Association between thyroid dysfunction and dysglycaemia: a prospective cohort study.

AIMS: To compare the incidence of hyperglycaemia among participants with low, elevated and normal serum thyroid-stimulating hormone concentration, as well as the incidence of abnormal thyroid function test results among participants with normal blood glucose and those with hyperglycaemia. METHODS: In a prospective study, a cohort of 72 003 participants with normal, low and elevated serum thyroid-stimulating hormone concentration were followed from the study beginning to the first report of diabetes and prediabetes. A proportional hazards regression model was used to calculate the hazard ratios and 95% CIs for each outcome, adjusting for age, sex, education level, smoking, alcohol consumption and obesity. Analyses for the association between dysglycaemia and incident abnormal thyroid function test were also conducted. RESULTS: During a median 2.6 year follow-up, the incident rates for dysglycaemia, particularly prediabetes, were substantially higher in participants with elevated thyroid-stimulating hormone concentrations at baseline, while the rates for participants with normal and low thyroid-stimulating hormone were similar. After controlling for risk factors, participants with elevated thyroid-stimulating hormone retained a 15% increase in risk of prediabetes (adjusted hazard ratio 1.15, 95% CI 1.04-1.26), but were not at greater risk of diabetes (adjusted hazard ratio 0.96, 95% CI 0.64-1.44). By contrast, participants with normal and low thyroid-stimulating hormone concentrations had similar dysglycaemia risks. Participants with diabetes and prediabetes were not at greater risks of developing abnormal thyroid function test results when compared with participants with euglycaemia. CONCLUSIONS: People with elevated serum thyroid-stimulating hormone concentration are at greater risk of developing prediabetes. Whether this includes a greater risk of developing frank diabetes may require an extended period of follow-up to clarify.

Repeated delta1-opioid receptor stimulation reduces delta2-opioid receptor responses in the SA node.

Ultra-low-dose methionine-enkephalin-arginine-phenylalanine improves vagal transmission (vagotonic) and decreases heart rate via delta(1)-opioid receptors within the sinoatrial (SA) node. Higher doses activate delta(2)-opioid receptors, interrupt vagal transmission (vagolytic), and reduce the bradycardia. Preconditioning-like occlusion of the nodal artery produced a vagotonic response that was reversed by the delta(1)-antagonist 7-benzylidenaltrexone (BNTX). The following study tested the hypothesis that extended delta(1)-opioid receptor stimulation reduces subsequent delta(2)-receptor responses. The delta(2)-agonist deltorphin II was introduced in the SA node by microdialysis to evaluate delta(2) responses before and after infusion of the delta(1)-agonist TAN-67. TAN-67 reduced the vagolytic effect of deltorphin by two-thirds. When the delta(1)-antagonist BNTX was combined with TAN-67, the deltorphin response was preserved, suggesting that attrition of the prior response was mediated by delta(1) activity. When TAN-67 was omitted in time control studies, some loss of delta(2) responses was apparent in the absence of the delta(1) treatment. This loss was also eliminated by BNTX, suggesting that the attenuation of the response after deltorphin alone was also the result of delta(1) activity. Additional studies tested TAN-67 alone in the absence of prior deltorphin. When time controls were conducted without the initial deltorphin treatment, a robust vagolytic response was observed. When TAN-67 preceded the delayed deltorphin, the vagolytic response was eroded, indicating an independent effect of TAN-67. BNTX infused afterward was unable to restore the delta(2) response. These data support the conclusion that the loss of the delta(2) response resulted from reduced delta(2) activity mediated by continued delta(1)-receptor stimulation and not the arithmetic consequence of increased competition from that same delta(1) receptor.

Bimodal delta-opioid receptors regulate vagal bradycardia in canine sinoatrial node.

Methionine-enkephalin-arginine-phenylalanine (MEAP) introduced into the interstitium of the canine sinoatrial (SA) node by microdialysis interrupts vagal bradycardia. In contrast, raising endogenous MEAP by occluding the SA node artery improves vagal bradycardia. Both are blocked by the same delta-selective antagonist, naltrindole. We tested the hypothesis that vagal responses to intranodal enkephalin are bimodal and that the polarity of the response is both dose- and opioid receptor subtype dependent. Ultralow doses of MEAP were introduced into the canine SA node by microdialysis. Heart rate frequency responses were constructed by stimulating the right vagus nerve at 1, 2, and 3 Hz. Ultralow MEAP infusions produced a 50-100% increase in bradycardia during vagal stimulation. Maximal improvement was observed at a dose rate of 500 fmol/min with an ED50 near 50 fmol/min. Vagal improvement was returned to control when MEAP was combined with the delta-antagonist naltrindole. The dose of naltrindole (500 fmol/min) was previously determined as ineffective vs. the vagolytic effect of higher dose MEAP. When MEAP was later reintroduced in the same animals at nanomoles per minute, a clear vagolytic response was observed. The delta1-selective antagonist 7-benzylidenenaltrexone (BNTX) reversed the vagal improvement with an ED50 near 1 x 10-21 mol/min, whereas the delta2-antagonist naltriben had no effect through 10-9 mol/min. Finally, the improved vagal bradycardia previously associated with nodal artery occlusion and endogenous MEAP was blocked by the selective delta1-antagonist BNTX. These data support the hypothesis that opioid effects within the SA node are bimodal in character, that low doses are vagotonic, acting on delta1-receptors, and that higher doses are vagolytic, acting on delta2-receptors.

Local opiate receptors in the sinoatrial node moderate vagal bradycardia.

Met-enkephalin-arg-phe (MEAP) interrupts vagal bradycardia when infused into the systemic circulation. This study was designed to locate the opiate receptors functionally responsible for this inhibition. Previous observations suggested that the receptors were most likely located in either intracardiac parasympathetic ganglia or the pre-junctional nerve terminals innervating the sinoatrial node. In this study 10 dogs were instrumented with a microdialysis probe inserted into the sinoatrial node. The functional position of the probe was tested by briefly introducing norepinephrine into the probe producing an increase in heart rate of more than 30 beats/min. Vagal stimulations were conducted at 0.5, 1.2 and 4 Hz during vehicle infusion (saline ascorbate). Cardiovascular responses during vagal stimulation were recorded on-line. MEAP was infused directly into the sinoatrial node via the microdialysis probe. The evaluation of vagal bradycardia was repeated during the nodal application of MEAP, diprenorphine (opiate antagonist), and diprenorphine co-infused with MEAP. MEAP introduced into the sinoatrial node via the microdialysis probe reduced vagal bradycardia by more than half. Simultaneous local nodal blockade of these receptors with the opiate antagonist, diprenorphine, eliminated the effect of MEAP demonstrating the participation by opiate receptors. Systemic infusions of MEAP produced a reduction in vagal bradycardia nearly identical to that observed during nodal administration. When local nodal opiate receptors were blocked with diprenorphine, the systemic effect of MEAP was eliminated. These data lead us to suggest that the opiate receptors responsible for the inhibition of vagal bradycardia are located within the sinoatrial node with few, if any, participating extra-nodal or ganglionic receptors.

Transient arterial occlusion raises enkephalin in the canine sinoatrial node and improves vagal bradycardia.

The C-terminal proenkephalin sequence, Methionine-enkephalin-arginine-phenylalanine (MEAP), is abundant in the myocardium and when delivered into the sinoatrial (SA) node by microdialysis, the peptide had significant vagolytic activity. The study that follows was conducted to determine if an increase in endogenous nodal MEAP could be demonstrated during reduced nodal blood flow and was endogenous MEAP similarly vagolytic. Microdialysis probes were placed in the canine SA node and perfused at 5 microl per min. The SA node artery was occluded and released four times at 10-min intervals. The intermittent occlusions were followed by one or two prolonged occlusions (30 min). Vagally mediated bradycardia was compared before, during, and after occlusion of the artery. An increase in recovered MEAP (70-220 fmol) was recorded during each of the initial 10-min occlusions. MEAP returned to baseline during each subsequent 10-min reperfusion. There was a sustained increase in MEAP (110-150 fmol) during longer occlusions. Contrary to the hypothesis, the increased MEAP during arterial occlusion was coincident with improved vagal bradycardia. The improvement in vagally mediated bradycardia was highly reproducible and was observed again during a second 30-min occlusion. The improved vagal function was reversed or reduced, respectively, when naltrindole or glibenclamide was included in the microdialysis inflow during arterial occlusion. Although these observations suggested that opioid receptors and ATP-sensitive K+ channels might have been involved, only a single dose of each agent was practical. Therefore, the specificity of these two responses remains to be confirmed. In summary, the recovery of endogenous opioids from the sinoatrial node increased during reduced arterial perfusion of the node. Contrary to expectations, the increase in recovered endogenous opioids was accompanied byimproved rather than impaired vagal bradycardia.

Delta opioid receptors inhibit vagal bradycardia in the sinoatrial node.

BACKGROUND: Methionine-enkephalin-arginine-phenylalanine (MEAP) is an endogenous opiate derived from the C-terminal sequence of the larger precursor molecule proenkephalin. This heptapeptide is abundant in the myocardium and has significant vagolytic activity when infused systemically. MEAP interrupted vagal bradycardia when it was delivered directly into the sinoatrial node by local microdialysis. This study was conducted to determine the opioid receptor responsible for the vagolytic effect of MEAP. METHODS AND RESULTS: Microdialysis probes were placed in the sinoatrial node of mongrel dogs and perfused at 5 microL/min. Increasing doses of MEAP were included in the nodal perfusate and approximately two thirds of the vagal bradycardia was inhibited with a maximal effect at 0.3 nmoles/microL and a half-maximal response near 0.1 nmoles/microL. When deltorphin II (a delta opioid receptor agonist) was infused into the sinoatrial node, more than 95% of the vagal bradycardia was eliminated at 0.3 nmoles/microL with the half-maximal response near 0.1 nmoles/microL, indicating that deltorphin II was more efficacious than MEAP. The maximal deltorphin II and MEAP effects were both similarly reversed by the paired infusion of increasing doses of the delta opiate receptor antagonist, naltrindole. Selected mu (endomorphin, super DALDA) and kappa (dynorphin, U50488) receptor agonists and mu (CTAP) and kappa (norBNI) receptor antagonists were completely ineffective in this system. CONCLUSIONS: These data suggest that the vagolytic effect of MEAP involves the activation of delta opiate receptors within the sinoatrial node.

Cardiac synthesis, processing, and coronary release of enkephalin-related peptides.

Although preproenkephalin mRNA is abundant in the heart, the myocardial synthesis and processing of proenkephalin is largely undefined. Isolated working rat hearts were perfused to determine the rate of myocardial proenkephalin synthesis, its processing into enkephalin-containing peptides, their subsequent release into the coronary arteries, and the influence of prior sympathectomy. Enkephalin-containing peptides were separated by gel filtration and quantified with antisera for specific COOH-terminal sequences. Proenkephalin, peptide B, and [Met(5)]enkephalin-Arg(6)-Phe(7) (MEAP) comprised 95% of the extracted myocardial enkephalins (35 pmol/g). Newly synthesized enkephalins, estimated during a 1-h perfusion with [(14)C]phenylalanine (4 pmol x h(-1) x g wet wt(-1)), were rapidly cleared from the heart during a second isotope-free hour. Despite a steady release of enkephalins into the coronary effluent (4 pmol x h(-1) x g wet wt(-1)), enkephalin replacement apparently exceeded its release, and tissue enkephalins actually accumulated during hour 2. In contrast to the tissue, methionine-enkephalin accounted for more than half of the released enkephalin. Chemical sympathectomy produced an increase in total enkephalin content similar to that observed after 2-h control perfusion. This observation suggested that the normal turnover of myocardial enkephalin may depend in part on continued sympathetic influences.

Canine cardiac muscarinic receptors, G proteins, and adenylate cyclase after long-term morphine.

Short-term morphine stimulates vagal bradycardia. This led us to propose the hypothesis that chronically administered morphine would down-regulate myocardial muscarinic receptor systems. Dogs received morphine continuously for 2 weeks through an s.c. catheter, and cellular aspects of parasympathetic control of the heart were examined. Contrary to expectations, morphine increased muscarinic receptor density in the right atrium and left ventricle by 17 and 34%, respectively, with no change in the apparent affinity of the receptor (K(D)). Morphine also increased the expression of the G protein G(ialpha) by 115 and 233%, respectively, in right atrial and left ventricular sarcolemmal membranes. Morphine increased ventricular and atrial G(salpha) to a much lesser degree (49 and 25%). Morphine failed to alter basal or maximally stimulated (forskolin plus MnCl(2)) adenylate cyclase activity. The maximum cyclase activation by isoproterenol and the maximum inhibition by carbachol were similarly unaltered by morphine. Morphine reduced the ventricular but not atrial norepinephrine. Both long- and short-term morphine lowered tissue epinephrine content, suggesting that short-term morphine reduces extraneuronal uptake. Potential systemic and cellular models for myocardial adaptation to morphine are proposed, including sequential sympathetic and parasympathetic compensations.

Antioxidant properties of pyruvate mediate its potentiation of beta-adrenergic inotropism in stunned myocardium.

UNLABELLED: This study tested the hypothesis that pyruvate's antioxidant actions, particularly its enhancement of the endogenous glutathione system, mediate its potentiation of beta-adrenergic inotropism in stunned myocardium. Isolated working guinea pig hearts, metabolizing 10 m M glucose and stunned by 45 min of low flow ischemia, were treated with 5 m M pyruvate, 5 m M N-acetylcysteine (NAC) and/or 2 n M isoproterenol beginning 15 min after reperfusion. The antioxidant NAC alone did not increase cardiac power (mJ/min/g wet: 11 +/- 1 in untreated and 15 +/- 2 in NAC treated stunned hearts), but NAC potentiated the increase in power produced by 2 n M isoproterenol (isoproterenol alone: 50+/-10; NAC plus isoproterenol: 133 +/- 24). Addition of NAC doubled cyclic AMP content but lowered cytosolic phosphorylation potential by 32% in isoproterenol-stimulated hearts. Stunning decreased the glutathione antioxidant ratio (GSH/GSSG) by 68%. The antioxidant ratio was completely restored by pyruvate alone or in combination with isoproterenol, but only partially restored by isoproterenol alone. Combining isoproterenol and NAC increased the GSH/GSSG ratio by an additional 36%. The combined treatment of pyruvate and isoproterenol increased the NADPH/NADP(+) ratio almost three-fold, and produced the greatest accumulation of glucose-6-phosphate of any treatment. CONCLUSIONS: like pyruvate, the antioxidant NAC potentiated beta-adrenergic inotropism of stunned myocardium. Unlike pyruvate, NAC did not increase cellular energy reserves, thus effectively limiting its potentiation of beta-adrenergic stimulation. Thus, pyruvate's potentiation of beta-adrenergic stimulation in stunned myocardium is most likely the result of the combined effects of its antioxidant and energetic properties.

Enkephalin inhibits vagal control of heart rate, contractile force and coronary blood flow in the canine heart in vivo.

The following studies were conducted to determine if the ability of the intrinsic cardiac opioid, met-enkephalin-arg-phe to interrupt vagal bradycardia can be generalized to include the disruption of vagal effects on atrial contraction and coronary blood flow. Anesthetized dogs were instrumented to measure heart rate and left atrial contractile force or heart rate and coronary blood flow. The response of each variable was recorded at rest and during vagal stimulation. During the evaluation of vagal effects on contractile activity and coronary blood flow, heart rate was maintained constant by electrically pacing the hearts above their resting heart rate. In the first protocol, vagal stimulation reduced both heart rate and atrial contractile force in a frequency dependent fashion. When met-enkephalin-arg-phe (MEAP) was infused systemically for three min at 3 nmol min(-1) kg(-1), there were no observed changes in resting heart rate or atrial contraction. However, when the vagal stimuli were reapplied during the peptide infusion, the previously observed vagal effects on rate and contractile force were reduced in magnitude by one-half to two-thirds. The ability of MEAP to interrupt the vagal control of heart rate and contractile activity involves opiate receptors since the effect was eliminated in both cases by prior opiate receptor blockade with the high affinity antagonist, diprenorphine. In the second protocol, vagal stimulation produced a transient increase in coronary blood flow and an accompanying increase in myocardial oxygen consumption. These effects were reduced by approximately 80% during the systemic infusion of MEAP. A similar increase in coronary blood flow mediated by the direct acting muscarinic agonist, methacholine, was unaltered by the infusion of peptide. In summary, these data suggest that the intrinsic cardiac enkephalin, MEAP, is capable of inhibiting the vagal control of heart rate, contractile force and coronary blood flow and probably does so through a common opiate receptor located prejunctionally on vagal nerve terminals or within nearby parasympathetic ganglia.

Autonomic control of heart rate in dogs treated chronically with morphine.

The vagotonic effect of chronic morphine on the parasympathetic control of the heart was examined in dogs treated with morphine for 2 wk. Because normal vagal function is critical to myocardial stability, the study was conducted to evaluate for potential impairments following chronic vagal stimulation. The hypothesis that persistent vagal outflow would result in a loss of vagal reserve and reduced vagal control of heart rate was tested. Heart rate and the high-frequency variation in heart rate (power spectral analysis) declined shortly after initiation of subcutaneous morphine infusion. A progressive bradycardia correlated well with the rising plasma morphine. The resting bradycardia (57 beats/min) was maintained through day 2 and was accompanied by a significant parallel increase in vagal effect and a decline in the intrinsic heart rate (160 vs. 182 beats/min). A compensatory increase in the ambient sympathetic control of heart rate was evident on day 2 and was supported by an increase in circulating catecholamines. The lowered intrinsic heart rate and elevated sympathetic activity were maintained through day 10 despite a return of the resting heart rate and plasma catecholamines to pretreatment values. These observations suggested that chronic morphine alters either the intrinsic function of the sinoatrial node or reduces the postvagal tachycardia normally attributed to nonadrenergic, noncholinergic agents. Both acute and chronic morphine depressed the rate of development of bradycardia during direct vagal nerve stimulation without altering the rate of recovery afterward. This last observation suggests that acute morphine reduces the rate of acetylcholine release. Results provide insight into the mechanisms that maintain vagal responsiveness. The results are also relevant clinically because opiates are increasingly prescribed for chronic pain and opiate abuse is currently in resurgence.

Pyruvate potentiates beta-adrenergic inotropism of stunned guinea-pig myocardium.

UNLABELLED: This study tested the hypotheses that the sensitivity of stunned myocardium to beta-adrenergic stimulation is diminished, and that metabolic intervention with pyruvate can restore beta-adrenergic responsiveness to pre-ischemic levels. Isolated working guinea-pig hearts metabolizing 10 mM glucose were stunned by 45 min of low flow ischemia, and pyruvate and/or isoproterenol treatments were initiated 15 and 30 min after reperfusion, respectively. The dose: response for cardiac power from 0.1-100 nM isoproterenol was significantly shifted to the right in stunned hearts: EC50 (nm) increased from 0.3 +/- 0.06 to 5.2 +/- 1.86. Pyruvate (5 mM) largely restored isoproterenol responsiveness of stunned myocardium, lowering EC50 to 1.1 +/- 0.34 nM. Maximum power was similar in each group. Additional stunned hearts were treated with intermediate (2 nM) or high (30 nM) isoproterenol concentrations with or without pyruvate. Combining treatments produced a significant interaction at the low dose of isoproterenol, increasing cardiac power (mJ x min(-1) x g(-1)) to 149 +/- 20, twice the sum of the individual treatments (2 nM isoproterenol: 34 +/- 11; pyruvate: 33 +/- 8). Cyclic AMP content was unaltered by isoproterenol or pyruvate alone but was increased 41% by the combination. Power was maximized by 30 nM isoproterenol, which tripled cyclic AMP content; pyruvate did not augment these responses, but lessened the isoproterenol-induced decline in cytosolic phosphorylation potential. CONCLUSIONS: Beta-adrenergic inotropism is attenuated in stunned myocardium, although the maximal response is unchanged. Pyruvate potentiated the effects of sub-maximal doses of isoproterenol without depleting cellular energy reserves further, and attenuated energy depletion by high doses of isoproterenol. Pyruvate may allow restoration of contractile performance with lower, energetically less costly doses of beta-adrenergic agents.

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.

Pericardial repair depresses canine cardiac catecholamines and met-enkephalin.

Decreased cardiac catecholamines were observed following incision and repair of the pericardium in sham-operated vs. unoperated control dogs. Animals were assigned to five groups: unoperated, sham-operated intact pericardia, open pericardia, sutured pericardia and complete ventricular sympathectomy. Hearts were collected four weeks after surgery. Sympathectomy decreased catecholamine content when compared to all other groups. Hearts with open/sutured pericardia contained significantly less catecholamines than controls. When the pericardium was intact or left open following incision, cardiac catecholamines were unchanged compared to unoperated controls. Since opioid peptides are colocalized with catecholamines, we measured met-enkephalin and met-enkephalin-arg-phe, proenkephalin A peptide products, in parallel samples. Similar to norepinephrine, met-enkephalin was decreased following both sympathectomy and pericardial repair. However, met-enkephalin-arg-phe, which may be more associated with the myocardium than its innervation, was not changed by any treatment. The sutured pericardium more than the stress of surgery apparently alters the tissue catecholamines and enkephalin. This may have resulted from the mechanical friction at the site of repair. Epinephrine and met-enkephalin contents in sympathectomized hearts were significantly lower than unoperated controls but were not significantly different from the intermediate values observed in the sutured group. The functional consequences of these changes on neuroendocrine status are unclear and will require further evaluation. The results also emphasize the need for careful attention to proper controls for surgical studies.

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.

Aging, cardiac proenkephalin mRNA and enkephalin peptides in the Fisher 344 rat.

Cardiac proenkephalin (PENK) mRNA, methionine-enkephalin (ME) and leucine-enkephalin (LE) were determined from 2 days of age through senescence in Fisher 344 rats. Tissues were collected at 2 days, 2 weeks, 1, 2, 3, 7, 19, and either 22 or 27 months of age. Hearts were dissected, extracted and assayed for ME and LE by radioimmunoassay (RIA) or for PENK mRNA by Northern blot analysis with a cDNA probe. Relative left ventricular (LV) PENK mRNA was low in 2 day animals and increased slowly between 2 weeks and 3 months of age. LV PENK mRNA then rose five to six-fold between 3 and 27 months of age. LV ME measurements were high in neonatal animals, declined to a nadir during development and then rose again as the animals matured and advanced in age. The pattern for right ventricular (RV) ME was similar. Atrial ME, also high at 2 days, declined thereafter and remained low. LE measurements in LV, RV and the atria followed patterns similar to those described for ME. To evaluate for peptides contributed by cardiac nerves, 3, 7 and 22-month-old animals were acutely sympathectomized for 24 h with 6-hydroxydopamine. No decline in LV ME and LE was observed in the 6-hydroxydopamine treated animals. These data suggest several conclusions regarding myocardial enkephalinergic systems: (a) tissue enkephalin and PENK mRNA increase with advancing age, (b) tissue enkephalins may not strictly correlate with the relative abundance of PENK mRNA, and (c) most myocardial enkephalins are non-adrenergic in origin. The age-associated patterns in both PENK mRNA, ME and LE suggest that physiological, maturational or behavioral events between 3 and 7 months of age initiate the up-regulation and subsequent expansion of cardiac enkephalinergic systems.

(+)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.