Age-associated reductions in cardiac beta1- and beta2-adrenergic responses without changes in inhibitory G proteins or receptor kinases.

While an age-associated diminution in myocardial contractile response to beta-adrenergic receptor (beta-AR) stimulation has been widely demonstrated to occur in the context of increased levels of plasma catecholamines, some critical mechanisms that govern beta-AR signaling must still be examined in aged hearts. Specifically, the contribution of beta-AR subtypes (beta1 versus beta2) to the overall reduction in contractile response with aging is unknown. Additionally, whether G protein-coupled receptor kinases (GRKs), which mediate receptor desensitization, or adenylyl cyclase inhibitory G proteins (Gi) are increased with aging has not been examined. Both these inhibitory mechanisms are upregulated in chronic heart failure, a condition also associated with diminished beta-AR responsiveness and increased circulatory catecholamines. In this study, the contractile responses to both beta1-AR and beta2-AR stimulation were examined in rat ventricular myocytes of a broad age range (2, 8, and 24 mo). A marked age-associated depression in contractile response to both beta-AR subtype stimulation was observed. This was associated with a nonselective reduction in the density of both beta-AR subtypes and a reduction in membrane adenylyl cyclase response to both beta-AR subtype agonists, NaF or forskolin. However, the age-associated diminutions in contractile responses to either beta1-AR or beta2-AR stimulation were not rescued by inhibiting Gi with pertussis toxin treatment. Further, the abundance or activity of beta-adrenergic receptor kinase, GRK5, or Gi did not significantly change with aging. Thus, we conclude that the positive inotropic effects of both beta1- and beta2-AR stimulation are markedly decreased with aging in rat ventricular myocytes and this is accompanied by decreases in both beta-AR subtype densities and a reduction in membrane adenylate cyclase activity. Neither GRKs nor Gi proteins appear to contribute to the age-associated reduction in cardiac beta-AR responsiveness.     

Protection against myocardial dysfunction induced by global ischemia-reperfusion by antisense-oligodeoxynucleotides directed at beta(1)-adrenoceptor mRNA

Plasma catecholamine levels rise, and myocardial beta(1)-adrenoceptor (beta(1)-AR) sensitivity increases during ischemia. These factors enhance myocardial injury and cardiac dysfunction. beta(1)-AR blockers are clinically used to protect heart against ischemia and to improve cardiac dysfunction in patients with ischemic heart disease, but these agents often cause intolerable side effects. To examine the potential cardioprotective effect of therapy with antisense-oligodeoxynucleotides directed at beta(1)-AR mRNA (beta(1)-AS-ODNs) during myocardial ischemia-reperfusion, Sprague-Dawley rats were treated with beta(1)-AS-ODNs or inverted-oligodeoxynucleotides (IN-ODNs), each 200 microg/rat. Hearts were excised, perfused, and subjected to global ischemia (30 min) followed by reperfusion (30 min). Other rats were given selective beta(1)-AR blocker atenolol (2 mg/kg) or saline before excising the hearts. Ischemia-reperfusion resulted in cardiac dysfunction, indicated by an increase in coronary perfusion pressure and left ventricular end-diastolic pressure and a decrease in developed left ventricular pressure, as well as evidence of lipid peroxidation in saline-treated rats (all P <.05 versus control values). Administration of AS-ODNs or atenolol, but not IN-ODNs, protected hearts against functional deterioration and lipid peroxidation (P <.05 versus saline or IN-ODNs treatment). AS-ODNs therapy appeared to be equivalent to atenolol in these effects. Expression of beta(1)-AR protein as well as mRNA in the myocardium were markedly up-regulated after ischemia-reperfusion, and treatment with beta(1)-AS-ODNs, but not atenolol, decreased the rise in enhanced expression of beta(1)-AR. These observations imply that beta(1)-AS-ODNs can ameliorate cardiac dysfunction after ischemia-reperfusion by reducing the expression of beta(1)-AR in the ischemic-reperfused myocardium.     

Positive inotropic stimulation

Adrenergic receptors transduce signals through the G proteins to regulate cardiac function. The catecholamines, via alpha- and beta-adrenergic receptor (beta-AR) stimulation, may play a role in the development of heart failure. Norepinephrine and isoproterenol can induce cardiac myocyte apoptosis. Studies suggest that alpha-, beta1-, and beta2-adrenergic pathways differentially regulate cardiac myocyte apoptosis. The stimulation of beta1-AR leads to cyclic AMP-dependent apoptosis, whereas that of the beta2-AR elicits concurrent apoptosis and survival signals in cardiac myocytes coupled to Gs protein. Overexpression of alpha1-adrenergic receptors does not induce apoptosis in wild-type mice. In contrast, the heart failure observed in some murine models has to be related to an enhanced beta-AR kinase expression. These recent advances make it possible to understand the beneficial effects of beta-blockers in the treatment of chronic heart failure and provide novel therapeutic modalities through the stimulation of beta2-ARs or the inhibition of beta-AR kinase expression.     

Enhanced cardiac L-type calcium current response to beta2-adrenergic stimulation in heart failure.

The beta2-adrenergic receptor (beta2-AR)-mediated increase in cardiac L-type Ca2+ current (I(Ca,L)) has been documented in normal subjects. However, the role and mechanism of beta2-AR activation on I(Ca,L) in heart failure (HF) are unclear. Accordingly, we compared the effect of zinterol (ZIN), a highly selective beta2-AR agonist, on I(Ca,L) in isolated left ventricular cardiomyocytes obtained from normal control and age-matched rats with HF induced by left coronary artery ligation (4 months). I(Ca,L) was measured by using the whole-cell voltage-clamp technique. In normal myocytes, superfusion of ZIN (10(-5) M) caused a 21% increase in I(Ca,L) (9.21 +/- 0.24 versus 7.59 +/- 0.20 pA/pF) (p < 0.05). In HF myocytes, the same concentration of ZIN produced a significantly greater increase (30%) in I(Ca,L) (6.20 +/- 0.24 versus 4.75 +/- 0.17 pA/pF) (p < 0.01). This ZIN-induced increase in I(Ca,L) was further augmented in both normal and HF myocytes (normal: 59 versus 21%; HF: 71 versus 30%) after the incubation of myocytes with pertussis toxin (PTX, 2 microg/ml, 36 degrees C, 6 h). These effects were not modified by the incubation of myocytes with CGP-20712A (3 x 10(-7) M), a beta1-AR antagonist, but were abolished by pretreatment of myocytes with ICI-118551 (10(-7) M), a beta2-AR antagonist. In addition, all of the effects induced by ZIN were completely prevented in the presence of an inhibitory cAMP analog, Rp-cAMPS (100 microM, in the patch-pipette solution). In conclusion, beta2-AR activation stimulates L-type Ca2+ channels and increases I(Ca,L) in both normal and HF myocytes. In HF, beta2-AR activation-induced augmentation of I(Ca,L) was increased. These effects are likely to be mediated through a cAMP-dependent mechanism and coupled with both stimulatory G protein and PTX-sensitive G protein.     

Aryloxypropanolamine and catecholamine ligand interactions with the beta(1)-adrenergic receptor: evidence for interaction with distinct conformations of beta(1)-adrenergic receptors

Pharmacological responses to aryloxypropanolamines were examined in cells expressing rat or human beta(1)-adrenergic receptors (ARs) using adenylyl cyclase assays. The aryloxypropanolamines CGP 12177 and LY 362884, originally developed as beta(3)-AR agonists, were found to stimulate the beta(1)-AR. Interestingly, both CGP 12177 and LY 362884 exhibited an anomalous biphasic effect on beta(1)-AR. Low concentrations of either CGP 12177 or LY 362884 potently blocked isoproterenol-induced stimulation of beta(1)-AR, whereas higher concentrations of these compounds stimulated the beta(1)-AR. The unusual interaction of these aryloxypropanolamine ligands with the beta(1)-AR was further characterized using beta-AR antagonists. Activation of beta(1)-AR by CGP 12177 or LY 362884 was observed to be significantly more resistant to blockade by beta-AR antagonists compared with activation by catecholamines. These results suggest that catecholamines and aryloxypropanolamines interact with distinct active conformations of the beta(1)-AR: a state that is responsive to catecholamines and is blocked with high affinity by CGP 12177 and LY 362884, and a novel state that is activated by aryloxypropanolamines but is resistant to blockade by standard beta-AR antagonists. Moreover, dependence of antagonist affinity on agonist structure is unprecedented, and its implications on the use of beta-AR agonists such as CGP 12177 in receptor classification are discussed.     

Pharmacological characterization of KUR-1246, a selective uterine relaxant

The aim of the present study was to evaluate the efficacy and beta 2-adrenoceptor (AR) selectivity of KUR-1246, a new uterine relaxant. Inhibition of spontaneous or drug-induced uterine contractions by KUR-1246 was evaluated in pregnant rats and rabbits by an organ bath method or by a balloon method. The selectivity of KUR-1246 was assessed simultaneously in organs isolated from late-pregnant rats. The affinity of KUR-1246 for human beta 1-, beta 2-, and beta 3-ARs was determined using two radioligands. KUR-1246 suppressed both spontaneous and drug-induced contractions in isolated uteri, the rank order of potency being isoproterenol > KUR-1246 > terbutaline > ritodrine. ICI-118551 (selective beta 2-AR antagonist) competitively antagonized the KUR-1246-induced inhibition of spontaneous uterine contractions, but CGP-20712A (selective beta 1-AR antagonist) and SR-58894A (selective beta 3-AR antagonist) did not. All beta-AR agonists tested produced significant inhibition of spontaneous uterine contractions in vivo: ED(30) value for KUR-1246 was 0.13 microg/kg/min, a potency about 6 times and 400 times greater than that of terbutaline and ritodrine, respectively. In contrast, the positive chronotropic effect was minimal in KUR-1246-treated rats. KUR-1246 displaced radioligand binding to beta 1-, beta 2-, and beta 3-ARs, the pK(i) values being 5.75 +/- 0.03, 7.59 +/- 0.08, and 4.75 +/- 0.03 for beta 1-, beta 2-, and beta 3-ARs, respectively. For the selectivity of KUR-1246 for human beta 2-AR, we obtained values of 39.2 ([IC(50) for beta 1-AR]/[IC(50) for beta 2-AR]) and 198.2 ([IC(50) for beta 3-AR]/[IC(50) for beta 2-AR]), indicating an apparently higher affinity for human beta 2-AR than for other beta-AR subtypes. The present study clearly demonstrated that KUR-1246 is a more selective beta 2-AR agonist than the drugs presently used for relaxing uterine muscle.     

Real-time optical recording of beta1-adrenergic receptor activation reveals supersensitivity of the Arg389 variant to carvedilol

Antagonists of beta-adrenergic receptors (beta-ARs) have become a main therapeutic regimen for the treatment of heart failure even though the mechanisms of their beneficial effects are still poorly understood. Here, we used fluorescent resonance energy transfer-based (FRET-based) approaches to directly monitor activation of the beta(1)-AR and downstream signaling. While the commonly used beta-AR antagonists metoprolol, bisoprolol, and carvedilol displayed varying degrees of inverse agonism on the Gly389 variant of the receptor (i.e., actively switching off the beta(1)-AR), surprisingly, only carvedilol showed very specific and marked inverse agonist effects on the more frequent Arg389 variant. These specific effects of carvedilol on the Arg389 variant of the beta(1)-AR were also seen for control of beating frequency in rat cardiac myocytes expressing the 2 receptor variants. This FRET sensor permitted direct observation of activation of the beta(1)-AR in living cells in real time. It revealed that beta(1)-AR variants dramatically differ in their responses to diverse beta blockers, with possible consequences for their clinical use.     

Enhanced inhibition of L-type Ca2+ current by beta3-adrenergic stimulation in failing rat heart

beta3-adrenergic receptors (AR) have recently been identified in mammalian hearts and shown to be up-regulated in heart failure (HF). beta3-AR stimulation reduces inotropic response associated with an inhibition of L-type Ca2+ channels in normal hearts; however, the effects of beta3-AR activation on Ca2+ channel in HF remain unknown. We compared the effects of beta(3)-AR activation on L-type Ca2+ current (ICa,L) in isolated left ventricular myocytes obtained from normal and age-matched rats with isoproterenol (ISO)-induced HF (4 months after 340 mg/kg s.c. for 2 days). ICa,L was measured using whole-cell voltage clamp and perforated-patch recording techniques. In normal myocytes, superfusion of 4-[-[2-hydroxy-(3-chlorophenyl)ethylamino]propyl]phenoxyacetate (BRL-37,344; BRL), a beta3-AR agonist, caused a dose-dependent decrease in ICa,L with maximal inhibition (21%, 1.1 +/- 0.2 versus 1.4 +/- 0.1 nA) (p < 0.01) at 10(-7) M. In HF myocytes, the same concentration of BRL produced a proportionately greater inhibition (31%) in ICa,L (1.1 +/- 0.2 versus 1.6 +/- 0.2 nA) (p < 0.05). A similar inhibition of ICa,L was also observed with ISO (10(-7) M) in the presence of a beta1- and beta2-AR antagonist, nadolol (10(-5) M). Inhibition was abolished by the beta3-AR antagonist (S)-N-[4-[2-[[3-[3-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide (L-748,337; 10(-6) M), but not by nadolol. The inhibitory effect of BRL was attenuated by a nitric-oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (10(-4) M), and was prevented by the incubation of myocytes with pertussis toxin (PTX; 2 microg/ml, 36 degrees C, 6 h). In conclusion, beta3-AR activation inhibits L-type Ca2+ channel in both normal and HF myocytes. In HF, beta3-AR stimulation-induced inhibition of Ca2+ channel is enhanced. These effects are likely coupled with PTX-sensitive G-protein and partially mediated through a NOS-dependent pathway.     

Mechanisms of impaired beta-adrenoceptor-induced airway relaxation by interleukin-1beta in vivo in the rat.

We studied the in vivo mechanism of beta-adrenergic receptor (beta-AR) hyporesponsiveness induced by intratracheal instillation of interleukin-1beta (IL-1beta, 500 U) in Brown-Norway rats. Tracheal and bronchial smooth muscle responses were measured under isometric conditions ex vivo. Contractile responses to electrical field stimulation and to carbachol were not altered, but maximal relaxation induced by isoproterenol (10(-6)-10(-5) M) was significantly reduced 24 h after IL-1beta treatment in tracheal tissues and to a lesser extent, in the main bronchi. Radioligand binding using [125I]iodocyanopindolol revealed a 32+/-7% reduction in beta-ARs in lung tissues from IL-1beta-treated rats, without any significant changes in beta2-AR mRNA level measured by Northern blot analysis. Autoradiographic studies also showed significant reduction in beta2-AR in the airways. Isoproterenol-stimulated cyclic AMP accumulation was reduced by IL-1beta at 24 h in trachea and lung tissues. Pertussis toxin reversed this hyporesponsiveness to isoproterenol but not to forskolin in lung tissues. Western blot analysis revealed an IL-1beta-induced increase in Gi(alpha) protein expression. Thus, IL-1beta induces an attenuation of beta-AR-induced airway relaxation through mechanisms involving a reduction in beta-ARs, an increase in Gi(alpha) subunit, and a defect in adenylyl cyclase activity.     

Myocardial protection from ischemia/reperfusion injury by endogenous and exogenous HGF

Using a rat model of ischemia/reperfusion injury, we demonstrate here that HGF is cardioprotective due to its antiapoptotic effect on cardiomyocytes. Following transient myocardial ischemia and reperfusion, c-Met/HGF receptor expression rapidly increased in the ischemic myocardium, an event accompanied by a dramatic increase in plasma HGF levels in the infarcted rats. When endogenous HGF was neutralized with a specific antibody, the number of myocyte cell deaths increased markedly, the infarct area expanded, and the mortality increased to 50%, as compared with a control group in which there was no mortality. Plasma from the myocardial infarcted rats had cardioprotective effects on primary cultured cardiomyocytes, but these effects were significantly diminished by neutralizing HGF. In contrast, recombinant HGF administration reduced the size of infarct area and improved cardiac function by suppressing apoptosis in cardiomyocytes. HGF rapidly augmented Bcl-xL expression in injured cardiomyocytes both in vitro and in vivo. As apoptosis of cardiomyocytes is one of the major contributors to the pathogenesis in subjects with ischemia/reperfusion injury, prevention of apoptosis may prove to be a reasonable therapeutic strategy. Supplements of HGF, an endogenous cardioprotective factor, may be found clinically suitable in treating subjects with myocardial infarction.     

Direct intra-cardiomuscular transfer of beta2-adrenergic receptor gene augments cardiac output in cardiomyopathic hamsters

In chronic heart failure, down-regulation of beta-adrenergic receptor (beta-AR) occurs in cardiomyocytes, resulting in low catecholamine response and impaired cardiac function. To correct the irregularity in the beta-AR system, beta-AR gene was transduced in vivo into failing cardiomyocytes. The Epstein-Barr virus (EBV)-based plasmid vector carrying human beta2-AR gene was injected into the left ventricular muscle of Bio14.6 cardiomyopathic hamsters whose beta-AR is down-regulated in the cardiomyocytes. The echocardiographic examinations revealed that stroke volume (SV) and cardiac output (CO) were significantly elevated at 2 to 4 days after the beta2-AR gene transfer. Systemic loading of isoproterenol increased the cardiac parameters more significantly on day 2 to day 7, indicating that the adrenergic response was augmented by the genetic transduction. The same procedure did not affect the cardiac function of normal hamsters. Immunohistochemical examinations demonstrated human beta2-AR expression in failing cardiomyocytes transduced with the gene. RT-PCR analysis detected mRNA for the transgene in the heart but not in the liver, spleen, or kidney. The procedures may provide a feasible strategy for gene therapy of severe heart failure. Gene Therapy (2000) 7, 2087-2093.     

Role of the beta(3)-adrenoceptor in urine storage in the rat: comparison between the selective beta(3)-adrenoceptor agonist, CL316, 243, and various smooth muscle relaxants

The objective of this study was to compare the effects of a beta(3)-adrenoceptor (beta(3)-AR) agonist on bladder function and cardiovascular parameters in rats with those of several drugs that act on smooth muscle. CL316,243 (beta(3)-AR agonist), isoproterenol (nonselective beta-AR agonist), procaterol (beta(2)-AR agonist), verapamil (Ca(2+) antagonist), and papaverine (antispastic drug) each evoked a concentration-dependent relaxation of the detrusor in vitro. They also reduced bladder pressure in anesthetized rats, the beta-AR agonists apparently being more potent than the other drugs. Atropine (muscarinic antagonist) neither relaxed detrusor strips nor reduced bladder pressure. In anesthetized rats, CL316,243 and atropine each had only a slight influence on blood pressure and heart rate, but isoproterenol, procaterol, verapamil, and papaverine significantly affected cardiovascular function at the same dose range as that required to reduce bladder pressure. In cystometry experiments, CL316,243 (10 microg/kg i.v.), verapamil (1 mg/kg i.v.), and papaverine (1 mg/kg i.v.) all significantly prolonged micturition interval and increased bladder capacity, but did not change the residual urine volume after a micturition contraction. Procaterol (100 microg/kg i.v.) prolonged the micturition interval and increased both bladder capacity and residual urine volume (all significantly). Atropine (100 microg/kg i.v.) reduced micturition pressure and increased residual urine volume (both significantly). Because the human detrusor, like the rat detrusor, relaxes on beta(3)-AR stimulation, we conclude that this beta(3)-AR agonist may have potential in pollakiuria (frequent urination) as a therapeutic agent without cardiovascular side effects.     

Adenoviral delivery of the beta2-adrenoceptor gene in sepsis: a subcutaneous approach in rat for kidney protection

Successful gene therapy requires gene delivery that is efficient, has an optimal route of administration and has biosafety. The aims of the present study were to evaluate the safety and applicability of the subcutaneous delivery route for adenoviral transgenes containing the human beta(2)-adrenoceptor (adeno-beta(2)-AR) and to investigate whether this approach prevented renal dysfunction in a rat model of endotoxaemic shock induced by LPS (lipopolysaccharide). Subcutaneous administration of adeno-beta(2)-AR (a total of 10(10) viral particles) significantly increased beta-AR density in the kidney, lung and liver, but was without effect on physiological and plasma biochemical parameters. Moreover, this dose of virus did not cause any of the potential toxic responses of viral administration, such as inflammation and tissue TNF (tumour necrosis factor)-alpha expression. Although the LPS challenge caused a decrease in glomerular filtration rate, fractional excretion of sodium and renal beta-AR density in all groups, the reduction in renal function was significantly less in the rats given adeno-beta(2)-AR compared with non-treated rats. Thus, although further evaluation will be required, this initial study demonstrated that the subcutaneous injection of adeno-beta(2)-AR was efficient, comparatively non-pathogenic and potentially therapeutic to deal with acute renal failure associated with sepsis.     

beta3-adrenoceptors in the cardiovascular system: putative roles in human pathologies

The sympathetic nervous system is central for the neurohumoral regulation of the cardiovascular system and is largely involved in many cardiovascular diseases affecting millions of people around the world. It is classically admitted that beta-adrenoceptors (beta-AR) of the beta1 and beta2 subtypes mediate the effects of catecholamines on the force of contraction of cardiac muscle, and on the relaxation of vascular smooth muscle. However, the molecular characterization in 1989 of a third beta-AR subtype, beta3, and later its identification in human heart has changed the classically admitted paradigm on the regulation of heart function by the beta-adrenergic system. In blood vessels, beta3-AR, like beta1 and beta2, produced a relaxation. But at the present time, the physiological role of beta3-AR is not clearly identified. Thus, the purpose of this review is to summarize the pharmacological and molecular evidence supporting the functional roles of beta3-AR in cardiovascular tissues of various species, including humans. In addition, this review discusses the potential role of beta3-AR in several cardiovascular diseases and emphasizes their putative involvement as new therapeutic targets.     

Steroid administration after myocardial infarction promotes early infarct expansion. A study in the rat.

Whether steroids lead to thinner scars and larger aneurysms by delaying collagen deposition or worsening infarct expansion before significant collagen deposition begins is unknown. Rats underwent either transmural infarction by left coronary ligation or sham operation. Both infarct and sham rats were randomized to methylprednisolone 50 mg/kg i.p. X 4 or saline treatment within 24 h after operation. Sacrifice occurred before (3 d) or after (7 d) collagen deposition typically begins. Despite similar infarct size, infarct wall thickness was 1.35 +/- 0.08 mm in the saline and 0.99 +/- 0.12 mm in the methylprednisolone group (P less than 0.001) at 3 d. This decrease in wall thickness was explained by a decrease in the number of myocytes across the infarct wall (r = 0.99; P less than 0.001), suggesting that steroids promote myocyte slippage. Furthermore, methylprednisolone caused no further infarct thinning or cavity dilatation beyond 3 d. Thus, high-dose methylprednisolone given within 24 h after transmural infarction worsens infarct expansion before collagen is laid down by promoting the slippage of necrotic myocytes.     

Selectivity and potency of agonists for the three subtypes of cloned human beta-adrenoceptors expressed in Chinese hamster ovary cells

The selectivities, potencies and efficacies of beta3-adrenoceptor (beta3-AR) agonists on human three beta-AR subtypes expressed in Chinese hamster ovary (CHO) cells were investigated using radioligand binding assay and cyclic AMP (cAMP) accumulation assay. The three beta-AR subtypes showed the nature of G protein-coupled receptors with the constitutive activity. BRL37344, CL-316,243 and a newly synthesized beta3-AR agonist N-5984, 6-[2-(R)-[[2-(R)-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-2,3-dihydro-1,4-benzodioxine-2-(R)-carboxylic acid, were compared for the potency and selectivity for the beta3-AR. In the radioligand binding assay, the affinity of N-5984 for beta3-ARs was 14, 70 and 220 times more potent than those of BRL37344, isoproterenol and CL-316,243, respectively. N-5984 had higher selectivity than BRL37344 for human beta3-ARs compared with either for beta1-ARs or beta2-ARs. N-5984 showed higher potency and intrinsic activity of cAMP production than BRL37344 in CHO cells expressing the beta3-ARs. CL-316,243 had almost no activity of cAMP production in CHO cells expressing any subtype of beta-ARs. These results indicate that N-5984 is the most potent and selective agonist for human beta3-ARs than any other agonists tested.     

Catecholamines relax detrusor through beta 2-adrenoceptors in mouse and beta 3-adrenoceptors in man

(-)-Isoproterenol [4-[1-hydroxy-2-[(1-methylethyl)amino]ethyl]-1,2-benzene diol hydrochloride] relaxes murine detrusor through beta-adrenoceptors (ARs); however, the beta-AR subtypes involved are unknown. beta(2)-ARs have been associated with caveolae, plasma-lemmal scaffolding domains that are absent in caveolin-1 (cav-1) knockout (KO) mice. Here, we studied detrusor responses in the absence and presence of beta-AR subtype-selective antagonists in wild-type (WT) and cav-1 KO mice. To inquire whether the murine detrusor model is relevant to man, beta-AR subtypes that mediate (-)-isoproterenol-evoked human detrusor relaxation were investigated. In WT mice, (-)-isoproterenol concentration-dependently relaxed the KCl (40 mM)-precontracted detrusor (-logEC(50)M = 8.04, E(max) = 62%). The effects of (-)-isoproterenol were surmountably antagonized by the beta(2)-AR-selective antagonist ICI 118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] (pK(B) = 9.28) but not affected by the beta(1)-AR-selective antagonist CGP 20712 [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol] and beta(3)-AR-selective L-748,337 [(S)-M-[4-[2-[3-[3-[acetamidomethyl)phenoxy)-2-hydroxypropyl]-amino]-ethyl]-phenylbenzsulfonamide)], suggesting involvement of beta(2)-AR only. The cav-1 KO detrusor displayed significant contractile dysfunction. (-)-Isoproterenol was less potent and efficient in relaxing detrusor from cav-1 KO (-logEC(50)M, 7.76; E(max) = 44%), but ICI 118,551 caused similar antagonism (pK(B) = 9.15), suggesting that beta(2)-AR function persisted in cav-1 KO. The beta(3)-AR-selective antagonist L-748,337 in the presence of ICI 118,551 and CGP 20712 caused additional blockade of (-)-isoproterenol effects in cav-1 KO, consistent with a beta(3)-AR involvement during relaxation and suppression of this effect in WT. (-)-Isoproterenol relaxed human detrusor muscle precontracted with carbachol (-logEC(50)M = 6.39, E(max) = 52%). However, the effects of (-)-isoproterenol in human detrusor were not blocked by CGP 20712 or ICI 118,551 but antagonized by L-748,337 (pK(B) = 7.65). We conclude that murine detrusor relaxation occurs via beta(2)-AR, and loss of caveolae does not perturb beta(2)-AR function but unmasks an additional activation of beta(3)-AR. In contrast, detrusor relaxation in man is mediated exclusively via beta(3)-AR.     

大鼠脑缺血再灌注后半影区葡萄糖转运体3表达的变化

背景近年来的研究表明,缺血缺氧导致脑内代谢异常乃至能量衰竭,是引起脑组织损伤坏死的重要原因,可见能量代谢障碍是脑缺血再灌注损伤的中心问题.在脑的能量代谢中葡萄糖转运体3中发挥着重要作用.目的观察大鼠局灶性脑缺血不同缺血时间和不同再灌注时间的脑梗死体积比、皮质半影区葡萄糖转运体3转录水平和蛋白水平的表达.设计随机对照实验.单位中山大学附属第二医院神经外科.材料实验于2002-08/10在中山大学附属第二医院医学研究中心动物试验室完成.选择SD大鼠56只,随机分成3组①缺血1 h再灌注组28只.②缺血3 h再灌注组24只.③假手术对照组4只.缺血1 h再灌注组自缺血开始分别选取1,3,6,12,24,72 h,1周7个时间点,每个时间点7只大鼠;缺血3 h再灌注组除无1 h时点外,其余时间点与缺血1 h再灌注组相同,假手术对照组只作切口,不作插线.方法用线栓法复制大鼠局灶性脑缺血模型,检测缺血中心区和缺血半影区脑梗死体积比;剥取缺血半影区皮质组织,采用反转录-聚合酶链反应测定葡萄糖转运体3 mRNA水平的变化;用免疫组织化学方法半定量测定葡萄糖转运体3蛋白水平的变化.主要观察指标①各组大鼠脑缺血再灌注后的脑梗死面积.②各组大鼠脑缺血再灌注后的葡萄糖转运体3 mRNA表达水平的变化.③各组大鼠脑缺血再灌注后的葡萄糖转运体3蛋白水平表达的变化.结果56只大鼠均进入结果分析.①脑缺血1 h后再灌注组的脑梗死体积明显小于缺血3 h再灌注组梗死体积.②葡萄糖转运体3 mRNA及蛋白水平表达变化葡萄糖转运体3自3 h即开始升高,24 h到达高峰,1周时仍高于假手术对照组;缺血3 h再灌注组在3 h有一下降点,然后升高,24 h到高峰,1周时接近正常水平.葡萄糖转运体3蛋白水平的表达与mRNA相符合.结论葡萄糖转运体3在缺血半影区的表达上调,可能是机体对缺血再灌注的保护性反应.