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Localization of pro-opiomelanocortin mRNA transcripts and peptide immunoreactivity in rat heart

William R Millington, David W Rosenthal, Can B Unal, Cynthia Nyquist-Battie
DOI: http://dx.doi.org/10.1016/S0008-6363(99)00076-0 107-116 First published online: 1 July 1999

Abstract

Objective: α-Melanocyte-stimulating hormone (α-MSH), β-endorphin and other pro-opiomelanocortin- (POMC) derived peptides have been detected in the heart, but it is uncertain whether they are synthesized by cardiomyocytes or by cardiac nerves innervating the heart. The objective of this study was to determine whether POMC peptides are synthesized by cardiomyocytes. Methods: Pro-opiomelanocortin peptides were localized in rat heart by immunohistochemistry using antisera against α-MSH, β-endorphin and αN-acetyl-β-endorphin, the predominant POMC peptides found in heart. Pro-opiomelanocortin mRNA was investigated by reverse transcription polymerase chain reaction (RT-PCR) using primers that discriminate between full-length POMC mRNA and a 5′ truncated POMC transcript that is presumed to be non-functional. Results: α-Melanocyte-stimulating hormone, β-endorphin and αN-acetyl-β-endorphin immunoreactivities were localized in atrial myocytes, particularly in the atrial appendages, but not to a significant extent in ventricular myocytes. Cardiac nerves were not immunostained. Atrial natriuretic peptide (ANP) immunoreactivity was similarly distributed in the adult heart. In neonatal heart, POMC-peptide and ANP immunoreactivities were present in both atrial and ventricular myocytes. RT-PCR amplification showed that full-length POMC mRNA transcripts were present in both atrial and ventricular tissue and provide evidence that 5′ truncated POMC mRNA is expressed in heart. Conclusions: These results support the hypothesis that cardiomyocytes synthesize POMC peptides.

Keywords
  • Atrial function
  • Gene expression
  • Hormones
  • Neurotransmitters
  • Ventricular function

Time for primary review, 24 days.

This article is referred to in the Editorial by B.A. Barron (pages 13–16) in this issue.

1 Introduction

The discovery of atrial natriuretic peptide (ANP) more than a decade ago led to the realization that atrial cardiomyocytes perform a dual role in mammalian heart as both muscle and endocrine cells [1,2]. Atrial myocytes store ANP in secretory vesicles and release it by the regulated secretory pathway, much like typical endocrine cells [3,4]. Ventricular cardiomyocytes also synthesize ANP, albeit in far lower amounts, but they secrete it by the constitutive secretory pathway, thus accounting for the very low ratio of ANP peptide to proANP mRNA concentrations in ventricular tissue [3,4]. More recent investigations have shown that cardiomyocytes also synthesize and secrete met-enkephalin- [5–8] and dynorphin-related peptides [8–11].

Cardiac tissue also contains adrenocorticotropic hormone (ACTH), β-endorphin and related pro-opiomelanocortin- (POMC) derived peptides, although far less is known about their cellular localization or function in the heart [12–17]. Pro-opiomelanocortin peptides were first identified in heart by Saito et al. [12] who reported that ACTH immunoreactivity is present in rat heart extracts. Forman et al. [14] later provided definitive evidence that β-endorphin and its immediate precursor, β-lipotropin, are detectable in cardiac tissue. They also reported that the ratio of β-endorphin to β-lipotropin is influenced by hemorrhagic shock, cardiac hypertrophy and other physiological stimuli, providing evidence that POMC processing can be regulated in heart [17]. Our laboratory extended these findings by showing that ACTH and β-endorphin undergo extensive post-translational processing in cardiac tissue [16]. ACTH is almost entirely converted to α-melanocyte-stimulating hormone- (α-MSH) related peptides. Correspondingly, virtually all of the immunoreactive β-endorphin isolated from heart is attributable to N-terminal acetylated and carboxy-terminal truncated β-endorphin peptides, rather than β-endorphin1–31, the opioid form of the peptide [16]. Heart tissue thus contains α-MSH and non-opioid β-endorphin peptides but little ACTH or opioid active β-endorphin1–31.

Forman and Bagasra [18] were also the first to provide evidence that POMC may be synthesized by cardiomyocytes. They reported that POMC mRNA is detectable in ventricular myocytes by in situ hybridization histochemistry using an oligonucleotide probe corresponding to the POMC gene sequence encoding β-endorphin’s N-terminus [18]. Our laboratory subsequently reported that POMC mRNA is also detectable in atrial myocytes by in situ hybridization [19] and McLaughlin and Wu [8] recently confirmed these findings. These data do not constitute definitive evidence that myocytes synthesize POMC, however, because they failed to discriminate between full-length POMC mRNA and a second, 5′ truncated POMC transcript (Fig. 1) expressed in brain [20–23] and many peripheral tissues [21,24–31]. The 5′ truncated POMC transcript lacks the POMC signal sequence encoded by exon 2 of the POMC gene and is thus unlikely to undergo translation [27–29]. This short POMC transcript is far more abundant than full-length POMC mRNA in the testes, ovaries, pancreas and other peripheral tissues that express the POMC gene [26,30,31]. However, POMC peptide concentrations are correlated with full-length, not 5′ truncated, POMC mRNA levels in these tissues, consistent with the conclusion that 5′ truncated POMC mRNA is not translated [26]. The oligonucleotides used in earlier studies to localize POMC mRNA in rat heart by in situ hybridization are unable to discriminate between 5′ truncated and full-length POMC mRNAs because they hybridize to a sequence common to both transcripts [8,18,19]. Hence, it remains to be determined whether full-length, 5′ truncated, or both POMC mRNAs are expressed by cardiac tissue.

Fig. 1

Schematic diagram of full-length and 5′ truncated POMC mRNA transcripts. The lightly shaded area illustrates the POMC protein-coding region and the vertical lines indicate the position of exons 1, 2 and 3. The darkly shaded area represents the PCR products. The horizontal arrows indicate the locations and sequences of PCR primers.

In the present study, we tested the hypothesis that POMC is synthesized by cardiomyocytes, rather than cardiac neurons, by using immunohistochemistry. This revealed that atrial myocytes express α-MSH, β-endorphin and αN-acetyl-β-endorphin immunoreactivities. Ventricular tissue exhibited little or no immunostaining, suggesting that ventricular myocytes do not synthesize POMC, or, alternatively, that they synthesize and release POMC peptides by the constitutive secretory pathway. To discriminate between these two alternatives, we used reverse transcription polymerase chain reaction (RT-PCR) to determine if POMC mRNA is present in the ventricles. Reverse transcription PCR revealed that both full-length and 5′ truncated POMC mRNAs are detectable in atrial and ventricular tissue. These findings indicate that both atrial and ventricular cardiomyocytes synthesize POMC.

2 Methods

2.1 Animals

Male and pregnant female Sprague-Dawley rats (250–300 g; Sasco, Omaha, NE, USA) were housed under a 12 h light–dark cycle with free access to food and water. Males were used exclusively for all experiments with adult rats; both males and females were used for experiments with neonatal animals. The animal research protocol conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Immunohistochemistry

Rats were anesthetized with pentobarbital (60 mg/kg i.p.) and perfused through the left ventricle with ice-cold phosphate-buffered saline (pH 7.4; PBS) followed by 8% periodate–lysine paraformaldehyde (PLP: 37.5 mM sodium phosphate buffer, pH 7.4, containing 10 mM NaIO4, 75 mM lysine and 2% paraformaldehyde). Hearts were removed, bisected longitudinally into right and left halves, immersed in PLP for two–three days at 4°C, infiltrated with 20% sucrose in PBS for 24 h and stored at −70°C.

Slide-mounted sections of heart tissue (15 μm) were incubated for 12–24 h at 4°C with one of several primary antisera diluted in PBS containing 3% normal goat serum and 0.3% Triton X-100. The primary antisera, which were generated in rabbits against rat peptide sequences, included α-MSH (1:300; Incstar, Stillwater, MN, USA), β-endorphin (1:300, Incstar), αN-acetyl-β-endorphin (1:300) [32,33] and ANP (1:1000, Peninsula Laboratories, Belmont, CA, USA). After incubation with primary antiserum, slide-mounted tissue sections were rinsed three times in PBS containing 0.01% Triton X-100 (PBS–TX) then incubated for 1 h at room temperature in goat anti-rabbit immunoglobulin G, diluted 1:50 in PBS–TX containing 3% normal goat serum, and rinsed three times in PBS–TX. The sections were incubated for 1 h in rabbit peroxidase–antiperoxidase, diluted 1:100 in PBS–TX containing 1% normal goat serum, then rinsed once in PBS and twice in 0.1 M Tris-buffered saline (pH 7.6; TBS). Finally, the sections were reacted for 6–10 min with freshly prepared TBS containing 22 mg of 3,3′-diaminobenzidine and 10 μl of 30% hydrogen peroxide in 100 ml of TBS. The reaction was terminated by rinsing once with PBS and once with water and the sections were coverslipped with or without counterstaining.

2.3 Reverse transcription polymerase chain reaction

For mRNA analyses, adult male rats were anesthetized with pentobarbital (60 mg/kg i.p.) and killed by decapitation. The heart and pituitary were removed from each rat, dissected, rinsed in ice-cold saline, frozen on dry ice and stored at −70°C. Cardiac and pituitary tissues were homogenized in 20 volumes of RNAzol B (Tel Test, Friendswood, TX, USA) and RNA was isolated by the method of Chomczynski and Sacchi [34].

Single-stranded cDNA was synthesized using mouse murine leukemia virus reverse transcriptase and primed with oligo dT (Perkin Elmer, Foster City, CA, USA) in 40 μl of 10 mM Tris–HCl (pH 9.0) containing 50 mM KCl, 6.5 mM MgCl2, 40 units of ribonuclease inhibitor, 1 μM deoxynucleotide triphosphates, 100 units of reverse transcriptase, 2.5 μM oligo dT (Perkin Elmer) and 2 μg of RNA [23]. The samples were overlaid with mineral oil and mRNA was reverse transcribed for 15 min at 42°C then enzyme-inactivated for 5 min at 99°C. The samples were then stored at 4°C until further use.

A region of rat POMC cDNA corresponding to a segment of exons 2 and 3 was amplified by using the sense primer sequence (LF) 5′ GAG ATT CTG CTA CAG TCG CTC 3′ and the antisense primer sequence (BR) 5′ TTG ATG ATG GCG TTC TTG AA 3′ (Fig. 1) [35]. To amplify the 5′ truncated POMC transcript, a different sense primer, BF, was used with the same antisense primer, BR. The BF sense primer, 5′ GGC CTT TCC CCT AGA GTT CA 3′, anneals to a sequence of POMC cDNA corresponding to the ACTH coding region; hence, the BF/BR primers amplify both 5′ truncated and full-length POMC cDNAs (Fig. 1). The primers were designed using PRIMER version 0.5 software and were compared for known genomic sequence homology with GenBank using a BLAST search.

Single-stranded cDNA was amplified by PCR in a model PTC-100 programmable thermocycler (MJ Research, Watertown, MA, USA) using 10 μl of the single-stranded RT-product from cardiac tissue in 50 μl of 10 mM Tris–HCl (pH 9.0) containing 50 mM KCl, 0.1% Triton X-100 and 10 pmol of the appropriate sense and antisense primers. The samples were overlaid with mineral oil, heat-denatured at 95°C for 5 min, and 1.6 units of Taq polymerase (Promega, Madison, WI, USA) were added to each sample. The samples were cycled 36 times at 95°C for 1 min to denature, 65°C for 1 min to anneal and at 72°C for 1 min to extend. When the amplification was finished, the samples were incubated for 10 min at 72°C for final extension and were stored at 4°C. PCR products (20 μl) were separated in 4% 3:1 agarose gels (Amresco, Solon, OH, USA) electrophoresed at 70 V for 1 h 15 min in 40 mM tris–acetate buffer containing 1 mM EDTA (pH 7.0). The gels were stained for 30 min in the same buffer containing 2.5 μg/ml of ethidium bromide and were then photographed.

3 Results

Pro-opiomelanocortin-derived peptides have been isolated from rat [12,14–16] and human [13] heart but it is uncertain whether or not POMC is actually synthesized by cardiomyocytes. To test the hypothesis that cardiomyocytes synthesize POMC, we conducted immunohistochemical experiments using antisera against α-MSH, β-endorphin and αN-acetyl-β-endorphin. We found that atrial myocytes were immunostained by all three antisera. The immunoreactivity was granular in appearance, particularly for αN-acetyl-β-endorphin (Fig. 2B), and was more intense in atrial appendages than in the rest of the atrium (Table 1). Importantly, nerve fibers did not contain α-MSH, β-endorphin or αN-acetyl-β-endorphin immunoreactivity; connective tissue was similarly unstained. Atrial natriuretic peptide immunoreactivity was similarly distributed, although it was greater in intensity and more granular in appearance than POMC-peptide immunoreactivity. In control experiments, immunostaining was eliminated completely by omitting the primary antiserum (Fig. 2A) or by preincubating each antiserum with 1 μM homologous antigen (data not shown).

Fig. 2

αN-Acetyl-β-endorphin immunoreactivity in rat atria. (A) Control section of the right ventricle (left) and atrium (right) incubated without primary antiserum. Magnification, ×160. Scale bar, 100 μm. (B) αN-Acetyl-β-endorphin immunoreactivity in the right atrial appendage of an adult rat. Magnification, ×640. Scale bar, 25 μm.

View this table:
Table 1

Relative distribution of α-MSH, αN-acetyl-β-endorphin and ANP immunoreactivities in adult rat hearta

RegionPeptide immunoreactivity
α-MSHαN-Acetyl-β-endorphinANP
Right atrium1.16±0.101.30±0.151.83±0.16
Right atrial appendage1.63±0.101.40±0.111.90±0.09
Left atrium1.12±0.111.10±0.121.63±0.06
Left atrial appendage1.53±0.061.40±0.061.87±0.09
Ventricle0.20±0.150.20±0.150.13±0.14
  • a Coronal sections from the hearts of three adult rats were immunostained with antisera against α-MSH, αN-acetyl-β-endorphin or ANP. Three sections from each heart were stained with each antiserum and the immunostaining intensity was scored as follows: 0, background staining; 0.5, minimal staining; 1.0, low staining; 1.5, moderate staining; 2.0 maximal staining (mean±SE).

In marked contrast to the atria, ventricular myocytes exhibited little or no immunoreactivity for α-MSH, αN-acetyl-β-endorphin or ANP (Table 1). Fig. 3A shows that only faint staining was produced by the αN-acetyl-β-endorphin antiserum in the left ventricle; comparable results were obtained in the right ventricle and inter-ventricular septum (data not shown). The α-MSH and ANP antisera also produced little discernable staining in adult ventricles (Table 1). Again, nerve fibers and connective tissue did not display α-MSH, β-endorphin, αN-acetyl-β-endorphin or ANP immunoreactivities. Hence, like ANP, POMC-peptide immunostaining was considerably lower in ventricles than atria.

Fig. 3

αN-Acetyl-β-endorphin immunoreactivity is present in ventricular cardiomyocytes of neonatal, but not in adult, rat heart. (A) αN-Acetyl-β-endorphin immunostaining of the left ventricle of an adult male rat. Magnification, ×640. Scale bar, 25 μm. (B) αN-Acetyl-β-endorphin immunoreactivity in the left ventricle of a 12-day-old rat. Magnification, ×320. Scale bar, 50 μm.

Unlike adult rats, relatively high immunoreactive ANP concentrations are found in the ventricles of neonatal rats [36]. To determine if POMC-peptide immunoreactivity is regulated similarly during development, we tested whether or not the ventricular cardiomyocytes of rats sacrificed on postnatal day twelve were immunostained by antisera against POMC peptides. Relatively intense αN-acetyl-β-endorphin (Fig. 3B) and α-MSH (Table 2) immunostaining was present, not only in the atria, but also in the ventricles of 12-day-old rats. Comparable ventricular αN-acetyl-β-endorphin immunostaining was observed during the first two postnatal weeks, but, by day 21, the staining intensity had returned to levels that were indistinguishable from those of the adult (data not shown). Atrial natriuretic peptide immunoreactivity was also present in ventricular cardiomyocytes during the postnatal period (Table 2) as previously reported [36].

View this table:
Table 2

Relative distribution of α-MSH, αN-acetyl-β-endorphin and ANP immunoreactivities in post-natal day 12 rat hearta

RegionPeptide immunoreactivity
α-MSHαN-Acetyl-β-endorphinANP
Right atrium1.10±0.061.13±0.071.40±0.06
Right atrial appendage1.13±0.071.10±0.061.43±0.07
Left atrium1.07±0.070.67±0.171.17±0.04
Left atrial appendage1.20±0.121.13±0.071.73±0.14
Ventricle0.83±0.170.87±0.141.20±0.10
  • a Coronal sections from the hearts of three 12-day-old rats were immunostained with antisera against α-MSH, αN-acetyl-β-endorphin or ANP. Three sections from each heart were stained with each antiserum and the immunostaining intensity was scored as follows: 0, background staining; 0.5, minimal staining; 1.0, low staining; 1.5, moderate staining; 2.0 maximal staining (mean±SE).

To investigate further whether atrial and/or ventricular cardiomyocytes are capable of synthesizing POMC, we determined if POMC mRNA is present in rat heart. Initially, we attempted to measure POMC mRNA by using Northern blot analysis. Pro-opiomelanocortin mRNA was not consistently detectable in heart extracts by Northern blot although it was easily measurable in the pituitary (data not shown). This means that either cardiac tissue does not express the POMC gene or that POMC mRNA concentrations are below the detection limit of Northern blot analysis.

To determine if small amounts of POMC mRNA are present in rat heart, we amplified POMC mRNA using RT-PCR [23]. For the initial experiments, RNA was extracted from the ventricle, ventricular septum, atrial appendage, atria and pituitary neurointermediate lobe. The RNA was reverse transcribed and POMC cDNA was amplified using a sense primer (LF) corresponding to part of the POMC signal sequence encoded by exon 2 and an antisense primer (BR) corresponding to part of the β-endorphin coding sequence in exon 3 (Fig. 1). These primers do not amplify the 5′ truncated POMC transcript, which lacks exons 1 and 2 [29].

Reverse transcription PCR amplification of RNA extracted from the left or right ventricle or atrial appendages generated an ethidium-bromide-stained band on agarose gels corresponding in size to the predicted 678 base pair POMC PCR product (Fig. 4). A 678-base-pair band was not consistently detected by RT-PCR amplification of RNA extracted from the atria or ventricular septum (Fig. 4), but repeated RT-PCR analysis of these tissues occasionally generated faint bands corresponding to the 678 base pair PCR product (data not shown). As expected, a PCR product of comparable size was also amplified from pituitary neurointermediate lobe cDNA. In control experiments, omission of cardiac RNA (Fig. 4, lane C) or reverse transcriptase (data not shown) abolished the 678 base pair band completely, which indicates that neither genomic DNA nor cDNA contamination accounts for the 678 base pair signal. These data suggest that cardiac tissue expresses full-length POMC mRNA.

Fig. 4

Detection of POMC mRNA in adult rat heart by RT-PCR. cDNA was prepared from 2 μg of total RNA and amplified using primers LF and BR. These primers generate a 678-base pair PCR product corresponding to a portion of exons 2 and 3 of the POMC gene (Fig. 1). Primers LF and BR do not amplify the 5′ truncated POMC transcript. NIL=neurointermediate lobe.

Subsequently, we determined if the 5′ truncated POMC transcript is expressed in cardiac tissue by using a sense primer (BF) that anneals to a cDNA sequence corresponding to the ACTH coding region of POMC mRNA. This, and the same antisense primer (BR) used previously, amplifies both 5′ truncated and full-length POMC transcripts. Fig. 5 shows that a signal corresponding in size to the predicted 215 base pair PCR product was generated by PCR amplification of cDNA prepared from the neurointermediate pituitary, ventricles, ventricular septum, atrial appendages and atria using the BF and BR primers. The 215 base pair band was present following amplification of cDNA from heart regions, including the atria and ventricular septum, in which the 678 base pair band was not consistently detectable, which suggests that the 215 base pair signal is derived primarily from the short POMC transcript. Omission of either RNA or reverse transcriptase eliminated the signal, again indicating that it was not attributable to genomic DNA or cDNA contamination. These data suggest that both full-length and 5′ truncated POMC transcripts are present in cardiac tissue.

Fig. 5

Evidence that a 5′ truncated POMC transcript is present in rat heart. cDNA was amplified using primers BF and BR, which generate a 215-base-pair product from exon 3 of both 5′ truncated and full-length POMC cDNAs (Fig. 1). NIL=neurointermediate lobe.

4 Discussion

Pro-opiomelanocortin-derived peptides have been detected in heart extracts, but their cellular location has not been determined conclusively [12–17]. In this study, we tested whether or not POMC peptides are located in cardiomyocytes by using immunohistochemistry. We found that α-MSH, β-endorphin and αN-acetyl-β-endorphin immunoreactivities are present in atrial cardiomyocytes of adult rats, particularly in the atrial appendages, but not to a significant extent in ventricular cardiomyocytes. Nonetheless, RT-PCR amplification revealed that full-length POMC mRNA is present in both atria and ventricles. This finding is consistent with evidence that ventricular myocytes lack secretory vesicles and secrete ANP and other peptides by using the constitutive secretory pathway [3,4,6]. Although virtually undetectable in the ventricles of adult rat heart, POMC-peptide immunoreactivity was readily detectable in neonatal ventricles (Fig. 3A) and in neonatal ventricular myocyte cultures [19], indicating that POMC peptide synthesis and/or storage is regulated during cardiac development, as reported previously for ANP [36]. These results support the hypothesis that cardiomyocytes synthesize and post-translationally process POMC.

Pro-opiomelanocortin-derived peptides and mRNA transcripts have also been detected in the ovary, testes and several other non-neural tissues [21,24–27,30,31]. Initial investigations of POMC mRNA in these tissues were difficult to interpret because the transcript detected was smaller in size than the 1200 nucleotide POMC mRNA isolated from the pituitary and brain, and was greater in abundance than predicted from the relatively low POMC-peptide concentrations found in most non-neural tissues [26]. This smaller, 800 nucleotide transcript was later found to lack the 5′ region of full-length POMC mRNA corresponding to exon 2 of the POMC gene. Because exon 2 encodes the POMC signal sequence, the 800 nucleotide transcript is unlikely to undergo appropriate translation, processing and secretion [27–29]. Subsequent analyses of POMC mRNAs using more sensitive analytical methods revealed that full-length POMC mRNA is present in non-neural tissues in amounts that correspond to tissue POMC-peptide concentrations [26]. The function of the 800 nucleotide transcript, if any, is not known.

Here, we report evidence that both full-length and 5′ truncated POMC mRNAs are present in rat heart. By using appropriate primers, we amplified a 678-base-pair segment of full-length POMC mRNA corresponding to a portion of exons 2 and 3 of the POMC gene and a 215-base-pair segment of the ACTH/β-lipotropin coding region common to both 5′ truncated and full-length POMC mRNAs. A relatively intense 678 base pair band was generated by PCR amplification of cDNA prepared from the atrial appendages but the signal was weak and inconsistently amplified from cDNA from the rest of the atria, consistent with immunohistochemical data (Table 1). Reverse transcription PCR generated a robust 215 base pair signal from all of the heart regions that we analyzed, including the atria and ventricular septum, which evidently express little or no full-length POMC mRNA. These data indicate that both full-length and 5′ truncated POMC transcripts are expressed in heart, although it is not possible to draw definitive conclusions about their relative concentrations from RT-PCR data alone.

Cardiomyocytes also synthesize proenkephalin [5–8] and prodynorphin [9–11]. In contrast to ANP, proenkephalin [5] and prodynorphin [9] mRNA levels are considerably higher in ventricles than in atria, even in the adult. Indeed, proenkephalin mRNA concentrations are unusually high in the ventricles, being higher than in the hypothalamus or any other tissue [5]. Nevertheless, ventricular met-enkephalin [5] and dynorphin B [9] concentrations are quite low, again consistent with evidence that ventricular cardiomyocytes secrete peptides constitutively [3,6].

The heart thus expresses all three of the major opioid peptide genes. The function of the peptides derived from the three opioid prohormones may differ, however. Met-enkephalin- and dynorphin-related peptides produce quite different effects on autonomic transmission, for example. Caffrey et al. [37] reported that intra-arterial met-enkephalin or met-enkephalin–Arg–Phe administration inhibits vagal bradycardia in dogs, apparently by activating presynaptic delta receptors that inhibit acetylcholine release, but does not affect the sympathetic component of vagal nerve stimulation [37]. By contrast, norepinephrine release from sympathetic neurons is inhibited by dynorphin1–9, a kappa receptor agonist, but apparently not by met-enkephalin [38]. These data are consistent with the hypothesis that presynaptic delta receptors inhibit acetylcholine release from parasympathetic neurons, whereas kappa receptors inhibit norepinephrine release from sympathetic nerves innervating the heart [37].

Delta and kappa receptor agonists also produce direct effects on cardiomyocytes. Delta receptor agonists reduce heart rate, cardiac output and stroke volume in isolated heart preparations [39] and lower the maximum chronotropic response to norepinephrine in isolated atria [40]. Delta receptor activation also inhibits norepinephrine- [41] and electrically stimulated [42] calcium influx in ventricular myocytes and reduces the amplitude of spontaneous contractions in unstimulated cells [43]. Kappa receptor agonists produce the opposite response in unstimulated cardiomyocytes, i.e., they increase myocyte contraction and calcium influx [43,44,45], although they lower calcium influx in electrically stimulated myocytes [42,44]. Mu receptor agonists have little effect on cardiomyocytes [42] or isolated heart preparations [39], consistent with data from receptor binding experiments showing that heart tissue contains delta and kappa, but not mu, receptors [46].

β-Endorphin is unlikely to participate in the opioid regulation of cardiac function because the β-endorphin peptides localized in the heart are essentially inactive as opioid receptor agonists [16]. β-Endorphin1–31, the opioid form of the peptide, is almost entirely converted to β-endorphin1–27, β-endorphin1–26 and their αN-acetylated congeners [16], which display little or no affinity for opioid receptors [47]. The function of the non-opioid β-endorphin peptides produced in the heart is unknown, although one β-endorphin derivative, glycyl–l-glutamine [β-endorphin30–31], has been shown to influence myocyte biochemistry. Glycyl–l-glutamine induces expression of the asymmetric forms of acetylcholinesterase in neonatal ventricular myocytes [48], a response also observed in sympathetic ganglia [49] and chick myotubes [50]. Other POMC-derived peptides, including ACTH- and α-MSH-related peptides, also influence cardiac function; they enhance sympathetic transmission in rat atria [51–53], increase heart rate in isolated guinea pig heart preparations [54] and are potent pressor and cardioaccelerator agents when injected intravenously [55]. Hence, the major cardioactive products of POMC processing in the heart may be melanocortin, rather than opioid peptides.

Nevertheless, the exact role of POMC-peptides in the normal or pathogenic regulation of myocyte function remains to be definitively elucidated. Cardiac α-MSH and β-endorphin concentrations are relatively low (1–10 fmol/mg protein) [14,16], which are 10% or less of whole brain concentrations. Low tissue concentrations are not unexpected for constitutively released peptides, but they generally denote a paracrine or autocrine function, rather than the endocrine role attributed to ANP [1,2,4]. The observation that ventricular α-MSH and β-endorphin immunoreactivities are regulated postnatally raises the possibility that POMC-peptides may play a role in heart development. In addition, we found, in preliminary studies, that ventricular α-MSH immunoreactivity was elevated during cardiac hypertrophy, produced by constricting the abdominal aorta of rats [19]. Atrial natriuretic peptide immunoreactivity was also increased [19], consistent with reports that cardiac hypertrophy induces proANF gene expression [2]. Cardiac hypertrophy also induces ventricular proenkephalin [56,57] and prodynorphin [58] gene expression, which suggests that all three opioid peptide families may be involved in either the pathogenesis or the compensatory response to cardiac hypertrophy.

In summary, these data show that immunoreactive α-MSH, β-endorphin and αN-acetyl-β-endorphin are present in atrial and, to a lesser extent, ventricular cardiomyocytes. Reverse transcription PCR analysis demonstrated that both atrial and ventricular tissue express full-length POMC mRNA and provide evidence that a 5′ truncated POMC transcript is also present in heart, as it is in brain [20,23] and a number of non-neural tissues [26]. Finally, we found that POMC peptide immunoreactivity changes during postnatal development. Together, these data indicate that cardiomyocytes synthesize POMC-peptides.

Acknowledgements

This research was supported by a grant from the American Heart Association, Kansas Affiliate (KS-94-GS-32). The authors thank Gregory P. Mueller, Ph.D., Department of Physiology, Uniformed Services University of the Health Sciences, for the αN-acetyl-β-endorphin antiserum.

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View Abstract