore be considered as significant. The persistence of the ouabain induced alterations of g Na (in rat and guinea pig) and calcium current (in rat) at 18 degrees C supports the hypothesis of two ouabain cell receptors in heart muscle.     

Organ hypertrophic signaling within caveolae membrane subdomains triggered by ouabain and antagonized by PST 2238

In addition to inhibition of the Na-K ATPase, ouabain activates a signal transduction function, triggering growth and proliferation of cultured cells even at nanomolar concentrations. An isomer of ouabain (EO) circulates in mammalians at subnanomolar concentrations, and increased levels are associated with cardiac hypertrophy and hypertension. We present here a study of cardiac and renal hypertrophy induced by ouabain infused into rats for prolonged periods and relate this effect to the recently described ouabain-induced activation of the Src-EGFr-ERK signaling pathway. Ouabain infusion into rats (15 microg/kg/day for 18 weeks) doubled plasma ouabain levels from 0.3 to 0.7 nm and increased blood pressure by 20 mm Hg (p < 0.001), cardiac left ventricle (+11%, p < 0.05), and kidney weight (+9%, p < 0.01). These effects in vivo are associated with a significant enrichment of alpha1, beta1, gammaa Na-K ATPase subunits together with Src and EGFr in isolated renal caveolae membranes and activation of ERK1/2. In caveolae, direct Na-K ATPase/Src interactions can be demonstrated by co-immunoprecipitation. The interaction is amplified by ouabain, at a high affinity binding site, detectable in caveolae but not in total rat renal membranes. The high affinity site for ouabain is associated with Src-dependent tyrosine phosphorylation of rat alpha1 Na-K ATPase. The antihypertensive compound, PST 2238, antagonized all ouabain-induced effects at 10 microg/kg/day in vivo or 10(-10)-10(-8) m in vitro. These findings provide a molecular mechanism for the in vivo pro-hypertrophic and hypertensinogenic activity of ouabain, or by analogy those of EO in humans. They also explain the pharmacological basis for PST 2238 treatment.