Alpha1-adrenergic receptors in human spinal cord: specific localized expression of mRNA encoding alpha1-adrenergic receptor subtypes at four distinct levels.

alpha1-Adrenergic receptors (alpha1ARs) are important in lower urinary tract syndromes such as benign prostatic hypertrophy and bladder irritability. Spinal cord alpha1ARs have been postulated to play a role in modulating these diseases, yet alpha1AR subtype (alpha1a, alpha1b, alpha1d) neuronal localization in human spinal cord has not been described. We therefore tested the hypothesis that alpha1AR subtype distribution varies according to specific spinal cord tract and level. In situ hybridization was performed to identify cell bodies containing alpha1AR subtype mRNA at four levels of human spinal cord (cervical enlargement, thoracic, lumbar, sacral). alpha1AR mRNA is present in ventral gray matter only (ventral>dorsal; sacral>lumbar=thoracic>cervical). Signaling cell bodies were detected in anterior horn motor neurons at all levels; dorsal nucleus of Clarke and intermediolateral columns in cervical enlargement, thoracic and lumbar spinal cord regions; and parasympathetic nucleus in sacral spinal cord. Although all three alpha1AR subtypes are present throughout human spinal cord, alpha1d mRNA predominates overall. If confirmed at a protein level, these findings may contribute to the development of new therapeutic strategies in the treatment of several human diseases.

Cardiopulmonary bypass and circulatory arrest increase endothelin-1 production and receptor expression in the lung.

BACKGROUND: Endothelin-1 has been shown to be a mediator of pulmonary hypertension after cardiopulmonary bypass and deep hypothermic circulatory arrest. It is not known whether the mechanism is increased production of endothelin-1 or alterations in expression of endothelin-1 receptors in the lung. This study was designed to test the hypothesis that circulatory arrest increases endothelin-1 mRNA levels and endothelin-1 receptor expression in the lung. METHODS AND RESULTS: Twenty-four piglets (7 to 30 days old) were studied randomly either at baseline (controls, n = 12) or after cardiopulmonary bypass with 30 minutes of circulatory arrest (deep hypothermic circulatory arrest, n = 12). Lungs and pulmonary arteries were harvested immediately after hemodynamic data collection. Deep hypothermic circulatory arrest significantly increased pulmonary vascular resistance (p < 0.01). Deep hypothermic circulatory arrest also produced a significant increase in endothelin-1 mRNA levels in the pulmonary arteries (149 +/- 55 pg vs 547 +/- 111 pg, p = 0.007). There was no significant change in the pulmonary parenchymal endothelin-1 mRNA levels (4102 +/- 379 pg vs 4623 +/- 308 pg, p = 0.32). Ligand binding studies of the lung parenchyma revealed a single specific endothelin-1 binding site with an EC50 value (effective concentration causing 50% of the maximum response) of about 1 x 10(-8) mol/L, consistent with the endothelin B subtype. Deep hypothermic circulatory arrest resulted in a significant increase in the number of endothelin-1 receptors in the lung (109 +/- 6 fmol/mg total protein to 135 +/- 9 fmol/mg total protein, p = 0.02). CONCLUSIONS: Deep hypothermic circulatory arrest increases production of endothelin-1 by the pulmonary vascular endothelium. Endothelin-1 production in the pulmonary parenchyma does not change. Expression of endothelin B receptors in the pulmonary parenchyma also increases after cardiopulmonary bypass with deep hypothermic circulatory arrest. This study supports the hypothesis that deep hypothermic circulatory arrest results in pulmonary vascular endothelial activation with increased endothelin-1 mRNA production.

alpha 2-Adrenergic receptors in human spinal cord: specific localized expression of mRNA encoding alpha 2-adrenergic receptor subtypes at four distinct levels.

alpha 2-Adrenergic receptor (AR) subtype mRNA (alpha 2a, alpha 2b, alpha 2c) 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 alpha 2AR subtype specific mRNA. alpha 2AR mRNA is present in gray matter only (ventral > dorsal; sacral > cervical > thoracic = lumbar). In addition to alpha 2AR 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 alpha 2AR mRNA in dorsal horn laminae III and IV, and dorsal nucleus of Clarke, where alpha 2ARs have not been described. Previously unreported heterogeneity in alpha 2AR subtype distribution (alpha 2a and alpha 2bAR mRNA present, alpha 2cAR mRNA virtually absent) is found at all sites of alpha 2AR mRNA expression in human spinal cord, including locations known to mediate effects of alpha 2AR agonist drugs on nociception, autonomic function and motor tone. Cervical spinal cord demonstrates a predominance of alpha 2a mRNA signal, while thoracic, lumbar, and sacral spinal cord demonstrate an increasing predominance of alpha 2bAR mRNA. If confirmed at a protein level, these findings have profound implications for therapeutic strategies in managing human pain.

Clinicopathologic and cytogenic features of CD34 (My 10)-positive acute nonlymphocytic leukemia.

The authors performed membrane antigen phenotyping on 75 patients with acute nonlymphocytic leukemia with a panel of myeloid-associated monoclonal antibodies. The 34 patients (45%) with CD34-positive leukemia were not significantly different from the 41 with CD34-negative leukemia with respect to age, hemoglobin, white blood cell count, or platelet count at presentation, but their blasts were more likely to lack the CD15 or CD33 antigens and to have FAB M1 or M2 morphologic characteristics. CD34-positive leukemia was more likely to arise after chemotherapy. Patients with CD34-positive leukemia were less likely to enter a complete remission even when analysis was limited to those patients receiving a high-dose induction-type chemotherapy regimen. Giemsa-banding karyotyping studies were obtained in 55 of the cases. In 30 of these cases (56%) clonal karyotypic abnormalities were demonstrated. Although the karyotypic abnormalities and phenotypes were varied, there was a high degree of association between the karyotypic abnormalities monosomy 7/del (7q) and the CD34-positive phenotype; this antigen was expressed on blasts from eight of the nine patients displaying this abnormality. Monoclonal antibody phenotyping of myeloid leukemia with reagents such as anti-CD34 may help to define biologically interesting subsets of ANLL with distinct clinicopathologic expression.

Effects of androgen deprivation on prostate alpha 1-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.

Purification of human interleukin 1.

Interleukin I (IL-1) is a lymphocyte stimulant released by human monocytes cultured for 18-24 hours in tissue culture medium containing 5% serum and the non-specific immunostimulant lipopolysaccharide (LPS). Human IL-1 is found in the conditioned medium in a low molecular weight (approximately 13,000) and a high molecular weight (approximately 85,000) form. The high MW activity may result from the formation of a complex between IL-1 and serum constituents. During the course of purification, the low MW IL-1 activity is often recovered in a high MW form. Hollow fiber diafiltration and membrane ultrafiltration has been found to rapidly separate low MW IL-1 from all measurable protein with a yield of 4% of the original activity. The IL-1 which converts to the high MW form during the purification is recoverable, 21% of the original activity, but contains small amounts of serum proteins. Isoelectric focusing (IEF) of the low MW IL-1 resulted in a very highly purified sample which was analyzed by polyacrylamide gel electrophoresis (PAGE). Utilizing a new staining procedure which detects less than 1 ng of protein per band, the IEF-purified IL-1 revealed trace quantities (less than 1 ng) of a slowly migrating protein similar to immunoglobulin and no other bands. There were no bands which corresponded with the known electrophoretic mobility of IL-1. Since the samples applied to the gel contained significant biological activity, this result implies that human IL-1 is biologically active in picogram quantities.

Functional effects of activation of alpha-1 adrenoceptors by dexmedetomidine: in vivo and in vitro studies.

Dexmedetomidine, an alpha-2 adrenoceptor (AR) agonist, produces a biphasic hypnotic response in rats. Since central alpha-1 AR stimulation may reverse the hypnotic response produced by central alpha-2 AR stimulation, we have investigated, in both in vivo and in vitro models, the functional effects of dexmedetomidine on alpha-1 AR. For in vivo studies, stainless steel cannulas were inserted stereotaxically into the lateral ventricle of halothane-anesthetized rats to facilitate i.c.v. drug administration. Four to 7 days later, the alpha-1 AR antagonist prazosin (1 mg/kg-1) or saline was administered i.p. 15 min before i.c.v. injections of dexmedetomidine (10-333 micrograms) and the sleep-time (duration of loss of righting reflex) was assessed. The sleep-time increased, in a linear fashion, up to 33 micrograms; above this dose, there was a decrease in sleep-time. Pretreatment with prazosin prevented the decrease in sleep-time which was seen at higher doses. For in vitro studies, binding parameters of dexmedetomidine and its anesthetically inert L-isomer were determined from competition binding curves using [125I]2-[beta-(4-hydroxy-3-[125I]iodo- phenyl)ethylaminomethyl]-tetralone as the radiolabeled ligand and membranes prepared from HeLa cell lines stably expressing either alpha-1B or alpha-1C AR subtypes. Dexmedetomidine bound with equal affinity to both the alpha-1B (1178 +/- 63 nM) and the alpha-1C (1344 +/- 230 nM) isoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

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 alpha1-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 alpha1-receptor, as no comparable effects were seen for total membrane protein or for alpha2-receptors. The role of thyroid hormone in alpha1-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 alpha1-receptors. Studies of mRNAs encoding the alpha1-receptor subtypes indicated that hypothyroidism targets the alpha1a-subtype, which has been implicated in the transduction of neurotrophic signals; alpha1a-receptor mRNA also showed the largest proportional developmental increase compared with those encoding other alpha1-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.

Pharmacology of tamsulosin: saturation-binding isotherms and competition analysis using cloned alpha 1-adrenergic receptor subtypes.

BACKGROUND: alpha 1-adrenergic receptors (alpha 1 ARs) are important in the dynamic component of benign prostatic hyperplasia (BPH). Currently, several alpha 1AR antagonists are being used in the treatment of BPH. METHODS: In order to more fully characterize the pharmacology of the alpha 1AR antagonist tamsulosin, we utilized saturation-binding isotherms with [3H] tamsulosin to determine the Kd of this compound at all three cloned alpha 1AR subtypes stably expressed in rat-1 fibroblasts. To confirm these results, we performed competition binding experiments, displacing [125I]HEAT with increasing concentrations of alfuzosin, doxazosin, 5-methyl-urapidil, prazosin, tamsulosin, terazosin, and (+)YM617 (stereoisomer of tamsulosin) in the same clonal cell lines. RESULTS: [3H]tamsulosin binds to cloned alpha 1AR subtypes with a rank order of affinity of alpha 1a = alpha 1d > alpha 1b. Competition experiments confirmed the relative nonselectivity of alfuzosin, doxazosin, and prazosin, but revealed slight alpha 1b = alpha 1d > alpha 1a selectivity for terazosin, and clear alpha 1a = alpha 1d > alpha 1b for (+)YM617 and tamsulosin([-]YM617); alpha 1a > alpha 1d > alpha 1b selectivity for 5-methyl-urapidil was confirmed. CONCLUSIONS: We conclude that tamsulosin displays selectivity for alpha 1a and alpha 1d ARs. This selectivity may contribute to the tamsulosin efficacy reported in several recent clinical studies in patients with BPH.

Localization of messenger RNA for three distinct alpha 2-adrenergic receptor subtypes in human tissues. Evidence for species heterogeneity and implications for human pharmacology.

BACKGROUND: alpha 2-Adrenergic receptor (alpha 2AR) agonists have become important adjuncts as anesthetic agents. They act by binding to alpha 2ARs on the surface of cell membranes and cause centrally mediated sedation and analgesia. alpha 2ARs also contribute to other aspects of physiologic regulation. Three subtypes of alpha 2ARs (alpha 2-C2, alpha 2-C4, and alpha 2-C10) have been described using molecular and pharmacologic techniques. We recently demonstrated species heterogeneity in the distribution of alpha 1-adrenergic receptor subtypes, therefore making it imperative to analyze the distribution of alpha 2AR subtypes in human tissues. This information may have importance in the understanding of potential side effects of administration of alpha 2AR subtype-selective agonists for anesthesia in humans. METHODS: RNA extracted from human tissues was analyzed by using quantitative ribonuclease protection assays to determine alpha 2AR subtype messenger RNA (mRNA) expression in cardiovascular, central nervous system, and peripheral tissues. RESULTS: alpha 2AR mRNA is present in greatest concentrations in human kidney, followed by aorta > spleen > heart = lung. alpha 2-C4 mRNA predominates in heart, lung, aorta, cerebral cortex, cerebellum, spleen, kidney, and adrenal gland; alpha 2-C2 mRNA in liver; and alpha 2-C10 mRNA in pancreas and small intestine. Hence alpha 2AR 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 alpha 2AR subtype mRNA distribution (rat vs. human) for all three alpha 2AR subtypes. (2) We demonstrate the presence and subtype heterogeneity of alpha 2AR subtype mRNA in both brain and peripheral tissues. (3) Significant concentrations of alpha 2AR 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 alpha 2AR subtypes, and may be important for the rational development of alpha 2AR subtype-selective anesthetics and other therapeutic agents for use in treating human diseases.

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

BACKGROUND: Cardiopulmonary 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 beta-adrenergic receptor (beta AR) desensitization. METHODS AND RESULTS: We obtained canine transmyocardial left ventricular biopsies before, during (155 minutes), and after CPB (pre-CPB, CPB, and post-CPB, respectively) and determined beta AR density, proportion of beta 1AR to beta 2AR, and beta AR coupling capacity to adenylyl cyclase. Beta AR 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 beta 1AR to beta 2AR (determined by two-site fit for [125I]-iodocyanopindolol competition binding with the beta 1AR selective antagonist ICI89.406) remained constant throughout (60 +/- 3: 40 +/- 3 pre-CPB, 55 +/- 3: 44 +/- 3 CPB, and 61 +/- 2: 39 +/- 2 post-CPB), revealing that both beta 1AR and beta 2AR 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 beta 2AR 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 beta AR density or adenylyl cyclase activity. CONCLUSIONS: These data suggest that myocardial beta AR 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 beta AR 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.

The alpha 1C-adrenergic receptor: characterization of signal transduction pathways and mammalian tissue heterogeneity.

We recently reported the cloning of a novel alpha 1-adrenergic receptor (AR), the alpha 1CAR. By transient and stable expression of the alpha 1CAR and the previously cloned alpha 1BAR in COS-7 and HeLa cells, respectively, we have now compared their ability to interact with major signal-transduction pathways (including polyphosphoinositide hydrolysis, intracellular calcium, and cAMP metabolism), as well as their mammalian tissue localization. Both alpha 1C- and alpha 1BARs primarily couple to phospholipase C via a pertussis toxin-insensitive GTP-binding protein, leading to the release of calcium from intracellular stores. Even though alpha 1C- and alpha 1BARs activate polyphosphoinositide hydrolysis by similar biochemical mechanisms, the alpha 1CAR couples to phospholipase C more efficiently than does the alpha 1BAR; activation of the alpha 1CAR results in a 2-3-fold greater increase in inositol phosphates, compared with the alpha 1BAR. Both alpha 1AR subtypes can also increase intracellular cAMP, by a mechanism that does not involve direct activation of adenylyl cyclase. In agreement with ligand binding data, the agonist methoxamine and the antagonist WB4101 are 10-fold more potent in activating or inhibiting, respectively, the ability of the alpha 1CAR to stimulate phospholipase C, compared with the alpha 1BAR. In addition, methoxamine is almost a full agonist at the alpha 1CAR, whereas it can only weakly activate the alpha 1BAR. Tissue localization, using Northern blot analysis of total and poly(A)+-selected RNA from rabbit tissues, revealed striking mammalian species heterogeneity. As previously described, the alpha 1BAR is present in several rat tissues, including heart, liver, brain, kidney, lung, and spleen, whereas the alpha 1CAR is not present in any rat tissue studied. The alpha 1BAR is also present in rabbit aorta, heart, spleen, and kidney (and absent in rabbit liver), whereas the alpha 1CAR is present in rabbit liver. Our results indicate that the cloning and expression of different alpha 1AR subtypes represents a valuable tool to elucidate functional correlates of alpha 1AR heterogeneity.

Cloning and pharmacological characterization of human alpha-1 adrenergic receptors: sequence corrections and direct comparison with other species homologues.

We have cloned cDNAs encoding three human alpha-1 adrenergic receptor (AR) subtypes and characterized pharmacological properties of the expressed receptor protein. A number of significant sequence corrections have been identified and compared with previously published data, at both nucleotide and amino acid levels; the most major differences occur for the human alpha-1a/dAR. Pharmacological characterization was performed simultaneously using six cloned alpha-1AR subtypes (human and rat alpha-1a/d, human and hamster alpha-1b, human and bovine alpha-1c) stably expressed in rat-1 fibroblasts at approximately equal receptor concentrations (1-2 pmol/mg of total protein). In general, human alpha-1AR subtypes have similar pharmacology compared to their rat, hamster and bovine homologs, although a few minor species differences important for alpha-1AR classification are noted. In addition, much lower inactivation (approximately 20%) by the alkylating agent chloroethylclonidine is noted in this study compared to previous reports for both human and bovine alpha-1cAR membrane preparations. All six alpha-1AR subtypes couple to phosphoinositide hydrolysis in a pertussis toxin-insensitive manner, including the cloned human alpha-1a/dAR which had not been expressed previously. In spite of significant sequence differences between human alpha-1ARs and their other species counterparts, previously established ligand selectivity remains fairly comparable. In summary, these data represent the first side-by-side comparison of pharmacological properties between species homologs of alpha-1AR subtypes and should facilitate the development of alpha-1AR subtype selective drugs for clinical use.

Subtype specific regulation of human vascular alpha(1)-adrenergic receptors by vessel bed and age.

BACKGROUND: alpha(1)-adrenergic receptors (alpha(1)ARs) regulate blood pressure, regional vascular resistance, and venous capacitance; the exact subtype (alpha(1a), alpha(1b), alpha(1 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 alpha(1)AR subtype expression differs with vascular bed, and (2) age influences human vascular alpha(1)AR subtype expression. METHODS AND RESULTS: Five hundred vessels from 384 patients were examined for alpha(1)AR subtype distribution at mRNA and protein levels (RNase protection assays, ligand binding, contraction assays). Overall vessel alpha(1)AR density is 16+/-2.3fmol/mg total protein. alpha(1a)AR predominates in arteries at mRNA (P<0.001) and protein (P<0.05) levels; all 3 subtypes are present in veins. Furthermore, alpha(1)AR mRNA subtype expression varies with vessel bed (alpha(1a) higher in splanchnic versus central arteries, P<0.05); competition analysis (selected vessels) and functional assays demonstrate alpha(1a) and alpha(1b)-mediated mammary artery contraction. Overall alpha(1)AR expression doubles with age (<55 versus > or = 65 years) in mammary artery (no change in saphenous vein), accompanied by increased alpha(1b)>alpha(1a) expression (P< = 0.001). CONCLUSIONS: Human vascular alpha(1)AR subtype distribution differs from animal models, varies with vessel bed, correlates with contraction in mammary artery, and is modulated by aging. These findings provide potential novel targets for therapeutic intervention in many clinical settings.