INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

ENDOGENOUS ENKEPHALIN PEPTIDES are involved in the normal regulation of cardiovascular function (14). These include actions at both central and peripheral locations. Enkephalin receptors are concentrated in the brain stem and hypothalamus in close proximity to the cardiovascular centers (2), and centrally administered enkephalins produce a variety of site-specific cardiovascular responses (12, 13). Some effects of peripherally released enkephalins may result from direct interaction with opiate receptors localized on cardiomyocytes (10, 18, 22-24, 26, 35, 39-41).

There is growing support for the rationale that endogenous cardiac opioids are potent modulators of cardiovascular function with significant physiological and pathological influences. The effects of opioids on heart rate, contractile strength, arterial tonus, and arterial pressure were initially summarized by Holaday (14). Although many opioid effects were attributed to the prejunctional inhibition of neurotransmitter release, newer studies indicate that they also modify myocardial function through postjunctional interactions. The stimulation of - and -opiate receptors in the cardiac sarcolemma modified both calcium homeostasis and associated intracellular signaling pathways (10, 18, 22-24, 26, 35, 39-41). We have recently shown that -receptor stimulation with the agonist leucine-enkephalin (LE) reversed the positive inotropic effect induced by norepinephrine in isolated ventricular cardiac myocytes and isolated, Langendorff-mode perfused hearts from adult rats (22, 39). This interaction involves a reduction in L-type Ca²⁺-channel current (38) and inhibition of cAMP formation via a pertussis toxin-sensitive G_i/o protein (22, 39). This effect may be considered to be protective during periods of metabolic stress, such as that due to ischemia or intense exercise, by preventing catecholamine-augmented metabolic demand from exceeding the supply of substrate. Further protection may be afforded by limiting intracellular Ca²⁺ overload and Ca²⁺-dependent arrhythmias (6, 21). The harmful effect of catecholamines in ischemia may be mediated by an increase in cAMP-dependent processes that result in degradation of high-energy phosphates and elevated intracellular Ca²⁺, which play a crucial role in developing myocardial necrosis (25). Catecholamine depletion by reserpine treatment prior to ischemia prevented the ischemia-induced decrease of myocardial ATP (16). Enkephalins may be implicated in this response as well, since reserpine also increased adrenal enkephalin content (37). Therefore, the protective role of enkephalins in ischemic preconditioning (7, 27-29) may result from their ability oppose both the release of norepinephrine and its postjunctional effect. Myocardial regulation of synthesis and release of opioids was altered in animal models of cardiac development (31), aging (4), myocardial infarction (21), cardiomyopathy (11, 33, 34), and hypertension (9, 41). Collectively, these prior studies suggest a specific adaptive role for opioids in the myocardium.

Although circulating enkephalins may, under some conditions, be sufficient to alter cardiac function directly, subnanomolar plasma concentrations at rest are presumed too low to explain their effects in the heart (8); thus other enkephalin sources are required to explain their direct action on heart tissues (22, 35, 39). However, locally synthesized cardiac enkephalins could produce interstitial enkephalin concentrations much higher than those usually observed in plasma. The concept of local enkephalin synthesis is supported by the finding that rat heart cells contain enkephalins and the mRNA for their precursor, proenkephalin (15). Furthermore, isolated cardiomyocytes translate the message effectively and release proenkephalin products into surrounding culture medium (31, 32).

Despite comparable transcript contents and translational status between the heart and brain, measured enkephalins recovered in myocardial extracts are much lower than those reported for the brain. Radioreceptor assay data indicated greater opioid activity in the heart (3), and when heart extracts were treated with trypsin and carboxypeptidase B, the amount of methionine-enkephalin (ME) recovered was increased (9). This suggests that the heart produces larger enkephalin-containing peptides, e.g., peptides B, E, F, or the heptapeptide [Met⁵]enkephalin-Arg⁶-Phe⁷ (MEAP), which are not adequately accessed by standard ME radioimmunoassays. Proenkephalin may be synthesized and processed into larger intermediates, such as peptides B, E, and F, at much greater rates in the heart than inferred previously from measurements of the fully processed pentapeptides. Therefore, we hypothesize that, independent of the nervous system, the isolated rat heart synthesizes proenkephalin and processes this protein into large intermediates rather than directly and completely into the pentapeptides ME and LE. Thus the aims of the present study were to 1) determine the myocardial content of proenkephalin-derived large peptide intermediates in the isolated rat heart; 2) assess the changes in tissue peptide content after 1 and 2 h of perfusion; 3) measure the rate of peptide release into coronary circulation; 4) estimate the rate of proenkephalin synthesis in isolated perfused rat heart by pulse chase methods; and 5) determine the effect of prior chemical sympathectomy on these measures.

	METHODS AND MATERIALS

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

Isolated rat heart preparation. The animal protocols were conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals. Hearts for isolated isovolumic perfusions were obtained from male Sprague-Dawley rats (300-400 g). The aorta was cannulated, and the coronary arteries were perfused by retrograde aortic flow (Langendorff mode) at 10 ml/min and paced at 3 Hz. Left ventricular systolic pressure was recorded from a pressure transducer and fluid-filled catheter attached to a latex balloon inserted via left atrium into the left ventricle. The volume in the balloon was adjusted to yield a left ventricular end-diastolic pressure less than 8 mmHg. Isolated hearts were perfused by filtered bicarbonate buffer consisting of (in mM) 3.48 KCl, 116.4 NaCl, 26.2 NaHCO₃, 1.67 NaH₂PO₄, 0.69 MgSO₄, 1.5 CaCl₂, and 11.1 glucose. The perfusate was saturated with 95% O₂-5% CO₂ and equilibrated to 30°C at pH 7.38-7.42. Where indicated, the perfusate was supplemented with normal plasma concentrations of 19 amino acids (36) plus 0.4 mM phenylalanine. An additional 0.1 µCi/ml [U-¹⁴C]phenylalanine was added to the perfusate when protein synthesis was estimated. This concentration of phenylalanine quickly equilibrates with intracellular tRNA pools and allows the estimation of specific rates of protein synthesis (20).

Experimental groups. Figure 1 illustrates the protocol for each of the seven experimental groups (groups A-G). In each case the heart or the heart plus perfusate were collected and extracted for peptide analysis as described below. In group A, the heart was excised from the rat and immediately subjected to the tissue extraction procedure, without buffer perfusion. In groups B and C, the heart was collected 15 min after the initiation of perfusion in the absence and presence of added amino acids, respectively, and served as a time 0 value for the other perfused heart groups. The 15-min equilibration period was based on the observation that the performance of the isolated rat heart routinely achieved a steady state within 10 min (Table 1). To estimate the rate of protein synthesis, group D was perfused with amino acids and received 0.4 mM [U-¹⁴C]phenylalanine (0.1 Ci/ml) for 1 h after the initial 15-min equilibration. To estimate clearance, group E was perfused as in group D but received a second hour of washout perfusion with unlabeled phenylalanine following the initial 1 h [¹⁴C]phenylalanine perfusion. To determine the influence of catecholamines on cardiac enkephalins prior to (group F) and following perfusion (group G), the adrenergic nerve terminals in groups F and G were destroyed by pretreating the animals with 6-hydroxydopamine (6-OHDA) for 36 h (5).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Isolated isovolumic perfused heart experimental protocols. Rats were allocated to either untreated groups (groups A-E) or groups pretreated 36 h earlier with 6-hydroxydopamine (6-OHDA), 20 mg/kg body wt ip (groups F and G). For groups A and F, hearts were excised and extracted without perfusion. In group B, hearts were perfused for 15 min in bicarbonate buffer (Krebs-Henseleit buffer) without added amino acids, and the tissues were extracted as described. Group C hearts were perfused for 15 min with bicarbonate buffer supplemented with a mixture of 20 amino acids. Groups D and E included [14C]phenylalanine in the 20 amino acid mixture. Group A was used to determine cardiac enkephalin content immediately after excision of the heart. Groups B and C were utilized to examine whether the initial equilibration (with and without amino acids) altered the starting cardiac enkephalin content. The 15-min equilibration period was selected, because the performance of the isolated rat heart reaches a steady state within 10 min (Table 1). Thus total perfusion was 15 min in groups B and C, 75 min for groups D and G, and 135 min for group E. The total effluent from perfused hearts (groups D-F) was collected between 15 and 75 min (hour 1 perfusion) and from group E between 75 and 135 min (hour 2 perfusion).

Tissue enkephalin extraction. Hearts were blotted, weighed, diced in 2.5 vol of 1 N acetic acid-0.2 N HCl, and boiled for 30 min. After cooling, 0.1% -mercaptoethanol was added, the tissue was homogenized (Polytron, Brinkmann), and the supernatant was collected after centrifugation at 25,000 g for 30 min. The pellet was rehomogenized in another 2.5 vol, and the centrifugation was repeated. The supernatants were combined and stored at 80°C. Extracts were later thawed, neutralized with 10 N NaOH, and filtered through 0.45-µm syringe filters. Peptides in 2 ml of filtered extract were separated by gel filtration chromatography on Bio-Gel P-10 columns (1.5 × 10 cm, 15 ml). The samples were eluted at pH 7.0 with 0.01 M PBS containing 0.1% gelatin. Fractions corresponding to small (ME, LE, and MEAP), intermediate (peptides B, E, F), and large (proenkephalin) enkephalin sequences were collected (5). The fractions were distributed and frozen in 0.5-ml aliquots for radioimmunoassay. When expected concentrations dictated, fractions were concentrated on Poropak-Q columns (Millipore/Waters, Bedford, MA). Absorbed peptides were eluted with ethanol-acetic acid-water (1:1:1), dried in a centrifugal evaporator (Savant), stored at 20°C, and later reconstituted in PBS for radioimmunoassay.

Perfusate enkephalin extraction. The total effluent from the perfused isolated hearts was continuously collected for 1 h in concentrated acetic acid and HCl, producing final concentrations of 1 N and 0.2 N, respectively. -Mercaptoethanol (0.1%) was added, and the constituent peptides were extracted on prewetted Sep-Pak C₁₈ cartridges (Millipore/Waters). The cartridges were washed with 10 ml of 0.1% trifluoroacetic acid; the peptides were eluted with 6 ml 60% acetonitrile in 0.1% trifluoroacetic acid, and the eluent was evaporated under vacuum. The dried extracts were reconstituted in 2 ml PBS and chromatographically processed as described above.

Radioimmunoassays. Samples were assayed with COOH-terminally directed antisera specific for ME, MEAP, and LE (Peninsula Laboratories, Belmont, CA). ¹²⁵I-ME and ¹²⁵I-MEAP were prepared with chloramine-T, and the products of the reaction were separated by combined gel filtration/anion exchange on Sephadex QAE-A25 as previously described (3). The MEAP assay was similar to that employed in the past (3). The new antisera was generated in rabbits against the COOH-terminal 15 amino acids in rat proenkephalin conjugated via an NH₂-terminal cysteine to keyhole limpet hemocyanin. The samples and standards were suspended in 0.5 ml PBS with 0.1% gelatin, and they were run in triplicate. The antisera (1:1,600) was diluted in 0.2 ml normal rabbit serum (1:400), and the ¹²⁵I-MEAP or ¹²⁵I-ME was added in 0.1 ml assay buffer with 0.1% Triton X-100. To increase the sensitivity of the MEAP assay, the samples and standards were oxidized by the addition of 1 µl of 30% H₂O₂ added to each MEAP assay tube. The reaction mixture was incubated overnight at 25°C. Sheep anti-rabbit -globulin (0.2 ml, 1:36) was added, and the reaction mixture was incubated overnight at 4°C. Bound and free radioligands were separated by centrifugation. The antiserum for LE, ME, or MEAP displayed less than 5% cross-reactivity for the reciprocal ligands (e.g., MEAP vs. the ME antiserum). Because standards, antisera, and radioligands for proenkephalin and peptides E, F, and B were unavailable, these peptides were quantified with antisera specific for COOH-terminal sequences that they share with readily available standards. Thus after chromatographic separation, peptide E was determined with an antisera specific for the COOH-terminal sequence it shares with LE. The resulting contents were then expressed as LE equivalents. Similarly, peptide F was expressed as ME equivalents, whereas peptide B and proenkephalin were expressed as MEAP equivalents

Determination of [¹⁴C]phenylalanine incorporation. Proenkephalin and peptide B fractions were separated from the myocardial fractions by gel filtration. These fractions were divided into aliquots and immunoprecipitated with the specific antiserum under the assay conditions described above. The radioligand (¹²⁵I-MEAP) was not added during the immunoprecipitation of the aliquots designated for the [¹⁴C]phenylalanine incorporation measurements. The immunoprecipitated pellet was dissolved in scintillation liquid and counted for ¹⁴C. Apparent rates of protein synthesis were calculated by dividing the radioactivity in the immunoprecipitate by the specific activity of phenylalanine in the perfusate. This calculation is based on the assumption that for 0.4 mM [¹⁴C]phenylalanine, the specific activities of phenylalanine in the perfusate and in the intracellular water and that bound to tRNA are the same (20) (see DISCUSSION for more details).

Statistical analysis. Data are expressed as means ± SE. A one-way ANOVA was employed to determine differences among groups at a constant perfusion time in the absence of 6-OHDA, and a two-way ANOVA was employed to test for differences due to time and 6-OHDA. The Bonferroni t-test was performed for post hoc comparisons of individual means.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

Isolated hearts were harvested and homogenized for enkephalin extractions before, or after, bicarbonate-buffer perfusion. Table 1 summarizes the main left ventricular performance data in isolated, isovolumic bicarbonate buffer-perfused hearts. No significant differences in contractile function were apparent among the groups.

The contents of myocardial enkephalins, which were separated by chromatography and measured by radioimmunoassay, are presented in Table 2. The values of myocardial ME were similar to those we measured previously by nonchromatographic means in adult rat hearts (31, 36). The ratio of ME to LE was approximately equal to the 4:1 expected from the known sequence of proenkephalin (Fig. 2). The majority of the immunoreactive enkephalins were, however, associated with proenkephalin, peptide B, and MEAP. Despite the large number of ME sequences in proenkephalin, the MEAP and peptide B contents were paradoxically each 10 times higher than that observed for ME. Since the antiserum does not distinguish well between MEAP and its des-tyrosyl metabolite (Fig. 3), one cannot determine with certainty from this data what proportion of the extracted MEAP remained biologically active. However, the large amount of MEAP and peptide B immunoreactivity suggests that MEAP and peptide B are preferentially retained or protected from metabolism in the heart.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   A schematic representation of potential enkephalin-containing polypeptides arising from posttranslational processing of preproenkephalin as previously identified in acid extracts of adrenal chromaffin granules. SP, signal peptide; ME, methionine-enkephalin; LE, Leu-enkephalin; MEAGL, Met-enkephalin-Arg-Gly-Leu; MEAP, Met-enkephalin-Arg-Phe; BAM 20, bovine adrenal medulla peptide 20; and PEP, peptide (for peptides I, B, E, and F).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   The displacement of 125I-MEAP from the from the antisera used to quantitate the COOH-terminal MEAP sequence in proenkephalin (), peptide B (), and MEAP (). Increasing concentrations of competing opiate sequences were incubated with 125I-MEAP, 1 µl of 30% H2O2, and the rabbit antiserum (1:6,400 final dilution) in 0.01 M PBS, pH 7.0. LE, ME, ME-Arg6, ME-Arg6-Gly7-Leu8, dynorphin-(1-8), bovine adrenal medulla peptide-12, and peptides E and F had less than 1% cross-reactivity and were omitted from the diagram for clarity. The peptides Phe-Met-Arg-Phe-amide (×) and des-Tyr-Met-enkephalin-Arg-Phe () were included to illustrate the specificity of the antisera for the Arg-Phe sequence. The term "%B/Bo" (percent binding) represents the 125I-MEAP bound at each added competing ligand concentration divided by the 125I-MEAP bound without competing ligand.

There was no significant difference in the peptide content of control hearts collected immediately and those mounted and preperfused for 15 min with or without added amino acids (groups A-C; Table 2). Steady-state perfusion of control hearts for an additional 60 min with amino acids resulted in a gradual though nonsignificant decline in total enkephalin content but very little change in the pattern of the individual myocardial enkephalins. The only exceptions were the LE-containing peptides, LE and peptide E, the contents of which were both very low initially and then declined by 25-50% during the 1-h perfusion. After an additional hour of perfusion (2 h + 15 min total, group E), however, total tissue enkephalin content increased significantly. Proenkephalin and peptide B contents were augmented by 67 and 51%, respectively (Table 2).

The rates of total enkephalin released into the perfusate are listed in Table 3. The rate of release (4-5 pmol · h¹ · g wet wt¹) was relatively constant when the hour 1 and the hour 2 perfusates were compared. The proportion of the constituent enkephalins also remained relatively uniform during hours 1 and 2. In contrast to tissue measurements, very little proenkephalin, peptide B, or MEAP was released into the perfusate. ME accounted for approximately one-half of the total enkephalins released. The sum of the ME recovered in the perfusate was more than double either its initial tissue concentration or the amount remaining in the tissue after 1 h of perfusion. This net production of ME would require either an increase in the extent of its synthesis or an increase in its processing from existing precursor. Total enkephalin release remained constant during hour 2, resulting in a doubling of total ME released. LE concentrations in the perfusate were, however, much lower. Although the amount of LE in the perfusate could reasonably account for the observed decline in tissue LE, the ratio of circulating ME to LE was much higher than expected.

Figure 4 (see also Table 4) summarizes the effects of chemical sympathectomy with 6-OHDA on the content and size distribution of myocardial enkephalins compared with the unperfused heart. Two-way ANOVAs, testing for differences in 75-min buffer perfusion, detected a significant effect of 6-OHDA pretreatment on the contents of proenkephalin (P = 0.02), peptide B (P = 0.008), MEAP (P = 0.04), ME (P = 0.002), and LE (P = 0.0005). In the absence of sympathetic nerve terminal transmitter stores, the tissue proenkephalin in hearts immediately excised without perfusion (group F) was increased significantly by 50% (P < 0.05 vs. group A), whereas the pentapeptides ME and LE were decreased by more than 75% (P < 0.05 vs. group A). This suggests that in vivo some of the end products may be stored in heart synaptosomal vesicles. After 75-min perfusion of the 6-OHDA-pretreated isolated hearts (group G), the content of ME, but not that of LE, was restored to that of the untreated heart. Notably, during this 75-min perfusion, the absence of sympathetic nerve terminal transmitter stores also led to increased heart content of peptide B and MEAP. A similar higher content was also observed when the perfusion duration was longer in hearts not treated with 6-OHDA (Table 2).

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effects of 6-OHDA treatment on myocardial enkephalin peptide content. Tissue was extracted from groups A and F (±6-OHDA pretreatment; both without buffer perfusion) and D and G (±6-OHDA pretreatment; with 75-min buffer perfusion), and peptides were fractionated by gel filtration and measured by radioimmunoassay. Results are expressed as percent values measured in group A. The main effects of 6-OHDA and perfusion and their interaction were tested for by two-way ANOVA. Post hoc comparisons were made between the following groups: A vs. F to examine the effect of 6-OHDA pretreatment on the amount of proenkephalin and derived peptides in heart immediately upon isolation before perfusion; D vs. G to examine the effect of 6-OHDA pretreatment on the amount of proenkephalin and derived peptides in heart after 75-min buffer perfusion; A vs. D to examine the effect of 75-min buffer perfusion; F vs. G to test the effect of perfusion in 6-OHDA-pretreated hearts. The results are summarized in Table 4.

The total perfusate enkephalin isolated (Table 3) during hour 1 closely approximated the amount of the decline in myocardial enkephalin content observed after 1 h. Since the heart continued to release enkephalins at a similar rate during the second hour of perfusion, an additional 4-5 pmol/g of total enkephalins decrease was expected after 2 h. Since a decline in total tissue enkephalin was expected during hour 2, the actual increase in the rate of proenkephalin synthesis may have been greater than the increase observed.

The data above indicated the active synthesis and processing of myocardial preproenkephalin. This observation was confirmed by immunoprecipitation (Table 5). The hearts were perfused for 1 h with labeled phenylalanine, and the extracts were chromatographed and assayed. The remaining column effluents from the proenkephalin and peptide B fractions were immunoprecipitated with the COOH-terminal specific antisera and counted. Approximately 1 and 12 pmol of phenylalanine per gram of heart tissue were incorporated into proenkephalin and peptide B, respectively (group D). When similarly treated hearts were perfused for hour 2 without labeled phenylalanine, the radiolabel incorporated into proenkephalin declined slightly, but this was not statistically significant. The radiolabel in peptide B, however, declined by more than 90% (P = 0.002, group E vs. D).

In summary, isolated hearts, when electrically paced and perfused at a constant flow rate, exhibited an active enkephalin metabolism characterized by a constant release of total enkephalins of ~4 pmol · h¹ · g wet wt¹ (Table 3). The mass of total enkephalins in the coronary effluent and the tissue content that accumulated during 2-h perfusion followed biphasic kinetics. After the first hour, total tissue enkephalins (30 pmol/g wet wt) were either unchanged or decreased slightly (nonsignificantly) from the content at 15 min (32 pmol/g), despite their constant release into coronary effluent. After 135-min perfusion, enkephalins accumulated in the myocardium while the perfused heart continued to release the same amount into coronary effluent (Fig. 5).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Total cardiac enkephalins (tissue content plus coronary effluent enkephalins) recovered during isolated heart perfusion are expressed as means ± SE in pmol/g wet wt. The points are represented sequentially by group A (n = 6) at t = 0 min; group C (n = 5) at t = 15 min; group D (n = 10) at t = 75 min; group E (n = 5) at t = 135 min. *Significantly different from t = 0 min (group A), P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

The present study reports the recovery of significant enkephalin content extracted from rat cardiac ventricle. Extracts were separated by molecular weight to determine the relative content of large (proenkephalin), intermediate (peptides B, E, F), and small (ME and LE, MEAP) peptide constituents. Total enkephalin content exceeded 35 pmol/g compared with 1 pmol/g ME, which we and others have previously reported (4, 5, 9, 21). Ninety-five percent of the recovered enkephalins was concentrated in precursor (20%), peptide B (43%), and MEAP (32%). Total enkephalin estimates may substantially underestimate the actual values for several reasons. For practical reasons, the radioimmunoassay for proenkephalin and peptide B used MEAP as both ligand and standard. When purified proenkephalin and peptide B were used as test standards, the resulting curves were substantially shifted to the right (Fig. 3). Since the antibody prefers MEAP, more proenkephalin and/or peptide B are required to displace it. As a result the assay employed in our study underestimates the content of proenkephalin and peptide B by 2- and 10-fold, respectively. Second, the total peptide measured does not include the potential or cryptic sequences integrated in the large and intermediate-sized peptides.

In comparing hearts following 1 and 2 h of perfusion, it appears that enkephalin synthesis follows a biphasic pattern. During the first hour, peptide synthesis, release, and degradation are in relative balance, so the sum of the tissue content plus the peptides released into the effluent remained relatively constant (Fig. 5). The rate of proenkephalin and peptide B metabolism can be estimated from the labeled [¹⁴C]phenylalanine incorporation measurements (Table 5). A definitive measurement of their rate of synthesis requires the determination of the specific activity of the pool of amino acids that are direct precursors of the synthetic pathway. Using the Langendorff technique to measure protein synthesis in isolated perfused rat heart, McKee et al. (20) demonstrated that accurate protein synthesis could be calculated from the specific activity of free amino acids. By combining high perfusate concentrations of labeled phenylalanine (0.4 mM) with the other 19 amino acids at normal plasma concentrations, they demonstrated that the specific activity for phenylalanine in the perfusate and that bound to tRNA were identical (20). Furthermore, [¹⁴C]phenylalanine was suitable for their studies, since it equilibrated with the intracellular pool within 10 min and was not converted to other amino acids. In the current study, conditions nearly identical those reported by McKee were employed, although the validity of assumptions regarding the equilibration of phenylalanine with the intracellular pool of tRNA was not confirmed directly. The incorporation of labeled phenylalanine was combined with specific immunoprecipitation as one estimate of enkephalin synthesis, and the rapid incorporation of [¹⁴C]phenylalanine (12 pmol · g¹ · h¹) into peptide B suggested a rate of peptide B synthesis of 4 pmol · g¹ · h¹, based on the three constituent phenylalanines of peptide B. Since there is one peptide B sequence in each proenkephalin, 4 pmol · h¹ · g wet wt¹ represented the minimum rate of preproenkephalin synthesis. Because of its large number of phenylalanines, a similar calculation for the immunoprecipitated proenkephalin suggested that labeled proenkephalin added little to the total on a molar basis, and most of the labeled proenkephalin had already been processed to peptide B. There are seven small enkephalin sequences (four MEs; and one each of LE, Met-enkephalin-Arg-Gly-Leu, and MEAP) in proenkephalin (Fig. 2). If the proenkephalin were fully processed, then as many as 28 pmol · g¹ · h¹ of newly synthesized enkephalin might be expected, based on the minimum estimate of 4 pmol · g¹ · h¹. This (28 pmol · g¹ · h¹) corresponded to a rate of total enkephalin synthesis nearly sufficient to replace the total measured enkephalin content of the heart (35 pmol/g) each hour. Since only 4-5 pmol of total enkephalins were recovered in the perfusate, either the remaining enkephalins are not processed to product or the resulting products are aggressively degraded locally within the tissue. The failure to find a similar rate of label incorporation into proenkephalin suggests that newly synthesized proenkephalin is immediately processed and does not mix freely with the existing tissue pool. The concept of two proenkephalin pools, new and preformed, is also supported by the washout kinetics observed during the second hour in the absence of label in the perfusate. More than 90% of the labeled peptide B was eliminated during hour 2, whereas the small mass of label recovered as proenkephalin was statistically unchanged. This is, again, consistent with an hourly peptide turnover equal or exceeding the initial tissue content.

The production of newly formed peptide B during the 1-h perfusion provides indirect evidence that the myocardium has prohormone convertases, similar to prohormone convertase (PC), PC1 and PC2 (17, 30), capable of processing precursor to peptide B and presumably peptide B to MEAP. Despite the nearly complete turnover of newly synthesized tissue peptide B during hour 2, little of the peptide B or its presumed product, MEAP, was recovered in the perfusate. This observation suggests that the myocardium may also be capable of actively removing the COOH-terminal Arg-Phe group, converting MEAP to ME (19). The failure to recover ME in either tissue or perfusate in concentrations equivalent to the peptide B or MEAP that had been processed suggested further that the heart most likely degrades ME at a much higher rate.

Prior to perfusion, the ratio of LE to ME in tissue was consistently 0.25, as predicted by the known sequence of proenkephalin (Fig. 2). However, the ratio of MEAP to ME was consistently greater than 10, i.e., more than 40-fold higher than predicted. If one reasonably includes peptide B in the calculation, then the deviation from predicted is more than 100-fold. This is consistent with our earlier findings in the dog heart in which the ventricular MEAP was consistently much higher than comparable ME determinations (3). This suggests that MEAP may be more resistant to degradation than ME or LE.

"Chemical sympathectomy" with 6-OHDA destroys sympathetic nerve endings in the heart, and in our hands, the dose used depleted more than 90% of measurable catecholamines from the heart (4). As opioid peptides have been colocalized with catecholamines and coreleased during synaptic transmission, 6-OHDA treatment may also have depleted opioids contained within myocardial neurons as well. Although we have not directly measured the effect of 6-OHDA on opioids within the cardiac sympathetic neurons, peptides of the proenkephalin family were reported in adrenergic nerve endings in the guinea pig heart (1). Following electrical stimulation of the accelerator nerve, the peptides were detected in the perfusate. Although this observation suggested that the peptides were released from sympathetic nerve terminals, these experiments did not distinguish between peptide released from neurons and peptide released from cardiomyocytes (or other cell types) in response to sympathetic neurotransmitters (1).

In the present study, chemical sympathectomy with 6-OHDA pretreatment 36 h prior to harvesting the heart had no significant effect on myocardial function, as illustrated in Table 1 (group G vs. E). This indicates that, under control conditions, endogenous catecholamines are not required for myocardial performance in vitro. However, pretreatment with 6-OHDA produced some notable changes in the content and size distribution of myocardial enkephalins (Table 2). Total enkephalin content was greater in the treated hearts than in their untreated controls, confirming that the majority of myocardial enkephalins are not enclosed in adrenergic nerve terminals. These data also suggest that adrenergic influences stimulate enkephalin turnover in vivo. The majority of this increase was attributable to proenkephalin, peptide B, and MEAP. As with the control hearts, myocardial enkephalin content following 6-OHDA pretreatment was largely maintained during perfusion for 60 min. Notably, however, the proenkephalin content declined and MEAP increased during the 1-h perfusion. The recovery for enkephalins in the perfusate from 6-OHDA-pretreated hearts was similar to that observed during control perfusions, as nearly 50% of the total enkephalins released were recovered as ME. The decline in tissue in large-molecular-weight enkephalin-containing peptides during isolated perfusion, in the absence of adrenergic influence, suggests a reduction in proenkephalin synthesis and/or an increased processing of proenkephalin.

In the present study, heart contraction rate was paced at a slightly lower rate (180 beats/min) than typically employed (~250 beats/min), to moderate coronary flow in the Langendorff perfusion model. The coronary flow rate was held at 10 ml/min to ensure constant coronary flow and a stable basal rate of performance while avoiding the shear stress that would have been imposed by higher rates of pump perfusion. How the technical conditions employed in the present study may have contributed to the enkephalin content is difficult to predict. However, the appropriate control groups were included to try to standardize for possible methodology effects and isolate the treatment effects of interest. For instance, when the heart was paced for a longer period (group E vs. C), the enkephalin content was greater. The consequences of in vitro perfusion could clearly result from a lot more than the pacing rate (e.g., shear stress, colloid osmotic pressure, pacing, workload, denervation, crystalloid perfusate, thrombocythemia). Indeed, enkephalin metabolism, in vivo, in innervated hearts actively pumping blood at higher heart rates and greater workloads might be expected to provide different qualitative and quantitative rates of enkephalin metabolism. Future investigation of a more complex animal model in vivo will be required to provide more specific details of rates and pathways of cardiac opioid metabolism under physiological conditions in vivo.

In summary, the present study indicates that the normoxic rat heart synthesizes, stores, and processes significant amounts of enkephalin-containing peptides represented primarily by proenkephalin, peptide B, and MEAP. In contrast, ME is the predominant postprocessing enkephalin product released into coronary effluent. The stoichiometry of the peptides suggests that ME must be metabolized very quickly. During the first hour of perfusion, released and/or degraded enkephalins are quickly replaced from newly synthesized precursor, which seems to be preferentially processed to products before mixing with the existing pools of proenkephalin. Despite the steady release of enkephalins into the perfusate, the total myocardial enkephalin content increased further when perfusion of the heart was continued for an additional hour. Chemical sympathectomy with 6-OHDA produced a similar peptide accumulation, which suggested that the withdrawal of sympathetic influences secondary to isolating the heart may play a role in the increase of myocardial enkephalin content during extended perfusion of the isolated rat heart. The spectrum of different enkephalins and enkephalin-containing intermediates observed in heart may serve discrete myocardial functions. Proenkephalin and its longer intermediates may serve as stored precursors and ready reserves. The precursors may then be processed on demand into an array of qualitatively distinct opioids specific to the appropriate physiological response. Some of the peptides may be destined for secretion, whereas others may be specifically designed to interact with opiate receptors locally positioned on cardiomyocytes and/or cardiac nerves. The specific conditions of their formation and the cardiac reactivity to each will necessarily constitute the subject of further study.