Cardiac contractile dysfunction in J2N-k cardiomyopathic hamsters is associated with impaired SR function and regulation

Although dilated cardiomyopathy (DCM) is known to result in cardiac contractile dysfunction, the underlying mechanisms are unclear. The sarcoplasmic reticulum (SR) is the main regulator of intracellular Ca²⁺ required for cardiac contraction and relaxation. We therefore hypothesized that abnormalities in both SR function and regulation will contribute to cardiac contractile dysfunction of the J2N-k cardiomyopathic hamster, an appropriate model of DCM. Echocardiographic assessment indicated contractile dysfunction, because the ejection fraction, fractional shortening, cardiac output, and heart rate were all significantly reduced in J2N-k hamsters compared with controls. Depressed cardiac function was associated with decreased cardiac SR Ca²⁺ uptake in the cardiomyopathic hamsters. Reduced SR Ca²⁺ uptake could be further linked to a decrease in the expression of the SR Ca²⁺-ATPase and cAMP-dependent protein kinase (PKA)-mediated phospholamban (PLB) phosphorylation at serine-16. Depressed PLB phosphorylation was paralleled with a reduction in the activity of SR-associated PKA, as well as an elevation in protein phosphatase activity in J2N-k hamster. The results of this study suggest that an alteration in SR function and its regulation contribute to cardiac contractile dysfunction in the J2N-k cardiomyopathic hamster. sarcoplasmic reticulum; cardiomyopathy; cAMP-dependent protein kinase; Ca²⁺/calmodulin-dependent protein kinase; sarco(endo)plasmic reticulum ATPase; phospholamban     

Changes in the expression of cardiac Na+-K+ ATPase subunits in the UM-X7.1 cardiomyopathic hamster

Previous studies have shown that cardiac Na+ -K+ ATPase activity in the UM-X7.1 hamster strain is decreased at an early stage of genetic cardiomyopathy and remains depressed; however, the mechanism for this decrease is unknown. The objective of the present study was to assess whether changes in the expression of cardiac Na+-K+ ATPase subunits in control and UM-X7.1 cardiomyopathic hamsters are associated with alterations in the enzyme activity. Accordingly, we examined sarcolemmal Na+-K+ ATPase activity as well as protein content and mRNA levels for the alpha1, alpha2, alpha3 and beta1-subunit of the Na+-K+ ATPase in 250-day-old UM-X7.1 and age-matched, control Syrian hamsters; this age corresponds to the severe stage of heart failure in the UM-X7.1 hamster. Na+-K+ ATPase activity in UM-X7.1 hearts was decreased compared to controls (9.0 +/- 0.8 versus 5.6 +/- 0.8 micromol Pi/mg protein/hr). Western blot analysis revealed that the protein content of Na+-K+ ATPase alpha1- and beta1-subunits were increased to 164 +/- 27% and 146 +/- 22% in UM-X7.1 hearts respectively, whereas that of the alpha2- and alpha3-subunits were decreased to 82 +/- 5% and 69 +/- 11% of control values. The results of Northern blot analysis for mRNA levels were consistent with the protein levels; mRNA levels for the alpha1- and beta1-subunits in UM-X7.1 hearts were elevated to 165 +/- 14% and 151 +/- 10%, but the alpha2-subunit was decreased to 60 +/- 8% of the control value. We were unable to detect mRNA for the alpha3-subunit in either UM-X7. 1 or control hearts. These data suggest that the marked depression of Na+-K+ ATPase activity in UM-X7.1 cardiomyopathic hearts may be due to changes in the expression of subunits for this enzyme.     

Cardiac-restricted expression of the carboxyl-terminal fragment of GRK3 Uncovers Distinct Functions of GRK3 in regulation of cardiac contractility and growth: GRK3 controls cardiac alpha1-adrenergic receptor responsiveness

G protein-coupled receptor kinase-2 and -3 (GRK2 and GRK3) in cardiac myocytes catalyze phosphorylation and desensitization of different G protein-coupled receptors through specificity controlled by their carboxyl-terminal pleckstrin homology domain. Although GRK2 has been extensively investigated, the function of cardiac GRK3 remains unknown. Thus, in this study cardiac function of GRK3 was investigated in transgenic (Tg) mice with cardiac-restricted expression of a competitive inhibitor of GRK3, i.e. the carboxyl-terminal plasma membrane targeting domain of GRK3 (GRK3ct). Cardiac myocytes from Tg-GRK3ct mice displayed significantly enhanced agonist-stimulated alpha(1)-adrenergic receptor-mediated activation of ERK1/2 versus cardiac myocytes from nontransgenic littermate control (NLC) mice consistent with inhibition of GRK3. Tg-GRK3ct mice did not display alterations of cardiac mass or left ventricular dimensions compared with NLC mice. Tail-cuff plethysmography of 3- and 9-month-old mice revealed elevated systolic blood pressure in Tg-GRK3ct mice versus control mice (3-month-old mice, 136.8 +/- 3.6 versus 118.3 +/- 4.7 mm Hg, p < 0.001), an observation confirmed by radiotelemetric recording of blood pressure of conscious, unrestrained mice. Simultaneous recording of left ventricular pressure and volume in vivo by miniaturized conductance micromanometry revealed increased systolic performance with significantly higher stroke volume and stroke work in Tg-GRK3ct mice than in NLC mice. This phenotype was corroborated in electrically paced ex vivo perfused working hearts. However, analysis of left ventricular function ex vivo as a function of increasing filling pressure disclosed significantly reduced (dP/dt)(min) and prolonged time constant of relaxation (tau) in Tg-GRK3ct hearts at elevated supraphysiological filling pressure compared with control hearts. Thus, inhibition of GRK3 apparently reduces tolerance to elevation of preload. In conclusion, inhibition of cardiac GRK3 causes hypertension because of hyperkinetic myocardium and increased cardiac output relying at least partially on cardiac myocyte alpha(1)-adrenergic receptor hyper-responsiveness. The reduced tolerance to elevation of preload may cause impaired ability to withstand pathophysiological mechanisms of heart failure.     

Different expression of adrenoceptors and GRKs in the human myocardium depends on heart failure ethiology and correlates to clinical variables

Downregulation of β(1)- adrenergic receptors (β(1)-ARs) and increased expression/function of G-protein-coupled receptor kinase 2 (GRK2) have been observed in human heart failure, but changes in expression of other ARs and GRKs have not been established. Another unresolved question is the incidence of these compensatory mechanisms depending on heart failure etiology and treatment. To analyze these questions, we quantified the mRNA/protein expressions of six ARs (α(1A), α(1B), α(1D), β(1), β(2), and β(3)) and three GRKs (GRK2, GRK3, and GRK5) in left (LV) and right ventricle (RV) from four donors, 10 patients with ischemic cardiomyopathy (IC), 14 patients with dilated cardiomyopathy (DC), and 10 patients with nonischemic, nondilated cardiopathies (NINDC). We correlated the changes in the expressions of ARs and GRKs with clinical variables such as left ventricular ejection fraction (LVEF) and left ventricular end-systolic and left ventricular end-diastolic diameter (LVESD and LVEDD, respectively). The main findings were 1) the expression of the α(1A)-AR in the LV positively correlates with LVEF; 2) the expression of GRK3 and GRK5 inversely correlates with LVESD and LVEDD, supporting previous observations about a protective role for both kinases in failing hearts; and 3) β(1)-AR expression is downregulated in the LV and RV of IC, in the LV of DC, and in the RV of NINDC. This difference, better than an increased expression of GRK2 (not observed in IC), determines the lower LVEF in IC and DC vs. NINDC.     

TNF‐α‐mediated signal transduction pathway is a major determinant of apoptosis in dilated cardiomyopathy

Although J2N-k strain of cardiomyopathic hamsters is an excellent model of dilated cardiomyopathy, the presence and mechanisms of apoptosis in the hearts of these genetically modified animals have not been investigated. This study examined the hypothesis that cardiac dysfunction and apoptosis in the cardiomyopathic hamsters were associated with tumour necrosis factor-alpha (TNF-α)-mediated signalling pathway involving the activation of some pro-apoptotic proteins and/or deactivation of some antiapoptotic proteins. Echocardiographic assessment of 31-week-old hamsters indicated an increase in the internal dimension of the left ventricle as well as decreases in the ejection fraction, fractional shortening and cardiac output without any evidence of cardiac hypertrophy. Increased level of TNF-α and apoptosis in cardiomyopathic hearts were accompanied by increased protein content for protein kinase C (PKC) -α and -ɛ isozymes as well as caspases 3 and 9. Phosphorylated protein content for p38 MAPK and NFκB was increased whereas that for Erk1/2, BAD and Bcl-2 was decreased in cardiomyopathic hearts. These results support the view that TNF-α and PKC isozymes may promote apoptosis due to the activation of p38 MAPK and deactivation of Erk1/2 pathways, and these changes may contribute toward the development of cardiac dysfunction in dilated cardiomyopathy.     

GRK3 mediates desensitization of CRF1 receptors: a potential mechanism regulating stress adaptation

Potential G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) mediation of homologous desensitization of corticotropin-releasing factor type 1 (CRF1) receptors was investigated in human retinoblastoma Y-79 cells. Inhibition of PKA activity by PKI(5-22) or H-89 failed to attenuate homologous desensitization of CRF1 receptors, and direct activation of PKA by forskolin or dibutyryl cAMP failed to desensitize CRF-induced cAMP accumulation. However, treatment of permeabilized Y-79 cells with heparin, a nonselective GRK inhibitor, reduced homologous desensitization of CRF1 receptors by approximately 35%. Furthermore, Y-79 cell uptake of a GRK3 antisense oligonucleotide (ODN), but not of a random or mismatched ODN, reduced GRK3 mRNA expression by approximately 50% without altering GRK2 mRNA expression and inhibited homologous desensitization of CRF1 receptors by approximately 55%. Finally, Y-79 cells transfected with a GRK3 antisense cDNA construct exhibited an approximately 50% reduction in GRK3 protein expression and an ~65% reduction in homologous desensitization of CRF1 receptors. We conclude that GRK3 contributes importantly to the homologous desensitization of CRF1 receptors in Y-79 cells, a brain-derived cell line.     

Cardiomyocyte-restricted inhibition of G protein-coupled receptor kinase-3 attenuates cardiac dysfunction after chronic pressure overload

Transgenic mice with cardiac-specific expression of a peptide inhibitor of G protein-coupled receptor kinase (GRK)3 [transgenic COOH-terminal GRK3 (GRK3ct) mice] display myocardial hypercontractility without hypertrophy and enhanced α(1)-adrenergic receptor signaling. A role for GRK3 in the pathogenesis of heart failure (HF) has not been investigated, but inhibition of its isozyme, GRK2, has been beneficial in several HF models. Here, we tested whether inhibition of GRK3 modulated evolving cardiac hypertrophy and dysfunction after pressure overload. Weight-matched male GRK3ct transgenic and nontransgenic littermate control (NLC) mice subjected to chronic pressure overload by abdominal aortic banding (AB) were compared with sham-operated (SH) mice. At 6 wk after AB, a significant increase of cardiac mass consistent with induction of hypertrophy was found, but no differences between GRK3ct-AB and NLC-AB mice were discerned. Simultaneous left ventricular (LV) pressure-volume analysis of electrically paced, ex vivo perfused working hearts revealed substantially reduced systolic and diastolic function in NLC-AB mice (n = 7), which was completely preserved in GRK3ct-AB mice (n = 7). An additional cohort was subjected to in vivo cardiac catheterization and LV pressure-volume analysis at 12 wk after AB. NLC-AB mice (n = 11) displayed elevated end-diastolic pressure (8.5 ± 3.1 vs. 2.9 ± 1.2 mmHg, P < 0.05), reduced cardiac output (3,448 ± 323 vs. 4,488 ± 342 μl/min, P < 0.05), and reduced dP/dt(max) and dP/dt(min) (both P < 0.05) compared with GRK3ct-AB mice (n = 16), corroborating the preserved cardiac structure and function observed in GRK3ct-AB hearts assessed ex vivo. Increased cardiac mass and myocardial mRNA expression of β-myosin heavy chain confirmed the similar induction of cardiac hypertrophy in both AB groups, but only NLC-AB hearts displayed significantly elevated mRNA levels of brain natriuretic peptide and myocardial collagen contents as well as reduced β(1)-adrenergic receptor responsiveness to isoproterenol, indicating increased LV wall stress and the transition to HF. Inhibition of cardiac GRK3 in mice does not alter the hypertrophic response but attenuates cardiac dysfunction and HF after chronic pressure overload.     

Increased expression of the gene for alpha-interferon-inducible protein in cardiomyopathic hamster heart

Cardiomyopathic (CM) hamsters have a disruption in the delta-sarcoglycan gene which leads to progressive cardiac necrosis by 30 to 40 days of age, hypertrophy by 120 days, and heart failure by 250 days. We used differential display to detect other changes in mRNA levels in 30-, 60-, and 90-day-old wild-type and CM hamsters. We identified a 400-bp cDNA with sequence similarity to the human alpha-interferon-inducible protein (p27). This cDNA annealed with a 570-base mRNA whose steady-state levels were increased in 30-, 60-, and 90-day-old CM compared to wild-type heart. Increased expression of this hamster homolog of p27 (p27-h) was detected in CM hamster cardiac and skeletal muscle at 60 days of age but not in liver, kidney, or brain. Thus, an inherited defect in CM hamsters leads to increased expression of p27-h in advance of the development of hypertrophy and heart failure.     

Selective Reduction in A2 Adenosine Receptor Desensitization Following Antisense-Induced Suppression of G Protein-Coupled Receptor Kinase 2 Expression

Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) is considered to play a critical role in the desensitization of responses mediated by these receptors. To explore the role of GRK2 in A2 adenosine receptor desensitization, we attempted to reduce specifically GRK2 expression in NG108-15 cells by stable transfection with an antisense rat GRK2 cDNA sequence. This yielded up to a 69% loss of GRK2 when compared with plasmid-transfected control cells, which correlated with a reduction in kinase activity when measured by the ability of cell lysates to promote light-dependent phosphorylation of rhodopsin. Levels of GRK3 were the same in antisense and plasmid-transfected controls. On addition of the A2 adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine, cyclic AMP accumulation was greater in GRK2 antisense cells as compared with plasmid control cells. In contrast, cyclic AMP accumulation via agonist stimulation of either IP-prostanoid or secretin receptors or by addition of forskolin was not significantly different among all clones examined. The increase in A2 adenosine receptor response could not be explained by changes in A2A adenosine receptor expression, as assessed by ligand binding experiments with the radioligand 2-3H-labelled 4-[2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-++ +ylamino]ethyl]phenol ([3H]ZM241385). These data show for the first time a direct correlation between expression of GRK2 and desensitization of natively expressed A2 adenosine receptors in intact cells, suggesting that GRK2 plays a major role in the regulation of these receptors. Key Words: G protein-coupled receptor kinase-G protein-coupled receptor-Antisense-NG108-15 cells-A2 adenosine receptors-Desensitization.     

Responses of chronically hypoxic rat hearts to ischemia: KATP channel blockade does not abolish increased RV tolerance to ischemia

Chronic hypoxia may precondition the myocardium and protect from ischemia-reperfusion damage. We therefore examined the recovery of left and right ventricular function after ischemia and reperfusion (15 min each) in isolated blood-perfused working hearts from normoxic (Norm) and hypoxic (Hypo; 14 days, 10.5% O(2)) adult rats. In addition, the mRNA expression of hypoxia-inducible factor (HIF)-1alpha and the protein expression of endothelial nitric oxide synthase (eNOS) were measured. Postischemic left ventricular function recovered to 66 +/- 6% and 67 +/- 5% of baseline in Norm and Hypo, respectively. In contrast, postischemic right ventricular function was 93 +/- 2% of baseline in Hypo vs. 67 +/- 3% in Norm (P < 0.05). Improved postischemic right ventricular function in Hypo (93 +/- 2% and 96 +/- 2% of baseline) was observed with 95% O(2) or 21% O(2) in the perfusate, and it was not attenuated by glibenclamide (5 and 10 micromol/l) (86 +/- 4% and 106 +/- 6% recovery). HIF-1alpha mRNA and eNOS protein expression were increased in both left and right hypoxic ventricles. In conclusion, postischemic right, but not left, ventricular function was improved by preceding chronic hypoxia. ATP-sensitive K(+) channels are not responsible for the increased right ventricular tolerance to ischemia after chronic hypoxia in adult rat hearts.     

Upregulation of beta-adrenergic receptors in heart failure due to volume overload

To examine the mechanisms of changes in beta-adrenergic signal transduction in heart failing due to volume overload, we studied the status of beta-adrenoceptors (beta-ARs), G protein-coupled receptor kinase (GRK), and beta-arrestin in heart failure due to aortocaval shunt (AVS). Heart failure in rats was induced by creating AVS for 16 wk, and beta-AR binding, GRK activity, as well as their protein content, and mRNA levels were determined in both left and right ventricles. The density and protein content for beta1-ARs, unlike those for beta2-ARs, were increased in the failing hearts. Furthermore, protein contents for GRK isoforms and beta-arrestin-1 were decreased in membranous fractions and increased in cytosolic fractions from the failing hearts. On the other hand, steady-state mRNA levels for beta1-ARs and GRK2, as well as protein content for Gbetagamma-subunits, did not change in the failing heart. Basal cardiac function was depressed; however, both in vivo and ex vivo positive inotropic responses of the failing hearts to isoproterenol were augmented. Treatment of AVS animals with imidapril (1 mg.kg(-1).day(-1)) or losartan (20 mg.kg(-1).day(-1)) retarded the progression of heart failure; partially prevented changes in beta1-ARs, GRKs, and beta-arrestin-1 in the failing myocardium; and attenuated the increase in positive inotropic effect of isoproterenol. These results indicate that upregulation of beta1-ARs is associated with subcellular redistribution of GRKs and beta-arrestin-1 in the failing heart due to volume overload. Furthermore, attenuation of alterations in beta-adrenergic system by imidapril or losartan may be due to blockade of the renin-angiotensin system in the AVS model of heart failure.     

MAP kinase protects G protein-coupled receptor kinase 2 from proteasomal degradation

The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and shuts down signaling from 7-transmembrane receptors (7TMs). Although, receptor activity controls GRK2 expression levels, the underlying molecular mechanisms are poorly understood. We have previously shown that extracellular signal-regulated kinase (ERK1/2) activation increases GRK2 expression [J. Theilade, J. Lerche Hansen, S. Haunso, S.P. Sheikh, Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2), FEBS Lett. 518 (2002) 195-199]. In the present study, we found that ERK1/2 regulates GRK2 degradation rather than synthesis. ERK1/2 blockade using PD98059 decreased GRK2 cellular levels to 0.25-fold of control in Cos7 cells. This effect was due to enhanced degradation of the GRK2 protein, since proteasome blockade prevented down-regulation of GRK2 protein levels in the presence of PD98059. Further, ERK blockade had no effect on GRK2 synthesis as probed using a reporter construct carrying the GRK2 promoter upstream of the luciferase gene. We predict ERK1/2 mediated GRK2 protection could be a general phenomenon as proteasome inhibition increased GRK2 expression in two other cell lines, HEK293 and NIH3T3.     

Apelin reduces myocardial reperfusion injury independently of PI3K/Akt and P70S6 kinase

Apelin, the endogenous ligand of the G protein-coupled APJ receptor, is a peptide mediator with emerging regulatory actions in the heart. The aim of the present studies was to explore potential roles of the apelin/APJ system in myocardial ischaemia/reperfusion injury. To determine the cardiac expression of apelin/APJ and potential regulation by acute ischaemic insult, Langendorff perfused rat hearts were subjected to regional ischaemia (left coronary artery occlusion, 35 min) or ischaemia followed by reperfusion (30 min). Apelin and APJ mRNA expression were then determined in ventricular myocardium by rt-PCR. Unlike APJ mRNA expression, which remained unchanged, apelin mRNA was upregulated 2.4 fold in ventricular myocardium from isolated rat hearts undergoing ischaemia alone, but returned back to control levels after 30 min reperfusion. We then proceeded to test the hypothesis that treatment with exogenous apelin is protective against ischaemia/reperfusion injury. Perfused hearts were subjected to 35 min left main coronary artery occlusion and 120 min reperfusion, after which infarct size was determined by tetrazolium staining. Exogenous Pyr(1)-apelin-13 (10(-8 )M) was perfused either from 5 min prior to 15 min after coronary occlusion, or from 5 min prior to 15 min after reperfusion. Whilst ineffective when used during ischaemia alone, apelin administered during reperfusion significantly reduced infarct size (47.6+/-2.6% of ischaemic risk zone compared to 62.6+/-2.8% in control, n=10 each, p<0.05) in hearts subject to temporary coronary occlusion followed by reperfusion. This protective effect was not abolished by co-administration of the PI3K inhibitor wortmannin (10(-7 )M, infarct size 49.8+/-4.1%, n=4) or the P70S6 kinase inhibitor rapamycin (10(-9 )M, 41.8+/-8.8%, n=4). In conclusion these results suggest that apelin may be a new and potentially important cardioprotective autacoid, upregulated rapidly after myocardial ischaemia and acting through an unknown pathway.     

Overlapping and Opposing Functions of G Protein-coupled Receptor Kinase 2 (GRK2) and GRK5 during Heart Development

G protein-coupled receptor kinases 2 (GRK2) and 5 (GRK5) are fundamental regulators of cardiac performance in adults but are less well characterized for their function in the hearts of embryos. GRK2 and -5 belong to different subfamilies and function as competitors in the control of certain receptors and signaling pathways. In this study, we used zebrafish to investigate whether the fish homologs of GRK2 and -5, Grk2/3 and Grk5, also have unique, complementary, or competitive roles during heart development. We found that they differentially regulate the heart rate of early embryos and equally facilitate heart function in older embryos and that both are required to develop proper cardiac morphology. A loss of Grk2/3 results in dilated atria and hypoplastic ventricles, and the hearts of embryos depleted in Grk5 present with a generalized atrophy. This Grk5 morphant phenotype was associated with an overall decrease of early cardiac progenitors as well as a reduction in the area occupied by myocardial progenitor cells. In the case of Grk2/3, the progenitor decrease was confined to a subset of precursor cells with a committed ventricular fate. We attempted to rescue the GRK loss-of-function heart phenotypes by downstream activation of Hedgehog signaling. The Grk2/3 loss-of-function embryos were rescued by this approach, but Grk5 embryos failed to respond. In summary, we found that GRK2 and GRK5 control cardiac function as well as morphogenesis during development although with different morphological outcomes.     

Cloning, expression, and regulation of lithocholic acid 6 beta-hydroxylase.

We have isolated a hamster liver cDNA whose expression is induced upon feeding hamsters with a cholic acid-rich diet. It was identified as a cytochrome P450 family 3 protein, by sequence homology, and named CYP3A10. The activity of CYP3A10 was determined by transient expression of its cDNA in transfected COS cells and was found to hydroxylate lithocholic acid at position 6 beta. CYP3A10 RNA is 50-fold higher in males than in female hamsters. In males, it appears to be regulated by age with expression highest after puberty. Shortly after weaning (28 days), cholic acid feeding of male hamsters elevates the level of message over that of hamsters fed with normal laboratory chow. Females do not exhibit regulation by cholic acid. In hamster liver, murideoxycholic acid, the 6 beta-metabolite of lithocholic acid, is the major hydroxylated product of lithocholic acid. Lithocholic acid 6 beta-hydroxylase (6 beta-hydroxylase) activity is greatly diminished in hamster female liver microsomes as would be expected due to the lack of CYP3A10 mRNA in females. Additionally, male liver microsomal 6 beta-hydroxylase activity was increased by cholic acid feeding, consistent with the cholic acid-mediated induction of its RNA. These results indicate that, in male hamsters, 6 beta-hydroxylation is the major pathway for detoxification of lithocholate and that, likely, CYP3A10 is responsible for that activity.     

Effect of ACTH on cholesterol and steroid synthesis in adrenocortical tissues

Previous studies have established that under normal conditions, adrenal HMG-CoA reductase activity is higher in hamsters than in rats and humans. The hamster reductase activity follows a diurnal rhythm corresponding to that of plasma ACTH and glucocorticoids [Endocrinology 107 (1980) 215] but not to that of aldosterone. ACTH treatments to hamsters increased reductase activity after a latency of 60 min; this enhancement was prevented by cycloheximide [J. steroid Biochem. 24 (1986) 325]. Immunotitration and immunoblotting studies confirmed that ACTH caused an increase in reductase protein synthesis. In rats, long-term (1-9 days) and short-term (3 h) treatments with ACTH also induced increase in adrenal HMG-CoA reductase activity and reductase protein. In the presence of iodoacetamide and inhibitors of proteolytic enzyme, a main specific band of enzyme was evinced in the area of 102 +/- 6 kDaMr, by Western blotting, for both hamster homogenate and microsomal preparations (Endocrinology, 120 (1987]. Similarly Mr values were found with rat adrenal preparations. The concentration of mRNA, analyzed using the c-DNA pRed-10 coding for the Chinese hamster ovary reductase, was increased in adrenals of hamsters treated with ACTH. The reductase mRNA levels also fluctuated during the day in parallel with those of reductase activity and reductase protein. In conclusion, these results indicate that ACTH and other conditions inducing a change in hamster adrenal HMG-CoA reductase activity provoke parallel changes in reductase mRNA and reductase protein content. ACTH acts on the adrenal reductase of species synthesizing large as well as small quantities of cholesterol, thus indicating the general importance of this hormonal control.     

Expression of mRNA of beta 1- and beta 2-adrenergic receptors in Xenopus oocytes results from structurally distinct receptor mRNAs

Poly(A)+-selected RNA prepared from cells or tissues that express a homogeneous population of either beta 1- or beta 2-adrenergic receptors was isolated and then microinjected into Xenopus laevis oocytes. Following microinjection, the expression of beta-adrenergic receptors was assessed by equilibrium radioligand binding analysis using the antagonist ligand [3H]dihydroalprenolol. The pharmacology of the newly- expressed beta-adrenergic receptors in oocyte membranes was the same as that of the original tissue used as a source of RNA. Hybridization of nick-translated cDNA of hamster beta 2-adrenergic receptor to poly(A)+-selected RNA from tissues containing beta 2-adrenergic receptors was to a mRNA species of 2.2 kilobases. In contrast, hybridization of the cDNA probe to poly(A)+-selected RNA from tissues containing beta 1-adrenergic receptors was to a mRNA species of 2.0 kilobases. A single-stranded fragment of hamster beta 2-adrenergic receptor cDNA corresponding to nucleotides 730-886 was isolated and uniformly radiolabeled. This region of the gene is predicted to encode for the entire second exofacial loop (L4-5), the entire fifth transmembrane-spanning region, and the first 5 amino acid residues of the third cytoplasmic loop (L5-6) of the beta 2-adrenergic receptor. Hybridization at 48 and 56 degrees C of poly(A)+-selected RNA prepared from sources that express either beta 1 or beta 2-adrenergic receptors to the antisense orientation strand of this region of the beta 2-adrenergic receptor cDNA was followed by S1 endonuclease digestion of nonhybridized sequences. At 48 degrees C, S1-resistant hybrids from both sources of RNA protected the probe from S1 endonuclease digestion. At 56 degrees C, however, only the RNA prepared from the source of beta 2-adrenergic receptors protected the probe from S1 endonuclease digestion. These results demonstrate that the mRNAs encoding for the structurally homologous beta 1- and beta 2-adrenergic receptors are distinct in the pharmacological specificity of their translation products and in their size and structure.