Progress toward elucidating the function of alpha1B-adrenergic receptors (alpha1BARs) in the central nervous system has been constrained by a lack of agonists and antagonists with adequate alpha1B-specificity. We have obviated this constraint by generating transgenic mice engineered to overexpress either wild-type or constitutively active alpha1BARs in tissues that normally express the receptor, including the brain. All transgenic lines showed granulovacular neurodegeneration, beginning in alpha1B-expressing domains of the brain and progressing with age to encompass all areas. The degeneration was apoptotic and did not occur in non-transgenic mice. Correspondingly, transgenic mice showed an age-progressive hindlimb disorder that was parkinsonian-like, as demonstrated by rescue of the dysfunction by 3, 4-dihydroxyphenylalanine and considerable dopaminergic-neuronal degeneration in the substantia nigra. Transgenic mice also had a grand mal seizure disorder accompanied by a corresponding dysplasia and neurodegeneration of the cerebral cortex. Both behavioral phenotypes (locomotor impairment and seizure) could be partially rescued with the alpha1AR antagonist terazosin, indicating that alpha1AR signaling participated directly in the pathology. Our results indicate that overstimulation of alpha1BAR leads to apoptotic neurodegeneration with a corresponding multiple system atrophy indicative of Shy-Drager syndrome, a disease whose etiology is unknown.

alpha(1)-Adrenergic receptors (ARs) are not well defined in the central nervous system. The particular cell types and areas that express these receptors are uncertain because of the lack of high avidity antibodies and selective ligands. We have developed transgenic mice that either systemically overexpress the human alpha(1A)-AR subtype fused with the enhanced green fluorescent protein (EGFP) or express the EGFP protein alone under the control of the mouse alpha(1A)-AR promoter. We confirm our transgenic model against the alpha(1A)-AR knockout mouse, which expresses the LacZ gene in place of the coding region for the alpha(1A)-AR. By using these models, we have now determined cellular localization of the alpha(1A)-AR in the brain, at the protein level. The alpha(1A)-AR or the EGFP protein is expressed prominently in neuronal cells in the cerebral cortex, hippocampus, hypothalamus, midbrain, pontine olivary nuclei, trigeminal nuclei, cerebellum, and spinal cord. The types of neurons were diverse, and the alpha(1A)-AR colocalized with markers for glutamic acid decarboxylase (GAD), gamma-aminobutyric acid (GABA), and N-methyl-D-aspartate (NMDA) receptors. Recordings from alpha(1A)-AR EGFP-expressing cells in the stratum oriens of the hippocampal CA1 region confirmed that these cells were interneurons. We could not detect expression of the alpha(1A)-AR in mature astrocytes, oligodendrocytes, or cerebral blood vessels, but we could detect the alpha(1A)-AR in oligodendrocyte progenitors. We conclude that the alpha(1A)-AR is abundant in the brain, expressed in various types of neurons, and may regulate the function of oligodendrocyte progenitors, interneurons, GABA, and NMDA receptor containing neurons.

␣ 1 -Adrenergic receptors (␣ 1A , ␣ 1B , and ␣ 1D ) are regulators of systemic arterial blood pressure and blood flow. Whereas vasoconstrictory action of the ␣ 1A and ␣ 1D subtypes is thought to be mainly responsible for this activity, the role of the ␣ 1B -adrenergic receptor (␣ 1B AR) in this process is controversial. We have generated transgenic mice that overexpress either wild type or constitutively active ␣ 1B ARs. Transgenic expression was under the control of the isogenic promoter, thus assuring appropriate developmental and tissue-specific expression. Cardiovascular phenotypes displayed by transgenic mice included myocardial hypertrophy and hypotension. Indicative of cardiac hypertrophy, transgenic mice displayed an increased heart to body weight ratio, which was confirmed by the echocardiographic finding of an increased thickness of the interventricular septum and posterior wall. Functional deficits included an increased isovolumetric relaxation time, a decreased heart rate, and cardiac output. Transgenic mice were hypotensive and exhibited a decreased pressor response. Vasoconstrictory regulation by ␣ 1B AR was absent as shown by the lack of phenylephrine-induced contractile differences between ex vivo mesenteric artery preparations. Plasma epinephrine, norepinephrine, and cortisol levels were also reduced in transgenic mice, suggesting a loss of sympathetic nerve activity. Reduced catecholamine levels together with basal hypotension, bradycardia, reproductive problems, and weight loss suggest autonomic failure, a phenotype that is consistent with the multiple system atrophy-like neurodegeneration that has been reported previously in these mice. These results also suggest that this receptor subtype is not involved in the classic vasoconstrictory action of ␣ 1 ARs that is important in systemic regulation of blood pressure.The adrenergic receptor family, which includes 3 ␣ 1 , 3 ␣ 2 , and 3 ␤-receptor subtypes, is a group of heptahelical G proteincoupled receptors that mediate the effects of the sympathetic nervous system. Extensive effort has been spent in classifying the three known ␣ 1 -adrenergic receptor (␣ 1 AR) 1 subtypes (␣ 1A , ␣ 1B , and ␣ 1D ) via molecular cloning techniques (1-4) and pharmacological analyses (5). The most well characterized cardiovascular regulatory actions associated with ␣ 1 AR activation include the contraction, growth and proliferation of vascular smooth muscle cells (6 -9), increased cardiac contractility (10), and regulation of the hypertrophic program in the myocardium (11,12). In other ␣ 1 AR-expressing tissues such as liver and kidney, the function of these receptors is to regulate metabolic processes (13) and sodium and water reabsorption (14), respectively. These responses are transduced primarily via receptor coupling to the G q /phospholipase C pathway (5), which leads to the subsequent activation of downstream signaling molecules including protein kinase C and inositol 1,4,5-trisphosphate.The progress toward elucidating the distinct regulatory role of each ␣ 1...

These data demonstrate that alpha1A-ARs protect the heart from ischemic injury through a staurosporine-sensitive signaling pathway that is independent of protein kinase C.

Although agonist binding in adrenergic receptors is fairly well understood and involves residues located in transmembrane domains 3 through 6, there are few residues reported that are involved in antagonist binding. In fact, a major docking site for antagonists has never been reported in any G-protein coupled receptor. It has been speculated that antagonist binding is quite diverse depending upon the chemical structure of the antagonist, which can be quite different from agonists. We now report the identification of two phenylalanine residues in transmembrane domain 7 of the ␣ 1a -adrenergic receptor (Phe-312 and Phe-308) that are a major site of antagonist affinity. Mutation of either Phe-308 or Phe-312 resulted in significant losses of affinity (4 -1200-fold) for the antagonists prazosin, WB4101, BMY7378, (؉) niguldipine, and 5-methylurapidil, with no changes in affinity for phenethylamine-type agonists such as epinephrine, methoxamine, or phenylephrine. Interestingly, both residues are involved in the binding of all imidazoline-type agonists such as oxymetazoline, cirazoline, and clonidine, confirming previous evidence that this class of ligand binds differently than phenethylamine-type agonists and may be more antagonist-like, which may explain their partial agonist properties. In modeling these interactions with previous mutagenesis studies and using the current backbone structure of rhodopsin, we conclude that antagonist binding is docked higher in the pocket closer to the extracellular surface than agonist binding and appears skewed toward transmembrane domain 7.Adrenergic receptors (ARs) 1 (␣ 1a , ␣ 1b , ␣ 1d , ␣ 2a , ␣ 2b , ␣ 2c , ␤ 1 , ␤ 2 , and ␤ 3 ) are members of the G-protein coupled receptor superfamily of membrane proteins that mediate the actions of the endogenous catecholamines, the neurotransmitter norepinephrine, and the hormone epinephrine. Similar to rhodopsin, these proteins are proposed to traverse the plasma membrane in a series of seven transmembrane-spanning ␣-helical domains linked by three intracellular and three extracellular loops (1). In accordance with the observation that the greatest structural conservation is localized to the transmembrane helical domains of the receptor, the catecholamine binding pocket is also localized to these regions. Mutagenesis studies in our laboratory and others have identified that the endogenous agonist in biogenic amine receptors is stabilized in the binding pocket by ionic, hydrogen bond, and aromatic/hydrophobic interactions involving residues on TM3, TM5, and TM6, although there are various modulations of these interactions between the families (2-5). We have also recently shown that ␣ 1 -ARs and perhaps some other biogenic amine receptors have additional aromatic/ hydrophobic interactions with the endogenous agonist to residues in TM4 and TM5 (6).However, our knowledge of how antagonists bind to the adrenergic receptor family is limited. Mutagenesis studies in our laboratory have identified that the subtype selectivity of two ␣ 1a -AR antagonis...