Desensitization Of Beta-adrenergic Receptors Research Articles

Targeted Inhibition of β-Adrenergic Receptor Kinase-1–Associated Phosphoinositide-3 Kinase Activity Preserves β-Adrenergic Receptor Signaling and Prolongs Survival in Heart Failure Induced by Calsequestrin Overexpression

Desensitization and down-regulation of beta-adrenergic receptors (betaARs) are prominent features of heart failure largely mediated by increased levels of betaAR kinase-1 (betaARK1). beta-adrenergic receptor kinase 1 interacts with phosphoinositide-3 kinase (PI3K), and upon agonist stimulation, the betaARK1/PI3K complex is recruited to agonist-stimulated betaARs. Here we tested the hypothesis that in vivo selective inhibition of betaARK1-associated PI3K activity would preserve betaAR signaling and, therefore, improve cardiac function and survival in experimental heart failure. We used a murine model of heart failure induced by calsequestrin (CSQ) cardiac-specific overexpression; CSQ mice were crossed with mice overexpressing in the heart a catalytically inactive PI3Kgamma (PI3Kgamma(inact)) to competitively displace endogenous PI3K from betaARK1. Catalytically inactive PI3KgammaPI3K overexpression in CSQ mice inhibited betaARK1-associated PI3K activity, normalized betaAR levels, and preserved betaAR responsiveness to isoproterenol (ISO). Restoration of betaAR signaling via PI3Kgamma(inact) overexpression resulted in marked improvement of cardiac function and a significant prolongation of survival. Importantly, the effects of PI3Kgamma(inact) overexpression were restricted to betaAR signaling, because cellular PI3K signaling was unaltered, as shown by the similar activation of multiple downstream signaling pathways in both CSQ and CSQ/PI3Kgamma(inact) mice. These data in the CSQ model of cardiac dysfunction indicate that membrane-targeted PI3K activity plays a detrimental role in heart failure, and its inhibition represents a novel therapeutic approach to ameliorate cardiac dysfunction and improve survival.

Tumor Necrosis Factor α, Rapid Ventricular Tachyarrhythmias, and Infarct Size in Canine Models of Myocardial Infarction

Etanercept (2 mg/kg), a TNFalpha sequestrant, was administered 24 hours and 1 hour before LAD coronary artery ligation to examine the role of TNFalpha on lethal ventricular tachyarrhythmias and myocardial necrosis. Dogs treated with etanercept had decreased very rapid (>360 bpm) ventricular triplets (6 +/- 1/h, n = 8) 2 to 24 hours following coronary artery ligation compared with saline (21 +/- 6/h, n = 10, P < 0.05). This was concordant with 8-fold salvage of beta-adrenergic receptor kinase 1 (betaARK) activity compared with control (33.8 +/- 7.2% versus 4.3 +/- 2.2% of unoperated control tissue, P < 0.01, n = 5). Salvage of betaARK occurred without change in the thickness of the epicardial tissue overlying the infarct. In dogs pretreated with etanercept before a 2-hour occlusion/4-hour reperfusion of the LAD coronary artery, infarct mass decreased by 61% (% area at risk) and 55% (% left ventricular mass) in the etanercept group (n = 8) compared with saline (n = 9, P < 0.05). This was concordant with an etanercept-mediated six-fold decrease in leukocyte accumulation within ischemically injured myocardium. TNFalpha antagonism decreases malignant ventricular tachyarrhythmias and may relate to partial protection of normal betaARK-mediated desensitization of beta-adrenergic receptors. TNFalpha sequestration also decreases infarct size in an occlusion/reperfusion model of myocardial ischemia.

Alterations of beta-Adrenergic Receptor Signaling in Cardiac Hypertrophy and Heart Failure: beta-Adrenergic Receptor Desensitization in Cardiac Disease

β-adrenergic receptors (βAR) belong to the large family of G protein-coupled receptors that form the interface between the sympathetic nervous and cardiovascular systems. G protein-coupled receptors undergo adaptation to repeated or prolonged agonist stimulation, which is termed desensitization. Significant βAR desensitization occurs with the development of cardiac hypertrophy and heart failure, and uncoupling of βARs and defects in this pathway might be primary elements underlying the transition from compensated to uncompensated cardiac failure. Decreasing the level of myocardial βARK1 in established heart failure is a novel approach to improving impaired βAR receptor function, and potentially alter the pathogenesis of this disease. (Korean Circulation J 2005;35:485-492)

Biologic Rationale for the Use of Beta-Blockers in the Treatment of Heart Failure

Enhanced and sustained cardiac adrenergic drive occurs in heart failure and contributes, in part, to the progression of left ventricular (LV) dysfunction and remodeling that are characteristic features of this disease state. Enhanced sympathetic drive in heart failure can lead to down-regulation and desensitization of cardiac beta-adrenergic receptors with a consequent impairment of myocardial reserve and exercise tolerance. This sympathoadrenergic maladaptation can also lead to cellular abnormalities in the failing heart manifested by defects in calcium cycling within the sarcoplasmic reticulum, by defects in myocardial energetics and by ongoing loss of cardiomyocytes through necrosis or apoptosis. Sympathoadrenergic overdrive in heart failure can also trigger the induction of the fetal gene program, a maladaptation that can lead to further compromise of the contractile state. Chronic treatment with beta-blockers in patients with heart failure and in animals with experimentally-induced heart failure has been shown to reverse, prevent, or at the very least, arrest many, if not all, of these adverse processes. Beta-blockers improve function of the failing LV, prevent or reverse progressive LV dilation, chamber sphericity and hypertrophy, and consequently positively impact cardiac remodeling. Beta-blockers also reduce heart rate and LV wall stress leading to reduced myocardial oxygen consumption, a clear benefit to the failing heart. Beta-blockers can also improve the intrinsic contractile function of cardiomyocytes and have also been shown to improve myocardial energetics in heart failure possibly through a desirable shift in substrate utilization. Recent studies from our laboratories have also shown that chronic therapy with beta-blockers in heart failure can attenuate cardiomyocyte apoptosis. Finally, chronic therapy with beta-blockers has been shown to abrogate induction of the fetal gene program. These benefits provide strong reinforcement to the clinical findings that beta-blockers are highly beneficial in the management of patients with chronic heart failure and, when properly used, afford unequivocal reductions in mortality and morbidity in this patient population.

Inhibition of receptor-localized PI3K preserves cardiac beta-adrenergic receptor function and ameliorates pressure overload heart failure.

beta-Adrenergic receptor (betaAR) downregulation and desensitization are hallmarks of the failing heart. However, whether abnormalities in betaAR function are mechanistically linked to the cause of heart failure is not known. We hypothesized that downregulation of cardiac betaARs can be prevented through inhibition of PI3K activity within the receptor complex, because PI3K is necessary for betaAR internalization. Here we show that in genetically modified mice, disrupting the recruitment of PI3K to agonist-activated betaARs in vivo prevents receptor downregulation in response to chronic catecholamine administration and ameliorates the development of heart failure with pressure overload. Disruption of PI3K/betaAR colocalization is required to preserve betaAR signaling, since deletion of a single PI3K isoform (PI3Kgamma knockout) is insufficient to prevent the recruitment of other PI3K isoforms and subsequent betaAR downregulation with catecholamine stress. These data demonstrate a specific role for receptor-localized PI3K in the regulation of betaAR turnover and show that abnormalities in betaAR function are associated with the development of heart failure. Thus, a strategy that blocks the membrane translocation of PI3K and leads to the inhibition of betaAR-localized PI3K activity represents a novel therapeutic approach to restore normal betaAR signaling and preserve cardiac function in the pressure overloaded failing heart.

Endogenous G protein-coupled receptor kinase 6 triggers homologous beta-adrenergic receptor desensitization in primary uterine smooth muscle cells.

We previously reported that G protein-coupled receptor kinase (GRK) may contribute to beta-adrenergic receptor (beta-AR) uncoupling occurring just before parturition in rat uterine muscle (myometrium). To identify the GRK involved, we set up in this study a primary cell culture retaining the morphological and functional characteristics of myometrial tissue as well as the in vivo pattern of GRK expression (GRK2, GRK5, and GRK6). In this model, homologous beta-AR desensitization was assessed by an approximately 60% decrease in cAMP production to a subsequent challenge with the beta-agonist, isoproterenol. Desensitization was reduced by 36% with a GRK inhibitor, heparin, and by 31% with a protein kinase A in-hibitor, H89. Using antibodies known to specifically inhibit either GRK2/3 or GRK4-6 families, we demonstrated that only the GRK4-6 family mediated beta-AR desensitization. To discriminate between endogenous GRK5 and GRK6, we attempted to inhibit their action by introducing, into myometrial cells, kinase-dead dominant-negative mutants ((K215R)GRK5 and (K215R)GRK6). Expression of (K215R)GRK6 increased by approximately 70% the cAMP response to isoproterenol without effect on forskolin stimulation. Conversely, expression of (K215R)GRK5 or (K220R)GRK2 had no effect on beta-adrenergic signaling. These results strongly suggest that endogenous GRK6 mediate homologous beta-AR desensitization in myometrial cells.

Cell-type specific effects of endocytosis inhibitors on 5-hydroxytryptamine(2A) receptor desensitization and resensitization reveal an arrestin-, GRK2-, and GRK5-independent mode of regulation in human embryonic kidney 293 cells.

The effect of endocytosis inhibitors on 5-hydroxytryptamine(2A) (5-HT(2A)) receptor desensitization and resensitization was examined in transiently transfected human embryonic kidney (HEK) 293 cells and in C6 glioma cells that endogenously express 5-HT(2A) receptors. In HEK-293 cells, 5-HT(2A) receptor desensitization was unaffected by cotransfection with a dominant-negative mutant of dynamin (DynK44A), a truncation mutant of arrestin-2 [Arr2(319-418)], or by two well-characterized chemical inhibitors of endocytosis: concanavalin A (conA) and phenylarsine oxide (PAO). In contrast, beta 2-adrenergic receptor desensitization was significantly potentiated by each of these treatments in HEK-293 cells. In C6 glioma cells, however, DynK44A, Arr2(319-418), conA, and PAO each resulted in the potentiation of 5-HT(2A) and beta-adrenergic receptor desensitization. The cell-type-specific effect of Arr2(319-418) on 5-HT(2A) receptor desensitization was not related to the level of GRK2 or GRK5 expression. Interestingly, although beta 2-adrenergic receptor resensitization was potently blocked by cotransfection with DynK44A, 5-HT(2A) receptor resensitization was enhanced, suggesting the existence of a novel cell-surface mechanism for 5-HT(2A) receptor resensitization in HEK-293 cells. In addition, Arr2(319-418) had no effect on 5-HT(2A) receptor resensitization in HEK-293 cells, although it attenuated the resensitization of the beta 2-adrenergic receptor. However, in C6 glioma cells, both DynK44A and Arr2(319-418) significantly reduced 5-HT(2A) receptor resensitization. Taken together, these results provide the first convincing evidence of cell-type-specific roles for endocytosis inhibitors in regulating GPCR activity. Additionally, these results imply that novel GRK and arrestin-independent mechanisms of 5-HT(2A) receptor desensitization and resensitization exist in HEK-293 cells.

Role of lysophosphatidylcholine in the desensitization of beta-adrenergic receptors by Ca(2+) sensitization in tracheal smooth muscle.

Lysophosphatidylcholine (Lyso-PC) is generally considered to promote tissue inflammation. To determine the involvement of exogenous Lyso-PC in the beta-adrenergic desensitization by phospholipase A2, we examined the inhibitory effects of isoproterenol (ISO) on tension and intracellular Ca(2+) concentration by methacholine (MCh) after continuous exposure to Lyso-PC in guinea-pig tracheal smooth muscle, using isometric tension recordings and fura-2 signal (F340/F380 ratio). Pre- exposure to 10 microM Lyso-PC markedly reduced subsequent inhibition by 0.3 microM ISO against 1 microM MCh-induced contraction in a time-dependent manner. In contrast, values of percent F340/F380 ratio for MCh with ISO were not affected after exposure to Lyso-PC. In the presence of Y-27632, a selective rho-kinase inhibitor, a reduction in subsequent relaxation by ISO after exposure to Lyso-PC was inhibited in a concentration-dependent manner. Preincubation with cholera toxin also inhibited reduced responsiveness to ISO by Lyso-PC. Pre-exposure to Lyso-PC did not attenuate subsequent relaxation by agents that bypass beta-adrenergic receptors. These results indicate that continuous exposure to Lyso-PC may cause homologous desensitization of beta-adrenergic receptors via an augmentation in sensitivity to Ca(2+) by rho, a small G protein, in airway smooth muscle, and that activation of the stimulatory G protein of adenylyl cyclase, G(s), may prevent this phenomenon.

Tubulin stimulates adenylyl cyclase activity in C6 glioma cells by bypassing the beta-adrenergic receptor: a potential mechanism of G protein activation.

While the cytoskeleton is known to play several roles in the biology of the cell, one role, which has been revealed only recently, is that of a participant in the signal transduction process. Tubulin binds specifically to the alpha subunits of Gs (stimulatory GTP-binding regulatory protein of adenylyl cyclase), Gi1 (inhibitory protein of adenylyl cyclase), and Gq and transactivates those molecules through direct transfer of GTP. The relevance of this transactivation process to G proteins which are normally activated by a neurotransmitter-occupied receptor is the subject of this study. C6 glioma cells, made permeable with saponin, retained tight coupling between Gs and the beta-adrenergic receptor. Although 5-guanylylimidodiphosphate (GppNHp) was incapable of activating Gs (and subsequently, adenylyl cyclase) in the absence of agonist, tubulin with GppNHp bound (tubulin-GppNHp) activated adenylyl cyclase with an EC(50) of 30 nM. Desensitization of beta-adrenergic receptors by isoproterenol exposure had no effect on the ability of tubulin-GppNHp to activate Gs and adenylyl cyclase. When the photoaffinity GTP analog, azidoanilido GTP (AAGTP; P3(4-azidoanilido)-P1-5'-GTP), was added to C6 membranes or permeable C6 cells, it was only weakly incorporated by G alpha s in the absence of isoproterenol. When the same concentration of dimeric tubulin with AAGTP bound was introduced, AAGTP was transferred from tubulin to G alpha s, activating the latter species. Similar 'preferential' activation of G alpha s by tubulin-AAGTP versus the free nucleotide was seen using purified components. Thus, membrane-associated tubulin may serve to activate G alpha s, independent of signals not normally coupled to that protein. Tubulin may act as an agent to link a variety of membrane-associated signalling systems.

Induction of cardiac β-adrenergic receptor kinase 1 in rat heart failure caused by coronary ligation.

Beta-adrenergic receptor kinase 1 (beta ARK1) participates in the desensitization of beta-adrenergic receptors by uncoupling the signal transduction. The present study was designed to examine whether neurohumoral increase is crucial for the activation of beta ARK1 in heart failure. Four weeks after the ligation of rat coronary artery, LV dP/d t max was reduced, cardiac response to isoproterenol was impaired, and ratio of right ventricular weight to body weight, an index of cardiac hypertrophy, was increased. At the same time, beta ARK1 expression and activity were augmented in the hypertrophied hearts. In addition, plasma norepinephrine content was enhanced in accordance with cardiac hypertrophy, cardiac beta ARK1 expression, LV dP/d t max, and LVEDP. These results of the present study suggest that beta ARK1 is augmented in concert with circulating norepinephrine level and that beta ARK1 may account for, at least in part, the cardiac dysfunction in rat with myocardial infarction.

The Effects of Hypoxia and Sleep Apnea on Isoproterenol Sensitivity

To determine the effects of both apnea and hypoxia on beta-adrenergic receptor sensitivity. Cross-sectional study. A clinical research center. Forty-five normotensive and hypertensive sleep apnea patients (respiratory disturbance index >20) and non-apneic controls. The chronotropic 25 dose (CD25), an in vivo measure of beta-adrenergic receptor sensitivity derived from the heart rate response to a graded infusion of isoproterenol, was determined while subjects breathed either a normoxic (21% O2, 79% N2) or a hypoxic (15% O2, 85% N2) gas mixture. Under normoxic conditions, apnea patients showed a significantly higher CD25 (lower beta-adrenergic receptor sensitivity) as compared to controls (5.9 microg, SD=2.1 versus 4.6 microg, SD=1.2, respectively; p=0.018). In response to hypoxia, apnea patients showed no change in CD25, while controls showed a significant increase in CD25 (beta-adrenergic receptor desensitization) (p=0.002), to a value comparable to the apneics' (5.6 microg, SD=2.0). The in vivo finding of reduced beta-adrenergic receptor sensitivity in sleep apnea patients is consistent with previous in vitro assessments of the beta-adrenergic receptor. The finding that apnea patients do not respond to hypoxia with a further receptor desensitization suggests that sleep apnea patients may have reached a threshold effect of hypoxia on the beta-adrenergic receptor. These findings may be relevant to the greater incidence of hypertension seen in patients with sleep apnea syndrome.

Inhibitory effects of Gs on desensitization of beta-adrenergic receptors in tracheal smooth muscle.

We examined the reduction of mechanical responsiveness to beta-agonists after continuous or repeated exposure to agonists in guinea pig tracheal smooth muscle, using an isometric tension record. The inhibitory action of 0.1 microM isoproterenol (Iso) on contraction induced by 1 microM methacholine (MCh) was suppressed markedly after incubation with 0.0003-3 microM Iso for 1 h. The effects of Iso on MCh-induced contraction gradually decreased after repeated application of these agents at intervals of 1 h. When Iso was perfused cumulatively to tissues repeatedly precontracted by MCh, the effects of Iso were reduced at the second application. This reduced responsiveness to beta-agonists was not mimicked by forskolin and did not occur after preexposure of the strips to 2 micrograms/ml cholera toxin for 6 h. We concluded that this reduced responsiveness to beta-agonists is homologous desensitization and that the irreversible activation of the stimulatory G proteins, Gs, may play an important role in prevention of the reduced responsiveness to beta-agonists.

A89 Beta-Adrenergic Receptor Desensitization Is Greater in "Fast Track" than in Narcotic-Based Cardiac Anesthesia

A89 Beta-Adrenergic Receptor Desensitization Is Greater in "Fast Track" than in Narcotic-Based Cardiac Anesthesia

Mechanism of beta-adrenergic receptor desensitization in cardiac hypertrophy is increased beta-adrenergic receptor kinase.

Pressure overload cardiac hypertrophy in the mouse was achieved following 7 days of transverse aortic constriction. This was associated with marked beta-adrenergic receptor (beta-AR) desensitization in vivo, as determined by a blunted inotropic response to dobutamine. Extracts from hypertrophied hearts had approximately 3-fold increase in cytosolic and membrane G protein-coupled receptor kinase (GRK) activity. Incubation with specific monoclonal antibodies to inhibit different GRK subtypes showed that the increase in activity could be attributed predominately to the beta-adrenergic receptor kinase (betaARK). Although overexpression of a betaARK inhibitor in hearts of transgenic mice did not alter the development of cardiac hypertrophy, the beta-AR desensitization associated with pressure overload hypertrophy was prevented. To determine whether the induction of betaARK occurred because of a generalized response to cellular hypertrophy, betaARK activity was measured in transgenic mice homozygous for oncogenic ras overexpression in the heart. Despite marked cardiac hypertrophy, no difference in betaARK activity was found in these mice overexpressing oncogenic ras compared with controls. Taken together, these data suggest that betaARK is a central molecule involved in alterations of beta-AR signaling in pressure overload hypertrophy. The mechanism for the increase in betaARK activity appears not to be related to the induction of cellular hypertrophy but to possibly be related to neurohumoral activation.

The neuronal calcium-sensor protein VILIP modulates cyclic AMP accumulation in stably transfected C6 glioma cells: amino-terminal myristoylation determines functional activity.

VILIP (visinin-like protein) is a member of the neuronal subfamily of EF-hand calcium sensor proteins. Members of this family are involved in the calcium-dependent regulation of the desensitization of signal cascades in retinal photoreceptors. To gain insight into the function of VILIP in cell signaling, we have transfected wild-type VILIP and mutant VILIP lacking the myristoylation consensus sequence into C6 glioma cells. Expression of wild-type VILIP did not significantly influence the desensitization of beta-adrenergic receptors, which are coupled to adenylyl cyclase in C6 cells. However, VILIP expression increased the beta-adrenergic receptor-stimulated cyclic AMP (cAMP) level in these cells severalfold. The stimulatory effect was also observed after direct stimulation of the adenylyl cyclase with forskolin, indicating that VILIP acts downstream of receptor and G protein in the beta-adrenergic signaling pathway in C6 cells. In contrast, the nonmyristoylated mutant of VILIP reduced cellular cAMP levels in C6 cells. Myristoylated wild-type VILIP was associated in a calcium-dependent manner with membrane fractions during subcellular fractionation, presumably owing to a calcium-myristoyl switch. In contrast, association of nonmyristoylated mutant VILIP with membranes was strongly reduced. Thus, myristoylation and most likely the calcium-dependent membrane association of VILIP are important prerequisites for the activating effect of wild-type VILIP on cAMP accumulation in C6 cells. These results suggest that VILIP acts as a calcium sensor molecule that modulates cell signaling cascades, possibly by direct or indirect regulation of adenylyl cyclase activity.

Perioperative lymphocyte adenylyl cyclase function in the pediatric cardiac surgical patient.

To examine intraoperative and postoperative lymphocyte adenylyl cyclase activities in children undergoing repair of congenital cardiac defects with hypothermic cardiopulmonary bypass. A prospective study. Tertiary university pediatric hospital. Twelve children were enrolled into the study to examine intraoperative lymphocyte adenylyl cyclase activities and 12 children were enrolled to examine postoperative lymphocyte adenylyl cyclase activities. None. Basal (unstimulated), isoproterenol, and prostaglandin E-1 stimulated adenylyl cyclase activities, and plasma norepinephrine and epinephrine concentrations were measured. Intraoperative basal (unstimulated), beta-adrenergic receptor-stimulated (in response to isoproterenol), and prostaglandin E1 (PGE1)-stimulated lymphocyte adenylyl cyclase activities all increased during cardiopulmonary bypass, then decreased immediately after cardiopulmonary bypass. In the postoperative group, a significant decrease in basal (unstimulated), beta-adrenergic receptor- and PGE1-stimulated adenylyl cyclase activities were observed on postoperative day 1 as compared with precardiopulmonary bypass values. In the pediatric cardiac surgical patient, there was an intraoperative enhancement of lymphocyte adenylyl cyclase activities. This increase in adenylyl cyclase activities was followed by reduced lymphocyte adenylyl cyclase activities, including beta-adrenergic receptor desensitization, postoperatively, as we have previously documented in adults.

Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice.

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 showed marked beta-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation was unchanged. In contrast, the angiotensin II-induced rise in contractility was significantly attenuated in transgenic mice overexpressing the beta-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype.

Desensitization of beta-adrenergic receptors in adipocytes causes increased insulin sensitivity of glucose transport.

To determine the effect of desensitization of adipocyte beta-adrenergic receptors on insulin sensitivity, rats were continuously infused with isoproterenol (50 or 100 micrograms.kg-1.h-1) for 3 days by osmotic minipumps. Epididymal adipocytes were isolated. The cells from treated animals were desensitized to isoproterenol, as determined by response of lipolysis (glycerol release). Binding of [125I]iodocyanopindolol was decreased by approximately 80% in adipocyte plasma membranes isolated from treated rats, indicating that beta-adrenergic receptors were downregulated. Cellular concentrations of Gn alpha and Gi alpha were not altered. Insulin sensitivity was determined by measuring the effect of insulin on glucose transport (2-deoxy-[3H]glucose uptake). Cells from the isoproterenol-infused rats were markedly more sensitive to insulin than those from control rats. This was evidenced by an approximately 50% increase in maximal glucose transport rate in cells from the high-dose isoproterenol-treated rats and by an approximately 40% decrease in the half-maximal effective concentration of insulin in both groups. 125I-labeled insulin binding to adipocytes was not altered by the isoproterenol infusions, indicating that desensitization of beta-adrenergic receptors results in tighter coupling between insulin receptors and stimulation of glucose transport.

Lactate production by white adipocytes in relation to insulin sensitivity.

Besides its lipogenic and lipolytic functions, adipose tissue has been recognized as an important site of lactate production by epididymal adipocytes under experimental conditions in which insulin sensitivity was increased (cold exposure) or reduced (glucose perfusion). Insulin sensitivity of adipocytes was assessed by its antilipolytic effect and its stimulation of glucose uptake. We showed that, in adipocytes isolated from control and cold-exposed animals, glycerol and lactate production rose simultaneously, in response to norepinephrine. On the other hand, in adipocytes from glucose-perfused animals, we observed a dissociation between lipolytic and glycolytic responses. Indeed, lactate production in response to norepinephrine reached 60% of maximal response (10(-7)M) while the lipolytic response was still basal. This phenomenon was the result of a desensitization of beta-adrenergic receptors. However, the sensitivity of adipocytes to stimulation of lactate production by catecholamines was not different between control and glucose-perfused rats. Because lactate production in response to norepinephrine was not affected by desensitization, it suggests that lactate production is under alpha 1-adrenergic control. We confirmed this hypothesis.

Mechanisms of beta-adrenergic receptor desensitization: from molecular biology to heart failure.

beta-Adrenergic receptors are often studied as prototypes of the large family of G-protein-coupled receptors, which includes many other well-known members such as the muscarinic acetylcholine receptors, but also the receptors for light, taste and olfaction. These receptors are regulated by multiple mechanisms which can affect either their function or their expression to a rapidly changing environment. The most obvious changes are effected by receptor agonists, and this process is called receptor desensitization. On the functional level, the most intriguing and important mechanism of desensitization involves the phosphorylation of beta-adrenergic and homologous receptors by specific receptor kinases, termed the G-protein-coupled receptor kinases (GRKs). This phosphorylation is followed by binding of arrestins to the receptors, which causes uncoupling of receptors and G-proteins and thus results in a loss of receptor function. On the expression level, there appear to be two major pathways leading to a reduction of the receptor number: degradation of the receptors themselves, or reduced receptor synthesis brought about by reduced receptor mRNA levels. Heart failure is accompanied by a markedly reduced responsiveness of the beta-adrenergic receptor system, which in many ways resembles the phenomena seen in agonist-induced receptor desensitization. The levels of beta 1-adrenergic receptors are reduced, and this reduction is paralleled by similar decreases in the levels of the corresponding mRNA. At the same time, the activity and the mRNA levels of one of the GRK-isoforms, GRK2 (which is identical to the beta-adrenergic receptor kinase 1) are increased. These alterations may contribute to the loss of beta-adrenergic receptor responsiveness in heart failure and result in further impairment of cardiac function.