 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
*Institute of Pharmacology and Toxicology and Department of Nephrology, Martin-Luther-University of Halle-Wittenberg, Halle, Germany.
Correspondence to Dr. Otto-Erich Brodde, Institute of Pharmacology and Toxicology, University Halle-Wittenberg, Magdeburgerstrasse 4, 06097 Halle, Germany. Phone: 49-345-557-1773; Fax.: 49-345-557-1835; E-mail: otto-erich.brodde{at}medizin.uni-halle.de
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have found that in patients on maintenance hemodialysis, isoprenaline-induced increase in heart rate and contractility mediated by cardiac ß-adrenoceptors were significantly decreased (14). To further elucidate the mechanism underlying this in vivo reduction in cardiac ß-adrenoceptor responsiveness, we performed additional studies in subtotally nephrectomized rats as a commonly used model of CRF (15). In this model, we found a selective reduction in ß-adrenoceptor–mediated activation of adenylyl cyclase, whereas the other components of the ß-adrenoceptor–G-protein(s)-adenylyl cyclase system were unchanged. This indicated that in CRF, cardiac ß-adrenoceptors are uncoupled from their effector systems.
The aim of this study was to further investigate the mechanisms underlying this uncoupling of the cardiac ß-adrenoceptor in CRF. For this purpose, we determined in the heart of subtotally nephrectomized rats the activity of G-protein–coupled receptor kinase (GRK; previously known as ß-adrenergic receptor kinase, or ßARK) because it could be demonstrated that expression and activity of GRK2, the most abundant GRK in the heart, is increased in various forms of heart failure (16). GRKs phosphorylate agonist-occupied ß-adrenoceptor and by this, uncouple the receptor from the G-protein(s)–adenylyl cyclase system (for review, see [17]). In addition, we examined the activity and density of the neuronal noradrenaline reuptake transporter because this system is involved in regulation of endogenous catecholamine concentrations at the ß-adrenoceptor.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Seven weeks after the second operation, the animals were killed by administration of pentobarbitone sodium (50 mg/kg intraperitoneally) followed by an intraperitoneal injection of heparin (2000 IU; Biochrom KG, Berlin, Germany), and the animals’ hearts were rapidly excised. To avoid additional effects on the myocardium (e.g., arrhythmias) by hyperkalemia, a common symptom of renal insufficiency (18), SNX rats received 7 d before death a K+-reduced diet (17% protein; 0.2% Na+, less than 0.03% K+; Altromin, Lage, Germany), whereas SOP rats received normal diet (22.5% protein, 0.2% Na+, 0.9% K+; Altromin) as usual. Within this time, the systolic BP of the animals was measured five times on three different days by tail-cuff plethysmography (Sphygmomanometer S-2; Hugo Sachs Elektronik, Freiburg, Germany). The excised hearts were divided into right ventricle (RV) and left ventricle (LV), including intraventricular septum (LV + S), and the ratio of the LV + S weight to body weight was calculated as an index for ventricular hypertrophy.
Because morphologic and functional studies of the myocardium of rats with experimental renal failure revealed morphologic changes and cardiac dysfunction predominantly in the basal half of the left ventricular wall (19), the following experiments were performed with tissue from the LV that excluded intraventricular septum.
Assessment of Plasma Noradrenaline, Creatinine, Urea, Na+, and K+
To determine the amount of plasma noradrenaline, 2 ml of blood was collected by puncture of the ophthalmic venous plexus with a heparinized glass pipette from the anesthestized rats before excision of the heart. Thereafter, 20 µl of a 50 mM glutathione solution (final concentration, 5 mM) was added, and the blood was centrifuged for 15 min at 1500 x g at 4°C. Plasma was removed and stored at -80°C until analysis by HPLC with fluorometric detection as described previously (20).
To determine the amount of plasma creatinine, urea, Na+, and K+, 2 ml of blood was withdrawn from the vena cava inferior from the anesthestized rats before excision of the heart. The blood was centrifuged for 10 min at 1000 x g at room temperature. Plasma was removed and stored at -80°C until further analysis could be performed by an autoanalyzer (Vitros; Jonsen & Jonsen Clinical Diagnostics; Rochester, NY).
Assessment of Tissue Noradrenaline Content in RV and LV
To determine the noradrenaline content in the RV and LV, the hearts of SOP and SNX rats were immediately frozen after excision in liquid nitrogen. The hearts were thawed in ice-cold 10% perchloric acid containing 5 mM glutathione and divided into RV and LV, excluding intraventricular septum (LV). Tissue samples (100 mg) were minced with scissors in ice-cold 5 ml perchloric acid/5 mM glutathione and homogenized with an Ultra Turrax (Ultra Turrax T25; Janke & Kunkel IKA, Staufen, Germany) for 2 min at 24,000 rpm on ice. The homogenates were centrifuged for 20 min at 10,000 x g at 4°C. The supernatants were removed and stored at -80°C until HPLC analysis could be performed as described above.
ß-Adrenoceptors
RV and LV (without intraventricular septum) stored at -80°C were thawed in 5 ml of ice-cold 1 mM KHCO3 solution, minced with scissors, and homogenized with an Ultra Turrax (Ultra Turrax T25; Janke & Kunkel IKA) for 10 s at maximal setting (24,000 rpm) and twice for 20 s at 17,500 rpm in 1-min intervals. The homogenates were diluted each to 20 ml with 1 mM KHCO3 solution and centrifuged at 1000 x g for 15 min at 4°C. The supernatants were filtered through 4 layers of cheesecloth and centrifuged at 50,000 x g for 30 min. The final crude membrane pellets were resuspended in ice-cold 10 mM Tris-HCl, 154 mM NaCl buffer (pH 7.4, 25°C), and 0.55 mM ascorbic acid, yielding a protein concentration of 0.2 mg/ml. Protein content was determined according to Bradford (21), with bovine 
-globulin used as a standard.
The density of ß-adrenoceptors in right and left ventricular membranes was determined by (-)-[125I]-iodocyanopindolol (ICYP) binding at 6 concentrations of ICYP ranging from 5 to 200 pmol/L, as previously described (14). Nonspecific binding of ICYP was defined as binding to membranes, which was not displaced by a high concentration of the nonselective ß-adrenoceptor antagonist (±)-CGP 12177 (1 µM). Specific binding of ICYP was defined as total binding minus nonspecific binding (usually 70 to 80% at 50 pmol/L of ICYP).
Determination of GRK Activity
Although GRK2, GRK3, and GRK5 are coexpressed in the heart (17) and all three GRK are able to phosphorylate agonist-occupied ß1-adrenoceptors in vitro (22), we believe that with our method, we might have assessed predominantly GRK2 activity because GRK2 is the most abundant GRK in the heart (17) and rhodopsin is phosphorylated with the relative order of potency: GRK2 >>: GRK3 = GRK5 (23). Therefore, we will refer to our rhodopsin phosphorylation assay as GRK activity, although it mostly measures GRK2. Enzymatic activity of GRK was measured as described by Benovic et al. (24) with minor modifications.
A total of 100 to 200 mg of frozen tissue samples taken from RV and LV, excluding intraventricular septum of SOP rats and subtotal nephrectomized rats, was thawed in 1 ml ice-cold lysis buffer (25 mM Tris-HCl, pH 7.5, 5 mM ethylenediaminetetraacetic acid, 5 mM ethylene glycol-bis(ß-aminoethyl ether)-N,N,N‘,N‘-tetraacetic acid, 40 µg/ml leupeptin, 40 µg/ml benzamidine, 40 µg/ml phenylmethylsulfonyl fluoride), minced with scissors and homogenized with an Ultra Turrax T25 (Janke & Kunkel IKA) for 60 s at 25,000 rpm. Before centrifugation for 30 min at 50,000 x g at 4°C, 10 µl of a 5 M NaCl solution (final concentration, 50 mM) was added, and the homogenate was incubated for 10 min on ice. After centrifugation, the supernatant (the cytosolic fraction) was transferred into new reaction tubes and stored on ice until further treatment. The pellet was resuspended in 950 µl lysis buffer plus 50 µl 5 M NaCl solution (final concentration, 250 mM) with the Ultra Turrax T25 for 60 s at 9500 rpm. After incubation for 15 min on ice, the homogenate was recentrifuged for 30 min at 50,000 x g at 4°C. The supernatant (the membrane fraction) was transferred into a new reaction tube and the pellet was discarded.
The cytosolic and membrane fractions were desalted in batch with 300 µl of a 50% (weight/volume) diethylaminoethyl-Sephacel solution (equilibrated with lysis buffer, pH 7.0) for 15 min on ice. The diethylaminoethyl-Sephacel matrix was removed by a centrifugation for 10 min at 2500 x g at 4°C. The volume of the supernatants was reduced by microfiltration (Ultracel-YM 30000; Millipore; Eschborn, Germany) to yield a final volume of 50 to 100 µl. Protein concentration was determined as described above.
A total of 100 µg of cytosolic protein and 50 µg of membrane protein were mixed in duplicates in the dark at 4°C with 400 pmol rhodopsin (prepared from bovine retina), 10 mM MgCl2, 100 µM ATP, 260 pmol ß-subunit, and -32P-ATP (20 µCi 
0.08 mM) in a total volume of 60 µl. Light-dependent phosphorylation of rhodopsin was initiated by incubating the samples under light for 10 min at 30°C. The reaction was terminated by addition of 250 µl ice-cold lysis buffer. The samples were immediately centrifuged for 10 min at 10,000 x g at room temperature, and the supernatants were discarded. The pellets were resuspended in 30 µl twice-concentrated Laemmli buffer (25) by vigorous shaking for 30 min at 30°C.
The samples were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gels, which were stained with Coomassie brilliant blue according to the method of Reissner et al. (26) and autoradiographed. The rhodopsin bands (35 kD) were cut off the gel, and their radioactivity was determined by Cerenkov counting.
Assessment of Noradrenaline Transporter Density
A total of 100 to 150 mg of frozen RV and LV, excluding intraventricular septum, was thawed on ice in 5-ml incubation buffer (10 mM Na2HPO4, 120 mM NaCl, 5 mM KCl, pH 7.4) containing 0.25 M sucrose. The tissue was minced with scissors and gradually homogenized with an Ultra-Turrax T25 (Janke & Kunkel IKA) at maximal setting (24,000 rpm) for 10 s and twice at 17,500 rpm for 20 s with 1-min intervals at 4°C. The homogenate was brought up to 20 ml with incubation buffer (+0.25 M sucrose) and centrifuged at 1200 x g for 10 min at 0°C. The supernatant was filtered through 4 layers of cheesecloth and centrifuged at 20,000 x g for 20 min at 4°C. The resulting pellet was resuspended in 20 ml incubation buffer (+0.25 M sucrose) and recentrifuged at 20,000 x g for 20 min at 4°C. The final crude membrane pellets were resuspended in ice-cold incubation buffer (without sucrose), yielding a protein concentration of 250 µg/ml. Protein content was determined as mentioned above.
[3H]-nisoxetine saturation analysis was performed as previously described (13) by incubating 100 µg protein per assay with 6 concentrations of [3H]-nisoxetine ranging from 0.3125 to 10 nM in a final assay volume of 500 µl for 3 h at 4°C. Nonspecific binding of [3H]-nisoxetine was defined as binding in the presence of 1 µM desipramine. Specific binding was defined as total binding minus nonspecific binding and amounted to approximately 85% at 2 nM [3H]-nisoxetine.
Determination of Uptake1 Activity
Uptake1 activity was assessed as described elsewhere (13). Briefly, 10 mg of RV and LV tissue slices excluding intraventricular septum taken from SOP and SNX rats were incubated at 37°C for 15 min in oxygenated Krebs-Henseleit solution (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 0.54 mM MgCl2, 25 mM NaHCO3, 1 mM NaH2PO4, 11 mM glucose, 0.094 mM ethylenediaminetetraacetic acid, 1.14 mM ascorbic acid, 0.067 mM nialamide) containing 1.56, 3.125, 6.25, 12.5, and 25 nM D,L-[7-3H(N)]-noradrenaline hydrochloride (13.5 Ci/mmol) in the presence of 40 µM corticosterone (specific uptake2 antagonist) in a final volume of 500 µl. Nonspecific accumulation of radioactivity was determined by parallel incubation at 4°C. Specific uptake was defined as total uptake (37°C) minus nonspecific uptake (4°C).
Statistical Analyses
The experimental data are expressed as the means ± SEM of n experiments. The equilibrium dissociation constants (KD) and the maximal number of binding sites (Bmax.) for ICYP and [3H]-nisoxetine binding were calculated by nonlinear regression analysis via the iterative curve-fitting program Prism (GraphPad Software, San Diego, CA). Statistical significance of differences was analyzed by unpaired two-tailed t test. P < 0.05 was considered to be significant. All statistical calculations were performed with the Prism program (GraphPad Software).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the study presented here, we have tried to further elucidate the mechanism underlying this defect. We found that SNX rats developed left ventricular hypertrophy and a very mild, but significant, rise in systolic BP. This was accompanied by an increase in the activity of left ventricular GRK and simultaneously by a decrease in left ventricular ß-adrenoceptor density. GRKs phosphorylate the agonist occupied receptor, and by this, they facilitate binding of arrestins to the phosphorylated receptor that uncouple the receptor from the Gs-protein-adenylyl cyclase system (for reviews, see [17,27]). The consequence of this chain of events is a decrease in ß-adrenoceptor responses to agonist stimulation. Thus, the increase in left ventricular GRK activity might well explain the blunted responsiveness of cardiac ß-adrenoceptors that we recently have observed in the SNX rats (14). A decrease in left ventricular ß-adrenoceptor density, as found in the study presented here, might contribute to this reduction. In this context, it should be noted that the decrease in left ventricular ß-adrenoceptor density observed in the study presented here is at variance with our data of unchanged left ventricular ß-adrenoceptors in SNX rats (14); this might be because in the study presented here, we assessed ß-adrenoceptor density in the LV without intraventricular septum, whereas in our previous experiments, left ventricular preparations including intraventricular septum were used (see Materials and Methods).
On the other hand, in the study presented here, right ventricular weight was unchanged in SNX rats; right ventricular ß-adrenoceptor density and GRK activity were not altered. These data are compatible with the view that in SNX rats, decreased cardiac ß-adrenoceptor responsiveness associated with increased GRK activity is found only in the hypertrophied LV, but not in RV. However, in the absence of any intervention studies that use angiotensin-converting enzyme inhibitors or ß-blockers, it is not possible to differentiate whether these changes are associated with the development of left ventricular hypertrophy or whether they are cause of the development of left ventricular hypertrophy.
The mechanism underlying the increase in GRK activity in the (hypertrophied) LV of SNX rats is not known at present. However, the following possibilities should be considered. First, data obtained in humans with CHF and in animal models of CHF have shown that an increase in the activity of the sympathetic nervous system—and by this, chronic activation of cardiac ß-adrenoceptors—is one important (patho)physiologic mechanism to increase GRK activity (for reviews, see [17,27]). Accordingly, chronic infusion of isoprenaline into mice has been shown to cause a desensitization of cardiac ß-adrenoceptors and an increase in the activity of GRK2 (28). Interestingly, in the same mouse model, chronic infusion of ß-adrenoceptor antagonists decreased GRK activity (28).
In the study presented here, plasma noradrenaline levels were approximately twofold higher in SNX rats than they were in SOP rats. Measurements of plasma noradrenaline are generally taken as an indicator of sympathetic activity (for review, see [29]); thus, it might well be that in SNX rats, sympathetic activity is also increased, and this may cause the increase in cardiac GRK activity. At least in patients with CRF, increased plasma noradrenaline levels (for reviews, see [30,31]) appear to reflect increased sympathetic activity, because in these patients, direct measurement of sympathetic drive by microneurography of the peroneal nerve showed an enhanced sympathetic firing rate (32,33).
In this context, it is worthwhile to note that Amann et al. (8) have suggested that the increase in plasma noradrenaline levels could be due to an enhanced release from the renal cortex, resulting in reduced renal tissue noradrenaline content but unchanged uptake1 activity. Interestingly, our data indicate similar effects for the hearts of SNX rats. Although we found no difference in [3H]-noradrenaline uptake1 activity between SOP and SNX rats, the cardiac release of noradrenaline appeared to be significantly increased because tissue noradrenaline content was decreased in RV and LV of SNX rats. In addition, we found in the RV and LV a significant reduction in noradrenaline transporter density, which leads to the assumption that the transport capacity of the remaining uptake1 carrier proteins might be even increased. However, nonexocytotic noradrenaline release via the myocardial neuronal uptake1 carrier associated with an increase in GRK activity have been described as typical indicators for cardiac ischemia (34), which is indeed a major cause of morbidity and mortality in patients with CRF (35).
Second, in the study presented here, SNX rats exhibited mild hypertension. Studies in hypertension have revealed an increase in GRK activity in the heart (36) as well as in vascular smooth muscle tissues (37,38). However, in all these studies, rats with hypertension had much higher BP levels than the SNX rats in the study presented here; thus, we believe it is quite unlikely that the increase in BP might contribute to the changes in cardiac GRK activity, although we cannot exclude this possibility.
Third, the increase in GRK might be linked to the development of left ventricular hypertrophy in the SNX rats. In fact, an elegant study by Choi et al. (39) showed that in mice with transverse aortic banding, cardiac hypertrophy was associated with a blunted cardiac ß-adrenoceptor responsiveness and a marked increase in cardiac GRK activity. However, these authors could also show that in another model of cardiac hypertrophy (the transgenic mice homozygous for oncogenic ras overexpression in the heart), cardiac GRK activity was unaltered despite marked cardiac hypertrophy. Moreover, Iaccarino et al. (40) showed that in mice, chronic stimulation of 
1-adrenoceptors (by phenylephrine) and ß-adrenoceptors (by isoprenaline) caused cardiac hypertrophy, but only the chronic ß-adrenoceptor stimulation was associated with increased GRK levels. These data indicate that induction of cellular hypertrophy per se appears not to be related to increases in GRK activity. Accordingly, it might well be that also in the aortic banding model neurohumoral activation via cardiac ß-adrenoceptor stimulation is the cause of the increase in GRK activity.
Taken together, it is possible that in SNX rats, an increased sympathetic drive to the heart (as indicated by the increased plasma noradrenaline levels and the simultaneously decreased cardiac tissue noradrenaline levels) that causes chronic activation of cardiac ß-adrenoceptors is responsible for the increase in left ventricular GRK activity. An increased sympathetic drive to the heart induces per se desensitization of cardiac ß-adrenoceptors, and this is enhanced by the increase in GRK. Such a pattern of pathophysiologic alterations is often seen in patients with CHF: they have increased plasma catecholamines, decreased cardiac tissue catecholamines, desensitized cardiac ß-adrenoceptors, and increased GRK levels (for review, see [41]). In addition, it has been observed in these patients that neurohumoral activation and increased activities of GRK are early events in the development, progression, or both of CHF (17,41). In this context, it is interesting to note that patients with CRF have also increased sympathetic activity (32,33) and exhibit in vivo a decreased cardiac ß-adrenoceptor responsiveness (14)—that is, patients with CRF show a similar pattern of alterations as do CHF patients. Thus, patients with CRF are more likely to develop chronic heart disease. In fact, patients with CRF have a high risk for cardiovascular diseases including hypertension, left ventricular hypertrophy, and cardiac insufficiency (42,43).
In conclusion, in several pathophysiologic conditions, such as heart failure, ischemia, hypertension, and cardiac hypertrophy, myocardial GRK is increased (17,41). All of these conditions are characterized by increased sympathetic activity, desensitization of cardiac ß-adrenoceptors, and, later, the development of heart failure. Thus, it might well be that in SNX rats with increased sympathetic activity, the increase in left ventricular GRK might contribute to the development or progression of CHF associated with CRF. Thus, inhibition of GRK (particularly GRK2) activity or lowering its expression could possibly present a novel target for treatment of CHF associated with CRF. This could be brought about by angiotensin-converting enzyme inhibitors (which can decrease sympathetic drive) (33) or ß-blockers (which can decrease GRK activity) (28).
|
Acknowledgments |
|---|
-subunit for the GRK assay and introduction into the methodology of the GRK assay. This work was supported by a grant of the Deutsche Forschungsgemeinschaft Bonn, Germany (DFG OS 131/3-2 to B.O. and O.-E.B.). |
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1-adrenergic stimulation. Hypertension 33[Part 2]: 396–401, 1999This article has been cited by other articles:
 |
|
 |
|
  X. J, L. G, W. P, V. H, Y. X, L. Y, W. Y, P. A, C. S, D. GV, et al. Kidney and Blood Pressure--The Story Unfolds: Renalase Is a Novel, Soluble Monoamine Oxidase That Regulates Cardiac Function and Blood Pressure. J Clin Invest J. Am. Soc. Nephrol., September 1, 2005; 16(9): 2521 - 2527. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Leineweber, P. Rohe, A. Beilfuss, C. Wolf, H. Sporkmann, H. Bruck, H.-G. Jakob, G. Heusch, T. Philipp, and O.-E. Brodde G-protein-coupled receptor kinase activity in human heart failure: Effects of {beta}-adrenoceptor blockade Cardiovasc Res, June 1, 2005; 66(3): 512 - 519. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. G. Bongartz, M. J. Cramer, P. A. Doevendans, J. A. Joles, and B. Braam The severe cardiorenal syndrome: 'Guyton revisited' Eur. Heart J., January 1, 2005; 26(1): 11 - 17. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Leineweber, S. Klapproth, A. Beilfuss, R.-E. Silber, G. Heusch, T. Philipp, and O.-E. Brodde Unchanged G-protein-coupled receptor kinase activity in the aging human heart J. Am. Coll. Cardiol., October 15, 2003; 42(8): 1487 - 1492. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Leineweber, K. Brandt, B. Wludyka, A. Beilfuss, K. Ponicke, I. Heinroth-Hoffmann, and O.-E. Brodde Ventricular Hypertrophy Plus Neurohumoral Activation Is Necessary to Alter the Cardiac {beta}-Adrenoceptor System in Experimental Heart Failure Circ. Res., November 29, 2002; 91(11): 1056 - 1062. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
HOME
CURRENT ISSUE
ARCHIVES
JASN Express
ONLINE SUBMISSION
AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP |
