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.

␣ 1a -Adrenergic receptors (␣ 1a ARs) are present intracellularly and at the cell surface in cultured and natural cell models, where they are subject to agonist-mediated desensitization and internalization. To explore ␣ 1a AR trafficking, a hemagglutinin (HA)-tagged ␣ 1a AR/enhanced green fluorescent protein (EGFP) fusion protein was expressed in rat-1 fibroblasts and tracked by EGFP fluorescence and antibody labeling of surface receptors. Confocal analysis of antibody-labeled surface receptors revealed unexpected constitutive internalization in the absence of agonist stimulation. In partial agreement, the inverse agonist prazosin also caused a modest 20 Ϯ 2% increase in surface receptor levels, suggesting a partial block of constitutive internalization caused by decreased basal activation. However, prazosin was unable to prevent internalization of antibody-tagged surface receptors observed by confocal microscopy or cause obvious redistribution of intracellular receptor to the surface, suggesting that the ␣ 1a AR is internalizing even in a basalinactive state. In contrast to the ␣ 1a AR, surface labeling of an HA-tagged ␣ 1b -EGFP fusion protein did not result in any apparent constitutive internalization. Constitutive internalization of the ␣ 1a AR seems to occur alongside reversible agonist-induced internalization, and both seem to involve clathrin-mediated endocytosis but not degradation in lysozymes. Surface receptor density must be maintained by recycling, because the protein synthesis inhibitor cycloheximide has no effect on total or surface receptor density in agonist-treated or untreated cells for 6 h. Constitutive agonist-independent trafficking of ␣ 1a ARs may provide a novel mechanism by which an internal pool of ␣ 1a ARs are maintained and recycled to allow continuous agonist-induced signaling.

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Despite important roles in myocardial hypertrophy and benign prostatic hyperplasia, little is known about acute effects of agonist stimulation on ␣ 1a -adrenergic receptor (␣ 1a AR) signaling and function. Regulatory mechanisms are likely complex since 12 distinct human ␣ 1a AR carboxyl-terminal splice variants have been isolated. After determining the predominance of the ␣ 1a-1 AR isoform in human heart and prostate, we stably expressed an epitope-tagged ␣ 1a-1 AR cDNA in rat-1 fibroblasts and subsequently examined regulation of signaling, phosphorylation, and internalization of the receptor. Human ␣ 1a AR-mediated inositol phosphate signaling is acutely desensitized in response to both agonist and phorbol 12-myristate 13-acetate (PMA) exposure. Concurrent with desensitization, ␣ 1a ARs in 32 P ilabeled cells are rapidly phosphorylated in response to both NE and PMA stimulation. Despite the ability of PKC to desensitize ␣ 1a ARs when directly activated with PMA, inhibitors of PKC have no effect on agonist-mediated desensitization. In contrast, involvement of GRK kinases is suggested by the ability of GRK2 to desensitize ␣ 1a ARs. Internalization of cell surface ␣ 1a ARs also occurs in response to agonist stimulation (but not PKC activation), but is initiated more slowly than receptor desensitization. Significantly, deletion of the ␣ 1a AR carboxyl terminus has no effect on receptor internalization or either agonist-induced or GRK-mediated receptor desensitization. Because mechanisms underlying acute agonist-mediated regulation of human ␣ 1a ARs are primarily independent of the carboxyl terminus, they may be common to all functional ␣ 1a AR isoforms.␣ 1a -Adrenergic receptors (␣ 1a ARs) 1 are G protein-coupled receptors (GPCR) that mediate sympathetic nervous system responses such as smooth muscle contraction and myocardial inotropy (1). NE stimulation of ␣ 1 ARs predominantly activates G q and results in membrane polyphosphoinositide hydrolysis by activation of phospholipase C␤; the resultant second messengers IP 3 and DAG mobilize intracellular calcium and activate protein kinase C (PKC), respectively (2). Three ␣ 1 AR subtypes (␣ 1a , ␣ 1b , and ␣ 1d ) have been cloned and pharmacologically characterized in several expression systems (for review, see Ref.2). Clinically, activation of ␣ 1a ARs has been implicated in the dynamic component of benign prostatic hyperplasia leading to bladder outlet obstruction and in the development of myocardial hypertrophy (3-5). Notwithstanding the importance of ␣ 1a ARs in several pathophysiological states, surprising little is known about mechanisms underlying ␣ 1a AR expression and function. Transcriptional mechanisms unique to ␣ 1a ARs have been shown to be important in maintaining full ␣ 1 AR responsiveness to agonist in rat neonatal myocytes where long term (24 -72 h) NE stimulation leads to up-regulation ␣ 1a AR mRNA and receptor protein expression, concurrent with down-regulation of ␣ 1b and ␣ 1d AR subtypes (5, 6). While important, such long-term studies do not examine...