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-Adrenergic and antiadrenergic modulation of cardiac adenylyl cyclase is influenced by phosphorylation
Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0127
Submitted 5 November 2002 ; accepted in final form 27 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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1-adrenergic receptor (1R) stimulation elicits myocardial protein phosphorylation, the present study investigated whether protein kinase A (PKA) catalyzed rat heart ventricular membrane phosphorylation affects the 1R adrenergic and A1R adenosinergic actions on adenylyl cyclase activity. Membranes were either phosphorylated with PKA in the absence/presence of a protein kinase inhibitor (PKI) or dephosphorylated with alkaline phosphatase (AP) and assayed for adenylyl cyclase activity (AC) in the presence of the 1R agonist isoproterenol (ISO) and/or the A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA). 32P incorporation into the protein substrates of 140–120, 43, and 29 kDa with PKA increased both the ISO-elicited activation of AC by 51–54% and the A1R-mediated reduction of the ISO-induced increase in AC by 29–50%, thereby yielding a total antiadrenergic effect of 
78%. These effects of PKA were prevented by PKI. AP reduced the ISO-induced increase in AC and eliminated the antiadrenergic effect of CCPA. Immunoprecipitation of the solubilized membrane adenylyl cyclase with the use of a polyclonal adenylyl cyclase VI antibody indicated that the enzyme is phosphorylated by PKA. These results indicate that the cardioprotective effect of adenosine afforded by its antiadrenergic action is facilitated by cardiac membrane phosphorylation.
adenosine; catecholamines; protein kinase A; adenosine A1 receptor; alkaline phosphatase
1-adrenergic catecholamine-induced increases in myocardial contractile and metabolic activity (7, 8, 13). Membrane adenylyl cyclase is an important focal point for the manifestation of the antiadrenergic action of A1R stimulation because the cyclase enzyme is a proximal component of the 1-adrenergic signaling pathway at which adenosine is known to exert its antiadrenergic effect (7, 8, 13).
In the rat myocardium, mRNA is present for adenylyl cyclase types V and VI (10, 29), suggesting the presence of these two isoforms of the enzyme. Phosphorylation of purified adenylyl cyclase and cell membranes has been reported to modify both the basal activity and the receptor-elicited activation or inhibition of the adenylyl cyclase V and VI enzymes (3, 18, 22). Protein kinase A (PKA)-induced phosphorylation of purified adenylyl cyclase V has been reported to reduce the catalytic activity of the enzyme (18) and a similar phosphorylation of overexpressed adenylyl cyclase VI in insect cell membranes inhibited both basal and stimulatory guanine nucleotide-
-induced enzyme activity (3). PKA also lowered the membrane adenylyl cyclase activity of hepatocytes cultured from 12- to 15-day-old chicks (22).
The importance of phosphorylation in the regulation of adenylyl cyclase activity is strongly suggestive, but the evidence for such a mechanism remains limited for intact myocardial membranes (15). Furthermore, the effect of PKA-induced phosphorylation on endogenous adenylyl cyclase activity of crude myocardial membranes has not been elucidated. When compared with purified or overexpressed adenylyl cyclase preparations, a crude myocardial membrane preparation represents more closely the actual functioning of the enzyme in the myocardial cell. The hypothesis for this study is that membrane phosphorylation influences the activity of adenylyl cyclase as modulated by -adrenergic and adenosinergic stimulation. With the use of crude myocardial membranes from the rat, the present study was undertaken to determine the effect of PKA-elicited myocardial membrane phosphorylation on the responsiveness of adenylyl cyclase to 1-adrenergic and A1-adenosinergic stimulation.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Enriched membrane preparation. Enriched preparations of membranes were prepared by homogenizing as described above 0.5 g of ventricular myocardium in 4 ml of a buffer containing 20 mM HEPES (pH 7.0), 1 mM EDTA, and 250 mM sucrose. The homogenate was layered on a self-forming 16% Percoll gradient containing 150 mM NaCl and 20 mM NaH2PO4 (pH 7.4) and centrifuged at 23,000 g for 35 min at 0–4°C. The membrane fraction ("buffy coat") was harvested and the Percoll eliminated by centrifugation at 100,000 g for 60 min. The enriched membrane preparation was used for phosphorylation studies and immunoblot analysis after determination of protein content as described above.
PKA. Approximately 250 µg of crude or enriched membrane protein was suspended in a buffer containing 20 mM HEPES (pH 7.4), 10 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 0.2 mM ATP, and 1.0 µM cAMP, and incubated in the absence or presence of 75 mU of catalytic subunit of PKA for 30 min at 30°C. Upon termination of the incubation, membranes were centrifuged at 44,000 g for 45 min at 0–4°C. The crude pelletized membranes were resuspended in 40 mM HEPES (pH 7.4) and immediately assayed for adenylyl cyclase activity. To assess membrane protein phosphorylation, as determined by 32P incorporation, [
-32P]ATP (100–250 µCi) was added to the above buffer of some preparations. After incubation, the resulting membrane pellet was solubilized with 2% SDS-PAGE, as previously described (11).
Alkaline phosphatase. Approximately 250 µg of crude membrane protein were incubated in a buffer containing (in mM) 20 HEPES (pH 7.4), 1 MgCl2, 0.1 ZnCl2, 0.5 EDTA, and 1 DTT in the absence or presence of 50 units of AP for 3 min at 30°C. In some experiments, the AP was boiled at 100°C for 5 min to denature the enzyme. Upon termination of the incubation, the membranes were centrifuged, resuspended, and assayed for adenylyl cyclase activity as described above for PKA.
Adenylyl cyclase. Crude membranes were assayed for adenylyl cyclase activity in the absence or presence of a 1-adrenergic agonist, isoproterenol (ISO), 2-chloro-N6-cyclopentyl-adenosine (CCPA), an adenosine A1 receptor agonist, or a combination of both (see RESULTS). The assay system for the measurement of adenylyl cyclase activity minimizes the formation of endogenous adenosine and has been previously described (20, 25). Briefly, membranes (15–25 µg protein) were incubated in 50 µl of a buffer containing 40 mM HEPES (pH 7.4), 5 mM MgCl2, 5.5 mM KCl, 1 mM DTT, 0.1 mM 2'-deoxy-cAMP (dcAMP), 0.1 mM 2'-deoxy ATP (dATP), 20 mM phosphoenolpyruvate, 2 units of pyruvate kinase, 0.25 units of adenosine deaminase, 1 mM ascorbic acid, 100 mM NaCl, 0.1 mM EGTA, 10 µM GTP, and 2 x 106 counts/min of [-32P]dATP for 10 min at 30°C. The reaction was stopped by the addition of a 50-µl solution composed of 2% SDS, 45 mM ATP, 1.3 mM cAMP, and [3H]dcAMP (4,000 counts/min) and by boiling for 2 min. The formed [-32P]dcAMP was separated from the [-32P]dATP by sequential chromatography with the use of columns of cation exchange resin AG 50W-X4 (200–400 mesh) and neutral alumina AG 7 (100–200 mesh) as previously described (20). All results were corrected for column recovery of [3H]dcAMP, which ranged between 60% and 80%. The activity of the adenylyl cyclase is expressed as picomoles [-32P]dcAMP formed per minute per milligram of protein.
PAGE and immunoblotting. Both membrane and enriched membrane preparations were dissolved in 10% glycerol, 2% SDS, 62 mM Tris (pH 7.5), 2.5% -mercaptoethanol, and 0.05% bromophenol blue. Approximately 15–20 µg of protein was subjected to PAGE, as previously described (11), except that 0.75-mm-thick minigels and 10% polyacrylamide were employed. Separated proteins were transferred to nitrocellulose membranes, and the latter were exposed to rabbit adenylyl cyclase V/VI antibody diluted 1:1,000 before use. The secondary antibody was goat anti-rabbit conjugated to horseradish peroxidase. The chemiluminescence was monitored with the use of X-ray film. In some experiments, an adenylyl cyclase V/VI blocking peptide was employed.
Protein digestion and identification. The molecular weight band of interest was cut from the polyacrylamide gel and subjected to tryptic digestion according to the method of Gharahdaghi et al. (15) with minor modifications. Briefly, 0.01% N-octylglucopyranoside was included in the digest buffer. The peptide digests were concentrated with the use of micropipette tips (model ZipTip C18, Millipore), according to recommended protocols, except that the ion-pairing agent trifluoroacetic acid was replaced with 1% formic acid. Electrospray ion trap-mass spectrometry (ESI-MS; Finnigan LCQ Deca) was performed, coupled in-line to a nanoflow HPLC (LC Packings Ultimate). Data were acquired with the use of a triple-play program that performed full mass range scans (400–2,000 amu) continuously, followed by a high resolution MS/MS scans for precursors, which passed a preset threshold. MS/MS scans were searched against a human/rodent version of the NCBI database with the use of the Sequest search algorithm. An identification was assumed positive when two or more MS/MS scans matched peptides of a particular protein with significant correlation scores.
Immunoprecipitation of membranes. Crude ventricular membranes (500 µg protein) were phosphorylated as described above in the absence or presence of 20–50 µl PKA (holoenzyme) prepared from rabbit skeletal muscle [DEAE peak 1 at 7 µg protein/ml (1)] along with 2 µM cAMP. After phosphorylation, the membranes were suspended in 100 µl of an immunoprecipitation buffer containing 150 mM NaCl, 10 mM HEPES, 1% Triton, 10 mM EDTA, 20 mM KF, 20 mM -glycerol phosphate, and 100 µg biotinylated avian adenylyl cyclase VI antibody. The cyclase was immunoprecipitated by centrifugation (12,000 g) with the use of streptavidin agarose. The 32P incorporated into the immunoprecipitated cyclase was determined and the protein subjected to SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose and immunoblotted with the avian adenylyl cyclase VI antibody diluted 1:1,000 before use either in the absence or presence of blocking peptide. The secondary antibody was sheep anti-avian conjugated to horseradish peroxidase, and the chemiluminescence was monitored as described above.
Statistical methods. All data are expressed as means ± SE. The concentration of agonist that produced EC50 was determined from nonlinear regression analysis with the use of sigmoid curve fitting (Prism 3.0; GraphPad Software, San Diego, CA). Statistical significance was determined with the use of one-way independent analysis of variance. A P value of <0.05 was accepted as indicating a statistically significant difference.
Animals. Male Sprague-Dawley rats 3–4 mo old and weighing 250–325 g were used to obtain myocardial tissues for these studies. The rats in this study were maintained and used in accordance with recommendations in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996) and the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School.
Materials. Buffer salts, acids, and general laboratory reagents were obtained from Fisher Scientific (Medford, MA). Phosphoenolpyruvate, a catalytic subunit of PKA, pyruvate kinase, AP, adenosine deaminase, PKA inhibitor (PKI), CCPA, GTP, ATP, and dATP were purchased from Roche (Indianapolis, IN). ISO, DTT, HEPES, Tris base, cAMP, dcAMP, EDTA, EGTA, DMSO, sodium orthovanadate, and BSA were obtained from RBI/Sigma (St. Louis, MO). AG 50W-X4, AG 7, SDS, and all gel electrophoretic reagents were obtained from Bio-Rad (Richmond, CA). Rabbit polyclonal adenylyl cyclase V/VI antibody and blocking peptide were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Avian polyclonal adenylyl cyclase VI antibody, blocking peptide, and the biotinylated antibody were purchased from J-QUE Biologics (Worcester, MA). Goat anti-rabbit and sheep anti-avian secondary antibodies conjugated to horseradish peroxidase were obtained from Santa Cruz and Calbiochem (La Jolla, CA), respectively. Streptavidin agarose was purchased from Oncogene (San Diego, CA). ISO (10 mM) was prepared in 0.1% Na2S2O5 with a final dilution in a solution of 1 mM ascorbic acid. CCPA (10 mM) was prepared in DMSO fresh daily. [-32P]dATP (800 Ci/mmol) was obtained from Amersham (Arlington Heights, IL). [3H]dcAMP (5.2 Ci/mmol) and [3H]cAMP (15 Ci/mmol) were purchased from ICN Pharmaceuticals (Irving, CA).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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PKA potentiates antiadrenergic effect of adenosine A1 receptor stimulation. CCPA, an adenosine A1 receptor agonist, inhibited the ISO-induced increase in adenylyl cyclase and the inhibition was enhanced by PKA (Fig. 3). The 10–7, 10–6, and 10–5 M ISO-induced increases in adenylyl cyclase activity were similar to those observed in Fig. 1 and were inhibited by 1 µM CCPA by 48, 40, and 35%, respectively. In the presence of PKA, CCPA inhibition of the 10–7, 10–6, and 10–5 M ISO-induced activation of the enzyme was augmented by an additional 50, 29, and 32%, respectively. Thus the total antiadrenergic effect of CCPA in the presence of PKA was 78 ± 7%. Neither CCPA nor CCPA plus PKA had a significant effect on adenylyl cyclase activity in the absence of ISO. Inclusion of PKI eliminated the PKA potentiation of the decrease in adenylyl cyclase activity caused by CCPA in the presence of ISO (Fig. 4). The PKI also prevented the ISO-induced increase in adenylyl cyclase activity for membranes pretreated with PKA as shown above.
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AP inhibits ISO-elicited increase in adenylyl cyclase activity. The stimulation of myocardial membrane adenylyl cyclase activity by ISO is decreased when the membranes are pretreated with increasing amounts of AP (Fig. 5). To dephosphorylate endogenous proteins as well as those phosphorylated by PKA, AP was employed because this phosphatase possesses broad substrate specificity. Because pretreatment with 100 units of AP caused a 54% decrease in the ISO-induced activation of adenylyl cyclase, this amount of the phosphatase was employed in the remaining studies.
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AP pretreatment of the membranes inhibited the dose-response ISO-induced increase in adenylyl cyclase activity without affecting enzyme activity in the absence of ISO (Fig. 6). The 10–7, 10–6, and 10–5 M ISO-induced increases in adenylyl cyclase were decreased by 60, 65, and 60%, respectively. When AP was denatured by being boiled, the enzyme did not inhibit the ISO-induced activation of adenylyl cyclase. Whereas AP decreased the ISO-induced increase in adenylyl cyclase activity, the phosphatase also reduced the enhancement of cyclase activity in membranes pretreated with PKA (Fig. 7). AP also eliminated the PKA potentiation of the CCPA elicited reduction of the ISO stimulation of adenylyl cyclase.
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PKA membrane protein phosphorylation. Ventricular membrane pretreatment with PKA resulted in 32P incorporation into three proteins as assessed by SDS-PAGE and radiography. The molecular masses of the proteins that incorporated 32P were 140–120, 43, and 29 kDa, respectively (Fig. 8A). The use of Percoll-enriched ventricular membranes revealed a similar protein phosphorylation pattern. PKI prevented the PKA-elicited phosphorylations. Gel tryptic digestion revealed that the 140–120, 43, and 29 kDa protein bands consisted primarily of -myosin/protein C/Ca2+-ATPase, cardiac troponin T/aldolase A protein, and troponin I/myosin light chain, respectively. This information was obtained by consideration of the greatest protein coverage based on amino acid count and mass. Immunoblot analysis with the use of a rabbit polyclonal adenylyl cyclase V/VI antibody in the absence and presence of blocking peptide indicated the presence of a 96-kDa protein (Fig. 8B).
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Adenylyl cyclase constitutes only 0.01–0.001% of the membrane protein (28) thus rendering it difficult to assess whether the enzyme is phosphorylated using conventional electrophoretic and autoradiographic techniques. Because immunoblotting with a rabbit polyclonal adenylyl cyclase antibody suggested the presence of the enzyme, solubilized membranes were subjected to immunoprecipitation with the use of an avian polyclonal adenylyl cyclase VI antibody in the following manner. Membrane adenylyl cyclase was phosphorylated, and a biotinylated avian adenylyl cyclase VI antibody was used with streptavidin agarose to harvest the enzyme by precipitation.
Immunoprecipitation of phosphorylated adenylyl cyclase. PKA caused a 2.2 ± 0.3-fold increase in 32P incorporation into immunoprecipitated protein with the use of the avian adenylyl cyclase VI antibody (Fig. 9A). A typical immunoblot of the precipitated protein resulting from the three different membrane preparations is presented (Fig. 9B) in the absence and presence of antibody blocking peptide. The precipitated protein had a molecular mass of 76 kDa. These results indicate that membrane adenylyl cyclase was immunoprecipitated by the specific cyclase antibody. Furthermore, the results indicate that adenylyl cyclase is one of the several membrane proteins phosphorylated by PKA.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-adrenergic receptor-mediated elevation of adenylyl cyclase activity. In addition, phosphorylation increases the antiadrenergic actions of A1R stimulation. It is interesting that the membrane phosphorylation catalyzed by PKA increases both the -adrenergic activation of adenylyl cyclase and the A1R antiadrenergic pathway inhibiting the activation of adenylyl cyclase. Because these results were obtained with the use of crude ventricular membranes, they most likely represent the actual biological functioning of the intact cardiac membrane adrenergic and adenosinergic systems operative in the heart.
Adenylyl cyclase activity and membrane protein phosphorylation. The -adrenergic receptor agonist ISO over a concentration range of 10–7 to 10–5 M caused an increase in membrane adenylyl cyclase activity that was enhanced by pretreatment of the membranes with PKA. However, pretreatment of the membranes with PKA did not appear to influence the EC50 for activation of adenylyl cyclase by ISO. Because the effect of PKA was eliminated by inclusion of a PKA inhibitor during the pretreatment protocol, the potentiation of the ISO-elicited activation of adenylyl cyclase by PKA is believed to result from enzyme catalyzed membrane protein phosphorylation.
The A1R agonist CCPA produced the expected attenuation of the ISO-induced increase in adenylyl cyclase activity (20, 25). However, PKA increased this antiadrenergic action of the A1R agonist and this effect was prevented with a PKA inhibitor. Because phosphorylation by PKA of the intact membranes increases the antiadrenergic action of CCPA, it might be concluded the activation of the -adrenergic system also facilitates the operation of the adenosinergic system via the activation of PKA. This may amplify the A1-adenosinergic-mediated antiadrenergic activity, which in turn limits the extent of -adrenergic stimulation.
Adenylyl cyclase activity and membrane protein dephosphorylation. The treatment of ventricular membranes with AP decreased the ISO-induced stimulation of adenylyl cyclase activity. However, CCPA did not further reduce the ISO-stimulated adenylyl cyclase activity in the presence of AP. The failure of CCPA to reduce the ISO-stimulated adenylyl cyclase activity in the presence of AP as it did in the absence of the phosphatase is not known. However, it could be because the stimulation of the cyclase was meager in the presence of AP. Thus when the adenylyl cyclase activity is rather low it may be difficult to demonstrate an inhibition by an adenosine A1 agonist.
These findings suggest that membrane protein phosphorylation is required to demonstrate a robust ISO-induced increase is adenylyl cyclase activity. More importantly, some level of membrane protein phosphorylation appears to be required for an A1R agonist to attenuate ISO-induced adenylyl cyclase activity. Because ISO stimulated adenylyl cyclase activity and CCPA inhibited it in the crude ventricular membranes prepared herein, the results suggest that the membrane isolation procedure employed preserved some degree of endogenous membrane protein phosphorylation. In addition, there is the possibility that proteins other than the ones observed here are also phosphorylated in the crude membranes endogenously or by PKA but that 32P incorporation is not observed because it is below the level of detection as a result of low protein abundance.
Membrane protein phosphorylation. PKA catalyzed 32P incorporation into membrane proteins having molecular masses of 140–120, 43, and 29 kDa. We reported (11, 14) comparable results with the use of isolated perfused hearts and ventricular myocytes. Gel tryptic digestion of the 140–120, 43, and 29 kDa protein bands suggests that the proteins present are -myosin/protein C/Ca2+-ATPase, troponin T/aldolase A protein, and troponin I/myosin light chain, respectively. Gel tryptic digestion was not sensitive enough to detect adenylyl cyclase in either the crude or enriched membrane preparations. However, immunoblotting of the crude or enriched ventricular membranes indicated the presence of adenylyl cyclase at 96 kDa and suggested the possibility of immunoprecipitation. Immunoblotting of the immunoprecipitated membrane protein revealed a 76-kDa protein that is most likely a monomer or fragment of adenylyl cyclase. This enzyme is a membrane glycoprotein ranging from 124 to 150 kDa (19, 21).
Types V and VI adenylyl cyclase have been shown by several investigators (3, 18, 22) to be capable of phosphorylation, and phosphorylation of Ser674 in the C1b domain of adenylyl cyclase VI appears to indeed regulate a low-affinity secondary binding site for guanine nucleotide- (3). However, the phosphorylation of purified (18), overexpressed (3), or chick-cultured hepatocyte membrane adenylyl cyclase generally appears to be inhibitory rather than facilatory, as presented herein. In the present study, the results obtained from preparations of crude ventricular membranes differ from those obtained with the use of purified adenylyl cyclase and overexpressed adenylyl cyclase systems. The present results may more accurately reflect the biological operation of the intact cardiac membrane adrenergic and adenosinergic systems. Because crude membranes were employed in the present study, the phosphorylation of minor proteins not detected in this study may have contributed to the facilitative effect of PKA. In addition to adenylyl cyclase, the 140–120 kDa (-myosin/protein C/Ca2+-ATPase), 43 kDa (troponin T/aldolase A protein), and 28 kDa (troponin I/myosin light chain) proteins were phosphorylated. However, it is unlikely that these latter proteins are involved in the facilitative effect of PKA on adenylyl cyclase activity. It is also possible that species differences may be considered as an explanation of the differences in this study and previous reports.
It is interesting that adenylyl cyclases II and VII have been reported to be phosphorylated by protein kinase C (2, 30, 31). This protein kinase C-mediated phosphorylation augments the stimulatory responsiveness of these adenylyl cyclase isoforms to guanine nucleotide-. Types II and VII adenylyl cyclase have been reported to be present in the heart, but types V and VI are the predominant isoforms present (17). Thus the possible roles of adenylyl cyclase II and/or VII in the present study are presumably minimal, if existent at all.
On the basis of the information in the literature and the new information presented herein, it may be concluded that whereas the study of purified and overexpressed adenylyl cyclase preparations is informative, it may not shed sufficient light on the functioning of the enzyme in the intact cell membrane. This may be especially important in the signal transduction schemes involving adenylyl cyclase, such as the adrenergic and adenosinergic pathways in the intact tissue. Recently, Simonis et al. (27) reported that in the intact heart there is a transient sensitization of adenylyl cyclase promoted by ischemic preconditioning. This, coupled with the results presented herein, suggests that the regulation of adenylyl cyclase in the intact cell membrane may not be accurately revealed with the use of purified preparations of the enzyme. The -adrenergic receptor-mediated enhancement of cardiac contractile and metabolic function is without question important in the heart (4, 11). The antiadrenergic action of adenosine caused by adenosine A1 receptor stimulation is also important in modulating the -adrenergic-mediated responsiveness so the myocardium does not overrespond to catecholamine stimulation (6, 12). Because the rate of myocardial adenosine released from the myocardium is increased with -adrenergic stimulation, adenosine is a negative feedback modulator that limits the responsiveness of the heart to the adrenergic stimulation (8, 9). In addition to facilitating the catecholamine activation of adenylyl cyclase, the activation of PKA resulting from -adrenergic stimulation also augments the antiadrenergic action of adenosine thereby providing balance to the response of the myocardium to adrenergic stimulation.
In summary, the results of this study indicate that the phosphorylation of heart ventricular membranes by PKA augments the -adrenergic catecholamine-induced increase in adenylyl cyclase activity. Furthermore, it is shown that the adenosine A1R-induced manifestation of an antiadrenergic action on the -adrenergic stimulation of cardiac adenylyl cyclase is also enhanced by membranous protein phosphorylation. Thus the evidence presented suggests that the phosphorylation of adenylyl cyclase and perhaps other unidentified proteins appears to be of importance in the functioning of both the adrenergic and adenosinergic systems in the heart.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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J. G. Dobson has financial investments in J-QUE Biologics.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-adrenergic contractile response in aged hearts. Circ Res 66: 1381–1390, 1990.-adrenergic contractile responses. Circ Res 72: 571–578, 1993.-adrenergic stimulation and contraction frequency on heart interstitial adenosine. Circ Res 66: 457–468, 1990.-adrenergic signal transduction in guinea pig heart ventricular membranes. J Mol Cell Cardiol 22: 1359–1370, 1990.[Web of Science][Medline]
-adrenergic receptor of rat myocardial membranes. Second Messengers Phosphoproteins 12: 29–43, 1988.[Web of Science][Medline]
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) or presence (
) of 75 mU of PKA before ISO stimulation (see METHODS). Adenylyl cyclase activity was determined at the concentrations of ISO as indicated. Data points represent means ± SE for 7 membrane preparations. *Significant increase from 0 ISO; 
significant difference from the corresponding value in the absence of PKA.
significant decrease from the appropriate value without PKI.
) or presence (
) or presence (
) of 100 units of AP (
) before ISO stimulation. Adenylyl cyclase activities were determined at the ISO concentrations indicated. Data points represent means ± SE for 15, 13, and 6 membrane preparations (
