Reinnervated nerves contribute to the secretion function and regeneration of denervated submandibular glands in rabbits.

This study aimed to investigate the contribution of redistributed nerves in the secretory function and regeneration of a denervated submandibular gland (SMG). The postganglionic parasympathetic and sympathetic denervated SMGs of rabbits were wrapped in polyester or acellular dermal matrices to block nerve regeneration either partially or completely. Submandibular glands were removed 4, 8, 16, and 24 wk after the operation and examined histologically. Furthermore, the aquaporin-5 (AQP5), muscarinic-3 (M3), and ß1-adrenergic receptors were evaluated by immunofluorescence and western blot analysis. After denervation, salivary flow was decreased and acinar cells were atrophic, and the expression levels of the M3, ß1-adrenergic, and AQP5 receptors were decreased. However, both impaired secretion function and atrophic parenchyma were gradually ameliorated with the growing redistribution of parasympathetic and sympathetic nerves. Apoptosis was markedly inhibited and expression of the M3, ß1-adrenergic, and AQP5 receptors was increased after reinnervation. In contrast, SMGs without reinnervated nerves maintained hyposecretion and atrophic parenchyma. In conclusion, reinnervated nerves in a rabbit's denervated SMG played an important role in the secretion function and regeneration of SMGs via up-regulation of the expression of neurotransmitter receptors and AQP5.

Expression of ß-adrenoceptors in human lower esophageal sphincter.

BACKGROUND/AIMS: Beta-adrenoceptor is considered to be an important modulator of smooth muscle function. It is widely present in the mammalian gastrointestinal tract and nervous system. The aim of this study was to explore the expression of 1-adrenoceptors (ß1-AR, ß2-AR, ß3-AR) in sling fibers and clasp fibers from human lower esophageal sphincter (LES). METHODOLOGY: Sling and clasp fibers from the LES were obtained from patients undergoing esophagogastrectomy; circular muscle strips from the esophagus and stomach were used as controls. Reverse transcription-polymerase chain reaction and western blotting were used to determine the expression of the three subtypes of ß-adrenoceptors. RESULTS: Messenger RNA and protein for three subtypes of ß-adrenoceptors were all identified in the sling and clasp fibers of the LES. Expression was highest for ß3-AR, then ß1-AR and ß2-AR in decreasing levels. CONCLUSIONS: ß1-AR, ß2-AR, ß3-AR can be detected in human lower esophageal sphincter and contribute to LES function.

Spatiotemporal control of heart rate in a rabbit heart.

Sinoatrial node is responsible for the origin of the wave of excitation, which spreads throughout the heart and orchestrates cardiac contraction via calcium-mediated excitation-contraction coupling. P wave represents the spread of excitation in the atria. It is well known that the autonomic nervous system controls the heart rate by dynamically altering both cellular ionic fluxes and the anatomical location of the leading pacemaker. In this study, we used isolated rabbit right atria and mathematical model of the pacemaker region of the rabbit heart. Application of isoproterenol resulted in dose-dependent acceleration of the heart rate and superior shift of the leading pacemaker. In the mathematical model, such behavior could be reproduced by a gradient of expression in ß1-adrenergic receptors along the superior-inferior axis. Application of acetylcholine resulted in preferentially inferior shift of pacemaker and slowing of the heart rate. The mathematical model reproduced this behavior with imposing a gradient of expression of acetylcholine-sensitive potassium channel. We conclude that anatomical shift of the leading pacemaker in the rabbit heart could be achieved through gradient of expression of ß1-adrenergic receptors and I(K,ACh).

Analysis of Density Changes of Selected Brain Receptors After a 14-Day Supply of Chromium(III) and Evaluation of Chromium(III) Affinity to Selected Receptors and Transporters.

Chromium(III) is one of the most controversial biometals. Although, it is no longer on the list of minerals necessary for the proper functioning of the human body, and its pharmacological effect is still under discussion. One of the purposes of Cr(III) administration is to use it in patients with mood disorders and it is strictly related to its pharmacological, not dietary effect. This is because its high doses are necessary to obtain the results and additionally, no deficiencies in human population have been noted. In this study, the affinity of chromium(III) to selected receptors and transporters in the rat brain was evaluated, and the effect of the 14-day administration of this metal was assessed on the density of selected receptors. All analyses were performed in vitro using radioligand binding assays, and the results indicated lack of affinity to ß1 and α1 receptors and serotonin transporter (SERT), furthermore very weak affinity to the 5-HT1A receptor (30% inhibition at 10-4 and 10-5 M). Analysis of the α1 and ß1 adrenergic receptor density indicated lack of any adaptive effects after 14 days of Cr(III) administration through intraperitoneal injections (doses 6 and 12 mg/kg). The antidepressant activity of chromium(III) indicated in clinical trials concerned patients with atypical, seasonal, or dystonic symptoms. This effect, as it seems based on the presented results, does not depend on direct affinity to serotonin receptors and transporter nor is the result of adaptive changes in the adrenoreceptor system.

Beta-blockers exert potent anti-tumor effects in cutaneous and uveal melanoma.

BACKGROUND: Melanoma is a life-threatening group of cancers mainly affecting the skin (cutaneous melanoma, CM) and the eyes (uveal melanoma, UM). Nearly half of patients with UM develop liver metastases regardless of the primary treatment. For this reason, adjuvant therapy to prevent disease progression is essential to improve survival of patients with melanoma. Beta-adrenoceptors (ß-AR) have emerged as novel targets to inhibit tumor growth and dissemination in CM, but have not been investigated in UM. METHODS: The aim of this study was to comprehensively evaluate the effects of a non-selective ß-blocker in UM and CM. Propranolol was tested on four UM and two CM cell lines to determine the effects of this beta-blocker. The expression of ß-AR in UM was assessed in enucleated eyes of 36 patients. RESULTS: The results showed that propranolol exerts potent anti-proliferative effects, attenuates migration, reduces VEGF and induces cell cycle arrest and apoptosis in both UM and CM in a dose-dependent manner. Furthermore, levels of cell-free DNA released from the cells correlated to propranolol treatment and may be an indicator of treatment response. Finally, immunohistochemical analysis revealed the expression of ß1 and ß2 adrenoceptors in all UM patients, with higher expression seen in the more aggressive epithelioid versus less aggressive spindle cells. CONCLUSIONS: Collectively our data suggest that a nonselective beta-blocker may be effective against melanoma. For the first time, we show potent anti-tumor effects in UM cells following propranolol administration and expression of ß1 and ß2 adrenoceptors in patient tissue.

Innervation and receptor profiles of the human apocrine (epitrichial) sweat gland: routes for intervention in bromhidrosis.

BACKGROUND: Human apocrine (epitrichial) sweat glands secrete in response to local or systemic administration of catecholamines and cholinergic agonists. As the process of secretion in human apocrine glands is not fully understood and no literature detailing the expression of adrenergic, cholinergic and purinergic receptors is available, there is a need to know the receptor types. Such data could provide new approaches for the treatment of axillary bromhidrosis. OBJECTIVES: To investigate the localization of nerve fibres, adrenergic, cholinergic and purinergic receptors in human axillary apocrine sweat glands by immunohistochemistry. METHODS: Human axillary apocrine sweat glands were investigated by serial sectioning of paraffin wax-embedded skin samples from volunteers. Sections were examined by light microscopy and immunohistochemistry, using antibodies against neurofilament, alpha- and beta-adrenoceptors, P2Y(1), P2Y(2) and P2Y(4) purinoceptors, and M(3) cholinoceptors. RESULTS: Neurofilaments were found near the eccrine but not the apocrine gland. Apocrine glands demonstrated the presence of beta-2 and beta-3 adrenoceptors in the secretory coil of the gland, but not alpha-1, beta-1 or M(3) receptors. Glandular purinergic staining (P2Y(1), P2Y(2) and P2Y(4)) was found in what looked like myoepithelial cells, while P2Y(1) and P2Y(2) staining was found on apical membranes and diffusely throughout secretory cells. Eccrine gland staining acted as internal positive controls. CONCLUSIONS: No nerve fibres were found near the apocrine gland, suggesting that any catecholamine influence is through humoral effects and that glands could be influenced by beta-adrenoceptor subtypes and purinoceptors. Blockage of both these types of receptors offers a route to controlling apocrine secretion from axillary glands and reducing the opportunity for the development of bromhidrosis.

Protective effects of a glucocorticoid on downregulation of pulmonary beta 2-adrenergic receptors in vivo.

We investigated the in vivo effects of a glucocorticoid on beta-agonist-induced downregulation of beta 1- and beta 2-adrenergic receptors (determined by [125I]iodocyanopindolol binding), mRNA expression (assessed by Northern blotting), and gene transcription (using nuclear run-on assays) in rat lung. Dexamethasone (Dex) (0.2 mg/kg/d, days 1-8) increased beta 1- and beta 2-receptor numbers by 70 and 69% above control, respectively, but did not change their mRNA expression. Isoproterenol (Iso) (0.96 mg/kg/d, days 2-8) decreased beta 1- and beta 2-receptor numbers by 48 and 51%, respectively, and also reduced mRNA expression by 69 and 57%, respectively. The combination of Dex and Iso resulted in no net change in beta 2-receptor number and its mRNA expression, although there was a significant reduction in beta 1-receptor number and mRNA expression. The mapping of beta 1- and beta 2-receptors by receptor autoradiography confirmed these findings over alveoli, epithelium, endothelium, and airway and vascular smooth muscle. We also measured the activation of the transcription factor, cyclic AMP response element binding protein (CREB) using an electrophoretic mobility shift assay. CREB-like DNA-binding activity was decreased after Iso treatment but this decrease was prevented after treatment with Dex. Nuclear run-on assays revealed that the transcription rate of the beta 1-receptor gene did not alter after Dex treatment, but was reduced after Iso treatment. The transcription rate of the beta 2-receptor gene was increased after Dex treatment by approximately twofold, but there was no change after Iso treatment. We conclude that glucocorticoids can prevent homologous downregulation of beta 2-receptor number and mRNA expression at the transcriptional level without affecting beta 1-receptors and that the transcription factor CREB may be involved in this phenomenon. Such an effect may have clinical implications for preventing the development of tolerance to beta 2-agonists in asthmatic patients treated with beta-agonist bronchodilators.

Heterodimerization of beta1- and beta2-adrenergic receptor subtypes optimizes beta-adrenergic modulation of cardiac contractility.

Intermolecular interactions between members of both similar and divergent G protein-coupled receptor subfamilies have been shown in various experimental systems. Here, we demonstrate heterodimerization of predominant beta-adrenergic receptor (betaAR) subtypes expressed in the heart, beta1AR, and beta2AR, and its physiological relevance. In intact adult-mouse cardiac myocytes lacking native beta1AR and beta2AR, coexpression of both betaAR subtypes led to receptor heterodimerization, as evidenced by their coimmunoprecipitation, colocalization at optical resolution, and markedly increased binding affinity for subtype-selective ligands. As a result, the dose-response curve of myocyte contraction to betaAR agonist stimulation with isoproterenol (ISO) was shifted leftward by approximately 1.5 orders of magnitude, and the response of cellular cAMP formation to ISO was enhanced concomitantly, indicating that intermolecular interactions of betaAR subtypes resulted in sensitization of these receptors in response to agonist stimulation. In contrast, the presence of beta1AR greatly suppressed ligand-independent spontaneous activity of coexisting beta2ARs. Thus, heterodimerization of beta1AR and beta2AR in intact cardiac myocytes creates a novel population of betaARs with distinct functional and pharmacological properties, resulting in enhanced signaling efficiency in response to agonist stimulation while silencing ligand-independent receptor activation, thereby optimizing beta-adrenergic modulation of cardiac contractility.

Crosstalk of beta-adrenergic receptor subtypes through Gi blunts beta-adrenergic stimulation of L-type Ca2+ channels in canine heart failure.

The mechanisms underlying the blunted contractile response to beta-adrenergic receptor (beta-AR) stimulation in heart failure (HF) are incompletely understood, especially with regard to beta-AR subtype-specific regulation of L-type Ca2+ channels. We evaluated the impact of HF induced by pacing tachycardia on beta-AR regulation of L-type Ca2+ channels in a canine model. To evaluate changes in the relative subcellular distribution of beta-AR subtypes, left ventricular membranes enriched in surface sarcolemma and T-tubular sarcolemma were prepared. Radioligand binding using [(125)I]cyanopindolol revealed that HF resulted in a comparable decrease in the density of beta1-ARs in both surface and T-tubule sarcolemma (55+/-4%, n=7, P<0.001; and 45+/-10%, n=7, P<0.01, respectively), but no significant change in beta2-AR density was observed. Whole-cell patch clamp studies demonstrated a markedly blunted increase in I(Ca,L) in response to saturating concentrations of the nonselective beta-AR agonist isoproterenol (0.1 micromol/L) in failing myocytes compared with control (129+/-20%, n=11, versus 332+/-35%, n=7; P<0.001). Experiments testing beta1-AR- and beta2-AR-selective stimulation showed that the major component of the blunted response to nonselective beta-AR stimulation in HF was caused by beta2-AR activation, resulting in a pertussis toxin-sensitive, Gi-mediated inhibition of the beta1-AR-induced increase in I(Ca,L). In conclusion, canine HF results in the following: (1) a uniform reduction in beta1-AR density in surface and T-tubule membrane fractions without a change in beta2-AR density; and (2) the emergence of distinct Gi-coupling to beta2-ARs resulting in accentuated antagonism of beta1-AR-mediated stimulation of I(Ca,L). These results have implications for optimizing the use of beta-AR drugs in HF.

Functional beta-adrenergic receptor signalling on nuclear membranes in adult rat and mouse ventricular cardiomyocytes.

OBJECTIVE: We sought to determine if different beta-adrenergic receptor (betaAR) subtypes, and their associated signalling machinery, are functionally localized to nuclear membranes. METHODS: Employing enriched nuclear preparations, we assayed the specific presence of betaAR by measuring 125I-cyanopindolol (CYP) binding, Western blotting, confocal microscopy and functional assays. RESULTS: Western blots of rat heart nuclear fractions and confocal immunofluorescent analysis of adult rat and mouse ventricular cardiomyocytes displayed the presence of beta 1AR and beta 3AR but, surprisingly, not the beta 2AR on nuclear membranes. Nuclear localization of downstream signalling partners Gs, Gi and adenylyl cyclases II and V/VI was also demonstrated. The functional relevance of nuclear betaAR was shown by receptor-mediated stimulation of adenylyl cyclase activity by isoproterenol but not the beta 3AR-selective agonist CL 316243 in enriched nuclear preparations. We also examined the effect of subtype-selective ligands on the initiation of RNA synthesis in isolated nuclei. Both isoproterenol and another beta 3AR-selective agonist, BRL 37344, increased RNA synthesis which was inhibited by pertussis toxin (PTX). Neither a beta 1AR-selective agonist, xamoterol, nor a beta 2AR-selective agonist, procaterol, was able to stimulate transcription. However, both CGP 20712A and ICI 118,551 blocked isoproterenol-mediated effects to varying extents. PTX treatment also revealed that nuclear betaAR may be coupled to other signalling pathways in addition to Gi, as stimulation under these conditions reduced initiation of transcription below basal levels. CONCLUSION: These results highlight differential subcellular localization for betaAR subtypes and indicate that betaAR may have specific roles in regulating nuclear function in cardiomyocytes.

Beta(2)-adrenergic and several other G protein-coupled receptors in human atrial membranes activate both G(s) and G(i).

Cardiac G protein-coupled receptors that couple to Galpha(s) and stimulate cAMP formation (eg, beta-adrenergic, histamine, serotonin, and glucagon receptors) play a key role in cardiac inotropy. Recent studies in rodent cardiac myocytes and transfected cells have revealed that one of these receptors, the beta(2)-adrenergic receptor (AR), also couples to the inhibitory G protein Galpha(i) (activation of which inhibits cAMP formation). If beta(2)ARs could be shown to couple to Galpha(i) in the human heart, it would have important ramifications, because levels of Galpha(i) increase with age and in failing human heart. Therefore, we investigated whether beta(2)ARs in the human heart activate Galpha(i). By photoaffinity labeling human atrial membranes with [(32)P]azidoanilido-GTP, followed by immunoprecipitation with antibodies specific for Galpha(i), we found that Galpha(i) is activated by stimulation of beta(2)ARs but not of beta(1)ARs. In addition, we found that other Galpha(s)-coupled receptors also couple to Galpha(i), including histamine, serotonin, and glucagon. When coupling of these receptors to Galpha(i) is disrupted by pertussis toxin, their ability to stimulate adenylyl cyclase is enhanced. These data provide the first evidence that beta(2)AR and many other Galpha(s)-coupled receptors in human atrium also couple to Galpha(i) and that abolishing the coupling of these receptors to Galpha(i) increases the receptor-mediated adenylyl cyclase activity.

Pharmacological evaluation of plasma membrane beta-adrenoceptors in rat hearts using the tissue segment binding method.

This study evaluates beta-adrenoceptors in rat atria and ventricle using the tissue segment binding method and compares the results with those obtained using conventional homogenate binding assays. In studies with tissue segment binding, the hydrophilic radioligand [(3)H]-CGP12177 selectively bound to plasma membrane beta-adrenoceptors, and the B(max) levels were significantly higher than those obtained with homogenate binding. However, both binding approaches revealed similar proportions of beta(1)- and beta(2)-adrenoceptors. The regional distribution of plasma membrane beta(1)- and beta(2)-adrenoceptors in rat hearts were also determined using tissue segment binding. Abundance of beta-adrenoceptors and proportion of beta(1)-adrenoceptors were higher in atria than in ventricle, but there was no significant difference between right and left atria or within ventricle (right and left ventricle free walls, apex, and interventricular septum). To establish the ability of the tissue segment binding method to study beta-adrenoceptor regulation such as the internalization of receptors, the effect of prolonged exposure of rat ventricle to (-)-isoprenaline was also investigated by using tissue segments and homogenate binding. Incubation with (-)-isoprenaline for 1 h in vitro caused a concentration-dependent decrease in the density of beta-adrenoceptors, predominantly beta(2)-adrenoceptors, when assessed with tissue segment binding method. In contrast, the subtype-specific change after treatment with (-)-isoprenaline was not detected using homogenate binding. In summary, the tissue segment binding method with [(3)H]-CGP12177 enables a more precise quantitation of plasma membrane beta(1)- and beta(2)-adrenoceptors in rat hearts and is suitable for studying their regulation.

High throughput bioassay for beta1-adrenoceptor autoantibody detection.

BACKGROUND: While the involvement of adrenergic beta1-autoantibodies (beta1-AABs) in pathogenesis of cardiomyopathies is well established as are the benefits associated with autoantibody removal by immunoapheresis, the development of drugs neutralizing beta1-AABs in-vivo has been slowed due to a lack of high throughput autoantibody analytics. Highly scalable routine diagnostics involving immobilized binding partners have mostly failed in comparison to the laborious bioassays, which are difficult to scale up, but present the most reliable and sensitive tools for detecting the beta1-autoantibodies. METHODS: A high throughput, image-based assay to measure cardiomyocyte beat rate and contractility was developed and tested for its applicability for detecting adrenergic beta1-autoantibodies. The classical bioassay of spontaneously beating neonatal rat cardiomyocytes was used for comparison. RESULTS: The high throughout assay using human iPSC-derived cardiomyocytes was able to detect beta1-AAB activity of biological sample material. The results from the high throughput assay were very similar to the data obtained from the original bioassay of spontaneously beating neonatal cardiomyocytes, with one exception, where a control antibody targeting the N-terminal end of the human beta1-receptor induced a response when tested with the high throughput imager, while none was observed by the classical bioassay. This discrepancy may be explained by the differences in host species of cardiomyocytes tested by the two methods. CONCLUSION: The high throughput system using iPSC-derived cardiomyocytes for the detection of beta1-AAB provides a realistic option to overcome the sample-size limitations of the bioassay-based diagnostics.

Enhanced G(i) signaling selectively negates beta2-adrenergic receptor (AR)--but not beta1-AR-mediated positive inotropic effect in myocytes from failing rat hearts.

BACKGROUND: Myocardial contractile response to beta1- and beta2-adrenergic receptor (AR) stimulation is severely impaired in chronic heart failure, in which G(i) signaling and the ratio of beta2/beta1 are often increased. Because beta2-AR but not beta1-AR couples to G(s) and G(i) with the G(i) coupling negating the G(s)-mediated contractile response, we determined whether the heart failure-associated augmentation of G(i) signaling contributes differentially to the defects of these beta-AR subtypes and, if so, whether inhibition of G(i) or selective activation of beta2-AR/G(s) by ligands restores beta2-AR contractile response in the failing heart. METHODS AND RESULTS: Cardiomyocytes were isolated from 18- to 24-month-old failing spontaneously hypertensive (SHR) or age-matched Wistar-Kyoto (WKY) rat hearts. In SHR cardiomyocytes, either beta-AR subtype-mediated inotropic effect was markedly diminished, whereas G(i) proteins and the beta2/beta1 ratio were increased. Disruption of G(i) signaling by pertussis toxin (PTX) enabled beta2- but not beta1-AR to induce a full positive inotropic response in SHR myocytes. Furthermore, screening of a panel of beta2-AR ligands revealed that the contractile response mediated by most beta2-AR agonists, including zinterol, salbutamol, and procaterol, was potentiated by PTX, indicating concurrent G(s) and G(i) activation. In contrast, fenoterol, another beta2-AR agonist, induced a full positive inotropic effect in SHR myocytes even in the absence of PTX. CONCLUSIONS: We conclude that enhanced G(i) signaling is selectively involved in the dysfunction of beta2- but not beta1-AR in failing SHR hearts and that disruption of G(i) signaling by PTX or selective activation of beta2-AR/G(s) signaling by fenoterol restores the blunted beta2-AR contractile response in the failing heart.

Involvement of the serotonin 5-HT2B receptor in cardiac hypertrophy linked to sympathetic stimulation: control of interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha cytokine production by ventricular fibroblasts.

BACKGROUND: The serotonergic 5-HT2B receptor regulates cardiomyocyte development and growth. A putative contribution of this receptor to fibroblast-dependent cardiac function has not been identified. METHODS AND RESULTS: By mimicking sympathetic stimulation with chronic isoproterenol perfusion in vivo, we found that mice developed a cardiac hypertrophy, which was prevented by exposure to the 5-HT2B receptor antagonists SB206553 or SB215505 or in 5-HT2B receptor-knockout mice. The isoproterenol-induced hypertrophy was associated with an increase in the plasma levels of interleukin-1beta and tumor necrosis factor-alpha but not interleukin-6. In contrast, the plasma isoproterenol-induced cytokine increase was not observed in either 5-HT2B receptor-mutant or wild-type mice perfused with isoproterenol+SB206553. We demonstrated that stimulation of wild-type cardiac fibroblasts by isoproterenol markedly increased the production of the interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha cytokines. Strikingly, we found that this isoproterenol-induced cytokine production was abolished by SB206553 or in 5-HT2B receptor-knockout fibroblasts. Serotonin also stimulated production of the 3 cytokines in wild-type fibroblasts, which was effectively reduced in 5-HT2B receptor-knockout fibroblasts. CONCLUSIONS: Our results demonstrate for the first time that 5-HT2B receptors are essential for isoproterenol-induced cardiac hypertrophy, which involves the regulation of interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha cytokine production by cardiac fibroblasts.

Up-regulation of beta 2-adrenergic receptors in previously transplanted, denervated nonfailing human hearts.

OBJECTIVES: The purpose of this study was to examine beta-adrenergic receptor signal transduction in denervated, previously transplanted human ventricular myocardium. BACKGROUND: In model systems, surgical denervation typically results in both presynaptic and postsynaptic supersensitivity in beta-adrenergic receptor pathways and alteration in G protein-mediated signal transduction. METHODS: We examined beta-adrenergic receptor signal transduction in the left and right ventricles removed from nine subjects with a previous transplant and surgical denervation 25 +/- 4 months after their first transplantation. Twenty-six hearts removed from organ donors served as control hearts. RESULTS: Total beta-adrenergic receptor density and stimulation of muscle contraction in isolated right ventricular trabeculae by the nonselective agonist isoproterenol were similar in the transplant and donor groups. Beta 1-receptor density was not different in the left ventricles of the two groups but tended to be reduced (by 29%, p = 0.09) in transplant right ventricles. By contrast, beta 2-receptor density was higher in transplant left and right ventricles relative to the respective values in donor ventricles by 33% in left ventricles and 97% in right ventricles (both p < 0.05). Isoproterenol, which in particulate fractions of human heart stimulates adenylyl cyclase primarily via beta 2-receptors, produced a greater increase in cyclic adenosine monophosphate generation in membranes prepared from transplant left ventricles and right ventricles compared with donors. In contrast, guanosine 5'-[beta,gamma-imido]triphosphate, sodium fluoride and forskolin, which stimulate adenylyl cyclase through nonreceptor/G protein-sensitive mechanisms, yielded similar degrees of adenylyl cyclase stimulation in the two groups, and both pertussis toxin- and cholera toxin-catalyzed adenosine diphosphate ribosylation were not altered in transplanted left ventricles. CONCLUSIONS: These data indicate that the transplanted human heart exhibits an up-regulation of functional beta 2-adrenergic receptors.

Role of beta2-adrenoceptors (beta-AR), but not beta1-, beta3-AR and endothelial nitric oxide, in beta-AR-mediated relaxation of rat intrapulmonary artery.

The aim of this study was to analyze beta-adrenoceptor (beta-AR)-mediated relaxation in rat intralobar pulmonary artery. The relaxant responses of beta-AR agonists were characterized using beta-AR antagonists in prostaglandin F2alpha (PGF2alpha)-precontracted arteries. The role of nitric oxide (NO) and endothelium in beta-AR-mediated relaxation was also investigated. Isoprenaline (a non-selective beta-AR agonist) and salbutamol (a selective beta2-AR agonist) induced vasorelaxation. ICI 118551 (a selective beta2-AR antagonist) antagonized the effect of both isoprenaline and salbutamol (pA2 values of 9.57 and 9.51 respectively). In contrast, atenolol (1 microM) and CGP 20712A (0.1 microM), two beta1-AR antagonists, did not modify the relaxing effect of isoprenaline. The response to isoprenaline obtained in the presence of nadolol (10 microM, a beta1/beta2-AR antagonist) was not further inhibited by SR 59230A (1 microM, a selective beta3-AR antagonist). The non-beta1/beta2-AR agonists studied (BRL 37344, SR 58611A, and CGP 12177A) did not elicit vasorelaxation. Relaxation to isoprenaline and salbutamol was unaffected by L-N(G)-nitro-arginine methyl ester (100 microM, an inhibitor of NO synthase) or after endothelium removal. These results demonstrate the role of beta2-AR in mediating relaxation in rat intralobar pulmonary artery precontracted with PGF2alpha. They indicate that beta2-AR-mediated relaxation in this artery is NO- and endothelium-independent. Furthermore, they do not provide evidence of a relaxant role of either beta1- or beta3-AR in PGF2alpha-precontracted rat intrapulmonary artery.

New beta-blocker: prolonged reduction in high blood pressure with beta(1) antisense oligodeoxynucleotides.

beta-Blockers are widely used for hypertension treatment but must be taken daily. We have developed a novel beta-blocker by targeting beta(1)-adrenergic receptor (beta(1)-AR) mRNA with antisense oligodeoxynucleotides (beta(1)-AS-ODN). A single intravenous injection of beta(1)-AS-ODN significantly reduced cardiac contractility and blood pressure (38+/-5 mm Hg, P<0.05) in spontaneously hypertensive rats for 3 weeks. In the present study, we improved the antihypertensive effect of beta(1)-AS-ODN by delivery with the cationic liposomes DOTAP/DOPE and studied its impact on the peripheral renin-angiotensin system. Five charge ratios (+/-) of liposome/ODN from 0 to 3.5 were tested to deliver 0. 5 mg/kg beta(1)-AS-ODN intravenously in spontaneously hypertensive rats (n=30). On the basis of the magnitude and duration of hypotension, 2.5 was determined to be the optimal charge ratio, which decreased blood pressure by up to 35 mm Hg for 20 to 33 days (P<0.05). The effects were specific for beta(1)-AR, because radioligand binding assay and quantitative autoradiography showed a 35% reduction in beta(1)-AR levels in kidney but no change in beta(2)-AR. beta(1)-AS-ODN diminished the preprorenin mRNA levels in renal cortex by 37% 4 days after administration. This transient effect was followed by a delayed yet marked diminution of plasma renin activity and plasma angiotensin II levels on days 10 and 17 (P<0.01). The results show that beta(1)-AS-ODN has an effective long-term antihypertensive effect up to 33 days with a single intravenous injection. The mechanism appears to be through reduced beta(1)-AR number specifically and reduced cardiac contractility. The inhibition of the renin-angiotensin system is probably a second mechanism to produce the sustained antihypertensive effect of beta(1)-AS-ODN.

Membrane biochips.

Membrane-bound proteins represent the single most important class of drug targets. This article discusses the issues surrounding fabrication of membrane-protein microarrays by conventional robotic pin printing techniques. Ligand binding selectivity and specificity to G protein-coupled receptor (GPCR) microarrays are presented. The potential applications of these arrays for drug screening are discussed.

High throughput fluorescence polarization: a homogeneous alternative to radioligand binding for cell surface receptors.

High throughput fluorescence polarization (FP) assays are described that offer a nonradioactive, homogeneous, and low-cost alternative to radioligand binding assays for cell surface receptors (G protein-coupled receptors and ligand-gated ion channels). FP assays were shown to work across a range of both peptide (vasopressin V1a and delta-opioid) and nonpeptide (beta1-adrenoceptor, 5-hydroxytryptamine3) receptors. Structure-activity relationships were investigated at beta1-receptors and were found to be consistent with radioligand binding assays. FP was shown to tolerate up to 5% DMSO with no loss in sensitivity or signal window. From a random set of 1,280 compounds, 1.9% were found to significantly interfere with FP measurement. If fluorescent or quenching compounds were eliminated (3% of all compounds), less than 0.4% of compounds were found to interfere with FP measurement. Assays could be run in 384-well plates with little loss of signal window or sensitivity compared to 96-well plate assays. New advances in FP measurement have therefore enabled FP to offer a high throughput alternative to radioligand binding for cell surface receptors.