Stella O. Page

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...

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.

Desensitization of myocardial beta-adrenergic receptors during cardiopulmonary bypass. Evidence for early uncoupling and late downregulation.

BackgroundCardiopulmonary bypass (CPB), a process routinely used during cardiac surgery, is a potent stimulant to the release of endogenous catecholamines. Hence, we tested the hypothesis that CPB results in myocardial β-adrenergic receptor (βAR) desensitization. Methods and ResultsWe obtained canine transmyocardial left ventricular biopsies before, during (155 minutes), and after CPB (pre-CPB, CPB, and post-CPB, respectively) and determined βAR density, proportion of β1AR to β2AR, and βAR coupling capacity to adenylyl cyclase. βAR density was stable at 112 ± 14 fmol/mg (pre-CPB) and 103 ± 9 fmol/mg (CPB) but decreased post-CPB to 84 % 7 fmol/mg. The ratio of β1AR to β2AR (determined by two-site fit for [1251]-iodocyanopindolol competition binding with the fI1AR selective antagonist 1C189.406) remained constant throughout (60 % 3:40 ± 3 pre-CPB, 55 ± 3:44 ± 3 CPB, and 61 ± 2:39 ± 2 post-CPB), revealing that both P1AR and β2AR subtypes were downregulated. A different pattern was noted in the functional properties of these receptors during CPB. Decreased maximal isoproterenol-stimulated adenylyl cyclase activity (252 ± 14 to 216 ± 12 pmol/30 min/mg), submaximal isoproterenol-stimulated adenylyl cyclase activity (183 ± 10 to 157 ± 11 pmol/30 min/mg), and zinterol-stimulated adenylyl cyclase activity (187 ± 11 to 159 ± 11 pmol/30 min/mg, a measure of βAR subtype activation) were noted during CPB, at the time when weaning from CPB takes place. However, this desensitized pattern was found to be completely reversed by 30 minutes post-CPB, with adenylyl cyclase activities returning to pre-CPB levels or slightly higher. Control dogs that did not receive CPB showed no change in βAR density or adenylyl cyclase activity. ConclusionsThese data suggest that myocardial βAR desensitization does occur during CPB in healthy, nonischemic canine myocardium and that this pattern is reversed 30 minutes after discontinuation of CPB. In addition, a slower process of βAR downregulation persists after discontinuation of CPB. Because successful weaning from CPB is a critical process during myocardial surgery, these findings have potentially important implications in the management of such patients.

Pharmacology of tamsulosin: Saturation-binding isotherms and competition analysis using cloned α1-adrenergic receptor subtypes

α1‐adrenergic receptors (α1 ARs) are important in the dynamic component of benign prostatic hyperplasia (BPH). Currently, several α1AR antagonists are being used in the treatment of BPH.

Localization of Messenger RNA for Three Distinct α2-Adrenergic Receptor Subtypes in Human Tissues: Evidence for Species Heterogeneity and Implications for Human Pharmacology

Backgroundα2-Adrenergic receptor (α2AR) agonists have become Important adjuncts as anesthetic agents. They act by binding to α2ARs on the surface of cell membranes and cause centrally mediated sedation and analgesia. α2ARs also contribute to other aspects of physiologic regulation. Three subtypes of α2ARs (α2-c2, α2-c4, and α2-c10) have been described using molecular and pharmacologic techniques. We recently demonstrated species heterogeneity in the distribution of α1-adrenergic receptor subtypes, therefore making it imperative to analyze the distribution of α2AR subtypes in human tissues. This information may have importance in the understanding of potential side effects of administration of α2AR subtype-selective agonists for anesthesia In humans. MethodsRNA extracted from human tissues was analyzed by using quantitative ribonuclease protection assays to determine α2AR subtype messenger RNA (mRNA) expression in cardiovascular, central nervous system, and peripheral tissues. Resultsα2AR mRNA is present in greatest concentrations in human kidney, followed by aorta > spleen > heart = lung. α2-c4 mRNA predominates in heart, lung, aorta, cerebral cortex, cerebellum, spleen, kidney, and adrenal gland; α2-c2 mRNA in liver; and α2-c10 mRNA in pancreas and small intestine. Hence α2AR subtype mRNA distribution is tissue-selective and differs from that reported for rat. Conclusions(1) On comparison with previous research we find possible species heterogeneity in α2AR subtype mRNA distribution (rat vs. human) for all three α2AR subtypes. (2) We demonstrate the presence and subtype heterogeneity of α2AR subtype mRNA in both brain and peripheral tissues. (3) Significant concentrations of α2AR mRNA are present in adult human heart. These findings have Important Implications for our understanding of human adrenergic physiology, provide a possible explanation for the existence of pharmacologically similar yet distinct α2AR subtypes, and may be important for the rational development of α2AR subtype-selective anesthetics and other therapeutic agents for use in treating human diseases.

Effects of androgen deprivation on prostate alpha1-adrenergic receptors*

OBJECTIVES Benign prostatic hyperplasia (BPH), the most common benign tumor in men, consists of two components-static (enlargement regulated by androgens) and dynamic (smooth muscle contraction through alpha 1-adrenergic receptors [alpha 1-ARs]). Because medical therapy of BPH involves tissue androgen deprivation, we studied the influence of androgen deprivation and replacement on regulation of rat ventral prostate alpha 1-ARs. METHODS Prostate weight, alpha 1-AR density, autoradiographic images, histologic features, and cell-specific protein were examined before and after castration and androgen replacement. RESULTS Castration decreases ventral prostate wet weight, a process reversed by testosterone administration. In contrast, there is an apparent increase in alpha 1-AR density (29 +/- 4 versus 65 +/- 6 fmol/mg total protein, mean +/- SEM) after castration, returning to baseline with testosterone replacement; alpha 1-AR density remains constant in control liver membranes. Alpha 1-ARs predominate in stroma throughout androgen deprivation therapy. Epithelially derived cells decrease (83% to 67%) after castration, resulting in a relative doubling in stroma (17% to 33%); the protein content of epithelial and stromal cells remains identical. Therefore, prostate-specific increases in alpha 1-ARs appear to result from relative increases in the ratio of smooth muscle to epithelium after castration rather than from direct upregulation of alpha 1-AR protein. CONCLUSIONS Because alpha 1-AR density does not decrease with androgen deprivation, these studies suggest that alpha 1-AR antagonists remain an important component in BPH therapy, even when 5-alpha-reductase inhibitors are utilized.

In situ hybridization: identification of rare mRNAs in human tissues

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non-selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32P or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

Partial Purification of Human Lymphocyte Activating Factor

Lymphocyte Activating Factor (LAF) is a T lymphocyte stimulant released by human monocytes cultured for 18-24 hours in tissue culture medium containing 5% human serum and the non-specific immunostimulant lipopolysaccharide. The purification of LAF is essentially the separation of low MW LAF (approximately 13,000) from the human serum proteins required for production of the activity. Hollow fiber ultrafiltration has been found to effect a rapid separation of low MW LAF from serum proteins, but with a yield of only 20% of the original activity. Isoelectric focusing (IEF) efficiently separates LAF from all traces of human serum, resulting in a purified sample containing no measurable protein and revealing no bands on polyacrylamide gels. The IEF purified material is about 2% of the low MW activity present in the unfractionated culture medium and is highly active in the biological assay system.

A Critical Period for the Role of Thyroid Hormone in Development of Renal|[alpha]|-Adrenergic Receptors

Adrenergic input influences renal cell replication/differentiation and the development of excretory function. Kidney cells make adrenoceptors before the arrival of the majority of nerve terminals, and the current study examines whether thyroid hormone plays a role in receptor development. Propylthiouracil(PTU) was given to pregnant and neonatal rats from gestational d 17 through postnatal d 5, a treatment that obtunds thyroid hormone levels throughout the first 2-3 wk postpartum. The PTU group showed significant deficits in the number of α1-receptors, and values resolved to normal in parallel with hormone level recovery. The effects were not secondary to alterations in cell differentiation or growth, as the period of receptor abnormalities did not correspond to that of growth inhibition. Similarly, the effects were selective for the α1-receptor, as no comparable effects were seen for total membrane protein or for α2-receptors. The role of thyroid hormone in α1-receptor ontogeny involved a critical developmental window; later in development neither treatment with PTU nor with large doses of thyroid hormone had any impact on α1-receptors. Studies of mRNAs encoding the α1-receptor subtypes indicated that hypothyroidism targets the α1a-subtype, which has been implicated in the transduction of neurotrophic signals; α1a-receptor mRNA also showed the largest proportional developmental increase compared with those encoding other α1-subtypes. Accordingly, thyroid hormone is likely to set the stage for the subsequent trophic control of renal development by neural input, and hypothyroidism during this critical window can be expected to result in abnormal renal functional development and increased perinatal risk.