Effects of naloxone on the mechanical activity of isolated rat hearts perfused with morphine or opioid peptides

In isolated rat hearts, the infusion for 10 min of 10(-10), 10(-8) or 10(-6) M (-)naloxone affected the cardiac function by markedly increasing the coronary pressure and by reducing both the heart rate and the developed tension. A lower dose of (-)naloxone (10(-11) M) or a dose of 10(-6) M (+)naloxone, did not modify the cardiac function. Morphine (10(-6) or 10(-5) M) and 10(-10), 10(-8) or 10(-6) M methionine-enkephalin or leucine-enkephalin, both significantly reduced the coronary pressure of the isolated rat hearts, during the first 4-6 min of perfusion, but the coronary pressure progressively increased above the control value in the last 4 min of perfusion. Each opioid also influenced the mechanical activity of the isolated rat heart, by significantly lowering both the heart rate and the developed tension. (-)Naloxone, at all the doses tested, was only able to antagonise the hypotensive effect induced by the opioids on the coronary pressure and was ineffective in counteracting the negative inotropic and chronotropic effects produced by each opioid. The perfusion in the presence of (+)naloxone (even at a dose of 10(-6) M) did not affect the opioid-induced changes on both the coronary pressure and the mechanical performance of the isolated heart.     

Cardiovascular activities of intravenous methionine-enkephalin-Arg6-Phe7 and methionine-enkephalin-Arg6-Gly7-Leu8 in the conscious dog

The preproenkephalin A molecule from the adrenal medulla contains the opioid peptides methionine-enkephalin (Met-ENK), leucine-enkephalin (Leu-ENK), methionine-enkephalin-Arg6-Phe7 (heptapeptide), and methionine-enkephalin-Arg6-Gly7-Leu8 (octapeptide). In the conscious, chronically instrumented dog, Met-ENK and Leu-ENK simultaneously increase heart rate and systemic arterial pressure following intravenous administration. In 19 of 23 dogs, heptapeptide produced a response identical to Met-ENK and Leu-ENK, which was inhibited by naloxone but unaffected by the dipeptidyl carboxypeptidase inhibitor SQ20881. However, in four dogs, heptapeptide produced only a fall in systemic pressure associated with an increase in heart rate despite characteristic Met-ENK responses in the same dogs; naloxone did not appear to alter this hypotensive response. Octapeptide produced slight increases in systemic pressure and heart rate. These data suggest that heptapeptide may possess intrinsic cardiovascular activity at opiate receptors; however, in certain dogs, non-opiate mechanisms, perhaps histamine release, may predominate.     

Enkephalins and pituitary hormone release: modification of responsiveness to LHRH.

An intraperitoneal injection of leucine-enkephalin into rats stimulates gonadotropin and prolactin release. To elucidate the mechanism of this releasing property of leucine-enkephalin, rat hemipituitaries were incubated with either enkephalin alone or enkephalin in combination with OHRH. Enkephalin alone had no effect on LH or prolactin release in vitro but potentiated the LH response to LHRH. Neither leucine-enkephalin nor LHRH alone had an effect on GH release; however, when combined, a GH response to LHRH occurred. These results suggest that leucine-enkephalin can modify the pituitary responsiveness to certain hypothalamic releasing hormones by a direct pituitary action.     

Distribution of methionine- and leucine-enkephalin within the rat pituitary gland measured by highly specific radioimmunoassays

The distribution of methionine- and leucine-enkephalin within the rat pituitary gland was measured using highly specific antisera in conjunction with purification by high performance liquid chromatography. The highest concentrations were found in the pars intermedia (7 pmole/mg for methionine-enkephalin, 4 pmole/mg for leucine-enkephalin), whilst the pars nervosa contained 2.2 pmole/mg of each and the pars anterior the least (methionine-enkephalin: 0.51 pmole/mg, leucine-enkephalin: 0.36 pmole/mg).     

Neuroimmunomodulation with enkephalins: in vitro enhancement of natural killer cell activity in peripheral blood lymphocytes from cancer patients

To initiate investigations into the effects of enkephalins on immune function in cancer patients, the effect of methionine-enkephalin and leucine-enkephalin on natural killer (NK) cell activity in isolated peripheral blood lymphocytes from cancer patients was investigated. Incubation of lymphocytes with either enkephalin resulted in significant increases in NK cell activity. At effector:target cell ratios of 100:1, 33:1 and 11:1 leucine-enkephalin significantly (p less than 0.05) enhanced NK activity at dilutions of 10(-6), 10(-8), 10(-10), and 10(-14) mg/ml. Similar results were obtained with methionine-enkephalin with the exception that the 10(-6) dilution gave insignificant changes at both the 33:1 and 11:1 cell ratios. The results indicate a difference in dose response to both enkephalins between lymphocytes from cancer patients and normal volunteers.     

Identification of dipeptidyl peptidase III in human neutrophils

We have found activity of dipeptidyl peptidase (DPP) III, one of the most important enkephalin-degrading enzymes in the central nervous system, in human neutrophils. HPLC analysis of the peptide fragments produced by treatment of leucine-enkephalin with isolated neutrophils in the presence of inhibitors of other enkephalin-degrading enzymes revealed that the enzyme in human neutrophils cleaved dipeptides from the NH(2) terminus of leucine-enkephalin, suggesting the presence of DPPIII activity in human neutrophils. Using a specific synthesized substrate and proteinase inhibitors, it was found that the neutrophils have 19.2 +/- 3.6 microM/h/5 x 10(6) cells of beta-naphthylamine for the enzyme. It was also confirmed that spinorphin and tynorphin, both reported to inhibit the activities of enkephalin-degrading enzymes, had potent inhibitory activities (IC(50): 4.0 and 0.029 microg/ml, respectively) against the enzyme. The presence of DPPIII activity in human neutrophils suggests that the biologically active peptides which are associated with enkephalin play a physiological role in regulating enkephalin or inflammatory mechanisms in peripheral tissues.     

Inhibition of cardiac Na+, K+-ATPase activity by dynorphin-A and ethylketocyclazocine

The effect of various opioids on Na+, K+ -ATPase partially purified from rat heart was examined. Dynorphin-A (1-13), dynorphin-A (1-17) and ethylketocyclazocine (EKC), which are k-type opiate agonists, markedly inhibited the enzyme activity in a dose-dependent manner; IC50 values were 12 microM, 21 microM and 0.38 mM, respectively. Morphine (mu-type agonist), methionine- and leucine-enkephalin (delta-type agonist) at the concentration of 1 mM did not affect the enzyme activity. The effect of dynorphin-A (1-13) and EKC was not antagonized by naloxone. Dynorphin-A (1-13) mainly decreased Vmax value without the change of Km value in the activation of Na+, K+-ATPase by ATP, Na+ and K+. Dynorphin-A(1-13) inhibited the partial reactions of Na+, K+-ATPase at the different degree of the potency; the inhibition of K+-stimulated phosphatase was greater than that of Na+-dependent phosphorylation. The present study suggests that dynorphin-A and EKC have an effect on cardiovascular system which is mediated by the inhibition of Na+, K+-ATPase in the heart.     

Inhibitory action of opioid peptides on ouabain-sensitive Na+-K+ and Ca2+-dependent ATPase activities in bovine cardiac sarcolemma

The present study demonstrates that morphine (10(-6) and 10(-5) M), methionine-enkephalin or leucine-enkephalin (10(-10), 10(-8), and 10(-6) M) were able to inhibit significantly, in a dose-dependent manner, both the sarcolemmal Ca2+-dependent ATPase and the ouabain-sensitive Na+-K+ ATPase activities. The inhibitory action of these opioids on the two ATPases was not antagonized by preincubation with naloxone (10(-6) M). Naloxone alone (10(-8), 10(-6) and 10(-5) M) did not affect both the sarcolemmal Ca2+-dependent ATPase and the ouabain-sensitive Na+-K+ ATPase activities. Heat-denatured methionine-enkephalin (10(-6) M) or leucine-enkephalin (10(-6) M) also unaffected both the ATPases. The possibility is also discussed that opioid peptides may regulate myocardial contractility by modulating the movement of ions across the heart sarcolemma.     

Effects of ischemia on the metabolism of cardiac enkephalins

The effect of ischemia on cardiac Leucine enkephalin (Leu-enk) content, degradation and coronary release was studied in the isolated perfused hearts of male Sprague Dawley rats. Hearts were electrically stimulated at 180 beats/min. Cardiac Leu-enk concentrations were increased when hearts were perfused (635 +/- 41 vs 301 +/- 60 fmol/g in control non-perfused hearts,) or during ischemia-reperfusion (520 +/- 78 vs 277 +/- 42 fmol/g in heart submitted to ischemia alone). The quantity of leucine-enkephalin released by the heart during perfusion was four times higher than the initial content measured in the heart tissue. The rate of this release was the same throughout the experiment (25.9 +/- 2.9 fmol/min/g during perfusion vs. 19.2 +/- 1.6 during ischemia-reperfusion). These findings suggested that cardiac enkephalin metabolism is regulated by cardiac events. In fact, enzymes involved in enkephalin degradation were decreased during perfusion (39%) and increased during ischemia (50%). The decrease in the enzyme activity during coronary perfusion depended on a reduced activity in the membrane fraction only while membrane and soluble fractions were interested in the increased enzyme activity after ischemia. Ischemia-reperfusion induced a larger release of Leu-enk than perfusion without ischemia. In view of the protective actions of enkephalin peptides against oxidative stress, we can infer from our results an implication of Leu-enk in ischemia-reperfusion and thus eventually in preconditioning phenomenon.     

Nonadrenergic modulation by clonidine of the cosecretion of catecholamines and enkephalins in adrenal chromaffin cells

Cultured bovine chromaffin cells cosecrete catecholamines and enkephalins following cholinergic nicotinic stimulation. Initial reports on the inhibitory effect of clonidine on catecholamine secretion raised the possibility of a modulation of chromaffin cell function through a presynaptic adrenergic mechanism. The purpose of this work was to investigate the pharmacological characteristics of this inhibitory effect of clonidine on the cosecretion of catecholamines and enkephalins in 4-day-old cultured chromaffin cells. We observed that clonidine completely inhibits nicotine-stimulated secretion of both leucine-enkephalin and catecholamines with an IC50 of 34 microM. Treatment of chromaffin cells for 3 days with 100 nM reserpine leads to a 67% increase in nicotine-stimulated secretion of leucine-enkephalin without any effect on the IC50 of clonidine. In reserpine-treated chromaffin cells, norepinephrine (100 microM) inhibits only by 27% nicotine-stimulated secretion of leucine-enkephalin with an IC50 of 50 microM. Neither the alpha 2-adrenergic antagonist yohimbine nor the alpha 1-adrenergic antagonist prazosin could fully reverse the inhibitory effect of clonidine on leucine-enkephalin secretion at 10 nM. These results tend to rule out the role of alpha-adrenergic receptors in the mediation of clonidine inhibition of cosecretion in chromaffin cells.     

Participation of leu-enkephalin in regulating carbohydrate metabolism

The experiments have been performed on 166 white male rats with a mass of 180-220 g. It has been revealed that leucine-enkephalin and its synthetic analogs prevent an increase in glucose blood levels and a decrease in glycogen liver levels caused by adrenaline and parathyroid hormone. At the same time the enkephalins inhibit the secretory activity of pancreatic beta-cells. The mechanisms of opioid peptide effect on carbohydrate metabolism are discussed.     

Chromatographic analysis of the enkephalin immunoreactivity in the nucleus locus coeruleus of the cat after local colchicine administration

Cell bodies immunoreactive for methionine- and leucine-enkephalin are found in the area of the locus coeruleus (dorsolateral pons) of the cat after injection of colchicine in the ascending projections of the nucleus. Using radioimmunoassay procedures, it is shown that colchicine induces a significant increase in methionine- and leucine-enkephalin-immunoreactive material in this area of the brain. High pressure liquid chromatography analysis demonstrated that the immunoreactive materials were authentic methionine- and leucine-enkephalin. The methionine- and leucine-enkephalin patterns were identical in the colchicine injected and non-injected sides of the dorsolateral pons. It is suggested that, in this area of the brain, colchicine (i) does not significantly modify the processing of proenkephalin to form the pentapeptides methionine- and leucine-enkephalin, and (ii) does not induce the appearance of new substances reactive to the enkephalin antisera employed.     

Isolation and structure identification of a morphine-like peptide "enkephalin" in bovine brain.

The ability of bovine brain extracts to compete in a selective fashion for opiate receptor binding is attributable to a small peptide. The substance has been purified to homogeneity and identified as comprising two penta-peptides HTyrGlyGlyPheLeuOH (Leucine-enkephalin) and HTyrGlyGlyPheMetOH (methionine enkephalin). Bovine brain contains 4 times as much leucine-enkephalin as methionine-enkephalin in contrast to pig brain in which these ratios are reversed. Competition for opiate receptor binding by leucine-enkephalin is reduced more by sodium and enhanced more by manganese than is the case for methionine-enkephalin, suggesting that leucine-enkephalin may be a “purer” agonist than methionine-enkephalin.     

Electrophysiology of the flounder heart (Platichthys flesus)—the effect of agents which modify transmembrane ion transport

1. A sucrose gap system was used to record action potentials and mechanical responses of flounder heart.2. Diltiazem eliminated mechanical responses and strongly inhibited the action potential plateau while nifedipine only slightly reduced cardiac contractions without significantly changing the action potential.3. Verapamil slightly hyperpolarized flounder heart but was without effect on either the action potential or mechanical activity except at very high concentrations.4. Lanthanum was ineffective at 2 mM on flounder heart, but manganese at 3 mM substantially inhibited electrical and mechanical responses accompanied by a small hyperpolarization. Substitution of manganese for calcium abolished all flounder cardiac activity.5. BAY K 8644 enhanced cardiac force and enhanced the action potential plateau while depolarizing the preparations. Calcium-free salines abolished heart contractions and the action potential plateau while the spike phase persisted.6. Low sodium salines enhanced while sodium-free salines abolished all heart activity as did tetrodotoxin above I μM. Tetrodotoxin abolished the action potential spike leaving only a small plateau phase.7. Substituting lithium for sodium hyperpolarized the heart, enhanced contractions and prolonged the action potential plateau. Ouabain enhanced cardiac activity and depolarized the heart but ferosemide was without effect on either electrical or mechanical activity.8. TEA at 6 mM had a modest positive inotropic effect and negative chronotropic effect on the heart while the action potential plateau phase was enhanced.9. These results indicate that extracellular sodium and calcium are crucial in flounder heart electrogenesis but such a major role for potassium could not be established.     

Mechanisms of electromechanical function disturbances in cardiomyocytes overloaded with calcium. The theoretical study

Arrhythmias and mechanical disturbances are simulated in a mathematical model of cardiomyocyte electromechanical activity. The simulated pattern is similar to that observed for acute heart failure associated with calcium overloading of myocardium cells. Special attention was paid to the calcium overloading resulting from the reduced Na+,K+ pump activity. In the framework of the model, it was shown that mechanical factors could promote arrhythmia initiation when the pump activity reduced. Different approaches to electrical and mechanical function restoration during acute heart failure associated with calcium overloading were suggested and analyzed in the model.     

Intraventricular self-administration of leucine-enkephalin by laboratory rats

Naive laboratory rats, without pre-exposure to operant training procedures or to opioids, were shown to self-administer directly into their cerebral ventricles the endogenous opiate peptide, leucine-enkephalin. They were shown to self-administer the peptide consistantly for six consecutive days with no indication of the development of tolerance. The results indicate that leucine-enkephalin may possess potent reinforcing properties and suggests that it may play a role as an endogenous reward transmitter.     

Enkephalin-binding protein in human blood

An enkephalin-binding protein was found in human plasma and serum. The protein was partially purified by DEAE-cellulose column chromatography. The binding of [3H]leucine-enkephalin to this protein was competitively inhibited by unlabeled leucine- and methionine-enkephalin and various peptide hormones such as beta-endorphin and glucagon, but not by Leu-enkephalin-amide. The fact that amide derivatives of leucine-enkephalin and methionine-enkephalin did not inhibit the binding suggests that c-terminuses of enkephalins might have an important part in binding the protein. From these results, physiological roles of the enkephalin-binding protein are discussed.     

Effect of mechanical oscillations in physiological solution on the contractile activity of the snail heart

The effect of perfusate on the contractile activity of an isolated internally perfused heart of Helix pomatia was studied. The changes in heart activity induced by the switching of the perfusate stream were more pronounced in a potassium-free solution when the Na+, K(+)-pump was inactivated. It was found that the decrease in the amplitude of contractions of snail heart by acetylcholine (5.10(-9) M) depends on the treatment of perfusate (Ringer solution) by mechanical vibrations (4, 10, 20, and 50 Hz; 90 dB). In the solution treated with 4 Hz mechanical vibrations, the inhibiting effect of acetylcholine decreased. A similar effect was observed after inactivating the Na+, K(+)-pump by ouabain (10(-4) M). Upon treating the solution by 10, 20, and 50 Hz mechanical vibrations, these changes were not observed. Based on the data, it is suggested that the water medium of the cell can serve as a target through which mechanical vibration can affect the cascade of cell metabolic processes.     

Heart action of the freshwater bivalve Anodonta anatina during activity.

1. Heart action of Anodonta anatina (L.) was investigated by recording the electrocardiogram (ECG), heart impedance, and ventricular and pericardial cavity pressure during different aspects of the normal behaviour. The contribution of mechanical and nervous mechanisms in controlling changes in heart action is discussed. 2. Pressure recordings were generally more reliable than the other methods and it is suggested that pericardial pressure pulses indicate the stroke volume output of the ventricle. 3. During spontaneous periods of prolonged shell closure there was an initial small increase in heart activity followed by a large reduction in both heart rate and systolic pressure, indicating that total heart output was considerably reduced. When the shell reopened, heart rate increased very rapidly with an initial overshoot of the normal level; systolic pressure increased more slowly with no overshoot. 4. These major changes in heart activity appear to be associated with respiratory changes and are controlled largely by the nervous regulatory system, but some minor rhythmic variations in the amplitude of heart beat are probably caused by mechanical factors. 5. Characteristic patterns of change in heart action were recorded during burrowing. These appear to result from haemodynamic changes associated with the muscular movements of the digging cycle. Control of the heart by the nervous regulatory system is apparently of much greater importance in relation to respiratory control than in relation to the haemodynamic functioning of the fluid-muscle system in locomotion.     

Dynorphin-Related Opioid Peptides in the Neurointermediate Pituitary of Rats Are Not α-N-Acetylated

Immunoreactive dynorphin in the neurointermediate pituitary of rats was found to consist of four different molecular weight forms. The three larger molecular weight forms, with apparent molecular weights of 4800, 3200, and 1700, constituted more than 80% of the total dynorphin immunoreactivity, and each liberated leucine-enkephalin but not alpha-N-acetyl-leucine-enkephalin upon enzymatic treatment with trypsin followed by carboxypeptidase B. Only a minor portion of the smallest dynorphin-related molecular weight form, dynorphin-(1-8), released alpha-N-acetyl-leucine-enkephalin upon enzymatic cleavage. This suggests that the vast majority of dynorphin-related peptides in the rat neurointermediate pituitary is not alpha-N-acetylated. The exceptionally high opiate-like activity of the molecular weight 1700 dynorphin suggests that this dynorphin-related opioid peptide may constitute the major part of opioid activity in the neurointermediate pituitary of rats.