Debra A. Schwinn

Identification, quantification, and localization of mRNA for three distinct alpha 1 adrenergic receptor subtypes in human prostate.

The dynamic component of bladder outlet obstruction caused by benign prostatic hyperplasia (BPH) is regulated by alpha 1 adrenergic receptors (alpha 1-AR) located in the prostatic stroma. Recently two alpha 1-AR subtypes (alpha 1A, alpha 1B) have been identified in the human prostate by both functional and pharmacological assays. However, the presence of the alpha 1C subtype has not been evaluated, presumably due to the lack of availability of selective ligands for this receptor subtype. We have used molecular techniques to investigate the mRNA expression of all three alpha 1-AR subtypes in the human prostate. RNA extracted from the prostate gland of 15 patients was used in ribonuclease protection assays to identify the expression of three alpha 1-AR subtype mRNAs. Quantitative solution hybridization assays further identify the predominant subtype of alpha 1-AR mRNA to be the alpha 1C, which represents approximately 70% of the total alpha 1-AR mRNA in the human prostate. Furthermore, in situ hybridization localizes the alpha 1C AR mRNA predominantly to the stromal compartment. The identification of a predominant alpha 1-AR mRNA in human prostate identifies a potential need for subtype selective pharmaceutical agents. These agents could be very important clinically in the treatment of diseases such as BPH.

Subtype Specific Regulation of Human Vascular α1-Adrenergic Receptors by Vessel Bed and Age

Background—α1-adrenergic receptors (α1ARs) regulate blood pressure, regional vascular resistance, and venous capacitance; the exact subtype (α1a, α1b, α1 d) mediating these effects is unknown and varies with species studied. In order to understand mechanisms underlying cardiovascular responses to acute stress and chronic catecholamine exposure (as seen with aging), we tested two hypotheses: (1) human α1AR subtype expression differs with vascular bed, and (2) age influences human vascular α1AR subtype expression. Methods and Results—Five hundred vessels from 384 patients were examined for α1AR subtype distribution at mRNA and protein levels (RNase protection assays, ligand binding, contraction assays). Overall vessel α1AR density is 16±2.3fmol/mg total protein. α1aAR predominates in arteries at mRNA (P<0.001) and protein (P<0.05) levels; all 3 subtypes are present in veins. Furthermore, α1AR mRNA subtype expression varies with vessel bed (α1a higher in splanchnic versus central arteries, P<0.05); competiti...

alpha1-ADRENERGIC RECEPTOR SUBTYPES IN HUMAN DETRUSOR

PURPOSE To identify and quantitate alpha1-adrenergic receptor (alpha1AR) subtype expression in human detrusor. MATERIALS AND METHODS Initial studies to determine alpha1AR expression in human detrusor were performed using saturation binding with [125I]HEAT. Once the presence of alpha1ARs was documented, subtype (alpha1a, alpha1b, alpha1d) expression at the mRNA level (and comparison with rat) was determined with RNase protection assays (human detrusor) and RT-PCR (human detrusor, rat whole bladder). Competition binding analysis with the alpha1dAR-selective ligand BMY7378 was used to measure alpha1AR subtype expression at a protein level in human detrusor. RESULTS Alpha1AR expression in human detrusor was low but reproducible (6.3 +/- 1.0 fmol./mg. total protein). RNase protection assays performed on total RNA extracted from human detrusor revealed the following alpha1AR subtype expression: alpha1d (66%) > alpha1a (34%), and no alpha1b. RT-PCR confirmed alpha1AR subtype mRNA distribution in human detrusor with alpha1d (approximately 60-70%) > alpha1a (approximately 30-40%), and a lack of alpha1b mRNA. Rat whole bladder expressed different alpha1AR subtype mRNA than human detrusor, with alpha1a approximately alpha1b approximately alpha1d. The presence of alpha1d > alpha1a expression in human detrusor was confirmed at a protein level by competition analysis utilizing BMY7378 which revealed a two-site fit, with Ki and high affinity binding (66%) consistent with the alpha1dAR subtype. CONCLUSIONS Human detrusor contained two alpha1AR subtypes (alpha1d > alpha1a), a finding that is different from rat, another commonly used animal model. Since non-subtype selective alpha1AR antagonists ameliorate irritative bladder symptoms (in men and women with/without outlet obstruction), and Rec 15/2739 (alpha1a selective antagonist) does not improve symptom scores in BPH, our findings suggest bladder alpha1dARs may provide a potentially novel mechanism underlying these therapeutic benefits.

α2-Adrenergic receptors in human spinal cord : specific localized expression of mRNA encoding α2-adrenergic receptor subtypes at four distinct levels

Abstract α2-Adrenergic receptor (AR) subtype mRNA (α2a, α2b, α2c) neuronal localization in human spinal cord has not been described. We therefore performed in situ hybridization to identify cell bodies at four levels of human spinal cord (cervical, thoracic, lumbar, sacral) containing α2AR subtype specific mRNA. α2AR mRNA is present in gray matter only (ventral > dorsal; sacral > cervical > thoracic = lumbar). In addition to α2AR mRNA in cell bodies in thoracic and lumbar intermediolateral (sympathetic) and sacral intermediate (parasympathetic) cell columns (lamina VII), all levels in dorsal horn laminae I, II, V, and ventral horn lamina IX, we demonstrate α2AR mRNA in dorsal horn laminae III and IV, and dorsal nucleus of Clarke, where α2ARs have not been described. Previously unreported heterogeneity in α2AR subtype distribution (α2a and α2bAR mRNA present, α2cAR mRNA virtually absent) is found at all sites of α2AR mRNA expression in human spinal cord, including locations known to mediate effects of α2AR agonist drugs on nociception, autonomic function and motor tone. Cervical spinal cord demonstrates a predominance of α2a mRNA signal, while thoracic, lumbar, and sacral spinal cord demonstrate an increasing predominance of α2bAR mRNA. If confirmed at a protein level, these findings have profound implications for therapeutic strategies in managing human pain.

α1-Adrenergic receptor regulation: basic science and clinical implications

Adrenergic receptors (ARs) are members of the G-protein-coupled receptor family, which includes alpha 1ARs, alpha 2ARs, beta 1ARs, beta 2ARs, beta 3ARs, adenosine, muscarinic, angiotensin, endothelin receptors, and many others that are responsible for a large variety of physiologic effects through G-protein coupling. This review focuses on alpha 1ARs and their regulation at both the mRNA and protein levels. Currently, three alpha 1AR subtypes have been characterized both pharmacologically and at the gene level: alpha 1aAR, alpha 1bAR, and alpha 1dAR. These are expressed in a species- and tissue-dependent manner. Mutagenesis approaches have been extremely valuable in the identification of key residues that govern alpha 1AR ligand binding and signaling. These studies reveal that alpha 1ARs have evolved an exquisitely sensitive regulation of their activity in which any disruption of the native structure has profound effects on subsequent function and effector coupling. Significant advances have also been made in the elucidation of signaling pathway components, resulting in the identification of novel pathways that can lead to pathologic conditions. Specific topics include mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and G-protein-coupled receptor cross-talk pathways. Within this context, recent studies identifying underlying transcriptional mechanisms involved in the regulation of the alpha 1AR subtypes are also discussed. Finally, given the potentially important role of alpha 1ARs in the vasculature, as well as in the pathology of many diseases, such as myocardial hypertrophy and benign prostatic hyperplasia, the clinical relevance of alpha 1AR distribution, pharmacology, and therapeutic intervention is reviewed.

Modulation of bladder α1-adrenergic receptor subtype expression by bladder outlet obstruction

Purpose: α1-Adrenergic receptor (α1AR) antagonists are effective for relieving obstructive and irritative symptoms in patients with bladder outlet obstruction. While the α1aAR is responsible for prostate smooth muscle relaxation and outlet obstruction relief, to our knowledge the mechanisms underlying the relief of irritative symptoms remain to be determined. Therefore, we investigated mechanisms by which bladder α1AR subtypes may be involved in this process.Materials and Methods: We studied 42 rats, including 6 unoperated controls, 17 sham operated controls and 19 obstructed animals. Animals were characterized for baseline voiding pattern, followed by surgical intervention or sham surgery to establish obstruction (1.09 mm. restricted opening). After 6 weeks to enable the development of detrusor hypertrophy, voiding behavior was reexamined, the animals were sacrificed and bladder tissue was immediately placed in liquid nitrogen. α1AR subtype messenger (m)RNA was quantitated using quantitative competitive ...

β2-Adrenergic and Several Other G Protein–Coupled Receptors in Human Atrial Membranes Activate Both Gs and Gi

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

Classification of α1-adrenoceptor subtypes

Two α1-adrenoceptor subtypes (α1A and α1B) have been detected in various tissues by pharmacological techniques, and three distinct cDNAs encoding α1-adrenoceptor subtypes have been cloned. The profile of an increasing number of subtype-selective compounds at cloned and endogenous receptors recently has facilitated alignment between cloned and pharmacologically defined α1-adrenoceptor subtypes. Thus, α1a-adrenoceptors (previously designated α1c), α1b-adrenoceptors and α1d-adrenoceptors (previously designated α1a, α1d or α1a/d) are now recognized. Since the α1d-adrenoceptor shares characteristics with both α1A-and α1B-adrenoceptors, tissues previously reported to express α1A- and/or α1B-adrenoceptors may additionally contain α1d-adrenoceptors. This article reviews the features of all three subtypes and discusses possible pitfalls in their pharmacological identification.

Distribution of β3-adrenoceptor mRNA in human tissues

The beta 3-adrenoceptor is a G protein-coupled receptor which mediates metabolic functions of the endogenous catecholamines epinephrine and norepinephrine. Questions exist regarding distribution of the beta 3-adrenoceptor in human tissue. In order to examine the distribution of beta 3-adrenoceptor mRNA in human tissues, we used sensitive and specific RNase protection assays without previous PCR amplification in an extensive list of human tissues. We confirm the presence of beta 3-adrenoceptor mRNA in human white fat from several locations, gall bladder, and small intestine, as well as extend the distribution of beta 3-adrenoceptor mRNA to previously uncharacterized human tissues such as stomach and prostate. The presence of beta 3-adrenoceptor mRNA in human white adipose tissue has important implications regarding possible use of beta 3-adrenoceptor selective agonists as anti-obesity agents, and the demonstration of beta 3-adrenoceptor mRNA in a number of gastrointestinal tissues and prostate raises the question of the role of the beta 3-adrenoceptor in motility and secretory processes.

α1-Adrenoceptor subtypes and lower urinary tract symptoms

Abstract:  Benign prostatic hyperplasia (BPH) is a common cause of urinary outflow obstruction in aging men leading to lower urinary tract symptoms (LUTS). α1‐Adrenoceptors (α1ARs) antagonists (blockers) have become a mainstay of LUTS treatment because they relax prostate smooth muscle and decrease urethral resistance, as well as relieving bladder LUTS symptoms. A review of key recent clinical trials suggests new insights into the role of specific α1AR subtypes in the treatment of LUTS.