Positive Inotropic Effect Of Ouabain Research Articles

Comparative effects of ouabain on isolated papillary muscle from tree shrews, guinea-pigs and rats

Inotropic effects of ouabain were investigated in the isolated papillary muscle preparations of the tree shrew (Tupaia glis), guinea-pigs and rats. In the guinea-pig and shrew papillary muscles, ouabain at concentrations of 10(-8) to 3 x 10(-7) M caused a concentration-dependent positive inotropic response in a similar magnitude, while in the rat, ouabain at concentrations of 10(-7) to 3 x 10(-6) M elicited negative inotropic one. Either phentolamine or pindolol in a concentration sufficient to block the alpha- and beta-adrenoceptors did not antagonize the positive inotropic effect of ouabain in the papillary muscle preparation of the shrews.

Factors underlying the variable inotropic effect of ouabain on isolated rat atria. Changes in contractile frequency and adrenergic mechanisms.

Abstract The effects of concentrations of ouabain on the isometric developed tension (IDT) of isolated left auricles driven at 0·4, 0·8 and 3·3 Hz, were explored. With 0·4 or 0·8 Hz, increasing concentrations of ouabain (3·4 or 6·8 times 10−6; 1·0; 3·4 or 6·8 times 10−6M) elicited a decreasing enhancement of atrial contractile peak tension, whereas with 3·3 Hz the effect was augmented with concentration. Ouabain (3·4 times 10−6 M) enhanced comparably the IDT at all the driving rates, but at higher concentrations the effect varied with the frequency. The positive inotropic action of ouabain at 3·4 times 10−6 M on preparations driven at faster frequencies was antagonized by Sotalol (MJ-1999) and also after catecholamine depletion by in vivo reserpinization followed by addition of tyramine in vitro. Cocaine or U-0521 significantly enhanced the positive inotropic effect of ouabain (3·4 times 10−5 M) in atria stimulated at low rates. On the contrary, Sotalol, cocaine, U-0521 or catecholamine depletion did not alter the positive inotropic influence of ouabain at 3·4 times 10−6 M at any of the frequencies used. It is suggested that ouabain influenced contractile peak tension of rat isolated atria through two mechanisms: one, seen after higher concentrations, which appeared to be associated with adrenergic factors and dependent on the frequency of stimulation; the other, observable with the lower concentrations, which did not appear to have a direct relationship with adrenergic processes and which was independent of the frequency of stimulation.

Role of the Na+,K+-ATPase α2 isoform in the positive inotropic effect of ouabain and marinobufagenin in the rat diaphragm

It was found that ouabain and marinobufagenin, specific inhibitors of Na+,K+-ATPase, increased the contraction of the isolated rat diaphragm by ∼15% (positive inotropic effect) at EC50 = 1.2 ± 0.3 nM and 0.3 ± 0.1 nM, respectively, which was indicative of the participation of the ouabain-sensitive Na+,K+-ATPase α2 isoform. Analysis of the dose-response curves for the effect of ouabain on the resting membrane potential of muscle fibers in the absence and in the presence of 100 nM acetylcholine (hyperpolarizing the membrane) showed the presence of two pools of Na+,K+-ATPase α2 that differed in affinity for ouabain. Only the high-affinity pool (IC50 ∼ 9 nM) mediates the hyperpolarizing effect of nanomolar concentrations of acetylcholine. Most likely, it is this pool of that is involved in the positive inotropic effect of ouabain, which can be a mechanism of regulation of the muscle function by circulating endogenous inhibitors of Na+,K+-ATPase.

Ouabain increases myofibrillar Ca2+ sensitivity but does not influence the Ca2+ release in human skinned fibres

The present study investigated the impact of the Na+ pump inhibitor ouabain (g-strophanthin) on Ca2+ sensitivity and Ca2+ release in human right auricular trabeculae (coronary bypass) and in skinned muscle fibres from left ventricular myocardium (cardiac transplantation, dilated cardiomyopathy). A time-dependent increase in force of contraction was observed in right auricular trabeculae in response to ouabain (100 nM) before the intracellular Ca2+ transient (fura-2) increased (n=6). In triton X-skinned fibres (no sarcoplasmic reticulum) of human failing myocardium, ouabain (1–100 nM) concentration-dependently increased tension at a free extracellular Ca2+ concentration of 1 μM and the Hill coefficient of the Ca2+-dependent tension development. Ouabain (1–100 nM) did not directly induce a Ca2+ release out of the sarcoplasmic reticulum, nor did it alter the caffeine (10 mM) induced sarcoplasmic reticulum Ca2+ release in saponin-skinned fibre preparations in which the sarcoplasmic reticulum had been Ca2+-loaded.In conclusion, ouabain increases myofibrillar Ca2+ sensitivity possibly due to an increase in the cooperativity of the thick and thin myofilaments. This mechanism may additionally contribute to the positive inotropic effect of ouabain.

Noradrenaline-induced desensitization in cultured heart cells as a model for the defects of the adenylate cyclase system in severe heart failure

Cultivation of rat heart muscle cells for up to 5 days in the presence of 10(-6) mol/l (-)noradrenaline leads to a desensitization of the cells to inotropic stimulation and cAMP formation at different levels of the cAMP-system: The decrease in the responsiveness to the beta-adrenoceptor agonist isoprenaline quantitatively parallels the down-regulation of beta 1-adrenoceptors. The impairment of the positive inotropic effect of the phosphodiesterase-inhibitor IBMX is associated with a diminished basal cAMP-formation but an unchanged phosphodiesterase-activity in noradrenaline-treated cells. This decrease in basal cAMP-accumulation as well as the attenuation of the forskolin-stimulated cAMP-formation indicate that a defect in the adenylate cyclase system beyond the receptor must be involved in noradrenaline-induced desensitization. In contrast to the diminished effectiveness of cAMP-increasing agents the positive inotropic effect of ouabain and the alpha-adrenoceptor-mediated positive inotropic effect of phenylephrine are unaltered in noradrenaline-treated cells. It is concluded from these results that in addition to the down-regulation of beta 1-adrenoceptors a defect on the post-receptor level of adenylate cyclase occurs in noradrenaline-induced desensitization not affecting mechanisms beyond the catalytic subunit of adenylate cyclase. As these findings are very similar to those observed in isolated preparations from severely failing human hearts, the presumption is confirmed that noradrenaline-induced desensitization might constitute an important etiological factor in the subsensitivity of failing human hearts to inotropic stimulation.

Positive inotropic effect of ouabain on isolated heart is accompanied by activation of signal pathways that link Na+/K+-ATPase to ERK1/2.

Exposure of cultured rat cardiac myocytes to ouabain is known to cause the interaction of Na+/K+-ATPase with adjacent proteins, leading to activation of multiple signal transduction pathways, regulation of growth-related genes, and hypertrophy. The aim of this work was to determine if the proximal signaling events identified in cultured myocytes also occur in isolated intact hearts of rat and guinea pig in response to positive inotropic doses of ouabain. Langendorff rat heart preparations were exposed to 50 microM ouabain to produce positive inotropy without toxicity, and assayed for Src kinase, protein kinase C, and extracellular signal-regulated kinases 1 and 2 (ERK(1/2)). These kinases were rapidly activated by ouabain as in cultured cells. In isolated guinea pig hearts, 1 microM ouabain caused ERK(1/2) activation comparable to the effect of 50 microM ouabain in rat heart and consistent with the higher ouabain sensitivity of the contractility of guinea pig heart. These data show that the proximal ouabain-induced signal pathways previously noted in cultured cells are not artifacts of dispersion/culturing of myocytes, and are not the peculiar properties of the rat heart with its relatively low ouabain sensitivity. They also suggest that treatment with positive inotropic doses of cardiac glycosides is likely to be associated with changes in the cardiac phenotype.

Longer term effects of ouabain on the contractility of rat isolated cardiomyocytes and on the expression of Ca and Na regulating proteins.

Cardiac glycosides like ouabain are used in the therapy of heart failure and atrial fibrillation. They exert a positive inotropic effect on cardiomyocytes by inhibiting the plasma membrane sodium pump (Na,K-ATPase), decreasing the Ca-extrusion by the sarcolemmal cardiac sodium/calcium exchanger (NCX) and increasing the intracellular Ca-concentration and Ca-release during subsequent contraction cycles.The longer term effects of ouabain treatment on the expression of proteins important for Ca- and Na-homeostasis are not well known and were investigated in this study. Isolated adult rat cardiomyocytes were cultured in the presence or absence of ouabain (30 microM). In these cells, the expression of the Na,K-ATPase, Na,Ca-exchanger (NCX), the sarcoplasmic reticulum Ca-ATPase (SERCA 2a) and phospholamban (PLB) were studied by Western blot. In addition, the contractile function of these cells was studied after electrical stimulation. After 2 days of ouabain treatment immunoreactivity of the NCX was increased significantly relative to control which was set 1 (1.78 +/- 0.16 vs. 1 +/- 0.13; n = 8; P = 0.003) and at day 4 (1.96 +/- 0.35 vs. 1 +/- 0.20; n = 6; P = 0.02). All other proteins (SERCA 2a, PLB and Na,K-ATPase a1 and b1) remained unchanged (n >/= 4). Ouabain treatment increased the fractional shortening of isolated cardiomyocytes at day 0 (1 Hz: 9.64 +/- 0.73 %, n = 24, vs. 7.18 +/- 0.60 %; n = 21; P = 0.01), whereas at day 2 the contractility was unchanged (1 Hz: 7.23 +/- 1.08 %, n = 9 vs. 7.70 +/- 0.63 %; n = 10, P = 0.71). The inhibition of SERCA 2a (10 microM cyclopiazonic acid (CPA)) decreased contractility in both the ouabain treated group and in controls, at day 0 and at day 2. These results show that chronic ouabain treatment increases the protein expression of the NCX. The positive inotropic effect of ouabain can no longer be observed after a chronic treatment for 2 days. Thus, both protein expression and contractile function of the cells are specifically altered by longer term cardiac glycoside exposure. Whether such regulation can be found in human cardiomyocytes and the resulting consequences in the clinical setting remain to be determined.

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase.

Ouabain binding to Na(+)/K(+)-ATPase activates Src/epidermal growth factor receptor (EGFR) to initiate multiple signal pathways that regulate growth. In cardiac myocytes and the intact heart, the early ouabain-induced pathways that cause rapid activations of ERK1/2 also regulate intracellular Ca(2+) concentration ([Ca(2+)](i)) and contractility. The goal of this study was to explore the role of caveolae in these early signaling events. Subunits of Na(+)/K(+)-ATPase were detected by immunoblot analysis in caveolae isolated from cardiac myocytes, cardiac ventricles, kidney cell lines, and kidney outer medulla by established detergent-free procedures. Isolated rat cardiac caveolae contained Src, EGFR, ERK1/2, and 20-30% of cellular contents of alpha(1)- and alpha(2)-isoforms of Na(+)/K(+)-ATPase, along with nearly all of cellular caveolin-3. Immunofluorescence microscopy of adult cardiac myocytes showed the presence of caveolin-3 and alpha-isoforms in peripheral sarcolemma and T tubules and suggested their partial colocalization. Exposure of contracting isolated rat hearts to a positive inotropic dose of ouabain and analysis of isolated cardiac caveolae showed that ouabain caused 1) no change in total caveolar ERK1/2, but a two- to threefold increase in caveolar phosphorylated/activated ERK1/2; 2) no change in caveolar alpha(1)-isoform and caveolin-3; and 3) 50-60% increases in caveolar Src and alpha(2)-isoform. These findings, in conjunction with previous observations, show that components of the pathways that link Na(+)/K(+)-ATPase to ERK1/2 and [Ca(2+)](i) are organized within cardiac caveolae microdomains. They also suggest that ouabain-induced recruitments of Src and alpha(2)-isoform to caveolae are involved in the manifestation of the positive inotropic effect of ouabain.

Positive inotropic effects of ouabain in isolated cat ventricular myocytes in sodium-free conditions.

The inotropic and toxic effects of cardiac steroids are thought to result from Na(+)-K(+)-ATPase inhibition, with elevated intracellular Na(+)(Na)causing increased intracellular Ca(2+)(Ca) via Na-Ca exchange. We studied the effects of ouabain on cat ventricular myocytes in Na(+)-free conditions where the exchanger is inhibited. Cell shortening and Ca transients (with fluo 4-AM fluorescence) were measured under voltage clamp during exposure to Na(+)-free solutions [LiCl or N-methyl-D-glucamine (NMDG) replacement]. Ouabain enhanced contractility by 121 +/- 55% at 1 micromol/l (n = 11) and 476 +/- 159% at 3 micromol/l (n = 8) (means +/- SE). Ca transient amplitude was also increased. The inotropic effects of ouabain were retained even after pretreatment with saxitoxin (5 micromol/l) or changing the holding potential to -40 mV (to inactivate Na(+) current). Similar results were obtained with both Li(+) and NMDG replacement and in the absence of external K(+), indicating that ouabain produced positive inotropy in the absence of functional Na-Ca exchange and Na(+)-K(+)-ATPase activity. In contrast, ouabain had no inotropic response in rat ventricular myocytes (10-100 micromol/l). Finally, ouabain reversibly increased Ca(2+) overload toxicity by accelerating the rate of spontaneous aftercontractions (n = 13). These results suggest that the cellular effects of ouabain on the heart may include actions independent of Na(+)-K(+)-ATPase inhibition, Na-Ca exchange, and changes in Na.

Preface

We observed that heterozygous knockout (+/-, KO) of either endothelin-A- (ET(A)) or -B- (ET(B)) receptors significantly reduced the pressor responses to systemically administered endothelin-1 (ET-1) in ET(A) or ET(B) (+/-) KO mice when compared to wild-type (WT) mice (data not shown). Also, we observed that basal mean arterial pressure (MAP) is significantly higher in ET(B) (+/-) (92.7 +/- 1.2 mmHg) (n = 53, p < 0.05) but not ET(A) (+/-) KO mice (70.6 +/- 1.8 mmHg) (n = 23) when compared to their anaesthetized WT littermates (70.1 +/- 0.7 mmHg) (n = 118). A 90 min treatment with either BQ-123 (10 mg/kg), an ET(A)-selective antagonist, or BQ-928 (10 mg/kg), a mixed ET(A)/ET(B) antagonist, administered intraperitoneally, significantly reduced basal MAP of ET(B) (+/-) KO mice almost to the level of their WT treated counterparts (94.9 +/- 4.9 mmHg) (n = 6) vs (+ BQ-123: 59.7 +/- 0.3 mmHg, n = 8); (+ BQ-928: 72.4 +/- 2.6 mmHg, n = 5). It is worthy of note that BQ-123 significantly reduced basal MAP in WT mice but to a lesser extent than in ET(B) (+/-) KO mice (69.6 +/- 2.3 mmHg, n = 8) vs (+ BQ-123: 57.3 +/- 1.4 mmHg, n = 8). In contrast, the ET(B)-selective antagonist, BQ-788 (10 mg/kg i.p.), had no significant effect on MAP even after 90 min of treatment (ET(B) (+/-) KO: (92.3 +/- 2.3 mmHg, n = 6) vs (+ BQ-788: 89.7 +/- 3.1 mmHg, n = 6); WT: (70.5 +/- 3.7 mmHg, n = 7) vs (+ BQ-788: 71.2 +/- 2.0 mmHg, n = 6). Therefore heterozygous KO of either ET(A)- or ET(B)-receptors significantly alters the phenotypic pressor properties of ET-1. We also suggest that there is less ET clearance in ET(B) (+/-) KO mice than in WT mice, which can explain the ET(A)-dependent hypertensive state of the former strain.

Intracellular Reactive Oxygen Species Mediate the Linkage of Na+/K+-ATPase to Hypertrophy and Its Marker Genes in Cardiac Myocytes

We showed before that in cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophy and transcriptional regulations of growth-related marker genes through multiple Ca2+-dependent signal pathways many of which involve Ras and p42/44 mitogen-activated protein kinases. The aim of this work was to explore the roles of intracellular reactive oxygen species (ROS) in these ouabain-initiated pathways. Ouabain caused a rapid generation of ROS within the myocytes that was prevented by preexposure of cells to N-acetylcysteine (NAC) or vitamin E. These antioxidants also blocked or attenuated the following actions of ouabain: inductions of the genes of skeletal alpha-actin and atrial natriuretic factor, repression of the gene of the alpha3-subunit of Na+/K+-ATPase, activation of mitogen-activated protein kinases, activation of Ras-dependent protein synthesis, and activation of transcription factor NF-kappaB. Induction of c-fos and activation of AP-1 by ouabain were not sensitive to NAC. Ouabain-induced inhibition of active Rb+ uptake through Na+/K+-ATPase and the resulting rise in intracellular Ca2+ were also not prevented by NAC. A phorbol ester that also causes myocyte hypertrophy did not increase ROS generation, and its effects on marker genes and protein synthesis were not affected by NAC. We conclude the following: (a) ROS are essential second messengers within some but not all signal pathways that are activated by the effect of ouabain on Na+/K+-ATPase; (b) the ROS-dependent pathways are involved in ouabain-induced hypertrophy; (c) increased ROS generation is not a common response of the myocyte to all hypertrophic stimuli; and (d) it may be possible to dissociate the positive inotropic effect of ouabain from its growth-related effects by alteration of the redox state of the cardiac myocyte.

Small doses of canrenone block the effects of ouabain on the mechanical activity of the heart and vessels of the rat.

Canrenone has been described as an antihypertensive drug that blocks endogenous ouabain effects in volume-dependent hypertensive models. Considering that some canrenone metabolites may be putative mutagenic factors, therapeutic dose reduction might be advantageous if the blockade of ouabain effects is maintained. In this study, the effects of low doses or concentrations of canrenone were investigated in rats by using isolated papillary muscles, Langendorff-perfused hearts, perfused rat-tail vascular bed, and anesthetized animals. Canrenone (0.5, 1, 2, and 5 mg/ml) produced a dose-dependent negative inotropic effect in papillary muscles contracting isometrically and blocked the positive inotropic effect produced by 660 microM ouabain. In Langendorff-perfused hearts beating spontaneously, a low concentration of canrenone (10 microg/ml) increased the isovolumic systolic pressure obtained at several diastolic pressures. Higher concentrations of canrenone (20, 30 microg/ml) brought the isovolumic pressure toward control values, and 100 microg/ml canrenone produced an isovolumic pressure reduction. In these preparations, 20 microg/ml canrenone reduced significantly the positive inotropic effects of 100 microM ouabain. Investigating the vascular smooth muscle reactivity to phenylephrine (PE; 0.5, 1, and 2 microg bolus injections) in the perfused rat-tail vascular bed, it was observed that canrenone blocked completely the enhancement of PE pressor effect produced by 1-h treatment with 100 microM ouabain. Similar results were obtained with the arterial blood pressure reactivity to PE in anesthetized rats. In these animals, canrenone (1 mg/kg) blocked the sensitizing effect of 18 microg/kg ouabain on PE reactivity. In conclusion, results presented here suggest that canrenone may block ouabain effects at very low concentrations. It blocked myocardial positive inotropic effects of ouabain on both papillary muscle and perfused hearts, and the sensitization of PE pressor effects. The results also suggest that canrenone at very small doses might be used to reduce arterial blood pressure in hypertensive conditions accompanied by increased ouabain plasma levels as the main therapeutic procedure or as an adjunct treatment to prevent ouabain sensitizing effects on pressor responses.

Dissociation of intracellular sodium from contractile state in guinea-pig hearts treated with ouabain.

The positive inotropic effect of cardiac glycosides has been attributed to inhibition of the Na-K-ATPase, accumulation of intracellular sodium and enhanced calcium availability due to Na-Ca exchange. However, few measurements of intracellular sodium in the functioning left ventricle following ouabain exposure at therapeutic doses are available. Our experimental objective was to quantitate the relationship between contractile state and intracellular sodium measured by 23Na nuclear magnetic resonance spectroscopy or atomic absorption in the intact heart. Isolated guinea-pig hearts, perfused in the Langendorff mode, were paced and then exposed to ouabain (3x10(-7)m) for 30 min. Left-ventricular pressure was monitored continuously. Intracellular sodium was measured either at 1-min intervals throughout the perfusion by shift reagent-aided 23Na nuclear magnetic resonance spectroscopy in the beating heart or following 30 minutes of perfusion by atomic absorption in myocardial tissue. While treatment with ouabain was associated with almost a two-fold rise in developed pressure, there was no significant increase in intracellular sodium measured by either technique. Thus, the positive inotropic effect of ouabain in this model is not associated with significant changes in bulk intracellular sodium. However, these results do not exclude the possibility of shifts between intracellular pools which would not be detected in bulk measurements, or changes in NMR-invisible intracellular pools which are not detectable by single quantum spectroscopy techniques.

Role of the Na+ /K+ /Cl− transporter in the positive inotropic effect of ouabain in cardiac myocytes

In this study we have characterized the bumetanide-sensitive K+/Na+/Cl- cotransport in cultured rat cardiac myocytes. 1) It carries about 10% of the total K+ influx. 2) It is sensitive to furosemide (Ki0.5 = 10(-6)M) and bumetanide (Ki0.5 = 10(-7)M). 3) It is strongly dependent on the extracellular concentrations of Na+ and Cl-. 4) It carries out influx of both ions, K+ and Na+. A therapeutic concentration of ouabain (10(-7) M) stimulated the bumetanide-sensitive K+ influx (as measured by 86Rb+), in the cultured myocytes, with no effect on the bumetanide-resistant K+ influx, which was mediated mostly by the Na+/K+ pump. Stimulation of the bumetanide-sensitive Rb+ influx by a low ouabain concentration was strongly dependent on Na+ and Cl- in the extracellular medium. A low concentration of ouabain (10(-7) M) was found to increase the steady-state level of cytosolic Na+ by 15%. This increase was abolished by the addition of bumetanide or furosemide. These findings suggest that ouabain, at a low (10(-7) M) concentration, induced its positive inotropic effect in rat cardiac myocytes by increasing Na+ influx into the cells through the bumetanide-sensitive Na+/K+/Cl- cotransporter. In order to examine this hypothesis, we measured the effect of bumetanide on the increased amplitude of systolic cell motion induced by ouabain. Bumetanide or furosemide, added to cultured cardiac myocytes, inhibited the increased amplitude of systolic cell motion induced by ouabain. Neither bumetanide nor furosemide alone has any significant effect on the basal amplitude of systolic cell motion. We propose that stimulation of bumetanide-sensitive Na+ influx plays an essential role in the positive inotropic effect in rat cardiac myocytes induced by low concentration of ouabain.

Role of Na‐H exchange in the inotropic action of Bay K 8644 and of ouabain in guinea‐pig isolated atria

1. The inotropic effects of two concentrations of ouabain and of Bay K 8644 have been studied in isolated left atria of the guinea-pig in physiological solutions at pH lowered from 7.4 to 6.0 and in the presence of ethylisopropylamiloride (EIPA) an inhibitor of Na+/H+ exchange. The low concentration of ouabain (300 nM) was chosen to saturate the high affinity binding sites (it occupied about 7% of the low affinity sites). The high concentration of ouabain saturated both high and low binding sites. Bay K 8644 evoked a positive inotropic effect of a magnitude similar to ouabain (300 nM). 2. When comparing the positive inotropic effects of equi-effective concentrations of ouabain (300 nM) and of Bay K 8644 (100 nM), it was observed that extracellular acidification specifically depressed the inotropic effect of ouabain 300 nM; the positive inotropic effect of the high concentration of ouabain (3 microM) was barely affected by extracellular acidification. 3. EIPA 10 microM depressed the positive inotropic effect of ouabain 300 nM, but did not affect the peak response to Bay K 8644. The depressant action of EIPA on the positive inotropic effect of ouabain was concentration-dependent and was much more obvious on the effect of ouabain 300 nm than on ouabain 3 microM. 4. An increase in diastolic tension was evoked by 3 microM but not by 300 nM ouabain. This increase in tone was reduced dose-dependently by EIPA (10-30 microM). It was also significantly reduced when the extracellular pH was equal to 6.4 or 6.0. 5. Ouabain (300 nM) evoked a gain in tissue Na and an equivalent loss in tissue K. Acidification of the extracellular pH down to pH 6.0 evoked a pH-dependent reduction of Na gain but left K loss unaltered. EIPA 10, 20 and 30 microM evoked a significant reduction of Na gain without significantly affecting K loss. 6. Ouabain (3 microM) evoked a large gain in tissue Na and an equivalent loss of K. Tissue Ca content was also increased. Acidification of the extracellular pH from pH 7.4 to pH 6.9 evoked a significant reduction of Na and Ca gain; changes in tissue K were not significant. Acidification down to pH 6.0 increased reduction of Na and Ca gain, but not that of K loss which nevertheless became statistically significant. 7. These results show that tissue Na gain observed after inhibition of the sodium pump by ouabain may be related to activation of Na-H exchange. They also indicate that blockade of Na-H exchange selectively reduced the inotropic effect of ouabain in guinea-pig atria resulting from interaction of the glycoside with high affinity binding sites. This confirms previous observations in rat heart.

Inotropic Effect of Meperidine

Meperidine increases developed force in isolated rat atria. We initiated this study to determine if this positive inotropic effect was attenuated by commonly used receptor- and ion channel blockers. Neither naloxone (opioid blocker), phentolamine (alpha-adrenoceptor blocker), propranolol (beta-adrenoceptor blocker), polaramine (H1-receptor blocker), ranitidine (H2-receptor blocker), verapamil (calcium-channel blocker), nor lidocaine (fast sodium-channel blocker) attenuated the positive inotropic effect of meperidine. However, after lidocaine pretreatment meperidine increased contractile force by 57% (27%-80%) (median and 95% confidence interval), which is significantly more (P less than 0.001) than the 21% (13%-35%) increase seen after pretreatment with saline. After Na,K-pump inhibition by ouabain, meperidine caused no further increase in contractility, but the atria still responded to isoproterenol with an increase in developed force. Conversely, meperidine prevented the positive inotropic effect of ouabain. These findings suggest that the positive inotropic effect of meperidine is mediated by an increase in intracellular sodium activity and not by regulation of the slow inward calcium channel.

Depressant effects of chloroquine on the isolated guinea-pig heart

Some anti-malarials have deleterious effects upon the heart. The actions of two of these, chloroquine and quinacrine, were compared on isolated guinea-pig atria and Langendorff preparations to assess their effects on several calcium pools. Both compounds decreased developed tension in a concentration (chloroquine 10-100 microM; quinacrine 3-100 microM) and time-dependent manner, with quinacrine being twice as potent as potent as chloroquine. Ventricular muscle was much more sensitive to chloroquine than was atrial muscle. Concentrations of chloroquine, comparable to that found in the serum of patients ingesting toxic doses, caused significant inhibition of developed tension. The effects of chloroquine were almost completely reversed on washout; quinacrine, however, was less readily reversible. Chloroquine also had a direct negative chronotropic effect, substantially reduced force-frequency relationships and developed tension in partially depolarized atrial preparations; while post-rest contraction and the positive inotropic effect of ouabain were unaffected. Increases in extracellular calcium antagonized the negative inotropic effect. Quinacrine had a marked effect on post-rest contraction and attenuated the positive inotropic action of ouabain. It is concluded that the action of chloroquine may involve a superficial calcium pool, while quinacrine may act upon several calcium pools.

Metabolic Requirement for the Inotropic Effects of Digitalis and BAY K 8644

The positive inotropic effects of ouabain and of BAY K 8644 are not apparent in rabbit atria suspended in substrate free medium or in a medium containing 5 mM pyruvate. Addition of glucose in graded concentrations (1-11 mM) during continued exposure of the preparations to the inotropic agents yields graded inotropic effects. The possible involvement of the glycolytic pathway to the development of the inotropic effect of ouabain and BAY K 8644 was tested by using inhibitors of glycolysis that act at two different steps. Iodoacetic acid completely blocks the inotropic effect of ouabain in atria at 0.1 mM and papillary muscles at 0.05 mM. The inotropic effect of BAY K 8644 was blocked partially in atria and papillary muscles. Iodoacetic acid did not change the inotropic effects of isoproterenol and Ca2+. Addition of 1 mM fluoride did not affect significantly the inotropic effect of either ouabain or BAY K 8644 in atria but partially blocked the effect of BAY K 8644 in papillary muscles. The response of atria to ouabain was not changed significantly when glyceraldehyde (10 mM) was substituted for glucose. We suggest that glycolytic ATP may be important for the full inotropic effect of ouabain and BAY K 8644.

Modification of the positive inotropic effects of catecholamines, cardiac glycosides and Ca 2+ by the orally active male contraceptive, gossypol, in isolated guinea-pig heart

Gossypol is an orally active male contraceptive with cardio-depressant side effects. To understand the mechanism of its cardiac actions, the interaction of gossypol with positive inotropic grugs was examined in isolated atrial muscle preparations obtained from guinea- pig heart. Gossypol delayed the onset of arrhythmias caused by digoxin. In the presence of gossypol, the positive inotropic effect of isoproterenol declined rapidly, and the effect of isoproterenol to increase tissue cyclic AMP concentrations was smaller. Pretreatment of atrial muscle with the combination of gossypol and isoproterenol markedly reduced effects of isoproterenol on developed tension and cyclic AMP concentrations when these effects were tested after the washout of the first dose of isoproterenol. These effects, however, were not specific to isoproterenol. The gossypol-isoproterenol pretreatment reduced the positive inotropic effect of ouabain or extracellular Ca 2+ These results indicate that gossypol has pharmacodynamic interactions with several positive inotropic agents that are known to enhance developed tension by increasing intracellular Ca 2+ transients.

O-065 - Two classes of ouabain binding sites and biphasic positive inotropic effects of ouabain observed in fitch heart.

O-065 - Two classes of ouabain binding sites and biphasic positive inotropic effects of ouabain observed in fitch heart.