Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary β2-adrenergic receptor gene delivery

Exogenous gene delivery to alter the function of the heart is a potential novel therapeutic strategy for treatment of cardiovascular diseases such as heart failure (HF). Before gene therapy approaches to alter cardiac function can be realized, efficient and reproducible in vivo gene techniques must be established to efficiently transfer transgenes globally to the myocardium. We have been testing the hypothesis that genetic manipulation of the myocardial beta-adrenergic receptor (beta-AR) system, which is impaired in HF, can enhance cardiac function. We have delivered adenoviral transgenes, including the human beta2-AR (Adeno-beta2AR), to the myocardium of rabbits using an intracoronary approach. Catheter-mediated Adeno-beta2AR delivery produced diffuse multichamber myocardial expression, peaking 1 week after gene transfer. A total of 5 x 10(11) viral particles of Adeno-beta2AR reproducibly produced 5- to 10-fold beta-AR overexpression in the heart, which, at 7 and 21 days after delivery, resulted in increased in vivo hemodynamic function compared with control rabbits that received an empty adenovirus. Several physiological parameters, including dP/dtmax as a measure of contractility, were significantly enhanced basally and showed increased responsiveness to the beta-agonist isoproterenol. Our results demonstrate that global myocardial in vivo gene delivery is possible and that genetic manipulation of beta-AR density can result in enhanced cardiac performance. Thus, replacement of lost receptors seen in HF may represent novel inotropic therapy.     

Estrogen reduces cardiac injury and expression of beta1-adrenoceptor upon ischemic insult in the rat heart

To test the hypothesis that estrogen confers cardioprotection by suppressing the expression of beta-adrenoceptor (beta-AR), we first correlated the infarct size in response to ischemic insult and beta-AR stimulation with the expression of beta(1)-AR in sham, ovariectomized (Ovx) and estrogen replaced (Ovx + E(2)) rats. When beta-AR is being activated during ischemia, the infarct size was significantly greater in Ovx than in the sham and Ovx + E(2) rats. There is a negative correlation between the infarct size and the expression level of beta(1)-AR as revealed by Western blotting and supported by binding analysis. Incubation of ventricular myocytes from Ovx rats with estrogen at 10(-9) M for 24 and 48 h, but not 12 h, significantly reduced lactate dehydrogenase release when the myocytes are subjected to simulated ischemia. The cardioprotective effect of 24 h estrogen incubation was accompanied by a reduction in the protein expression level of beta(1)-AR, which is estrogen receptor-dependent, whereas the lack of protection of 12-h estrogen incubation was not accompanied by any alterations in the expression level of beta(1)-AR. Together, the result from present study suggested that it is most likely that the cardioprotective effect of long-term estrogen replacement is due to suppressing the enhanced expression of cardiac beta(1)-AR in the Ovx rats, which in turn reduces cardiac injury when beta-AR is activated by sympathetic hyperactivity during ischemia. Therefore, suppression of the enhanced expression of cardiac beta(1)-AR in Ovx rats represents a novel cardioprotective mechanism of estrogen replacement therapy.     

Adenovirus-mediated gene transfer of the beta2-adrenergic receptor to donor hearts enhances cardiac function.

Gene transfer to modify donor heart function during transplantation has significant therapeutic implications. Recent studies by our laboratory in transgenic mice have shown that overexpression of beta2-adrenergic receptors (beta2-ARs) leads to significantly enhanced cardiac function. Thus, we investigated the functional consequences of adenovirus-mediated gene transfer of the human beta2-AR in a rat heterotopic heart transplant model. Donor hearts received 1 ml of solution containing 1 x 1010 p.f.u. of adenovirus encoding the beta2-AR or an empty adenovirus as a control. Five days after transplantation, basal left ventricular (LV) pressure was measured using an isolated, isovolumic heart perfusion apparatus. A subset of hearts was stimulated with the beta2-AR agonist, zinterol. Treatment with the beta2-AR virus resulted in global myocardial gene transfer with a six-fold increase in mean beta-AR density which corresponded to a significant increase in basal contractility (LV + dP/dtmax, control: 3152.1 +/- 286 versus beta2-AR, 6250.6* +/- 432.5 mmHg/s; n = 10, *P < 0.02). beta2-AR overexpressing hearts also had higher contractility after zinterol administration compared with control hearts. Our results indicate that myocardial function of the transplanted heart can be enhanced by the adenovirus-mediated delivery of beta2-ARs. Thus, genetic manipulation may offer a novel therapeutic strategy to improve donor heart function in the post- operative setting.     

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.     

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.     

Patterns of gene expression from in utero delivery of adenoviral-associated vector serotype 1

Adenoviral-associated viral vectors (AAV) have shown significant promise for efficient gene delivery to multiple tissues. Studies of different serotypes of AAV revealed different expression patterns provided by gene delivery in postnatal mice. Previous in utero gene delivery studies of AAV serotype 2 (AAV2) demonstrated efficient gene expression in certain fetal tissues depending on route of administration. We studied the pattern of gene expression from AAV serotype 1 (AAV1) using intramuscular, intraperitoneal, and intravascular routes of administration in embryonic day 16 C57BL/6 mice. Limb skeletal muscle transduction was only achieved with AAV1 by intramuscular administration. The levels of gene expression were 20-fold higher than a comparable administration of AAV2. Diaphragm muscle transduction by AAV1 was achieved at the highest level by intraperitoneal administration, and to a lesser degree by intravascular administration. All delivery routes resulted in transgene expression in the lung. Our results indicate that AAV1 can offer higher transgene expression in fetal skeletal muscle than AAV2 with intramuscular administration. The transgene expression pattern in different tissues, which depends on vector serotype and route of administration, will need to be considered in planning therapeutic studies for specific disorders.     

Endotoxin tolerance enhances interleukin-10 renal expression and decreases ischemia-reperfusion renal injury in rats

The potential implication of interleukin (IL) 6, tumor necrosis factor alpha (TNF-alpha), and IL-10 in the protective effect of low-dose lipopolysaccharide (LPS) administration against renal ischemia-reperfusion injury was evaluated in a rat model. Eighteen male Sprague-Dawley rats were injected intravenously with either 0.5 mg/kg of LPS (tolerant group) or saline (control group) 2 days before surgery. Ischemic renal injury was induced by clamping the left renal artery for 60 min on rats immediately after right-side nephrectomy. Reperfusion was obtained by clamp removal and was studied at R0 (no reperfusion), 2H (R2), and 24H (R24) by renal tubular disorder characterization and by plasma creatinine as well as renal cytokine (IL-6, IL-10, and TNF-alpha) studies. No differences were observed between the two groups as concerns the period immediately after renal ischemia (R0). The endotoxin-tolerant group was associated with a significantly lower creatinine level at R24 (231 +/- 28 vs 315 +/- 36 micromol/L; P = 0.007). Pretreatment with LPS significantly reduced the degree of proximal tubule necrosis and outer medulla congestion. In such tolerant animals, renal IL-6 production was decreased, whereas IL-10 production was significantly increased at R2 and R24. There were no differences in TNF-alpha renal production. In this study, we demonstrated that administration of low doses of LPS to rats had a protective effect from renal reperfusion injury, and our data suggest that IL-10 might play a role in this phenomenon.     

Distribution of retroviral vectors and vector producer cells using two routes of administration in rats.

The clinical use of retroviral vector producer cells (VPCs) to deliver retroviral vectors efficiently to target cells has been investigated as a method to increase efficiency of gene delivery, presumably as a result of continued vector production in vivo. Studies were conducted in rats to evaluate the distribution of vector to distal organs and tissues as measured by transduction. Rats were treated with two doses of VPCs using two routes of administration: (1) subcutaneous injection, chosen to maximize both the dose and exposure of animals, thereby enabling identification of potential target organs under worst-case conditions; and (2) direct injection into brain parenchyma, chosen to mimic the intended clinical route of administration and provide an estimate of risk to patients receiving this therapy. Twelve organs or tissues were collected 7 days after administration of VPCs and analyzed by PCR for the presence of vector and vector producer cell sequences. Vector was detected most frequently at the site of injection by either route of administration. Less frequently, vector was detected in draining lymph nodes at the higher dose only using either route of injection. Single specimens of lung and contralateral skin were positive for vector following subcutaneous administration only. Vector was detected in gonadal tissue from a single low-dose male following subcutaneous administration, but this finding was not reproduced in any high-dose male or any males injected intracerebrally. In contrast, VPCs were detected only at the site of administration. The frequency of detection of VPCs 7 days after administration was higher when rats were injected by the intracerebral route. Based on these studies, gene transfer to distal organs or gonadal tissue following intracerebral administration of VPCs is not considered to be a risk to patients undergoing retroviral vector gene therapy for the treatment of brain cancer (glioblastoma multiforme; GBM).     

Enhanced myocardial relaxation in vivo in transgenic mice overexpressing the beta2-adrenergic receptor is associated with reduced phospholamban protein.

To assess the effect of targeted myocardial beta-adrenergic receptor (AR) stimulation on relaxation and phospholamban regulation, we studied the physiological and biochemical alterations associated with overexpression of the human beta2-AR gene in transgenic mice. These mice have an approximately 200-fold increase in beta-AR density and a 2-fold increase in basal adenylyl cyclase activity relative to negative littermate controls. Mice were catheterized with a high fidelity micromanometer and hemodynamic recordings were obtained in vivo. Overexpression of the beta2-AR altered parameters of relaxation. At baseline, LV dP/dt(min) and the time constant of LV pressure isovolumic decay (Tau) in the transgenic mice were significantly shorter compared with controls, indicating markedly enhanced myocardial relaxation. Isoproterenol stimulation resulted in shortening of relaxation velocity in control mice but not in the transgenic mice, indicating maximal relaxation in these animals. Immunoblotting analysis revealed a selective decrease in the amount of phospholamban protein, without a significant change in the content for either sarcoplasmic reticulum Ca2+ ATPase or calsequestrin, in the transgenic hearts compared with controls. This study indicates that myocardial relaxation is both markedly enhanced and maximal in these mice and that conditions associated with chronic beta-AR stimulation can result in a selective reduction of phospholamban protein.     

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.     

Recent developments in gene therapy for cardiac disease

Cardiovascular[TRACE;del] disease is the leading cause of death in the US and world-wide. Advances in molecular biology and the human genome project have revealed opportunities for novel strategies for cardiac gene therapy. This review discusses general and specific aspects of gene transfer strategies in cardiac tissues. These include 1) the selection and/or optimization of the vector for gene transfer; 2) the identification of the target gene(s); 3) the use of cardiac-specific promoters; and 4) the use of an appropriate delivery system for administration. Currently, several vectors (e.g., viral and nonviral vectors) have been developed and many target genes have been identified (e.g., VEGF, FGF, beta-AR, etc.). Many investigations have provided experimental models for gene delivery systems but the most efficient cardiac gene transfer was obtained from intramyocardial injection or perfusion of explanted myocardium. The data available thus far have suggested favorable immediate effects following gene transfer, but long-term value of cardiac gene therapy has not been proven. Further refinements in appropriate vectors that provide cell or tissue selectivity and long-lasting effects are necessary as well as the development of minimally invasive procedures for gene transfer.     

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.     

Novel oral insulin delivery systems based on complexation polymer hydrogels: Single and multiple administration studies in type 1 and 2 diabetic rats

Insulin-loaded polymer microparticles (ILP) composed of crosslinked poly(methacrylic acid) and poly(ethylene glycol) are multi-functional carriers showing high insulin incorporation efficiency, a rapid insulin release in the intestine based on their pH-dependent complexation properties, enzyme-inhibiting effects and mucoadhesive characteristics. Thus, they are potential carriers for insulin delivery via an oral route. Recent studies suggest that the polymer composition and particle size of ILP strongly influenced insulin bioavailability. Therefore, the present study aimed at finding an optimal formulation and designing carriers for oral insulin delivery using in vivo experiments. Various types of ILPs were prepared and administered orally to healthy and type 1 and 2 diabetic rats. The most promising formulation was subsequently used for in vivo multiple oral administration studies using diabetic rats. The microparticles of diameters of < 53 μm (SS-ILP) composed of a 1 : 1 molar ratio of methacrylic acid / ethylene glycol units showed the most pronounced hypoglycaemic effects following oral administration to healthy rats, achieving a 9.5% pharmacological availability compared to subcutaneous insulin injection. Their usefulness was also confirmed with both type 1 and 2 diabetic rat groups. In a multiple administration study, SS-ILP significantly suppressed the postprandial rise in blood glucose and showed continuous hypoglycaemic effects following 3 times/day oral administration to both diabetic rat groups in the presence of foods. These results indicate that the blood glucose levels of diabetic rats can be effectively controlled by oral SS-ILP administration, and thus SS-ILP would be a promising delivery carrier of insulin via the oral route.     

Effects of mesenteric lymph duct ligation on apoptosis of renal tubule epithelial cells in rats after two-hits]

OBJECTIVE: To explore the effect of mesenteric lymph duct ligation in preventing apoptosis of renal tubule epithelial cells in rats by two-hits including hemorrhage and lipopolysaccharide (LPS) challenge. METHODS: Forty-five Wistar rats were randomly divided into three groups: the ligation group, the non-ligation group and sham operation group. The two-hits model was established by withdrawing the blood via the right side carotid artery of rats and intragastric administration 4 mg/kg LPS 6 hours after hemorrhage, and mesenteric lymph flow was blocked by ligating mesenteric lymph duct in ligation group. Twenty-four hours after operation, kidney was harvested for pathological examination, and the apoptosis cells rate of renal tubule epithelial cells were determined by method of terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), the expression of apoptosis-correlating gene bcl-2 and bax protein were determined by streptavidin-biotin complex (SABC) immunohistochemical method. RESULTS: After two-hits, the apoptosis rate and expression of bax protein of renal tubule epithelial cells in non-ligation group were significantly increased, and expression of bcl-2 protein was significantly lower as compared with those of sham operation group (all P<0.01). But the apoptosis rate and expression of bax protein of renal tubule epithelial cells in ligation group were significantly lower, and expression of bcl-2 protein was significantly increased as compared with non-ligation group (all P<0.01). CONCLUSION: The results demonstrate that the ligation of mesenteric lymph duct could ameliorate the apoptosis of renal tubule epithelial cells in rats as produced by hemorrhage and LPS, and its mechanism might relate to the reduction of the down regulation of gene expression of bax protein and enhancement of the expression of bcl-2 protein after ligation of mesenteric lymph duct.     

Influence of nifedipine on cyclosporin A nephrotoxicity after unilateral nephrectomy in the spontaneously hypertensive rat.

1. Nifedipine ameliorates cyclosporin A-induced renal impairment in surgically intact (two-kidney) rats. This study investigates the effect of nifedipine on cyclosporin A nephrotoxicity in spontaneously hypertensive rats after either uninephrectomy or uninephrectomy with contralateral renal denervation. 2. Fourteen days after uninephrectomy pair-fed rats were injected for 14 days with cyclosporin A (25 mg/kg body weight) via the subcutaneous route and with nifedipine (0.1 mg/kg body weight) via the intraperitoneal route. Renal and systemic haemodynamics were measured in conscious unrestrained rats. 3. Whole-blood levels of cyclosporin A did not differ between groups (overall 352 +/- 22 ng/ml, means +/- SEM). After uninephrectomy, cyclosporin A decreased the glomerular filtration rate (olive oil versus cyclosporin A: 0.96 +/- 0.04 versus 0.70 +/- 0.06 ml min-1 100 g body weight, P less than 0.02) and effective renal plasma flow (1.94 +/- 0.10 versus 1.38 +/- 0.13, P less than 0.01), and increased renal vascular resistance [(20.2 +/- 1.8) x 10(4) versus (31.6 +/- 3.3) x 10(4) kPa l-1 s [(20.2 +/- 1.8) x 10(3) versus (31.6 +/- 3.3) x 10(3) dyn s cm-5], P less than 0.02] and mean arterial pressure (146.7 +/- 6.7 versus 167.3 +/- 2.9 mmHg, P less than 0.05). Neither renal denervation nor nifedipine prevented the reduction in glomerular filtration rate or effective renal plasma flow induced by cyclosporin A. 4. This study infers that the sympathetic nervous system does not play an active role in cyclosporin A nephrotoxicity and demonstrates that the concomitant administration of nifedipine to rats with reduced renal mass does not ameliorate cyclosporin A-induced renal impairment.     

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.     

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.     

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.     

Low-dose dexamethasone ameliorates circulatory failure and renal dysfunction in conscious rats with endotoxemia

Corticosteroids have long been proposed as a therapeutic adjuvant in septic renal dysfunction because of their anti-inflammatory properties and favorable results from animal experiments. However, some reports suggested the potential for harm associated with the administration of early high-dose corticosteroids in patients with severe sepsis and septic shock. Thus, we examined the effects of low-dose dexamethasone (0.01 and 0.1 mg/kg) on hemodynamics and renal function in conscious rats with endotoxemia. Intravenous injection of rats with endotoxin (E. coli lipopolysaccharide, LPS, 1 mg/kg) caused hypotension, vascular hyporeactivity, and tachycardia as well as renal dysfunction. Circulatory failure and renal dysfunction caused by LPS were significantly attenuated in the dexamethasone 0.1 mg/kg-treated group. The nitric oxide (NO) production in plasma and renal tissue and the iNOS protein expression in the kidney were suppressed by cotreatment of LPS rats with dexamethasone, 0.1 mg/kg. Light microscopy showed that 0.1 mg/kg dexamethasone reduced marked infiltration of neutrophils in renal tissues from LPS rats. Moreover, the survival rate at 18 h was significantly increased in the dexamethasone 0.1 mg/kg-treated group when compared with the LPS group. These results suggest that the beneficial effects of low-dose dexamethasone (0.1 mg/kg) in conscious rats with endotoxic shock are associated with amelioration of circulatory failure and renal dysfunction, and this is attributed to inhibition of NO production.