Rapid desensitization of neonatal rat liver beta-adrenergic receptors. A role for beta-adrenergic receptor kinase.

Exposure of beta-adrenergic receptors (BAR) to agonists often leads to a rapid loss of receptor responsiveness. The proposed mechanisms of such rapid receptor desensitization include receptor phosphorylation by either cAMP-dependent protein kinase or the specific beta-adrenergic receptor kinase (BARK), leading to functional uncoupling from adenylyl cyclase and sequestration of the receptors away from the cell surface. To evaluate the physiological role of such mechanisms, we have investigated whether rapid regulation of BAR occurs in the neonatal rat liver immediately after birth, a physiological situation characterized by a dramatic but transient increase in plasma catecholamines. We have detected a rapid, transient uncoupling of liver plasma membrane BARs from adenylyl cyclase (corresponding to a desensitization of approximately 45%) within the first minutes of extrauterine life, followed by a transient sequestration of approximately 40% of the BARs away from the plasma membrane. In agreement with such pattern of desensitization, we have detected (by enzymatic and immunological assays) rapid changes in BARK specific activity in different neonatal rat liver subcellular fractions that take place within the same time frame of BAR uncoupling and sequestration. Our results provide new evidence on the potential role of BAR desensitization mechanisms in vivo and suggest that they are involved in modulating catecholamines actions at the moment of birth. Furthermore, our data indicate that in addition to its suggested role as a rapid modulator of adrenergic receptor function at synapse, rapid BARK-mediated receptor regulation may have functional relevance in other tissues in response to high circulating or local levels of agonists.     

Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling associated with chronic reduction in beta-adrenergic receptor agonist activation were found. First, there was coordinate downregulation of Gi alpha 2 and Gs alpha mRNA and protein expression in the left ventricle; reduced G-protein content was also found in the right atrium. Second, in the left ventricle, there was a twofold increase in beta-adrenergic receptor-dependent stimulation of adenylyl cyclase and a persistent high affinity state of the beta-adrenergic receptor. Finally, there was a reduction in left ventricular beta-adrenergic receptor kinase activity, suggesting a previously unrecognized association between the degree of adrenergic activation and myocardial beta-adrenergic receptor kinase expression. The heart appears to adapt in response to chronic beta-adrenergic receptor antagonist administration in a manner that would be expected to offset reduced agonist stimulation. The mechanisms for achieving this extend beyond beta-adrenergic receptor upregulation and include alterations in G-protein expression, beta-adrenergic receptor-Gs interaction, and myocardial beta-adrenergic receptor kinase activity.     

Uncoupling of the beta-adrenergic receptor as a mechanism of in vitro neutrophil desensitization

Human leukocytes have been useful in studying desensitization phenomena to beta-adrenergic agonists in a number of clinical conditions. For example, we have previously shown that oral terbutaline causes a time-dependent decrease in neutrophil (PMN) beta receptor number, using the beta antagonist ligand [3H]dihydroalprenolol (DHA), in conjunction with a significant loss of isoproterenol-induced adenylate cyclase activity. In the present in vitro study we have explored the mechanism for beta-adrenergic desensitization and have compared conditions for homologous and heterologous desensitization, using the intact PMN model. PMN preincubated with isoproterenol (10(-4)M), washed thoroughly, then restimulated, desensitize rapidly so that within 10 min 80% of control isoproterenol-induced cyclic AMP stimulation is lost. Cells washed free of isoproterenol recover full responsiveness in 1 to 2 hr. The estimated isoproterenol desensitization EC50 in cells washed and then restimulated is 1 X 10(-5)M, and the EC50 in unwashed cells that are restimulated is 9 X 10(-8)M. Rank-order potency studies of catecholamine desensitization show isoproterenol greater than epinephrine greater than norepinephrine, a beta-2 pattern. Isoproterenol-induced desensitization results in a small reduction in [3H]DHA binding sites, which becomes statistically significant (p less than 0.05) from control values at 1 hr (67% of control) and 3 hr (64%). Since the change in number of beta receptors did not explain the profound, rapid loss of beta agonist-induced cyclic AMP responsiveness, we explored the possibility of an uncoupling phenomenon. In the absence of GTP, isoproterenol binding is characterized by an EC50 of 6.6 +/- 2.6 X 10(-7)M, which is significantly different (p less than 0.05) from the EC50 of 38.1 +/- 9.1 X 10(-7)M found when cells are previously desensitized with isoproterenol for 10 min. GTP does not affect the EC50 of desensitized cells. These findings are consistent with the uncoupled receptor state fitting the model described by Su et al. Finally, prolonged (3 hr) isoproterenol preincubation results in a small but significant (p less than 0.05) loss of cyclic AMP responsiveness to histamine (67.7% +/- 11.7 of control) and PGE1 (59.3% +/- 7.4), suggesting heterologous desensitization. These studies suggest that the human PMN is a suitable model to study both homologous and heterologous desensitization in vitro.     

G-protein-coupled receptor kinase activity is increased in hypertension.

Impaired vascular beta-adrenergic responsiveness may play an important role in the development and/or maintenance of hypertension. This defect has been associated with an alteration in receptor/guanine nucleotide regulatory protein (G-protein) interactions. However, the locus of this defect remains unclear. G-Protein-coupled receptor kinases (GRKs) phosphorylate serine/threonine residues on G-protein-linked receptors in an agonist-dependent manner. GRK activation mediates reduced receptor responsiveness and impaired receptor/G-protein coupling. To determine whether the impairment in beta-adrenergic response in human hypertension might be associated with altered GRK activity, we studied lymphocytes from younger hypertensive subjects as compared with older and younger normotensive subjects. We assessed GRK activity by rhodopsin phosphorylation and GRK expression by immunoblot. GRK activity was significantly increased in lymphocytes from younger hypertensive subjects and paralleled an increase in GRK-2 (beta ARK-1) protein expression. In contrast, no alterations in cAMP-dependent kinase (A-kinase) activity or GRK-5/6 expression were noted. GRK activity was not increased in lymphocytes from older normotensive subjects who demonstrated a similar impairment in beta-adrenergic-mediated adenylyl cyclase activation. These studies indicate that GRK activity is selectively increased in lymphocytes from hypertensive subjects. The increase in GRK activity may underlie the reduction in beta-adrenergic responsiveness characteristic of the hypertensive state.     

Regulation of G protein-coupled receptor kinase subtypes in activated T lymphocytes. Selective increase of beta-adrenergic receptor kinase 1 and 2.

Beta-adrenergic receptor kinase (beta ARK) is a serine-threonine kinase involved in the process of homologous desensitization of G-coupled receptors. beta ARK is a member of a multigene family, consisting of six known subtypes, also named G protein-coupled receptor kinases (GRK 1-6). In this study we investigated the expression of GRKs during the process of T cell activation, which is of fundamental importance in regulating immune responses. T cell activation was induced by exposing mononuclear leukocytes (MNL) to PHA and confirmed by tritiated thymidine incorporation measurement. A substantial increase of GRK activity (as measured by in vitro phosphorylation of rhodopsin) was found after 48 h (331 +/- 80% of controls) and 72 h (347 +/- 86% of controls) of exposure to PHA. A threefold increase of beta ARK1 immunoreactivity was found in MNL exposed to PHA for 72 h. Persistent activation of protein kinase C (PKC) by 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) was able to increase beta ARK activity to the same extent as PHA, suggesting a PKC-mediated mechanism. The kinetic of beta-adrenergic-stimulated cAMP production was substantially modified in TPA and PHA-activated cells, indicating that the increased GRK activity resulted in an increased beta-adrenergic homologous desensitization. A three- to fourfold increase in GRK activity was also observed in a population of T cell blasts (> 97% CD3+) exposed to PHA for 48-72 h. A significant increase in beta ARK1 and beta ARK2 mRNA expression was observed 48 h after mitogen stimulation, while mRNA expression of GRK5 and GRK6 was not changed. In conclusion our data show that the expression of GRK subtypes is actively and selectively modulated according to the functional state of T lymphocytes.     

G-protein-coupled receptor kinase expression in hypertension.

In human hypertension we have recently identified an increase in lymphocyte G-protein receptor kinase-2 (GRK-2) protein expression, the key protein regulating the interaction between G-protein-coupled receptors and activation of adenylyl cyclase. However, it was not known whether this increase in GRK-2 protein expression was attributable to regulation at the level of translation. Furthermore, the relationship between extent of GRK-2 expression, receptor activation of adenylyl cyclase, and blood pressure was unclear. We therefore studied lymphocytes from 7 young subjects with borderline hypertension and 14 young normotensive subjects. Immunodetectable GRK-2 protein expression in lymphocytes from subjects with hypertension was increased (155%+/-7% of normotensive subjects; P < .05). In addition, GRK-2 protein expression was positively correlated with blood pressure (r = 0.53; P = .013) and inversely correlated with beta-adrenergic-mediated adenylyl cyclase activity (r = -0.54, P = .012). However, lymphocyte GRK-2 messenger ribonucleic acid (mRNA) content was not altered (110%+/-13% of that observed in normotensive control subjects). Increased GRK-2 protein expression may be an important factor in the impairment of beta-adrenergic-mediated vasodilation, characteristic of the hypertensive state.     

Ectopic beta-adrenergic receptor binding sites. possible molecular basis of aberrant catecholamine responsiveness of an adrenocortical tumor adenylate cyclase.

The molecular basis for the aberrant catecholamine responsiveness of the adenylate cyclase of adrenocortical carcinoma 494 was explored. The adenylate cyclase of this corticosteroid-producing, transplanted, adrenal cancer of the rat was stimulated not only by adrenocorticotropic hormone and fluoride, but also by the beta-adrenergic agonist, isoproterenol. The adenylate cyclase of normal adrenal tissue was unresponsive to isoproterenol. Direct binding studies with the specific high affinity B-adrenergic ligand, (-)[3H]dihydroalprenolol, demonstrated the pressure of 0.094 pmol of specific binding sites per milligram of tumor membrane protein. By contrast, normal adrenal membranes contained too few binding sites to accurately measure and study using these techniques. The tumor binding sites had high affinity for (-)[3H] dihydroalprenolol with an equilibrium dissociation constant of 2.1 nM. Adrenergic agonists competed for the binding sites in an order of potency, [(-) isoproterenol greater than (-) epinephrine (-) norepinephrine], paralleling their order of potency as beta-adrenergic agonists. The beta-adrenergic antagonist, (-) propranolol, competed for binding, causing half-mzximal inhibition of specific binding at a concentration of 6 nM. The alpha-adrenergic antagonist, phentolamine, and several catecholamine metabolites and precursors did not effectively compete for the binding sites at high concentrations. Binding was stereospecific, the (+) stereoisomers of beta-adrenergic agonists and antagonists requiring 40- to 300-fold higher concentrations than the corresponding (-) stereoisomers to half maximally inhibit (-) [3H] dihydroalprenolol binding. These results indicate that adrenocortical carcinoma 494 membranes contain beta-adrenergic receptor-binding sites which are not normally present in membranes of adrenal tissue. These ectopic beta-adrenergic receptors presumably confer on the neoplastic tissue the catecholamine sensitivity of its adenylate cyclase.     

Overexpression of hormone-sensitive lipase prevents triglyceride accumulation in adipocytes.

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that hydrolyzes intracellular stores of triglycerides within adipocytes and is thought to be the rate limiting enzyme in lipolysis; however, direct evidence to prove this concept has been lacking. The present study was designed to establish the function of HSL in adipocytes. A 2360-bp fragment containing the entire HSL coding region was cloned into the vector pCEP4 and was used to transfect the 3T3-F442A adipogenic cell line. Nondifferentiated, transfected cells were screened for HSL overexpression by indirect immunofluorescence microscopy and confirmed by immunoblotting cell extracts with anti-HSL/fusion protein antibodies and by Northern blots for HSL mRNA. Stable transfectants overexpressing HSL were obtained and cloned. Compared with undifferentiated 3T3-F442A cells transfected with pCEP4 not containing the insert (vector alone) where HSL expression was very low, undifferentiated HSL transfectants had up to a 100-fold increase in HSL activity. Likewise, immunoreactive HSL protein and HSL mRNA levels were increased up to 100-fold in HSL transfectants. When confluent cells were allowed to differentiate by exposure to insulin, HSL expression increased in vector alone transfected cells, but remained below that observed in HSL transfectants. A similar degree of differentiation was seen in both vector alone and HSL transfectants when based on the induction of lipoprotein lipase. Cellular triglyceride content increased dramatically in the vector alone transfected cells while triglyceride content was markedly reduced in the HSL transfectants. The expression of late markers of adipocyte differentiation, such as aP2 and GPDH, was diminished and appeared to vary with the degree to which HSL was overexpressed and the cellular triglyceride content was reduced. Thus, the overexpression of HSL in 3T3-F442A cells prevents differentiated adipocytes from taking on the appearance of fat cells, i.e., accumulating triglyceride. Furthermore, the overexpression of HSL directly or indirectly attenuates the expression of several genes that appear during late adipocyte differentiation.     

Role of beta-adrenergic receptor signaling and desensitization in heart failure: new concepts and prospects for treatment

The use of beta-blockers to antagonize beta-adrenergic receptor signaling in the heart has become a standard method of treatment for heart failure, resulting in positive clinical outcomes alone and in conjunction with other modulators of cardiomyocyte contractility. However, an entire explanation for improved cardiac function in patients using beta-blockers is unknown, and in fact may be quite complicated, considering the numerous intracellular signaling pathways associated with beta-adrenergic receptors. Stimulation of beta-adrenergic receptors during both normal conditions and during heart failure activate several distinct signaling cascades, which influence cardiomyocyte contraction, hypertrophy and apoptosis. This review explores the signaling cascades induced by beta-adrenergic receptor activation in normal and desensitized states to provide new insight into the effective treatment of cardiac dysfunction.     

Transgenic expression of the endothelin-B receptor prevents congenital intestinal aganglionosis in a rat model of Hirschsprung disease.

The spotting lethal rat, a naturally occurring rodent model of Hirschsprung disease, carries a deletion in the endothelin-B receptor (EDNRB) gene that abrogates expression of functional EDNRB receptors. Rats homozygous for this mutation (sl) exhibit coat color spotting and congenital intestinal aganglionosis. These deficits result from failure of the neural crest-derived epidermal melanoblasts and enteric nervous system (ENS) precursors to completely colonize the skin and intestine, respectively. We demonstrate that during normal rat development, the EDNRB mRNA expression pattern is consistent with expression by ENS precursors throughout gut colonization. We used the human dopamine-beta-hydroxylase (DbetaH) promoter to direct transgenic expression of EDNRB to colonizing ENS precursors in the sl/sl rat. The DbetaH-EDNRB transgene compensates for deficient endogenous EDNRB in these rats and prevents the intestinal defect. The transgene has no effect on coat color spotting, indicating the critical time for EDNRB expression in enteric nervous system development begins after separation of the melanocyte lineage from the ENS lineage and their common precursor. The transgene dosage affects both the incidence and severity of the congenital intestinal defect, suggesting dosage-dependent events downstream of EDNRB activation in ENS development.     

Myocardial beta-adrenergic receptor function during the development of pacing-induced heart failure.

The development of pacing-induced heart failure was studied in chronically instrumented, conscious dogs paced at a rate of 240 beats/min for 1 d (n = 6), 1 wk (n = 6), and 3-4 wk (n = 7). Left ventricular (LV) dP/dt was decreased (P < 0.0125) at 1 d, LV end-diastolic pressure and heart rate were increased (P < 0.0125) at 1 wk, but clinical signs of heart failure were only observed after 3-4 wk of pacing. Plasma norepinephrine rose (P < 0.0125) after 1 d of pacing, whereas LV norepinephrine was reduced (P < 0.0125) only after 3-4 wk of pacing. Both the fraction of beta-adrenergic receptors binding agonist with high affinity and adenylyl cyclase activity decreased (P < 0.0125) after 1 d of pacing. Total beta-adrenergic receptor density was not changed at any time point, but beta 1-adrenergic receptor density was decreased (P < 0.0125) after 1 wk. The functional activity of the guanine nucleotide binding protein, Gs, was not reduced, but the Gi alpha 2 isoform of the alpha subunit of the GTP-inhibitory protein rose after 3-4 wk of pacing. Thus, myocardial beta-adrenergic signal transduction undergoes change shortly (1d) after the initiation of pacing, before heart failure develops. The mechanism of beta-adrenergic receptor dysfunction in pacing-induced heart failure is characterized initially by elevated plasma levels of catecholamines, uncoupling of beta-adrenergic receptors, and a defect in the adenylyl cyclase catalytic unit. Selective down-regulation of beta 1-adrenergic receptors, increases in Gi alpha 2, and decreases in myocardial catecholamine levels occur as later events.     

Elevation of beta-adrenergic receptor density in human lymphocytes after propranolol administration.

Abrupt withdrawal after the chronic administration of propranolol has resulted in clinical syndromes that suggest adrenergic hypersensitivity. The effect of propranolol administration and withdrawal on beta-adrenergic receptors was studied in human lymphocyte membranes. Receptor density was quantitated by direct binding assays with the radioligand [125I]iodohydroxybenzylpindolol. Administration of propranolol (160 mg/d) for 8 d resulted in trough plasma levels of approximately 35 ng/ml. By day 5 of propranolol administration the density of beta-adrenergic receptors had increased 43 +/- 4% (P less than 0.01) above pretreatment levels. Abrupt withdrawal of propranolol was followed by the disappearance of propranolol from the plasma within 24 h. The density of beta-adrenergic receptors did not return to pretreatment level for several days. Physiologic supersensitivity of beta-adrenergic receptor-mediated responses was suggested by the appearance of significant increases in the orthostatic change in heart rate (P less than 0.05) and the orthostatic change in the heart rate-systolic blood pressure product (P less than 0.01) during the first 48 h after propranolol withdrawal. These data show that propranolol administration leads to an increase in the density of beta-adrenergic receptors in human tissue. The results are consistent with the hypothesis that some of the untoward effects observed after abrupt discontinuation of propranolol are caused by beta-receptor-mediated adrenergic hypersensitivity.     

sMiMi-CK cDNA的克隆、表达及活性检测

目的本实验拟通过ＲＴ－ＰＣＲ技术,克隆及表达大鼠心肌线粒体ｓＭｉＭｉ－ＣＫ基因。方法从大鼠心肌线粒体总ＲＮＡ中扩增出ｓＭｉＭｉ－ＣＫＤＮＡ片段（１３３ｋｂ）,将该片段克隆到载体ｐＵＣ１９上,对其进行酶切和测序,然后将其克隆到ＰＢＶ２２０上,经温度诱导,使其在大肠杆菌中得以表达,并用ＮＡＤＨ法测其活性。结果ｓＭｉＭｉ－ＣＫ基因序列与国外报导的序列完全一致,表达的ｓＭｉＭｉ－ＣＫ分子量为４５８ＫＤ,占菌体总蛋白的１０７％,其活性为２７４×１０５ＩＵ／Ｌ菌液。结论ｓＭｉＭｉ－ＣＫ基因的克隆及表达,为今后从基因水平上研究心肌缺血性损伤的机制及治疗提供了依据。     

The N terminus of the human alpha1D-adrenergic receptor prevents cell surface expression

We previously reported that truncation of the N-terminal 79 amino acids of alpha(1D)-adrenoceptors (Delta(1-79)alpha(1D)-ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in alpha(1D)-AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length alpha(1D)-ARs were found primarily in intracellular compartments, whereas Delta(1-79)alpha(1D)-ARs were translocated to the plasma membrane. This resulted in a 3- to 4-fold increase in intrinsic activity for stimulation of inositol phosphate formation by norepinephrine. We determined whether this effect was transplantable by creating N-terminal chimeras of alpha(1)-ARs containing the body of one subtype and the N terminus of another (alpha(1A)NT-D, alpha(1B)NT-D, alpha(1D)NT-A, and alpha(1D)NT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of alpha(1A)-or alpha(1B)-ARs containing the alpha(1D)-N terminus decreased by 86 to 93%, whereas substitution of alpha(1A)- or alpha(1B)-N termini increased alpha(1D)-AR binding site density by 2- to 3-fold. Confocal microscopy showed that GFP-tagged alpha(1D)NT-B-ARs were found only on the cell surface, whereas GFP-tagged alpha(1B)NT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged alpha(1D)- and Delta(1-79)alpha(1D)-ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express alpha(1D)-ARs. These findings demonstrate that the N-terminal region of alpha(1D)-ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization.     

Effects of two Gbetagamma-binding proteins--N-terminally truncated phosducin and beta-adrenergic receptor kinase C terminus (betaARKct)--in heart failure

Myocardial overexpression of the C-terminus of beta-adrenergic receptor kinase (betaARKct) has been shown to result in a positive inotropic effect or an improvement of survival in heart failure. However, it is not clear whether this beneficial effect is mainly because of dominant-negative inhibition of betaARK1, and a consecutive resensitization of beta-adrenergic receptors (betaAR), or rather due to inhibition of other Gbetagamma-mediated effects. In this study, we tested whether overexpression of N-terminally truncated phosducin (nt-del-phosducin), another Gbetagamma-binding protein that does not resensitize betaARs owing to simultaneous inhibition of GDP release from Galpha subunits, shows the same effects as betaARKct. Adenoviral gene transfer was used to express nt-del-phosducin and betaARKct in isolated ventricular cardiomyocytes and in myocardium of rabbits, which suffered from heart failure because of rapid ventricular pacing. BetaAR-stimulated cAMP formation was increased by betaARKct, but not by nt-del-phosducin, whereas both proteins inhibited Gbetagamma-mediated effects. Both transgenes also increased contractility of normal and failing isolated cardiomyocytes and improved contractility in rabbits with heart failure after gene transfer in vivo. In conclusion, overexpression of nt-del-phosducin enhances the contractility of cardiomyocytes to the same extent as betaARKct, suggesting that the effects of betaARKct might be owing to inhibition of Gbetagamma rather than to betaAR resensitization.     

Role of nitric oxide in parasympathetic modulation of beta-adrenergic myocardial contractility in normal dogs.

In vitro studies indicate that muscarinic cholinergic inhibition of beta-adrenergic cardiac responses may be modulated in part by nitric oxide (NO). To evaluate the role of NO in parasympathetic inhibition of the beta-adrenergic contractile response in vivo, we assessed the inotropic response to dobutamine before and during bilateral vagus nerve stimulation in closed-chest dogs. Dobutamine administration and vagal stimulation were repeated during intracoronary infusion of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 10 mumol/min) and again following infusion of L-arginine (100 mg/kg). In eight dogs, intracoronary dobutamine infusion at rates of 25 and 50 micrograms/min increased peak +dP/dt by 131 +/- 24 and 168 +/- 22%, respectively (P < 0.0001). Vagal stimulation (2.5 Hz) attenuated the responses to dobutamine (25 and 50 micrograms/min) by 23 +/- 4 and 21 +/- 4%, respectively (P < 0.001). L-NMMA reduced (by 44-62%; P < 0.001) and L-arginine restored vagal inhibition of the dobutamine-stimulated inotropic response. In a second group of nine dogs, dobutamine was administered systemically to assure a constant concentration in the coronary circulation. Vagal stimulation (2.5 Hz) attenuated the dobutamine-stimulated inotropic response (2.5 and 5.0 micrograms/kg per min) by 40 +/- 12% and 57 +/- 8%, respectively (P < 0.004). As with intracoronary dobutamine, L-NMMA diminished and L-arginine restored vagal inhibition of the inotropic response to dobutamine. Intracoronary infusion of atropine (12 micrograms/min) abolished the vagal inhibitory effect, and intracoronary infusion of 8-bromo-cyclic GMP (1 and 10 mM) caused a dose-dependent attenuation of the dobutamine-stimulated increase in +dP/dt. These data suggest that NO mediates, at least in part, vagal inhibition of the inotropic response to beta-adrenergic stimulation by dobutamine, and thus may play a role in normal physiologic regulation of myocardial autonomic responses.     

葛根素对儿茶酚胺诱导大鼠心肌损伤时凋亡相关蛋白的影响

目的 :探讨在儿茶酚胺诱导的心肌损伤过程中 ,心肌细胞的凋亡相关基因 Fas和 Bcl 2的蛋白表达改变及葛根素干预的影响。方法 :5 0只健康雄性 SD大鼠随机分成 3组 :心肌损伤组 (2 0只 )、葛根素干预组 (2 0只 )和对照组 (10只 )。动物模型仿用异丙肾上腺素 (ISO)皮下注射致心肌损伤方法。心肌组织切片分别行 HE及 Fas和 Bcl 2蛋白表达的免疫组织化学染色检测。结果 :心肌损伤组 :Fas蛋白和 Bcl 2蛋白的表达等级均上调 (P均 <0 .0 0 1) ,Fas蛋白的表达水平更高 (P<0 .0 0 1)。葛根素干预组 :心肌组织的 HE病理损害等级明显减轻 (P<0 .0 5 ) ;Fas蛋白表达等级下调 (P<0 .0 5 )。结论 :在儿茶酚胺诱导的心肌损伤过程中 ,Fas和 Bcl 2的蛋白表达水平表现出高水平的差异 ,葛根素能抑制损伤过程中 Fas蛋白的表达。     

诱导型一氧化氮合酶对β-肾上腺受体兴奋诱导老年大鼠心脏功能和心肌细胞凋亡的影响

目的:探讨心脏中诱导性一氧化氮合酶(induciblenitricoxidesynthase,iNOS)与心肌缺血性损伤和β-肾上腺受体兴奋之间的关系。方法:实验选用雄性Fisher大鼠50只。随机将其中20只大鼠(鼠龄3～5个月)分为青年大鼠对照组和青年大鼠干预组各10只;20只老年大鼠(鼠龄24～25个月)分为老年大鼠对照组和老年大鼠干预组各10只。余下年轻和老年大鼠各5只,处死后,取心肌组织作一氧化氮合酶Westernblot研究。青年大鼠对照组大鼠和老年大鼠对照组大鼠实验前接受生理盐水干预,青年大鼠干预组和老年大鼠干预组大鼠接受特异性iNOS阻断剂-1400W干预。4组大鼠的离体心脏分别接受生理盐水和1400W干预及50%正常冠状动脉流量灌注30min后,泵入异丙肾上腺素(isoproterenol,Iso)30min。结果:心肌梗死后30min,所有组左心室形成压leftventriculardeveloped(pressure,LVDP),左室内压最大上升下降速率rateofriseofleftventric-(ularpressure,±dp/dt)均较基础值下降13%～45%。Iso泵入青年大鼠对照组和青年大鼠干预组大鼠30min后,心室功能部分恢复,表现为LVDP和±dp/dt上升。与此明显对比,Iso泵入老年大鼠对照组大鼠后,非但未改善缺血所致的心功能不全,反倒进一步加重心功能损伤。表现为LVDP和±dp/dt进一步下降43%～60%。Westernblot研     

Mechanisms of ligand-induced desensitization of the 5-hydroxytryptamine(2A) receptor.

We have examined the cellular processes underlying the desensitization of the 5-hydroxytryptamine (5-HT)(2A) receptor induced by agonist or antagonist exposure. Treatment of C6 glioma cells with either 5-HT or the 5-HT(2A) receptor antagonist ketanserin resulted in an attenuation in 5-HT(2A) receptor function, specifically the accumulation of inositol phosphates stimulated by the partial agonist quipazine. 5-HT-induced desensitization of the 5-HT(2A) receptor involved receptor internalization through a clathrin- and dynamin-dependent process because it was prevented by concanavalin A, monodansylcadaverine, and by expression of the dominant negative mutants beta-arrestin (319-418) and dynamin K44A. Although short-term (i.e., 10 min) 5-HT and ketanserin exposure resulted in the same degree of desensitization, ketanserin-induced desensitization was not prevented by these agents and did not involve receptor internalization. In contrast, prolonged ketanserin exposure (i.e., 2 h) resulted in 5-HT(2A) receptor internalization through a clathrin- and dynamin-dependent process, as was observed after agonist treatment. Inhibitors of protein kinase C or calcium-calmodulin kinase II did not attenuate or prevent 5-HT-induced desensitization of the receptor. 5-HT(2A) receptor desensitization induced by 5-HT and prolonged ketanserin treatment, but not by short-term ketanserin treatment, was prevented by the expression of the dominant negative mutant of G protein-coupled receptor kinase (GRK)2, GRK2-K220R, and by an anti-GRK2/3 antibody. Our data indicate a dual mechanism of early and late desensitization by the antagonist ketanserin. Short-term ketanserin treatment reduced the specific binding of the agonist radioligand [(125)I](+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane ([(125)I]DOI) and the ability of 5'-guanylylimidodiphosphate to attenuate this binding, suggesting that at the early stage of antagonist-induced desensitization the capacity of the 5-HT(2A) receptor to couple to G protein is impaired.