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(Circulation Research. 1996;79:455-460.)
© 1996 American Heart Association, Inc.

Articles

Activation of ß-Adrenergic Receptor Kinase During Myocardial Ischemia

Martin Ungerer, Kerstin Kessebohm, Kai Kronsbein, Martin J. Lohse, Gert Richardt

the 1. Medizinische Klinik der Technischen Universitat Munchen (M.U., K. Kessebohm,, K. Kronsbein, G.R.) and Pharmakologisches Institut der Universitat Wurzburg (M.J.L.) (Germany).

Correspondence to Dr Martin Ungerer, 1. Medizinische Klinik der Technischen Universitat Munchen, Klinikum rechts der Isar, Ismaningerstr 22, 81675 Munchen, Germany. E-mail ungerer@med1.med.tu-muenchen.de.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
During myocardial ischemia, a local release of noradrenaline coincides with an increased density of ß-adrenergic receptors. The functional activity of these receptors, however, is mainly determined by their state of phosphorylation. The ß-adrenergic receptor kinase (ßARK) specifically phosphorylates and thereby inactivates ß-adrenergic receptors after stimulation by receptor agonists, facilitating the binding of the inhibitor protein ß-arrestin to the receptors. ßARK activation involves a translocation of the enzyme to the membrane. In the present study, we investigated the density and the functional activity of ß-adrenergic receptors, the enzymatic activity of ßARK in membranes and cytosol, the mRNA levels of ßARK-1, and the expression of ß-arrestin during stop-flow and low-flow ischemia in the isolated perfused rat heart. After 60 minutes of stop-flow ischemia, ß-adrenergic receptor density was upregulated, but ß-agonist–mediated adenylate cyclase activity was blunted. Simultaneously, ßARK activity in the particulate fraction was significantly induced. The increase in ßARK activity was reversible after inhibition of ischemia-evoked noradrenaline release by desipramine. Also, exposure to externally given noradrenaline increased ßARK activity in the particulate fraction. Cytosolic ßARK activity remained largely unchanged during stop-flow or low-flow ischemia. The steady state concentration of ßARK-1 mRNA increased after 20 minutes of stop-flow ischemia and then returned to baseline values after another 20 minutes. Cardiac ischemia did not alter ß-arrestin levels. During myocardial ischemia, an increase in the number of ß-adrenergic receptors is paralleled by increased membrane activity of the receptor kinase ßARK. This increased membrane activity may contribute to enhanced receptor phosphorylation and inactivation.

Key Words: ischemia • ß-adrenergic receptor kinase • rat heart

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In myocardial ischemia, a local metabolic release of large amounts of noradrenaline occurs^{1 2} together with an increased density of ß-adrenergic receptors.^{3 4 5 6 7} Consecutively, the capacity of ß-adrenergic agonists to stimulate adenylate cyclase activity is enhanced during the first 15 minutes of ischemia.⁷ With progressive ischemia, however, isoproterenol-stimulated activity of adenylate cyclase decreases to below the control value, although the density of ß-receptors remains elevated.^{5 7} This dissociation of receptor number and functional activity has been found in different models of cardiac ischemia,^{5 6} including the isolated perfused rat heart,⁷ and in human myocardium subjected to hypoxia during cardiopulmonary bypass surgery.⁸ We hypothesized that this apparent paradox might be caused by increased ß-receptor desensitization during progressive ischemia.

Several biochemical mechanisms contribute to ß-adrenergic receptor desensitization.⁹ Most important, agonist-occupied active receptors become phosphorylated by the specific ßARK,^{9 10} facilitating the subsequent binding of the inhibitor protein ß-arrestin to the receptors.¹¹ Receptor function is thereby inhibited by up to 70%.^{12 13} This process occurs within 10 to 15 minutes, and it coincides with a translocation of ßARK from the cytosol to the cell membrane.¹⁴ Phosphorylated ß-adrenergic receptors lose their capacity to activate subsequent second messenger systems, such as adenylate cyclase.^{9 10} Alternative mechanisms, such as the reduction of the receptor number by downregulation, become operative only after longer periods of 4 to 12 hours.⁹ ßARK can use different G protein–coupled receptors as substrates.^{10 15 16} The specificity of the different G protein–coupled receptor kinases for different receptors is still subject to investigation. According to a recent review, ßARK-1 should now be termed GRK-2.¹⁷ Among the different subtypes of GRKs known to date, GRK-2 and GRK-5¹⁸ appear to be dominantly expressed in the heart.¹⁹ Whereas ßARK-1 (GRK-2) activation invariably involves a translocation from the cytosol to the cell membrane, which is targeted by G protein ß subunits,²⁰ GRK-5 seems to rest constitutively in the membrane and does not undergo translocation.¹⁸

Various experimental approaches have been used to prove the pivotal importance of ßARK for the functional activity of ß-adrenergic receptors. In an in vitro model, inhibition of ßARK activity can indeed prevent or reduce ß-adrenergic receptor desensitization.²¹ Overexpression of ßARK-1 or of ß-arrestin-1 leads to enhanced rapid ß-adrenergic receptor desensitization.²² A recent study examined cardiac contractility in vivo in transgenic mice that overexpressed ßARK-1 in their hearts.²³ In these animals, the inotropic and chronotropic responses to isoproterenol were markedly attenuated. Vice versa, cardiac contractility was increased in animals in which the overexpression of a ßARK-inhibitor protein had been induced.²³

In summary, ßARK-1 plays a major role in ß-adrenergic desensitization and has been shown to functionally inactivate cardiac ß-adrenergic receptors. Therefore, we investigated ßARK activity in the cytosol and in membrane homogenates as well as the mRNA concentrations of ßARK-1 during experimental ischemia in isolated perfused rat hearts. In order to test the comparability of our model of cardiac ischemia, we also measured the density of ß-receptors and the activity of adenylate cyclase. Because increased levels of ß-arrestin may also contribute to enhanced ß-adrenergic desensitization, we assessed ß-arrestin levels immunologically.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Perfused Rat Heart
Male Wistar rats (200 to 250 g) were anesthetized with thiopental (100 mg/kg IP). The thorax was opened, and the hearts were rapidly cut out, rinsed in ice-cold buffer, and weighed. Thereafter, coronary perfusion was performed according to Langendorff²⁴ at an initial flow rate of 5 mL/min in the absence of external cardiac work. During each experiment, six hearts were perfused simultaneously by a multichannel peristaltic pump at a constant flow rate. Perfusion was started using a modified Krebs-Henseleit solution composed of (mmol/L) NaCl 125, NaHCO₃ 16.9, Na₂HPO₄ 0.2, KCl 4.0, CaCl₂ 1.85, MgCl₂ 1.0, glucose 11, and EDTA 0.0027. The buffer was gassed with 95% O₂/5% CO₂. pH was adjusted to 7.4 by small variations of the gas flow. The temperature of the perfusion medium was regulated to 37.5°C. After an equilibration period of 20 to 30 minutes, ischemia was induced either by stopping perfusion or by limiting flow to 0.5 mL/min. During ischemia, the hearts were covered by a chamber with the temperature adjusted to 37.5°C. Low-flow ischemia led to a progressive release of creatine kinase and lactate dehydrogenase from the hearts. After 6 hours of low-flow ischemia, we detected lactate dehydrogenase activity of 1244±342 U/L and creatine kinase activity of 841±289 U/L in the effluent, whereas normal perfusion did not result in any detectable overflow of the two enzymes. For each experimental condition, six hearts were subjected to a specific intervention, whereas six other hearts were perfused normally for the same period of time.

Density of ß-Adrenergic Receptors
Cardiac membranes were prepared, and ß-adrenergic receptor density was determined as described before.²⁵ Membranes were incubated with ³H-CGP 12177 (Amersham) in concentrations ranging from 0.1 to 2.8 nmol/L in triplicate in 50 mmol/L Tris-HCl, pH 7.5, and 10 mmol/L MgCl₂, with or without 12 µmol/L propranolol to define nonspecific binding, for 1 hour at 23°C in a volume of 200 µL. The reaction was terminated by filtration through GF/B filters and washing with ice-cold incubation buffer. Filter radioactivity was determined by liquid scintillation counting.

Adenylate Cyclase Assays
For adenylate cyclase assays, ventricular membranes (n=6 hearts in each experimental group) were incubated with 0.1 mmol/L [³²P]ATP (0.15 to 0.2 µCi per tube), 1 mmol/L MgCl₂, 10 µmol/L GTP, 150 mmol/L NaCl, 0.1 mmol/L cAMP, 5 mmol/L creatine phosphate as Tris salt, 0.4 mg/mL creatine kinase, 1 mg/mL bovine serum albumin, and 50 mmol/L Tris-HCl, pH 7.4, in a final volume of 100 µL. Incubations were initiated by the addition of membrane preparations (50 µg protein per tube) to reaction mixtures that had been preincubated for 5 minutes at 37°C and were conducted for 20 minutes at 37°C. Reactions were stopped by the addition of 0.4 mL of 125 mmol/L zinc acetate. cAMP was purified by coprecipitation of other nucleotides with ZnCO₃ and chromatography on neutral alumina. ZnCO₃ was formed by the addition of 0.5 mL of 144 mmol/L Na₂CO_3. After centrifugation for 5 minutes at 12 000g, 0.8 mL of the supernatant was applied to neutral alumina columns (1.2 g), followed by equilibration with 0.1 mol/L Tris-HCl, pH 7.5, and application of two 2-mL portions of the same buffer. The effluent was collected, and cyclic [³²P]cAMP was determined by measuring Cerenkov radiation in a liquid scintillation counter.

ßARK Activity
Enzymatic activity of ßARK was measured according to the method described by Benovic et al.¹⁰ Rod outer segments containing >95% rhodopsin were prepared from bovine retinas as described previously.¹⁰ The preparation was treated with urea to inactivate endogenous rhodopsin-kinase.¹⁰ Tissue samples of 100 mg were taken from the left ventricles and placed in 1 mL lysis buffer (25 mmol/L Tris-HCl, pH 7.5, 5 mmol/L EDTA, 5 mmol/L EGTA, 20 mg/mL leupeptin, 20 mg/mL benzamidine, and 40 mg/mL phenylmethylsulfonyl fluoride). The tissue was homogenized for 30 seconds in a polytron tissue mincer and centrifuged for 30 minutes at 50 000g. The supernatant represented the cytosolic fraction. The pellet was resuspended in lysis buffer with 250 mmol/L NaCl and homogenized again. The pellet suspension was then recentrifuged, purified, and used as membrane fraction.

NaCl was added to a final concentration of 50 mmol/L, and all samples were equilibrated with 0.5 mL 50% (vol/vol) DEAE-Sephacel, pH 7.0, for 15 minutes on ice. The slurry was then placed into small columns and eluted with 0.5 mL lysis buffer. The volume of this eluate was reduced by filtration in a microconcentrator (Centricon 30, Amicon) to yield 100 µL.

Protein concentration in the concentrates was determined according to the method of Bradford.²⁶ Then cytosolic or membrane eluates containing 50 µg of protein were incubated with 500 pmol rhodopsin from rod outer segments, 10 mmol/L MgCl₂, and 0.3 mmol/L [-³²P]ATP, in a total volume of 60 µL lysis buffer. Reactions were terminated by addition of 250 µL ice-cold lysis buffer. All vials were centrifuged for 15 minutes at 11 000g. Free radioactivity in the supernatant was discarded, and the pellet was resuspended in 30 µL of 2x Laemmli buffer by vigorous shaking for 20 minutes. The samples were electrophoresed on 10% SDS-polyacrylamide gels. Gels were stained with Coomassie blue and autoradiographed. In the next step, rhodopsin bands were cut out from the gel, and Cerenkov radiation was counted in a beta scintillation counter. Kinetic assays showed that the reaction was linear for up to 10 minutes and depended linearly upon the amount of heart preparations used (not shown).

Quantitative Reverse Transcription and PCR
Tissues for RNA preparation were taken from rat ventricular myocardium. RNA was isolated from the frozen tissues as described previously.^{27 28 29} The amount of RNA in the samples was determined by UV absorption. RNA of each heart was reverse transcribed by MMLV reverse transcriptase (BRL) into cDNA.

Total RNA (1 µg) was then used for reverse transcription into cDNA. RNA was denatured for 3 minutes at 65°C and incubated for 90 minutes at 42°C in the presence of 0.5 mmol/L dNTP, 0.01 mol/L dithiothreitol, 10 pmol random hexamers, 1 U MMLV Superscript reverse transcriptase (BRL), and 1 U RNAsin (Promega) in 50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, and 3 mmol/L MgCl₂. The mixture was then diluted with 50 µL TE buffer (10 mmol/L Tris-HCl, pH 8.0, and 1 mmol/L EDTA) to 70 µL and heated to 70°C for 10 minutes. Sense and antisense oligonucleotide primer pairs were synthesized to match the cDNA sequences of rat ßARK-1.³⁰ The primers were as follows: 5'-AGA TGG CCG ACC TGG AGG C-3' (sense, base pairs -2 to 17 of coding sequence) and 5'-GGA GTC AAA GAT CTC CCG-3' (antisense, base pairs 332 to 316 of coding sequence). PCR with these primers amplified specific products from cloned plasmid DNA containing ßARK-1 but not from a ßARK-2 plasmid. For rat GAPDH, we used primers as described previously.²⁵

Quantitative PCR reactions were performed with 7 µL of cDNA (1/10 of total cDNA resulting from reverse transcription of 1 µg RNA). The assay mix contained 2.5 U Taq DNA polymerase (Boehringer-Mannheim), 200 µmol/L dNTP, 0.8 µmol/L respective oligonucleotide primers, 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, and 1.5 mmol/L MgCl₂ in a volume of 100 µL. [-³²P]dCTP (1 µCi) was present to allow quantification of PCR products. The mixture was overlaid with mineral oil and amplified in a thermal cycler. Denaturation was carried out at 94°C for 1 minute, followed by an annealing step at 55°C for 1 minute and an extension step at 72°C for 1.5 minutes. Initially, the number of cycles needed for sufficient but still exponential amplification was titrated. Aliquots of 10 µL were removed from the reaction mix during successive cycles of amplification. The aliquots were electrophoresed on a 1% agarose (BRL) gel containing ethidium bromide. The appropriate PCR products were cut out under UV irradiation and counted in liquid scintillation fluid (Roth) in a beta counter. In all experiments, a negative H₂O control and known amounts of plasmids containing the respective gene sequences were amplified to check the efficiency of the PCR. The yield of ßARK-1 PCR product was proportional to the amount of template cDNA and increased exponentially up to 35 cycles. The method is sufficiently sensitive and accurate to detect changes in mRNA levels of <30%. Titration with known amounts of standard ßARK cDNA showed that the cDNA derived from 0.1 µg of total RNA contained 5x10^-20 mol of ßARK cDNA. On the basis of these titrations, 33 cycles of PCR were used for measurement of ßARK-1, and 26 cycles were used for GAPDH mRNA. Multiple samples from ischemic and control hearts were assayed for each gene using a single master reaction mixture.

Determination of ß-Arrestin Immunoreactivity
The antibody S6H8 recognizes the highly conserved epitope PVDGVVLVDP (amino acids 36 to 45 of both rat ß-arrestin-1 and rat ß-arrestin-2). Specificity of this antibody for ß-arrestin-1 in cardiac cytosol has been shown in a previous study.²⁸ Cytosolic preparations were carried out as follows: Rat hearts were placed into 500 µL of TE buffer (20 mmol/L Tris-HCl, pH 8.0, and 1 mmol/L EDTA) and minced with an ultraturrax. The homogenate was centrifuged at 1000g for 10 minutes. The supernatant from this centrifugation was again centrifuged at 200 000g for 30 minutes. The resulting supernatant was used as a crude cytosolic preparation. The protein concentration was determined according to Bradford.²⁶ Samples containing 100 µg of protein were suspended in Laemmli buffer by gentle shaking for 15 minutes and electrophoresed on 12% SDS-polyacrylamide gels. Proteins were then blotted onto Amersham Hybond-C membranes. Efficiency of transfer was verified by Ponceau red staining of the blots. The blots were blocked with 3% nonfat dried milk and 1% ovalbumin in PBS. ß-Arrestin was detected with the anti–ß-arrestin antibody plus peroxidase-coupled second antibodies using chemiluminescent substrate (ECL, Amersham) and Kodak XAR films. The resulting films were scanned with an Apple Eagle View video system and analyzed densitometrically with WinCam software. Results are expressed as arbitrary units, referring to the calculated area of density. Variability between single blots was eliminated by normalization to a standard of pooled samples.

Statistics
Results for ß-receptor binding and adenylate cyclase activity are expressed as mean±SEM. Data for ßARK activity and mRNA and for ß-arrestin immunoreactivity are given as mean±SD. Results of time-matched groups were compared by one-way ANOVA and tested by Scheffe's test. A value of P<.05 was regarded as significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
ß-Adrenoceptor Density and Adenylate Cyclase Activity
The cardiac density of ß-adrenergic receptors amounted to 80±8 fmol/mg protein after 60 minutes of normal perfusion and 110±9 fmol/mg protein after 60 minutes of stop-flow ischemia (P<.05). After 60 minutes of normal perfusion, cardiac adenylate cyclase activity was raised from 45±3 pmol/min per milligram protein (basal value) to 80±6 pmol/min per milligram protein by 10 µmol/L isoproterenol and to 340±22 pmol/min per milligram protein by 100 µmol/L forskolin. In contrast, isoproterenol-stimulated adenylate cyclase activity was significantly reduced after 60 minutes of stop-flow ischemia to 50±10 pmol/min per milligram protein (-38%, P<.02 ischemia versus normoxia), whereas forskolin-stimulated cyclase activity was markedly less reduced, reaching 273±21 pmol/min per milligram protein (-19%, P<.05 for ischemia versus normoxia).

ßARK Activity
ßARK activity was measured in cardiac membranes and cytosol by testing their capacity to phosphorylate light-activated rhodopsin. Fig 1 shows an autoradiogram of an SDS-PAGE of rhodopsin, which had been incubated with 50 µg of cardiac membranes from normally perfused hearts (lane 1). Addition of 1 mmol/L heparin, a specific inhibitor of ßARK activity, inhibited the phosphorylation of rhodopsin almost completely (lane 2). In contrast, 10 µmol/L staurosporine (an inhibitor of protein kinase C) or 1 µmol/L protein kinase A inhibitor did not inhibit phosphorylation of rhodopsin (not shown). On lanes 3 and 4, 50 µg of membranes from ischemic hearts were tested for comparison. These membrane fractions had markedly enhanced capacities to phosphorylate rhodopsin.

View larger version (50K): [in this window] [in a new window]   Figure 1. Autoradiogram of SDS-PAGE of ßARK assays as described in "Materials and Methods." Purified membrane preparations (100 µg) from different rat hearts were incubated with 0.3 mmol/L [32P]ATP and 500 pmol rhodopsin (Rho) for 10 minutes. Reaction was stopped, free radioactivity was eliminated, and phosphorylated Rho was run on gels. Lanes are as follows: 1, sample from a normally perfused heart; 2, sample from the same heart, incubated in the presence of 1 mmol/L heparin (Hep); 3, sample from a heart subjected to 20-minute stop-flow ischemia; and 4, sample from a heart subjected to 40-minute stop-flow ischemia.

Similar experiments were used to determine ßARK activity after various interventions. After stop-flow ischemia (0 mL/min), we observed a rapid increase of ßARK activity in the particulate fraction (Fig 2A). The membrane activity of the receptor kinase rose to values almost twice as high as those of normally perfused hearts (5 mL/min) after 20 minutes of stop-flow ischemia. ßARK activity remained elevated during longer periods of stop-flow ischemia and after 6 hours of low-flow ischemia (0.5 mL/min) (Fig 2A).

View larger version (17K): [in this window] [in a new window]   Figure 2. Time course of ßARK activation in cardiac membranes (A) and cytosol (B) during myocardial ischemia. Isolated perfused rat hearts were subjected to normal perfusion (open circles), stop-flow ischemia (solid circles), or low-flow ischemia (hatched circles) for the indicated periods of time. Activities were calculated as 32P incorporation in picomoles per minute per milligram of membrane protein. All interventions were carried out in six hearts. Values are mean±SD. *P<.05, **P<.0005.

Total cytosolic ßARK activity amounted to values almost three times those of membrane activity (Fig 2B). During stop-flow ischemia, the cytosolic activity of ßARK did not markedly change. Only after longer periods of stop-flow ischemia was cytosolic activity slightly, though not significantly, reduced (by 20%, P=NS, Fig 2B). Likewise, after a longer period of low-flow ischemia (6 hours), ßARK activity was not significantly altered (Fig 2B).

Exposure to external noradrenaline during normal perfusion induced ßARK activity in the particulate fraction to 165% of the control values (P<.0001) (Fig 3). When the hearts were exposed to desipramine before stop-flow ischemia, ßARK activity was markedly less induced and did not differ significantly from control values (Fig 3). Perfusion with 10 µmol/L propranolol during normal flow did not alter ßARK activity in the particulate fraction (not shown).

View larger version (33K): [in this window] [in a new window]   Figure 3. Bar graph of ßARK activity in cardiac membranes after various interventions, which were performed for 40 minutes. Isolated perfused rat hearts were subjected to normal perfusion (open bars), perfusion with 10 µmol/L noradrenaline (gray bars), stop-flow ischemia (hatched bars), or perfusion with 10 µmol/L desipramine (DMI) before 40 minutes of stop-flow ischemia (dark hatched bars). Activities were calculated as 32P incorporation in picomoles per minute per milligram of membrane protein. All interventions were carried out in six hearts. Values are mean±SD. *P<.0005, **P<.0001.

mRNA for ßARK-1
In addition, we also determined the concentration of specific mRNA for ßARK-1 during various interventions (Fig 4A). We found that ßARK-1 mRNA concentration was induced to almost threefold the control value after 20 minutes of stop-flow ischemia. Later, ßARK-1 mRNA concentration was reduced to control values 40 minutes after the beginning of ischemia. Longer periods of low-flow or stop-flow ischemia did not change ßARK-1 mRNA concentration (Fig 4A). Exposure to external noradrenaline for 60 minutes also induced a significant upregulation of ßARK-1 mRNA (to 1630±210 cpm, not shown). Control message GAPDH was not affected by any of these interventions (Fig 4B).

View larger version (16K): [in this window] [in a new window]   Figure 4. Time course of cardiac ßARK-1 mRNA concentration (A) and GAPDH mRNA concentration (B) during myocardial ischemia. Isolated perfused rat hearts were subjected to normal perfusion (open circles), stop-flow ischemia (solid circles), or low-flow ischemia (hatched circles) for the indicated periods of time. RNA (1 µg) was prepared. Reverse transcription/PCR assays were carried out in the presence of [32P]dCTP as described in "Materials and Methods." PCR products appeared as single bands, which were cut out and counted in a beta counter. All interventions were carried out in six hearts. Values are mean±SD. *P<.02, **P<.0001.

ß-Arrestin Immunoreactivity
Immunoblotting with antibodies directed against ß-arrestin yielded a dominant signal at the expected protein size of 48 kD, corresponding to rat ß-arrestin-1. In contrast, no signal was found for ß-arrestin-2, corresponding to ß-arrestin immunoreactivity in human cardiac cytosolic preparations.²⁹ Quantification did not reveal any significant alteration of ß-arrestin expression during cardiac ischemia. After 20 minutes of stop-flow ischemia, ß-arrestin immunoreactivity was 456±44 arbitrary units (537±67 arbitrary units in normally perfused hearts), whereas it amounted to 512±122 arbitrary units after 60 minutes of stop-flow ischemia (585±13 arbitrary units in normally perfused controls, P=NS for all values).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study documents that after cardiac ischemia in isolated rat hearts, the density of ß-adrenergic receptors is increased, but their capacity to stimulate adenylate cyclase is blunted. Simultaneously, ßARK activity in cardiac membranes is induced. This induction occurred after both stop-flow and low-flow ischemia. Increased ßARK activity is probably caused by local noradrenaline release and may be the underlying mechanism of ß-receptor uncoupling during cardiac ischemia. The levels of ß-arrestin do not change during cardiac ischemia.

During myocardial ischemia, the density of ß-adrenergic receptors is increased.^{3 4 5 6 7} The capacity of ß-adrenergic agonists to stimulate adenylate cyclase activity is enhanced during the first 15 minutes of ischemia.⁷ With sustained ischemia, however, isoproterenol-stimulated activity decreases to below control values, although the density of ß-receptors remains elevated.^{5 7} This dissociation of receptor number and functional activity has been found in different models of cardiac ischemia^{5 6 7} and has been confirmed in the present study. Likewise, a 30-minute period of anoxia during cardiopulmonary bypass surgery induced a reversible functional impairment of ß-adrenergic receptors in human and dog myocardium in vivo. Two studies found blunted adenylate cyclase responses to ß-adrenergic agonists 30 minutes after the onset of cardioplegia, despite stable ß-receptor densities.^{8 31} Our findings suggest that the activation of ßARK may explain why cardiac ß-receptors lose their functional potency. The time course of ßARK activation (10 to 15 minutes) corresponds quite well to the functional inactivation of the ß-adrenergic system, which starts from the 15th to the 30th minute of ischemia.⁷ In the same time frame, other serin/threonine kinases, such as protein kinase C, are also activated during cardiac ischemia.³²

The present study describes a rapid induction of ßARK activity in the membranes of ischemic rat hearts. The finding that an externally added receptor agonist, noradrenaline, also induced cardiac membrane activity during normoxia implies that the receptor agonist itself triggers the intracellular translocation of the enzyme. We know from many previous experiments that noradrenaline is massively released during ischemia in isolated rat hearts² ; therefore, we assumed that ßARK induction during ischemia was caused by this release of the receptor agonist. In order to test this hypothesis, we perfused the hearts with desipramine before ischemia. This intervention is known to suppress ischemic noradrenaline release by almost 75%.² Under this condition, ßARK activity did not change significantly in the particulate fraction compared with normally perfused hearts. Thus, we conclude that agonist occupation of cardiac ß-receptors during ischemia (and the subsequent activation of G proteins including the liberation of ß subunits) leads to the intracellular translocation of ßARK-1 and the induction of membrane ßARK activity.

In a previous study, we have already described an upregulation of cardiac ßARK activity and mRNA levels in human heart failure.²⁸ This activation specifically involved the mRNA for ßARK-1 but not for ßARK-2 or for ß-arrestin.²⁹ The activation of ßARK seems to be a very early event during the development of heart failure, since it preceded the alterations of ß-receptor density or G proteins in the model of pacing-induced heart failure in the pig.³³ Chronic exposure to ß-blockers in turn decreased cardiac ßARK activity in the cytosol and membranes.³⁴ Therefore, regulation of ßARK activity might be a more generalized mechanism to regulate ß-adrenergic responsiveness in the heart. In contrast, an altered expression of ß-arrestin does not seem to contribute to increased receptor desensitization during cardiac ischemia or heart failure.²⁹

We have also documented an early induction of ßARK-1 mRNA in cardiac membranes that is independent of the increase in ßARK activity. This increase was shown to be transient and disappeared during progressive ischemia. At present, we cannot determine which mechanisms trigger cardiac ßARK-1 gene transcription in ischemia or heart failure.²⁸ The promoter region and the genomic sequence of ßARK-1 were recently published, and they do not seem to contain a typical cAMP response element.³⁵ The contrasting regulation of ßARK-1 mRNA and protein levels during sustained ischemia is in line with the regulation of other cardiac proteins, which are differentially regulated on message and protein levels. However, the induction of ßARK-1 mRNA (and the subsequent translation into cytosolic enzyme) may explain why there is no decrease in cytosolic ßARK activity occurring simultaneously with the increase in membrane activity. Alternatively, the ischemia-induced activation of protein kinase C³² might enhance cytosolic ßARK activity, as suggested recently.³⁶ Additionally, the induction of other subtypes of cardiac GRKs, especially GRK-5, might also contribute to the regulation of total ßARK activity, since the phosphorylation assay cannot distinguish between individual subtypes. Because the specific rat mRNA sequence for GRK-5 has not been cloned to date, we could not quantify this mRNA by PCR. Additionally, GRK-5 seems to rest constitutively in the membrane, so that a contribution of this subtype to ßARK translocation seems less probable.

In conclusion, translocation of the receptor kinase ßARK may contribute to desensitization of the ß-adrenergic receptor system during cardiac ischemia. This mechanism may be responsible for the rapid blunting of ß-adrenergic and other G protein–coupled receptor responsiveness during ischemia.

	Selected Abbreviations and Acronyms

 

ßARK = ß-adrenergic receptor kinase GRK = G protein–coupled receptor kinase MMLV = mouse multiple lymphoma virus PCR = polymerase chain reaction

	Acknowledgments

 
This study was supported by a grant of the Deutsche Forschungsgemeinschaft (Un 103/1-1).

Received January 31, 1996; accepted June 4, 1996.

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