Positive inotropic effect of porcine left ventricular extract on canine ventricular muscle.

1. We previously isolated an extract from porcine left ventricle that possessed digitalis-like properties such as inhibition of cardiac and kidney Na+, K(+)-ATPase, displacement of [3H]-ouabain from its binding sites and cross reactivity with digoxin antibodies. The extract also had a positive inotropic effect on the guinea-pig heart. 2. In the present study the positive inotropic response of the extract was characterized in canine right ventricular trabeculae. Maximum inotropic response (501 +/- 20%) was produced by 300 microliters and the half maximal increase occurred with 125 microliters of the extract. 3. Ouabagenin produced aftercontractions in rapidly paced trabeculae. Equipotent and even greater amounts of the extract did not produce aftercontractions. 4. The extract increased the amplitude of the delayed component (P2) of biphasic contractions produced by replacing about 92-96% of the external Ca with Sr. A smaller increase in the size of the early component (P1) was also seen. 5. The extract decreased post-rest potentiation after rest for 30s and 2 min. After 8 min of rest, post-rest potentiation was converted to post-rest depression. 6. The extract (20 microliters) produced a decrease in the amplitude of the post-rest rapid cooling contracture (RCC) at all rest intervals. The steady state RCC, although greater than that in the control muscle, was increased to a lesser extent than the size of the steady state electrically driven contractions. 7. It is suggested that the extract from porcine left ventricle produces a positive inotropic response by increasing the trans-sarcolemmal influx of Ca. It also has additional effect(s) on the sarcoplasmic reticulum in that it may facilitate the loss of Ca from the sarcoplasmic reticulum and/or inhibit the uptake of Ca by the organelle.

Mechanisms of increased digitalis tolerance in streptozotocin-induced diabetic rat myocardium.

Our earlier studies revealed that the inotropic and cardiotoxic responses to ouabain are depressed significantly in chronically-induced diabetic rats (Navaratnam S and Khatter JC, Arch Int Pharmacodyn 301: 151-164, 1989). In the present study, we examined the Na(-)-Ca2+ exchange mechanism in sarcolemmal membrane vesicles (right-side out orientation) isolated from chronically-induced diabetic rat hearts. The apparent initial rates of Na(+)-dependent 45Ca2+ uptake were substantially lower in vesicles from 6- and 12-week diabetic rat hearts when compared to non-diabetic controls. These rates were reduced further in vesicles of 24-week diabetic rat hearts. Associated with the progressive reduction in initial rates was also a progressive reduction in the maximum amount of 45Ca2+ accumulated by the vesicles. A kinetic analysis revealed a significant reduction in the maximum initial rate of 45Ca2+ uptake (Vmax) in vesicles of 24-week diabetic rat hearts. Affinity for 45Ca2+, however, was the same for both diabetic and control groups. The efflux rate of 45Ca2+ was also depressed in these vesicles, and they retained significantly more 45Ca2+ than controls after 2-4 min of initiation of Na(+)-dependent 45Ca2+ efflux. These data demonstrate that the trans-sarcolemmal Ca2+ flux through Na(+)-Ca2+ exchange is depressed and may explain the observed increase in digitalis tolerance of the myocardium in diabetic rats.

Mechanisms of reduced digitalis tolerance with advancing age in the guinea pig.

In the present study, arrhythmogenic toxicity of cardiac glycoside ouabain was investigated in guinea pigs after intravenous infusion (5 micrograms/kg/min). Guinea pigs of 18-24 months of age required significantly (P less than 0.05) lower doses of ouabain that 3-month-old animals (72 +/- 3 vs 100 +/- 3 micrograms/kg) for the initiation of cardiac arrhythmias. Investigation of Na(+)-Ca2+ exchange in the isolated sarcolemmal vesicles revealed a marked reduction in the Na(+)-dependent Ca2+ uptake. Kinetic analysis of these data has demonstrated a 70% reduction in Vmax and reduced affinity for Ca2+ in vesicles from 18-month-old as compared to 3-month-old guinea pigs. The rate of Na(+)-dependent Ca2+ efflux was also markedly lower in the vesicles of older animals, and the vesicles retained more Ca2+ after 3 min of Na(+)-dependent Ca2+ extrusion than did those from 3-month-old animals. The results suggest that the sensitivity to cardiac glycocide increases with age and may be associated with altered sarcolemmal Na(+)-Ca2+ exchange activity.

Endogenous inotropic substance from heart tissue has digitalis-like properties.

In the past few years, we developed an extraction procedure which we successfully used to isolate a crude fraction containing digitalis-like substance (DLS) from porcine left ventricular tissue. In this study, the crude fraction was found to cross-react with digoxin antibodies and showed immunoreactivity of 4.25 +/- 0.6 ng digoxin equivalent/ml. On further purification of the crude fraction using silica gel G column chromatography, a fraction C was obtained, which was highly positive inotropic on canine trabeculae and it dose-dependently inhibited ouabain sensitive 86Rb+ uptake in rat heart slices. A 50% inhibition of uptake was obtained by 25 microliters of fraction C. Fraction C also inhibited canine kidney Na+, K(+)-ATPase (Sigma, U.S.A.) dose-dependently and a 50% inhibition of this enzyme required 17 microliters of fraction C. Ashing of the fraction C at 500 degrees C resulted in loss of inotropic and enzyme inhibitory activities, indicating an organic nature of the unknown digitalis-like substance.

Endogenous digitalis-like substance in pig left ventricle.

A crude fraction was isolated from pig heart left ventricle (150 g) homogenates after extraction of lipids, chromatographic separation and desalting. The extract contained an ionic content of 0.21, 0.27, 0.33 and 1.7 mM respectively for Mg2+, Ca2+, K+, and Na+. The albumin extract, used as a reference control, contained an ionic content of 0.88 and 2.1 mM respectively for K+ and Na+ and negligible amounts of Mg2+ and Ca2+. The isolated fraction exhibited digitalis-like properties in the inhibition of sarcolemmal Na+, K+-ATPase in a dose dependent manner, the displacement of [3H]-ouabain binding from membrane receptor sites and produced +ve inotropic response in isolated perfused heart in a dose dependent manner. The albumin extract tested in the same manner showed no digitalis-like properties. The ventricular fraction was unable to displace (-) 3H-DHA binding from membrane sites and its inotropic action was not blocked by propranolol. The data suggests that the fraction isolated from pig heart left ventricle contains a substance which has some properties like digitalis.

Incorporation of 3-deoxy-3-fluoro-D-glucose into glycogen and trehalose in fat body and flight muscle in Locusta migratoria.

Flight muscle and fat body extracts from Locusta migratoria were incubated with D-[U-14C]-glucose or D-[3-3H]-3-deoxy-3-fluoroglucose and the products were analyzed. In the case of the latter compound, radio-chromatographic analysis yielded glycogen and trehalose fractions that were shown by 19F nuclear magnetic resonance to contain fluorine. Acid hydrolysis of these fractions liberated tritium labelled 3-deoxy-3-fluoro-D-glucose. In addition to the formation of "fluoroglycogen" and "fluorotrehalose" in these tissue extracts, there was an accumulation of tritium labelled fructose.

Purification and characterization of endogenous digitalis-like substance (DLS) from pig heart.

This study was undertaken to further purify and characterize a crude DLS-containing fraction isolated from left ventricular tissue of pig heart. The crude fraction, on purification, separated into three fractions designated as C1, C2 and C3. Only fraction C1 produced positive inotropic response on isolated perfused guinea pig heart. Fraction C1 specifically inhibited rat heart Na+, K(+)-ATPase, whereas fractions C2 and C3 were found to be non-specific inhibitors. Studies with cardiac Na+, K(+)-ATPase, its interaction in the presence of increasing concentration of [K+] and competitive displacement of [3H]-ouabain binding, demonstrated fraction C1 to have ouabain-like properties. C1 also inhibited dog kidney Na+, K(+)-ATPase activity in a dose-dependent manner and was found to be devoid of catecholamines, peptides and lipids. Fraction C1 was found to be heat sensitive and its inotropic activity was destroyed on ashing at 500 degrees C. These studies provide further evidence for the presence of an endogenous DLS in mammalian heart.

Pharmacological characterization of the activity of endogenous inotropic factor from porcine left ventricle.

We report some of the unique pharmacological properties of a semipurified endogenous inotropic factor (EIF) present in the extract of the porcine left ventricle. EIF produced the following effects: (a) increase in isometric contractile force developed by electrically driven canine right ventricular trabecula, reaching a maximum with 60-100 microliters/ml concentration; (b) inhibition of Na-pump activity in canine portal vein; (c) no digitalis-like cardiac toxicity, e.g., increased diastolic tension or spontaneous diastolic mechanical oscillatory activity, despite inhibition of the sodium pump; (d) a small increase in sarcoplasmic reticular Ca release from the heart but a large increase in transsarcolemmal Ca influx as seen in biphasic contractions, an action similar to that produced by digitalis-like substances; and (e) prolongation of the action potential duration and refractory period of the canine isolated trabeculae. This latter action may confer a unique antiarrhythmic property to EIF.

Digitalis cardiotoxicity: cellular calcium overload a possible mechanism.

Isolated perfused guinea pig (Langendorff) heart was employed to determine if the myocardial mechanical dysfunction (mechanical toxicity) produced by toxic concentration of ouabain (1 microM) was accompanied by alterations in mitochondrial function. Ouabain (1 microM) produces a transient increase in the myocardial contractile force and then a continuous decline in the left ventricular mechanical function. Mitochondria isolated from ouabain perfused hearts showed a significantly higher rate of 45Ca2+ uptake and reduction in oxidative phosphorylation. The rate of ATP generation was reduced by almost 50% at the time of contracture development. Verapamil or nifedipine, when combined with ouabain in the perfusion medium, delayed or abolished the mechanical toxicity in a dose dependent manner. The mitochondria isolated from these hearts demonstrated normal rate of Ca2+ uptake and ATP generation capacity. The data indicate that the cardiac mechanical dysfunction induced by toxic doses of ouabain may be associated with mitochondrial Ca2+ overload and dysfunction and that the Ca2+ channel blockers may have a protective effect.

Mechanisms of developmental increase in the sensitivity to ouabain.

In the present study we examined (1) the arrhythmogenic toxicity of ouabain (5 micrograms/kg/min intravenously) in anesthetized guinea pigs of 1-5 days and 3 months of age, and (2) the state of Na+-Ca2+ in the sarcolemmal vesicles isolated from these age groups. Guinea pigs of 1-5 days old tolerated 75% more ouabain than the young adults without significant alteration in the maximal inotropic response. Sodium-dependent 45Ca2+ uptake was substantially lower in the vesicles isolated from hearts of 3-day-old animals, which was characterized by a lower rate of 45Ca2+ uptake (60% Vmax), as compared to the young adults. There was no significant difference in the affinity for 45Ca2+ (Km). The apparent rate of Na+-dependent 45Ca2+ efflux was also lower in the vesicles from 3-day-old guinea pigs. However, the percent extrusion of 45Ca2+ appeared to be unchanged.

An endogenous positive inotropic factor (EPIF) from porcine heart: its effects on sarcoplasmic reticular (SR) Ca2+ metabolism.

We have isolated an endogenous positive inotropic factor (EPIF) from porcine left heart ventricular tissue, which demonstrated to have only weak digitalis-like properties including the inhibition of myocardial Na+,K(+)-ATPase. EPIF completely lacks digitalis-like toxicity such as after-contractions in larger doses. In our recent studies, we have demonstrated that EPIF produces a decrease in the amplitude of the post-rest rapid cooling contracture which indicated that EPIF may release Ca2+ from the sarcoplasmic reticulum. In the present study, the effects of EPIF were investigated on the Ca2+ uptake and release properties of SR enriched membrane vesicles from rat heart. At pH 6.8 and in the presence of oxalate, EPIF dose-dependently inhibited the ATP-dependent uptake of Ca2+ by SR vesicles. Concentrations as low as 25 ul (in 1 mL uptake medium) of EPIF caused a 45-47% reduction in the uptake of Ca2+ within 3-4 min. Increases in EPIF concentration to 50 ul/mL caused additional reduction of only 15-20% in the uptake of Ca2+. Concentrations of 25 ul/mL of EPIF had little or no effects on passive release of actively loaded Ca2+ in SR vesicles. On doubling the concentrations to 50 ul/mL EPIF, however, enhanced the release of Ca2+ by 25-28% during 1-2 min and 44-48% after 4 min of incubation of Ca2+ loaded vesicles in the release medium. Relatively smaller effects of EPIF on Ca2+ release implies that EPIF may mainly lower the uptake of Ca2+ in SR. This reduced uptake of Ca2+ may be explained by the EPIF-induced inhibition of Ca2+ pump.

Digitalis-induced mechanical toxicity: protection by slow Ca++ channel blockers.

In the in vitro perfusion of the isolated heart, toxic doses of cardiac glycosides produce an inotropic response which is followed by a decline in contractile force and an increase in the resting tension. Several reports in the literature indicate that the subsequent decline in contractile force may be related to cardiac cellular Ca++ overload. The purpose of the present study was to determine if the slow Ca++ channel blockers such as verapamil and nifedipine, which block Ca++ influx through voltage-dependent gated channels, can reduce or prevent the digitalis-induced decline in contractile force (mechanical toxicity). Langendorff preparations of isolated perfused guinea pig heart were used for the present study. The data obtained demonstrate that 1 to 2 microM ouabain in the perfusion medium produced mechanical toxicity in the hearts after an initial inotropic response. Verapamil or nifedipine, when combined with ouabain in the perfusion medium, increased the magnitude of the inotropic response and delayed or abolished the mechanical toxicity in a dose-dependent manner. No changes in the sarcolemmal Na+,K+-adenosine triphosphatase or ouabain binding were observed in the presence of verapamil or nifedipine. The data suggest that simultaneous use of verapamil or nifedipine may protect against digitalis-induced mechanical toxicity.