Hemorrhage alters plasma and cardiac enkephalins and catecholamines in anesthetized dogs.

Intrinsic cardiac enkephalins participate in circulatory regulation either through the modification of vagal control or vasomotor sympathetic control. We extracted, chromatographed, and assayed plasma and myocardial enkephalins from anesthetized dogs under control conditions and during hemorrhagic hypotension (2 h at 40 mmHg). Blood samples were collected at intervals during the experiment. Blood gases were stable, pH declined to 7.1, and heart rate rose. Plasma catecholamines increased and remained high throughout hypotension. Catecholamine and enkephalin immunoreactivities (ir) were unchanged in time controls. Plasma methionine-enkephalin (ME) and peptide F increased twofold and methionine-enkephalin-arginine-phenylalanine (MEAP) and peptide B increased 10- to 30-fold during hypotension. Plasma proenkephalin (ProEnk) and other large enkephalins were unchanged during hypotension. Myocardial norepinephrine was greater in the atria and both atrial and ventricular contents were decreased after hypotension. ProEnk and peptide B accounted for > 60% of the cardiac enkephalins, and their ventricular concentrations were three to four times atrial concentrations. Myocardial MEAP concentrations were 15-25 times the ME concentrations in the same tissue extracts. Hypotension increased myocardial peptide B and ProEnk, and ME, MEAP, and peptide F were unchanged. The data demonstrate a preferential processing to or retention of MEAP rather than ME-ir enkephalins in the heart. The data also indicate that myocardial MEAP-ir enkephalins respond to changes in the circulatory environment and appear in the plasma during hemorrhagic hypotension.

Intrinsic cardiac enkephalins inhibit vagal bradycardia in the dog.

Met-enkephalin-Arg-Phe (MEAP) has been identified in acid extracts of canine heart tissue. The effects of synthetic MEAP on the vagal control of heart rate were investigated in anesthetized dogs. The arterial infusion of MEAP (3 nmol.min-1.kg-1) inhibited the bradycardia observed during electrical stimulation of the right vagus nerve by 72%. After the infusion was stopped, the responsiveness to vagal stimulation returned to normal, with a half-time between 2 and 3 min. The inhibition by MEAP was reversed by the high-affinity opiate antagonist diprenorphine (100 micrograms/kg). MEAP did not alter the negative chronotropic effect of the direct-acting muscarinic agonist methacholine. This observation suggested that MEAP exerted its effect at a site in the efferent vagal tract proximal to nodal muscarinic receptors. Increasing MEAP infusions (0.09-3.00 nmol.min-1.kg-1) produced a graded suppression of vagal bradycardia, with a half-maximal effect near 0.3 nmol.min-1.kg-1. Met-enkephalin (ME) produced responses very similar to those obtained with MEAP. The effects of ME were also blocked by prior administration of diprenorphine. Dose responses to ME were shifted to the right of those for MEAP, and half-maximal responses for ME were obtained at two to four times the dose required for MEAP. The data suggest that the intrinsic cardiac enkephalin MEAP can regulate vagal control of heart rate at physiologically achievable concentrations and may serve as a local regulator of the parasympathetic-myocardial interface.

Methionine-enkephalin-Arg-Phe immunoreactivity in heart tissue.

Previous findings of enkephalins in cardiac tissue led us to investigate enkephalin distribution in animal models used for cardiovascular research. Canine cardiac methionine-enkephalin (ME) concentrations are low and evenly distributed between atria (4.2 +/- 0.6 fmol/mg protein, n = 30) and ventricles (4.4 +/- 0.5). In contrast, methionine-enkephalyl-arginyl-phenylalanine (MEAP) immunoreactivity (IR) is higher and preferentially concentrated in the ventricle (112 +/- 12) vs. the atria (23.2 +/- 2.6 fmol/mg protein). HPLC analysis suggests the atrial/ventricular difference is partly due to altered posttranslational processing. Nearly 90% of ventricular IR is comprised of MEAP (46%) and peptide B (40%) whereas these peptides represent less than half of the atrial content. A nonneuronal localization is indicated because the peptide distribution does not correspond to the catecholamine distribution. Canine left ventricular tissue sections were processed for immunohistochemistry with the MEAP antibody. Fluorescence was distributed throughout the myocytes and concentrated in ordered lines perpendicular to the myocyte longitudinal axis corresponding to the area of the intercalated disc. This suggests opioids may be important in communication between cardiomyocytes, and possibly the presence of a unique peptide secretory mechanism utilizing the intercalated disc. The relative peptide content in cat and pig hearts was similar to the dog; however, the distribution was different. Feline cardiac ME content was distributed 2:1 in favor of the ventricles and corresponded with a preferential ventricular norepinephrine distribution. The MEAP-IR pattern gave a ventricular/atrial ratio lower (3.5:1) in cat heart vs. dog (5:1). In contrast, pig heart ME and MEAP-IR ventricular/atrial ratios were reversed for both ME (1:10) and MEAP (1:2). HPLC of pig left ventricle showed that MEAP and peptide B represented 33% and 39% of the MEAP-IR, respectively. These species variations may correlate to the differences observed in cardiac function.

(+)Naloxone potentiates the inotropic effect of epinephrine in the isolated dog heart.

Naloxone potentiates the inotropic effect of circulating catecholamines in the isolated canine heart. The stereospecificity of this response was evaluated with the aid of the less active (+)enantiomer of naloxone. The more common (-)isomer of naloxone increased the contractile response to epinephrine only at the higher dose tested (4 mg). This effect of naloxone was not observed at a tenfold lower dose (0.4 mg), indicating a very narrow dose-response range. (+)Naloxone was effective at the lower dose and was, therefore, equal to or better than (-)naloxone in potentiating the inotropic effect of epinephrine. When introduced afterward, (-)naloxone did not add to the effect of (+)naloxone. These data suggest that naloxone modifies cellular responsiveness to catecholamines through a nontraditional opiate receptor, through a nonopiate receptor, or through a nonreceptor mechanism.

Arteriovenous-shunt-mediated increase in venous return causes apparent right coronary arterial autoregulation.

OBJECTIVE: Reports of autoregulation in the right coronary vasculature have varied from non-existent to almost perfect. At least some of this discrepancy may be due to failure to account for changes in myocardial metabolism secondary to the method used to vary perfusion pressure. The aim of this study was to determine if the potent autoregulation reported when right coronary perfusion pressure was lowered by opening a large arteriovenous shunt was due to increased right ventricular myocardial oxygen consumption (MVO2) induced by augmented preload and afterload. METHODS: Two protocols were used to produce right coronary perfusion pressures of 100, 80, and 60 mm Hg in anaesthetised dogs. In both protocols the right coronary artery was cannulated and supplied with blood from a pressurised chamber. In protocol 1, right coronary perfusion pressure was decreased independently of aortic pressure, and in protocol 2, aortic pressure was decreased in parallel with right coronary perfusion pressure by opening a large arteriovenous shunt. Right coronary blood flow, central venous pressure, and pulmonary arterial pressure were measured, and right ventricular oxygen extraction and MVO2. Central venous pressure (right ventricular preload) and pulmonary arterial pressure (right ventricular afterload) did not change. In protocol 2, opening the arteriovenous shunt increased venous return, as shown by increased central venous pressure and pulmonary arterial pressure. This increased right ventricular MVO2 at the lower right coronary perfusion pressures and maintained right coronary blood flow at the level recorded when right coronary perfusion pressure was 100 mm Hg. CONCLUSIONS: This apparently potent autoregulation resulted from the shunt induced increase in oxygen consumption at low right coronary perfusion pressures, in contrast to the decreased right ventricular oxygen consumption and right coronary blood flow observed when right coronary perfusion pressure is selectively decreased.

Dynorphin, naloxone, and overflow of norepinephrine during cardiac nerve stimulation in dogs.

The effects of dynorphin-(1-9) and naloxone on norepinephrine (NE) overflow and myocardial contractility were determined during left cardiac nerve stimulation in the anesthetized dog. Stimulation-induced increases in NE overflow from the left ventricle were monitored during control conditions, during infusion of dynorphin-(1-9), during dynorphin plus naloxone, and after naloxone alone. Four electrical stimulations were applied for 1 min at 20-min intervals. Repeated left cardiac nerve stimulations (control group) reduced stimulated NE overflow 50-60% by 1 h. If stimulations were only conducted at 0 and 1 h, the decline in NE overflow was not observed. Intracoronary dynorphin (2 nmol.min-1.kg-1, 20 min) lowered the stimulation-induced increase in NE overflow further and reduced first time derivative of left ventricular pressure (dP/dt) and myocardial O2 consumption responses. Naloxone (100 micrograms/kg) prevented all of the dynorphin-mediated effects. When given alone, naloxone increased both NE overflow and left ventricular dP/dt during stimulation and prevented or significantly delayed the gradual decline in overflow observed in stimulated controls. A postjunctional effect of dynorphin was evaluated by comparing contractile responses to the intracoronary infusion of NE before and during dynorphin. Dynorphin did not alter contractile function at rest or during NE infusion. In summary, dynorphin-(1-9) depresses nerve stimulation-induced, cardiac NE overflow, and myocardial contractility in a naloxone-reversible fashion. Alone, naloxone appears to regulate stimulated NE overflow through a qualitatively different mechanism. Endogenous opioids may normally moderate myocardial function during cardiac nerve stimulation by regulating junctional NE concentrations through a combination of effects on NE release and/or its subsequent reuptake.

Screening for opioids in dog heart.

Dog hearts divided into right and left atria, right and left ventricles and intraventricular septum were homogenized in acid for extraction. Total opioids, and specific peptides (methionine-enkephalin, methionine-enkephalin-arg6-gly7-leu8) were determined by radioreceptor and radioimmunoassay, respectively. Catecholamines were quantitated amperometrically following HPLC. The effects of anesthetic agents (pentobarbital, alpha-chloralose), hemorrhage and ganglionic blockade (hexamethonium and atropine) were evaluated. Total opioids, enkephalins and epinephrine were distributed uniformly throughout the myocardium, while norepinephrine was preferentially concentrated in the atria. Immunoreactive methionine-enkephalin accounted for only 1 to 2% of the total cardiac opioids estimated by radioreceptor assay. Hemorrhage lowered methionine-enkephalin content throughout the myocardium with no significant effect on total opioids or catecholamines. Ganglionic blockade increased total opioid, methionine-enkephalin-arg6-gly7-leu8 and catecholamine content without altering methionine-enkephalin content. HPLC of left ventricular extracts demonstrated that 50% of met-enkephalin-immunoreactivity eluted at retention times equal to synthetic metenkephalin. In summary, there appears to be substantive opioid concentrations within canine myocardium which respond to physiological and pharmacological interventions. These cardiac opioid responses do not parallel changes observed for catecholamines under the same conditions.

Enkephalin lowers vascular resistance in dog hindlimb via a peripheral nonlimb site.

The intravenous administration of methionine enkephalin in anesthetized dogs produces an abrupt decline in mean arterial pressure, left ventricular pressure, and the maximal rate of left ventricular pressure development. All of these changes are prevented by receptor blockade with the opiate antagonist, naloxone. To evaluate peripheral vascular contributions to these responses, experiments were conducted in a constant pressure-isolated perfused hindlimb. In this model, the sharp decline in mean arterial pressure associated with enkephalin injection (5 micrograms/kg iv) coincided with an equally sharp decline in vascular resistance (rise in blood flow) in the hindlimb. Both were blocked by naloxone pretreatment (1 mg/kg). When equal doses of enkephalin were administered directly into the femoral inflow (external iliac artery), both arterial pressure and hindlimb flow responses were all but eliminated. This observation ruled out significant direct vascular interactions in the response and indicated a site of action outside the hindlimb. Additional catheters were placed in the bracheocephalic artery and descending aorta to permit the comparison of arterial injections conducted, respectively, into the cerebral or abdominal circulations. Injections introduced into the descending aorta consistently produced the greatest response, followed by injections (in descending order of effectiveness) into the jugular, the brachiocephalic, and external iliac. The response in the hindlimb vasculature was initiated at a site somewhere between the diaphragm and terminal aorta. The vascular response to enkephalin was subsequently eliminated by blocking ganglionic transmission with the nicotinic antagonist mecamylamine. These observations suggest that the opioids probably interrupt local vasomotor traffic via opiate receptors in regional sympathetic ganglia or in the spinal cord.

Coronary and myocardial adaptations to high altitude in dogs.

The objective of this study was to determine whether exposure to high altitude (hypoxic hypoxemia) induces coronary and/or collateral growth. Fourteen mongrel dogs were maintained at a simulated altitude of 18,000 ft for 1 mo and 7 dogs maintained for 3 mo. Within 2 days after their sojourn, the following data were obtained at ambient pressure: pulmonary, right heart chamber, and wedge pressures as well as cardiac output. On an isolated heart preparation, coronary and collateral flows were determined; each vessel was injected with a different color tracer; and the heart was sliced, separated by perfusion territories, and examined for myocardial hypertrophy. We found that pulmonary artery pressures in altitude-adapted animals were higher compared with controls, and coronary flow per gram was increased after 1 mo of exposure but not different from control after 3 mo. Collateral flows were not significantly different from that of control animals, and biventricular hypertrophy occurred with right ventricular dominance. Comparing these results with those that we obtained previously from anemic animals, we favor the hypothesis that oxygen availability rather than blood flow velocity is most likely linked to vascular growth.

Naloxone enhances cardiac contractile responses to epinephrine without altering epinephrine uptake from plasma.

Naloxone potentiates the inotropic effect of selected beta-agonists in the canine isolated heart. This could be accomplished by elevating circulating catecholamines through a reduction in their disposal or by the facilitation of events at or subsequent to the cardiac beta-receptor. To evaluate the first hypothesis, epinephrine was infused intravenously into a blood-perfused isolated heart-lung preparation. Catecholamines were determined and myocardial and pulmonary epinephrine uptakes were calculated. Naloxone enhanced the inotropic effect (peak +dP/dt) during epinephrine infusion. Coronary blood flow and coronary venous epinephrine concentrations were also elevated after naloxone. Calculated myocardial and pulmonary uptake of epinephrine were, however, unaltered by naloxone. The increased coronary sinus epinephrine after naloxone was evaluated further in experiments redesigned to eliminate the influence of changing coronary blood flow. Epinephrine was infused into the left common coronary and coronary blood flow as maintained constant, 100% above the resting flow rate. Naloxone enhanced the contractile response to epinephrine without altering coronary artery or coronary sinus epinephrine concentrations or myocardial epinephrine uptake. By comparison, corticosterone, an extra-neuronal uptake inhibitor, also potentiated the inotropic effect of infused epinephrine under identical conditions. However, corticosterone was accompanied by a significant increase in coronary sinus epinephrine concentration and a decrease in myocardial epinephrine uptake. We therefore concluded that the ability of naloxone to enhance the inotropic effect of epinephrine is not mediate through an increase in plasma epinephrine concentration secondary to a decrease in the disposal of circulating catecholamines.

Mechanism of attenuated pressure-flow autoregulation in right coronary circulation of dogs.

Right coronary autoregulation was assessed in 14 open-chest, anesthetized dogs. In Group 1 (n = 5), the left common and right coronary arteries were cannulated and perfused independently. As coronary perfusion pressures varied simultaneously between 70 and 120 mm Hg, right coronary blood flow changed by 48%, whereas left coronary flow changed by 13%. In this pressure range, the autoregulatory closed-loop gain of the right coronary circulation averaged 0.37 +/- 0.01, reflecting a modest autoregulatory capability but significantly less than that of the left coronary circulation, 0.78 +/- 0.08. In Group 2 (n = 9), only the right coronary artery was perfused, and right coronary venous blood was collected for determining arteriovenous oxygen extraction. Autoregulatory gain was similar to that of Group 1, indicating that collateral flow associated with intercoronary pressure gradients does not mask right coronary autoregulation. Right ventricular myocardial oxygen consumption varied directly with perfusion pressure, ranging from 7.1 +/- 1.0 to 2.9 +/- 0.8 ml O2/min/100 g as pressure was reduced from 160 to 40 mm Hg. Thus, right coronary autoregulation is masked by an opposing change in oxygen demand. When right ventricular oxygen consumption was altered by pacing, a linear flow-oxygen consumption relationship was observed (8.2 +/- 0.4 ml/min/100 g per ml O2/min/100 g). Subtraction of flows associated with pressure-induced changes in metabolism revealed a potential autoregulatory capability of the right coronary circulation similar to that manifested by the left coronary circulation.

Naloxone enhances myocardial responses to isoproterenol in dog isolated heart-lung.

Intracoronary injection of naloxone produces rapid local increases in myocardial performance. The role of beta-adrenergic activation in this response was investigated. Naloxone was injected into the left anterior descending artery (LAD) of anesthetized dogs with the adjacent circumflex territory serving as control. Naloxone increased the contractile state locally in the LAD region. Pretreatment with propranolol eliminated the regional inotropic response. An isolated heart-lung model was set up, and isoproterenol dose responses were determined. When repeated after naloxone, contractile (dP/dt) responses to isoproterenol were both augmented and prolonged. In a third study imipramine was used to determine whether naloxone might act by reducing neuronal uptake of the isoproterenol. Imipramine extended the effect of isoproterenol temporally but did not alter the peak response. Adding naloxone increased the peak response to isoproterenol and maintained it above the extended imipramine response. The data support the concept that the blockade of opiate receptors facilitates adrenergic activity mediated by myocardial beta 1-receptors 1) directly through postsynaptic mechanisms, 2) indirectly through beta 2-mediated release of norepinephrine, or 3) via interference with nonneuronal disposal mechanisms.

Local endogenous opiate activity in dog myocardium: receptor blockade with naloxone.

The participation of endogenous opiates in myocardial performance and coronary blood flow was investigated. Heart rate, left ventricular contractile force (LVCF), left coronary blood flow (LCBF), and left ventricular oxygen extraction were monitored in anesthetized dogs before and after intracoronary opiate receptor blockade with naloxone. LVCF consistently increased in a dose-dependent fashion following intracoronary naloxone. The increasing LVCF was accompanied by significant increases in LCBF and myocardial oxygen consumption, without changes in heart rate. Rapid onset of responses suggested the presence of endogenous opiates operating locally within the myocardium. Similar effects did not follow right atrial injection of naloxone, ruling out a systemic mechanism. Furthermore, naloxone injected into the isolated left anterior descending artery selectively increased contractile force in that perfusion territory while the adjacent untreated circumflex territory showed no change. The administration of dynorphin into the coronaries produced a depression of LVCF qualitatively consistent with these effects. The effect of dynorphin was subsequently reversed with naloxone. These results support the concept that endogenous opiates participate in the regulation of myocardial function through local mechanisms at the myocardial level.

The interaction of endogenous opiates with autonomic circulatory control in the dog.

The intravenous injection of either methionine or leucine enkephalin sharply reduces blood pressure, peak left ventricular pressure, and peak LV dP/dt in anesthetized dogs. The magnitude of the hypotensive response increases in proportion to the severity of the preceding surgical stress. The peptides are relatively ineffective after only simple surgical procedures but become highly effective when the autonomic balance is shifted toward sympathetic dependence after more complicated procedures or following bilateral carotid occlusion. The greater the animal's dependence upon sympathetic outflow to maintain blood pressure, the more effective is the opiate peptide. This suggests that the peripherally administered opiates may act by opposing existing adrenergic tone. Such antagonism of adrenergic tone during circulatory shock may help to explain some of the benefit of opiate receptor blockade in this condition. The rapid decline in blood pressure can be demonstrated in response to a variety of the proenkephalin-A derived peptides expected to circulate during physiological stresses. Based on a comparison of the responses to a series of peptides, the hypotensive effect is most likely mediated through activating opiate receptors of the delta subtype.

Cardiopulmonary response to sodium fluoride infusion in the dog.

Because humans are occasionally acutely exposed to high levels of fluoride (F-), and cardiac and especially pulmonary tissue accumulate higher concentrations of F- than do the other soft tissues, the present study was undertaken to investigate the effects of acute exposure to toxic plasma levels of F- on cardiopulmonary hemodynamics. Anesthetized dogs were instrumented with right and left cardiac catheters to measure pulmonary arterial and wedge pressures, left ventricular and aortic pressures, left ventricular dP/dt, and cardiac output. An intravenous loading dose of NaF followed by a 3-h infusion produced a plasma F- level of 800 microM in the "low" group of 6 animals, and 1300 microM in the "high" group. The mean pulmonary arterial pressure peaked at 1 h, 83% above preinfusion values in the high group, while that of the low group attained the same level by the end of the infusion period. Impaired pulmonary gas exchange, as indicated by an increased alveolar-arterial PO2 gradient, occurred in half the animals, and an obvious hyperventilation was reflected in a decreased PCO2 value; there was no change in arterial pH, ECG T-wave peaking was common. The central venous pressure declined steadily, while there were no significant changes from controls in systemic arterial pressure, heart rate, cardiac output, or myocardial contractility (dP/dt). Thus, pulmonary hemodynamics and the systemic capacitance vessels are more affected by acute exposure to F- than is cardiac function.