Translation of heart preproenkephalin of enkephalin peptides from cultured
mRNA and secretion cardiac myocytes
JEREMY P. SPRINGHORN AND WILLIAM C. CLAYCOMB Department of Biochemistry and Molecular Biology, Louisiana State University New Orleans, Louisiana 70112 Springhorn, Jeremy P., and William C. Claycomb. Translation of heart preproenkephalin mRNA and secretion of enkephalin peptides from cultured cardiac myocytes. Am. J. Physiol. 263 (Heart Circ. PhysioL. 32): H1560-H1566, 1992.Rat ventricular cardiac muscle has previously been shown to contain exceptionally high levels of preproenkephalin mRNA (ppEnk mRNA). We have recently determined that the level of ppEnk mRNA is developmentally and hormonally regulated in rat ventricular cardiac muscle tissue and in cultured myocytes (J. P. Springhorn and W. C. Claycomb. Biochem. J. 258: 73-77, 1989). We demonstrate in the current study that heart ppEnk mRNA is structurally identical at the 5’ end to brain ppEnk mRNA using a ribonuclease protection assay and that heart ppEnk mRNA can be translated in vitro using a rabbit reticulocyte lysate system. In vitro synthesized preproenkephalin peptides were immunoprecipitated with a polyclonal antibody directed to the carboxy-terminal seven amino acids of preproenkephalin. We have also established by radioimmunoassay that enkephalin-containing peptides are secreted from cultured neonatal and adult rat ventricular cardiac muscle cells. This secretion is linear with respect to time and can be stimulated by phorbol 12-myristate 13-acetate (PMA) and adenosine 3’,5’cyclic monophosphate (CAMP). It was determined by column chromatography that CAMP induced neonatal rat ventricular cardiac muscle cells to secrete Met”-enkephalin-Arg6-Phe7, whereas PMA plus 3-isobutyl-l-methylxanthine induced adult rat ventricular cardiac muscle cells to secrete Mets-enkephalin. These studies establish that ventricular heart muscle ppEnk mRNA can be translated and that enkephalin peptides are secreted from ventricular cardiac muscle cells. cardiac muscle cells; enkephalin-containing peptides
is the precursor of many enkephalin-containing peptides. Each bioactive segment of the precursor is flanked by pairs of basic amino acids, which are thought to be the sites of proteolytic cleavage in the prohormone activation pathway (10). In addition, any processed bioactive peptides that are secreted from ventricular cardiac muscle must be secreted by a constitutive pathway because of the inherent lack of secretory granules in this tissue (11). We have recently reported that preproenkephalin mRNA (ppEnk mRNA) is both developmentally and hormonally regulated in rat ventricular cardiac muscle tissue and cultured cells (22). Although the original report (9) describing the presence of ppEnk mRNA in heart suggested that the very low levels of protein present within the heart cell were due to an inability to translate ppEnk mRNA into protein, a more recent report has shown that enkephalin mRNA in heart tissue was found to be associated with polysomes (16). This latter study is suggestive of translational feasibility. The role that enkephalin-containing peptides may play in regulating cardiac function by acting directly on the ventricular cardiac muscle cell has been little studied. Early reports have indicated that enkephalin-conPREPROENKEPHALIN
HI560
0363-6135/92
$2.00
Copyright
Medical
Center,
taining peptides can mediate the calcium influx, the inotropic response, and the adenylate cyclase activity of cultured neonatal ventricular cardiac muscle cells (14). Moreover, this response was shown to be specifically linked to the presence of opiate receptors on these cells (15). Although extensive studies have indicated that opiates and opioid peptides can dramatically influence cardiac function through sympathetic and parasympathetic neurons that innervate the heart, the site of action in these studies appears to be higher centers (8). The findings that enkephalin-containing peptides can act on cardiac myocytes and directly influence function and that enkephalin-containing peptides have a very short half-life, and the fact that ppEnk mRNA is present as well as hormonally regulated within the ventricular cardiac myocyte, suggest that cardiac function may be regulated by locally produced enkephalin-containing peptides. Therefore, it was the purpose of this study to determine whether ppEnk mRNA from ventricular cardiac muscle tissue can be translated and determine which enkephalin-containing peptides are secreted from this tissue. These issues were addressed by investigating: 1) the 5’ structure of the mRNA encoding ppEnk mRNA, i.e., whether the cap site between brain and heart is similar; 2) whether ppEnk mRNA from ventricular cardiac tissue could be translated in an in vitro translation system; 3) whether Mets-enkephalincontaining peptides can be detected in the cells and in the media of cultured ventricular cardiac muscle cells; and finally 4) the identity of Mets-enkephalin-containing peptide that are secreted from ventricular cardiac muscle cell cultures. MATERIALS
AND METHODS
Total and poly(A) + RNA isolation. RNA was isolated exactly as previously described (22) and oligo(dT) chromatography was performed to obtain poly(A)+ mRNA exactly as previously described (18). Ribonuclease (RNase) protection assay. Twelve micrograms of total RNA from ventricular cardiac muscle, brain, and liver were used as samples. A genomic clone of the rat preproenkephalin gene spanning the 5’ flanking and exon 1 sequence was obtained from Dr. James Douglass (Vollum Institute for the Advancement of Biomedical Research, Portland, OR). A 134-bp Pst I-Sst I fragment was subcloned into pBluescriptIIsk+ (Stratagene, LaJolla, CA). This subclone was linearized using EcoR I and was used as a template for T3 RNA polymerase (Stratagene) to generate a radiolabeled antisense cRNA. The probe that contained 18 bases of plasmid sequence, for a total length of 152 bases, was gel purified using a nondenaturing 5% acrylamide gel. The probe was eluted for 3 h, ethanol precipitated, and resuspended in a volume to give - 1.0 X 10” counts. min- l. ~1~ l of cRNA.
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
ENKEPHALIN
BIOSYNTHESIS
The sample RNA and the radiolabeled cRNA were resuspended in 25 ~1 of hybridization buffer [40 mM piperazineN,N’-bis(2-ethanesulfonic acid) (PIPES; pH 6.4), 1.0 mM EDTA, 0.4 M sodium chloride, and 80% deionized formamide]. The samples were centrifuged briefly, denatured at ~85°C for 5 min, and rapidly transferred to a 51 “C water bath for hybridization (8 h). After the incubation, 350 ~1 of RNase digestion buffer [ 10 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.4, 5 mM EDTA, 0.3 M sodium chloride, 40 pg/ml RNase A, and 0.65 pg/ml RNase Tl] were added to each sample and allowed to incubate for 45 min at 30°C. The digestion was terminated by the addition of 10 ~1 of 20% sodium dodecyl sulfate (SDS) and 2.5 ~1 of 10 mg/ml proteinase K and by incubation at 37°C for 15 min. The samples were phenol-chloroform extracted, ethanol precipitated, and analyzed by electrophoresis through a 10% denaturing urea-acrylamide gel. The dried gel was then exposed overnight to Kodak X-ray film. In vitro translation and immunoprecipitation. Ethanol-precipitated poly(A)+ mRNA isolated by oligo(dT) chromatography was resuspended in diethyl pyrocarbonate (DEPC) -treated water. Approximately 3 pg of poly(A)+ mRNA were used in each reaction with the addition of 300 U RNasin (Promega Biotech) and 1 mM dithiothreitol. Aliquots of each mRNA sample were heated to 65°C for 5 min. The [?S]methionine, rabbit reticulocyte lysate, and cocktail were all supplied in a kit from New England Nuclear (Boston, MA). Each reaction contained 3 pg of poly(A)+ mRNA in 30 ~1 of DEPC water, 30 ~1 [:V]methionine (>800 Ci/mmol), 33 ~1 translation cocktail, 11.4 ~1 of 1 M potassium acetate, 3.6 ~1 of 32.5 mM magnesium acetate, and 60 ~1 lysate. The samples were incubated at 37°C for 1 h. Incorporation of radiolabel into polypeptides was determined by trichloroacetic acid-precipitable counts. Each in vitro translation reaction was aliquoted into two samples, and 170 ~1 of immunoprecipitation buffer [ 10 mM sodium phosphate (pH 7.4), 75 mM sodium chloride, 0.5 mM EDTA, 0.25 mg/ml bovine serum albumin (BSA), 0.5 mM methionine, 0.5% Triton X-100, 0.1% SDS, 0.7 tissue inhibitory units/ml aprotinin, and 0.03 mg/ml bacitracin] were added to each. Immunocompetition was performed by adding 50 PM heptapeptide in 10 mM acetic acid to one set of samples and vehicle alone into the other. Two antibodies that have been previously reported to be immunoreactive to the carboxy-terminal seven amino acids of preproenkephalin were used for immunoprecipitation of the in vitro translation samples. Twelve microliters of RB-13 (anti-heptapeptide antibody) and 5 ~1 of MEAP-I (antiheptapeptide antibody) were added to all the samples and incubated overnight at 4°C (20). Anti-heptapeptide antibodies MEAP-I and RB-13 were generous gifts of Drs. J. S. Hong (National Institute of Environmental Health Sciences, Research Triangle Park, NC) and S. L. Sabol (National Heart, Lung, and Blood Institute, Bethesda, MD), respectively. The combination of these two antibodies was in following a previously published protocol whereby preproenkephalin was immunoprecipitated from in vitro translated mRNA isolated from adrenal tissue (20). Sixty microliters of a 0.1 g/ml protein A-Sepharose CL-4B (Pharmacia) in immunoprecipitation buffer were added to each sample and agitated on a mechanical rocker for 2 h at 4°C. The suspension was layered onto 800 ~1 of 1 M sucrose-buffer A [ 10 mM sodium phosphate (pH 7.4), 150 mM sodium chloride, 1.0 mM EDTA, 0.5 mg/ml BSA, 1.0 mM methionine, 1.0% Triton X-100, and 0.1% SDS] in an Eppendorf tube and centrifuged for 5 min. The supernatant was removed, and the Sepharose-protein A pellet was resuspended in 400 ~1 of buffer A. This was layered again on top of 800 ~1 of 1 M sucrose-buffer A and centrifuged for 5 min. This step was repeated a third time. The pellet was then resuspended in 500 ~1 buffer A, inverted a few times, and centrifuged. The pellet was resuspended in 500 ~1 of
IN CARDIAC
MUSCLE
HE61
ice-cold phosphate-buffered saline (PBS), inverted a few times and centrifuged, resuspended in 500 ~1 of release solution (0.2% SDS and 20 PM heptapeptide) and boiled for 5 min, and centrifuged for 5 min; the supernatant was collected in a 15-ml centrifuge tube. The pellet was resuspended in 500 ~1 of water and boiled again for 5 min and centrifuged for 5 min, and the supernatant was collected and pooled with the previous fraction. Each sample was frozen at -70°C and lyophilized overnight. The sample was reconstituted in 50 ~1 of sample buffer (2% SDS, 62 mM Tris, pH 7.2, 2% P-mercaptoethanol, and 20% glycerol), boiled, sonicated briefly, and electrophoresed through a discontinuous 5-10% SDS-polyacrylamide gel (13). Before fluorography the gel was treated for 1 h with EnHance (New England Nuclear). The dried gel was exposed to Kodak X-ray film for 1 wk at -70°C. Determination of Met”-enkephalin-containingpeptides within ventricular cardiac muscle cell cultures and in culture medium. Neonatal and adult ventricular cardiac muscle cells were cultured as previously described (4, 5, 22) for either 2 or 9 days, respectively, before adding the following compounds: control (nothing), N”,2’-0-dibutyryladenosine 3’,5’-cyclic monophosphate (DBcAMP; 1 mM), PMA (100 rig/ml), 3isobutyl+methylxanthine (IBMX; 0.1 mM), forskolin (1 PM), norepinephrine (Arterenol; 1 PM), and isoproterenol (1 PM). Nonmuscle cell contamination of neonatal primary cultures was minimized in the neonatal myocyte cultures by aggressive preplating and maintenance in serum-free media during the experiment, as well as initiation of the experiment within 48 h of isolation, which together reduced the percentage of nonmuscle cells to
mRNA and secretion cardiac myocytes
JEREMY P. SPRINGHORN AND WILLIAM C. CLAYCOMB Department of Biochemistry and Molecular Biology, Louisiana State University New Orleans, Louisiana 70112 Springhorn, Jeremy P., and William C. Claycomb. Translation of heart preproenkephalin mRNA and secretion of enkephalin peptides from cultured cardiac myocytes. Am. J. Physiol. 263 (Heart Circ. PhysioL. 32): H1560-H1566, 1992.Rat ventricular cardiac muscle has previously been shown to contain exceptionally high levels of preproenkephalin mRNA (ppEnk mRNA). We have recently determined that the level of ppEnk mRNA is developmentally and hormonally regulated in rat ventricular cardiac muscle tissue and in cultured myocytes (J. P. Springhorn and W. C. Claycomb. Biochem. J. 258: 73-77, 1989). We demonstrate in the current study that heart ppEnk mRNA is structurally identical at the 5’ end to brain ppEnk mRNA using a ribonuclease protection assay and that heart ppEnk mRNA can be translated in vitro using a rabbit reticulocyte lysate system. In vitro synthesized preproenkephalin peptides were immunoprecipitated with a polyclonal antibody directed to the carboxy-terminal seven amino acids of preproenkephalin. We have also established by radioimmunoassay that enkephalin-containing peptides are secreted from cultured neonatal and adult rat ventricular cardiac muscle cells. This secretion is linear with respect to time and can be stimulated by phorbol 12-myristate 13-acetate (PMA) and adenosine 3’,5’cyclic monophosphate (CAMP). It was determined by column chromatography that CAMP induced neonatal rat ventricular cardiac muscle cells to secrete Met”-enkephalin-Arg6-Phe7, whereas PMA plus 3-isobutyl-l-methylxanthine induced adult rat ventricular cardiac muscle cells to secrete Mets-enkephalin. These studies establish that ventricular heart muscle ppEnk mRNA can be translated and that enkephalin peptides are secreted from ventricular cardiac muscle cells. cardiac muscle cells; enkephalin-containing peptides
is the precursor of many enkephalin-containing peptides. Each bioactive segment of the precursor is flanked by pairs of basic amino acids, which are thought to be the sites of proteolytic cleavage in the prohormone activation pathway (10). In addition, any processed bioactive peptides that are secreted from ventricular cardiac muscle must be secreted by a constitutive pathway because of the inherent lack of secretory granules in this tissue (11). We have recently reported that preproenkephalin mRNA (ppEnk mRNA) is both developmentally and hormonally regulated in rat ventricular cardiac muscle tissue and cultured cells (22). Although the original report (9) describing the presence of ppEnk mRNA in heart suggested that the very low levels of protein present within the heart cell were due to an inability to translate ppEnk mRNA into protein, a more recent report has shown that enkephalin mRNA in heart tissue was found to be associated with polysomes (16). This latter study is suggestive of translational feasibility. The role that enkephalin-containing peptides may play in regulating cardiac function by acting directly on the ventricular cardiac muscle cell has been little studied. Early reports have indicated that enkephalin-conPREPROENKEPHALIN
HI560
0363-6135/92
$2.00
Copyright
Medical
Center,
taining peptides can mediate the calcium influx, the inotropic response, and the adenylate cyclase activity of cultured neonatal ventricular cardiac muscle cells (14). Moreover, this response was shown to be specifically linked to the presence of opiate receptors on these cells (15). Although extensive studies have indicated that opiates and opioid peptides can dramatically influence cardiac function through sympathetic and parasympathetic neurons that innervate the heart, the site of action in these studies appears to be higher centers (8). The findings that enkephalin-containing peptides can act on cardiac myocytes and directly influence function and that enkephalin-containing peptides have a very short half-life, and the fact that ppEnk mRNA is present as well as hormonally regulated within the ventricular cardiac myocyte, suggest that cardiac function may be regulated by locally produced enkephalin-containing peptides. Therefore, it was the purpose of this study to determine whether ppEnk mRNA from ventricular cardiac muscle tissue can be translated and determine which enkephalin-containing peptides are secreted from this tissue. These issues were addressed by investigating: 1) the 5’ structure of the mRNA encoding ppEnk mRNA, i.e., whether the cap site between brain and heart is similar; 2) whether ppEnk mRNA from ventricular cardiac tissue could be translated in an in vitro translation system; 3) whether Mets-enkephalincontaining peptides can be detected in the cells and in the media of cultured ventricular cardiac muscle cells; and finally 4) the identity of Mets-enkephalin-containing peptide that are secreted from ventricular cardiac muscle cell cultures. MATERIALS
AND METHODS
Total and poly(A) + RNA isolation. RNA was isolated exactly as previously described (22) and oligo(dT) chromatography was performed to obtain poly(A)+ mRNA exactly as previously described (18). Ribonuclease (RNase) protection assay. Twelve micrograms of total RNA from ventricular cardiac muscle, brain, and liver were used as samples. A genomic clone of the rat preproenkephalin gene spanning the 5’ flanking and exon 1 sequence was obtained from Dr. James Douglass (Vollum Institute for the Advancement of Biomedical Research, Portland, OR). A 134-bp Pst I-Sst I fragment was subcloned into pBluescriptIIsk+ (Stratagene, LaJolla, CA). This subclone was linearized using EcoR I and was used as a template for T3 RNA polymerase (Stratagene) to generate a radiolabeled antisense cRNA. The probe that contained 18 bases of plasmid sequence, for a total length of 152 bases, was gel purified using a nondenaturing 5% acrylamide gel. The probe was eluted for 3 h, ethanol precipitated, and resuspended in a volume to give - 1.0 X 10” counts. min- l. ~1~ l of cRNA.
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
ENKEPHALIN
BIOSYNTHESIS
The sample RNA and the radiolabeled cRNA were resuspended in 25 ~1 of hybridization buffer [40 mM piperazineN,N’-bis(2-ethanesulfonic acid) (PIPES; pH 6.4), 1.0 mM EDTA, 0.4 M sodium chloride, and 80% deionized formamide]. The samples were centrifuged briefly, denatured at ~85°C for 5 min, and rapidly transferred to a 51 “C water bath for hybridization (8 h). After the incubation, 350 ~1 of RNase digestion buffer [ 10 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.4, 5 mM EDTA, 0.3 M sodium chloride, 40 pg/ml RNase A, and 0.65 pg/ml RNase Tl] were added to each sample and allowed to incubate for 45 min at 30°C. The digestion was terminated by the addition of 10 ~1 of 20% sodium dodecyl sulfate (SDS) and 2.5 ~1 of 10 mg/ml proteinase K and by incubation at 37°C for 15 min. The samples were phenol-chloroform extracted, ethanol precipitated, and analyzed by electrophoresis through a 10% denaturing urea-acrylamide gel. The dried gel was then exposed overnight to Kodak X-ray film. In vitro translation and immunoprecipitation. Ethanol-precipitated poly(A)+ mRNA isolated by oligo(dT) chromatography was resuspended in diethyl pyrocarbonate (DEPC) -treated water. Approximately 3 pg of poly(A)+ mRNA were used in each reaction with the addition of 300 U RNasin (Promega Biotech) and 1 mM dithiothreitol. Aliquots of each mRNA sample were heated to 65°C for 5 min. The [?S]methionine, rabbit reticulocyte lysate, and cocktail were all supplied in a kit from New England Nuclear (Boston, MA). Each reaction contained 3 pg of poly(A)+ mRNA in 30 ~1 of DEPC water, 30 ~1 [:V]methionine (>800 Ci/mmol), 33 ~1 translation cocktail, 11.4 ~1 of 1 M potassium acetate, 3.6 ~1 of 32.5 mM magnesium acetate, and 60 ~1 lysate. The samples were incubated at 37°C for 1 h. Incorporation of radiolabel into polypeptides was determined by trichloroacetic acid-precipitable counts. Each in vitro translation reaction was aliquoted into two samples, and 170 ~1 of immunoprecipitation buffer [ 10 mM sodium phosphate (pH 7.4), 75 mM sodium chloride, 0.5 mM EDTA, 0.25 mg/ml bovine serum albumin (BSA), 0.5 mM methionine, 0.5% Triton X-100, 0.1% SDS, 0.7 tissue inhibitory units/ml aprotinin, and 0.03 mg/ml bacitracin] were added to each. Immunocompetition was performed by adding 50 PM heptapeptide in 10 mM acetic acid to one set of samples and vehicle alone into the other. Two antibodies that have been previously reported to be immunoreactive to the carboxy-terminal seven amino acids of preproenkephalin were used for immunoprecipitation of the in vitro translation samples. Twelve microliters of RB-13 (anti-heptapeptide antibody) and 5 ~1 of MEAP-I (antiheptapeptide antibody) were added to all the samples and incubated overnight at 4°C (20). Anti-heptapeptide antibodies MEAP-I and RB-13 were generous gifts of Drs. J. S. Hong (National Institute of Environmental Health Sciences, Research Triangle Park, NC) and S. L. Sabol (National Heart, Lung, and Blood Institute, Bethesda, MD), respectively. The combination of these two antibodies was in following a previously published protocol whereby preproenkephalin was immunoprecipitated from in vitro translated mRNA isolated from adrenal tissue (20). Sixty microliters of a 0.1 g/ml protein A-Sepharose CL-4B (Pharmacia) in immunoprecipitation buffer were added to each sample and agitated on a mechanical rocker for 2 h at 4°C. The suspension was layered onto 800 ~1 of 1 M sucrose-buffer A [ 10 mM sodium phosphate (pH 7.4), 150 mM sodium chloride, 1.0 mM EDTA, 0.5 mg/ml BSA, 1.0 mM methionine, 1.0% Triton X-100, and 0.1% SDS] in an Eppendorf tube and centrifuged for 5 min. The supernatant was removed, and the Sepharose-protein A pellet was resuspended in 400 ~1 of buffer A. This was layered again on top of 800 ~1 of 1 M sucrose-buffer A and centrifuged for 5 min. This step was repeated a third time. The pellet was then resuspended in 500 ~1 buffer A, inverted a few times, and centrifuged. The pellet was resuspended in 500 ~1 of
IN CARDIAC
MUSCLE
HE61
ice-cold phosphate-buffered saline (PBS), inverted a few times and centrifuged, resuspended in 500 ~1 of release solution (0.2% SDS and 20 PM heptapeptide) and boiled for 5 min, and centrifuged for 5 min; the supernatant was collected in a 15-ml centrifuge tube. The pellet was resuspended in 500 ~1 of water and boiled again for 5 min and centrifuged for 5 min, and the supernatant was collected and pooled with the previous fraction. Each sample was frozen at -70°C and lyophilized overnight. The sample was reconstituted in 50 ~1 of sample buffer (2% SDS, 62 mM Tris, pH 7.2, 2% P-mercaptoethanol, and 20% glycerol), boiled, sonicated briefly, and electrophoresed through a discontinuous 5-10% SDS-polyacrylamide gel (13). Before fluorography the gel was treated for 1 h with EnHance (New England Nuclear). The dried gel was exposed to Kodak X-ray film for 1 wk at -70°C. Determination of Met”-enkephalin-containingpeptides within ventricular cardiac muscle cell cultures and in culture medium. Neonatal and adult ventricular cardiac muscle cells were cultured as previously described (4, 5, 22) for either 2 or 9 days, respectively, before adding the following compounds: control (nothing), N”,2’-0-dibutyryladenosine 3’,5’-cyclic monophosphate (DBcAMP; 1 mM), PMA (100 rig/ml), 3isobutyl+methylxanthine (IBMX; 0.1 mM), forskolin (1 PM), norepinephrine (Arterenol; 1 PM), and isoproterenol (1 PM). Nonmuscle cell contamination of neonatal primary cultures was minimized in the neonatal myocyte cultures by aggressive preplating and maintenance in serum-free media during the experiment, as well as initiation of the experiment within 48 h of isolation, which together reduced the percentage of nonmuscle cells to
