alpha1-AR Subtypes Research Articles

Two α1-Adrenergic Receptor Subtypes Regulating the Vasopressor Response Have Differential Roles in Blood Pressure Regulation

To study the functional role of individual alpha1-adrenergic (AR) subtypes in blood pressure (BP) regulation, we used mice lacking the alpha1B-AR and/or alpha1D-AR with the same genetic background and further studied their hemodynamic and vasoconstrictive responses. Both the alpha1D-AR knockout and alpha1B-/alpha1D-AR double knockout mice, but not the alpha1B-AR knockout mice, had significantly (p < 0.05) lower levels of basal systolic and mean arterial BP than wild-type mice in nonanesthetized condition, and they showed no significant change in heart rate or in cardiac function, as assessed by echocardiogram. All mutants showed a significantly (p < 0.05) reduced catecholamine-induced pressor and vasoconstriction responses. It is noteworthy that the infusion of norepinephrine did not elicit any pressor response at all in alpha1B-/alpha1D-AR double knockout mice. In an attempt to further examine alpha1-AR subtype, which is involved in the genesis or maintenance of hypertension, BP after salt loading was monitored by tail-cuff readings and confirmed at the endpoint by direct intra-arterial recording. After salt loading, alpha1B-AR knockout mice developed a comparable level of hypertension to wild-type mice, whereas mice lacking alpha1D-AR had significantly (p < 0.05) attenuated BP and lower levels of circulating catecholamines. Our data indicated that alpha1B- and alpha1D-AR subtypes participate cooperatively in BP regulation; however, the deletion of the functional alpha1D-AR, not alpha1B-AR, leads to an antihypertensive effect. The study shows differential contributions of alpha1B- and alpha1D-ARs in BP regulation.

EFFECTS OF CHRONIC ADMINISTRATION OF DOXAZOSIN ON α 1-ADRENOCEPTORS IN THE RAT PROSTATE

Although the clinical efficacy of alpha1-adrenoceptor (alpha1-AR) antagonists for the treatment of benign prostatic hyperplasia is not disputed, their mechanism of action and ability to maintain long-term effectiveness have only recently been investigated. Since it is known that chronic administration of receptor antagonists causes up-regulation in the targeted receptor, we examined the effects of chronic administration of doxazosin, a nonspecific long acting alpha1-AR antagonist, on the properties of alpha1-AR subtypes in the rat prostate. Rats were treated with doxazosin (2 or 4 mg/kg daily subcutaneously, supplemented with 4 mg/kg daily orally) for 8 or 12 weeks. Prostatic alpha1-AR properties at the protein and gene transcript levels were quantified by radioligand receptor binding and real-time reverse transcriptase-polymerase chain reaction, respectively. Treated rats that received the highest levels of doxazosin had significantly heavier prostates compared with age matched controls. After 12 weeks of treatment radioligand binding studies with radiolabeled alpha1-AR antagonist demonstrated no significant differences in the density of total alpha1-ARs in the prostate, whereas the results of real-time reverse transcriptase-polymerase chain reaction showed up-regulation in the mRNA expression levels of all 3 alpha1-AR subtypes (alpha1A, alpha1B and alpha1D) in the ventral and dorsolateral regions of the rat prostate. These data demonstrate that chronic treatment with doxazosin causes an alteration in the properties of the alpha1-AR system in the rat prostate. These findings may provide insight into the long-term effectiveness of alpha1-AR antagonists in the treatment of benign prostatic hyperplasia.

Mechanistic insights into the role of alpha1adrenergic receptors in lower urinary tract symptoms.

Although alpha(1)AR antagonists have been used for more than two decades to treat lower urinary tract symptoms (LUTS), we have little understanding of the mechanistic basis of their efficacy and their role in the development of LUTS. It is clear that alpha(1)ARs play a critical role in bladder dysfunction and recent data suggest that alpha(1)AR subtype switching may play a key role in this pathophysiology, providing support for use of alpha(1)(d)AR-selective antagonists in treating irritative symptoms. This review seeks to summarize current levels of understanding in this field and discusses new concepts that suggest increased levels of complexity involving cross-talk in multiple receptor systems. Effective therapeutic modalities likely will involve increased subtype selective alpha(1)AR antagonists and other pharmacodynamic factors.

Changes in the receptor profile of the aging bladder

Future demographic developments will challenge urology with a steadily increasing incidence of lower urinary tract symptoms (LUTS) derived from the aging bladder. Obstruction, instability and hypocontractility, which may be caused by changes in the receptor profile of the detrusor, are typical pathophysiologic findings in geriatric bladder dysfunction. Benign prostatic hyperplasia and diabetes mellitus are age-associated comorbidities with an additional influence on bladder receptors. Muscarinic (M(2), M(3)), purinergic (P2X, P2Y) and adrenergic receptors (alpha(1), beta(3)) are targets of efferent sympathetic and parasympathetic bladder innervation. Although the results from animal experiments are somewhat inconsistent, aging and bladder outlet obstruction (BOO) probably cause partial cholinergic denervation of the detrusor with a subsequent upregulation of muscarinic receptor sensitivity leading to bladder instability. The non-cholinergic (atropine-resistant) component of the detrusor contraction rises with aging and BOO to 50%, emphasizing the increasing impact of purinergic receptors in geriatric LUTS. alpha(1)-adrenergic receptors are modulated in the aging bladder by a shift from the predominant alpha(1a) subtype to the alpha(1d) subtype, which shows 100-fold higher affinity towards norepinephrine and increases alpha-adrenergic bladder susceptibility. No data are available on the changes in beta(3) receptor density or sensitivity with aging. Moreover, the role of sensory C-fiber receptors in geriatric LUTS remains completely unclear, although specific C-fiber blockers are already under clinical evaluation (capsaicin, resiniferatoxin).

Involvement of signal transducing GTP-binding proteins in renal artery alpha 1-adrenoceptor mediated smooth muscle contraction.

To determine the participation of GTP-binding proteins (G-proteins) in the cellular mechanism of the phenylephrine-induced renal artery vasospasm by using swine renal artery smooth muscle rings in a standard organ baths, as increased noradrenaline release from perivasal and intramural sympathetic nerve endings during renal ischaemia results in increased vascular smooth muscle tone that is important in the loss of kidney function during renal transplantation and nephron-sparing surgery. Fresh swine kidneys were transported in cold calcium-free Tyrode solution to the laboratory. Adipose tissue around the arteries was removed, the organ de-capsulated and interlobar arteries dissected. The contractile properties of renal artery smooth muscle rings were assessed in a standard organ bath, the rings pre-tensioned at 2 g. Contractions were evoked by applying the alpha 1-adrenoceptor selective agonist phenylephrine (1 nmol/L to 0.3 mmol/L). Isometric contractions of the tissue were registered and stored digitally. Dose-response curves were obtained sequentially with a wash-out of 20 min between each concentration; the maximum contractility of an individual muscle ring was set at 100%. Dose-response curves of inhibitory agents (e.g. WB4101, cholera and pertussis toxins) were determined by comparing the remaining contractility after incubating with the respective drug with a control contraction that was evoked three times (10 mumol/L phenylephrine) and the mean set at 100%. Phenylephrine induced dose-dependent and fully reversible isometric contractions with a threshold concentration of 100 nmol/L and an EC50 of 0.8 mumol/L. The receptor was identified as the alpha 1A-subtype by the selective antagonist WB4101. Pre-treatment of tissue rings with 5 micrograms/mL pertussis toxin (120 min, 37 degrees C) inhibited the control contraction by a mean (SEM) of 52.0 (4.6)%, whereas pre-treatment with 1 microgram/mL cholera toxin (60 min, 37 degrees C), leading to a permanent activation of the Gs-protein via blockade of the GTPase activity, decreased the response by 39.0 (8.2%). These results suggest a coupling of alpha 1A-adrenoceptors in renal vascular tissue to the heterotrimeric Gs-protein and to heterotrimeric G-proteins of the G1- and/or G0-family in the phenylephrine-induced contraction.

Excitatory α 1-Adrenergic Receptors Predominate Over Inhibitory β-Receptors in Rabbit Dorsal Detrusor

We mapped the regional distribution of alpha1 and beta-adrenergic receptors (ARs) in rabbit ventral and dorsal bladder, and characterized the alpha1-AR subtypes responsible for norepinephrine induced contraction of rabbit dorsal detrusor smooth muscle. Responses to norepinephrine in the absence and presence of adrenergic antagonists were measured in strips taken from clearly defined regions of male rabbit ventral and dorsal bladder. In the absence of antagonists ventral strips from the bladder body relaxed in response to norepinephrine, while those from the ventral base contracted. Dorsal strips from the bladder dome also relaxed in response to norepinephrine but dorsal strips from the mid and lower body, and the base contracted. All contractile responses were antagonized by incubation with prazosin. Characterization of the alpha-AR subtypes present in dorsal strips from the mid and lower bladder body using BMY 7378 (8-[2-[4 to 2(-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro [4,5] decane-7,9-dione dihydrochloride), chloroethylclonidine, 5-methylurapidil and WB 4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane) suggested that the alpha1-AR subtype responsible for the contraction is the alpha1A or alpha1L-AR. The male rabbit bladder contains at least 4 heterogeneous regions with differing functional responses to adrenergic stimulation, that is 1) the dorsal and ventral dome, where beta-ARs predominate, 2) the ventral detrusor, where beta-ARs predominate, 3) the dorsal detrusor, where alpha1-ARs predominate, and 4) the dorsal and ventral bladder neck, where alpha1-ARs predominate.

Oligomerization of the α1a- and α1b-Adrenergic Receptor Subtypes: POTENTIAL IMPLICATIONS IN RECEPTOR INTERNALIZATION

We combined biophysical, biochemical, and pharmacological approaches to investigate the ability of the alpha 1a- and alpha 1b-adrenergic receptor (AR) subtypes to form homo- and hetero-oligomers. Receptors tagged with different epitopes (hemagglutinin and Myc) or fluorescent proteins (cyan and green fluorescent proteins) were transiently expressed in HEK-293 cells either individually or in different combinations. Fluorescence resonance energy transfer measurements provided evidence that both the alpha 1a- and alpha 1b-AR can form homo-oligomers with similar transfer efficiency of approximately 0.10. Hetero-oligomers could also be observed between the alpha 1b- and the alpha 1a-AR subtypes but not between the alpha 1b-AR and the beta2-AR, the NK1 tachykinin, or the CCR5 chemokine receptors. Oligomerization of the alpha 1b-AR did not require the integrity of its C-tail, of two glycophorin motifs, or of the N-linked glycosylation sites at its N terminus. In contrast, helix I and, to a lesser extent, helix VII were found to play a role in the alpha 1b-AR homo-oligomerization. Receptor oligomerization was not influenced by the agonist epinephrine or by the inverse agonist prazosin. A constitutively active (A293E) as well as a signaling-deficient (R143E) mutant displayed oligomerization features similar to those of the wild type alpha 1b-AR. Confocal imaging revealed that oligomerization of the alpha1-AR subtypes correlated with their ability to co-internalize upon exposure to the agonist. The alpha 1a-selective agonist oxymetazoline induced the co-internalization of the alpha 1a- and alpha 1b-AR, whereas the alpha 1b-AR could not co-internalize with the NK1 tachykinin or CCR5 chemokine receptors. Oligomerization might therefore represent an additional mechanism regulating the physiological responses mediated by the alpha 1a- and alpha 1b-AR subtypes.

Gene expression profile of neurodegeneration induced by alpha1B-adrenergic receptor overactivity: NMDA/GABAA dysregulation and apoptosis.

The alpha1-adrenergic receptors (alpha1ARs) play an important role in mediating sympathetic neurotransmission in peripheral organ systems; however, central alpha1ARs are not well characterized. Additionally, due to the lack of sufficiently subtype-selective drugs or high avidity antibodies, the contribution of each alpha1AR subtype to various central functions is currently unclear. Transcription regulation through alpha1AR subtypes in the CNS is also unknown. Of interest, transgenic mice that systemically overexpress the alpha1BAR show central symptoms that include age-progressive impaired mobility, neurodegeneration and susceptibility to epileptic seizure. To investigate the molecular basis of this phenotype, oligonucleotide microarray studies of whole brains of various ages were carried out to compare gene expression profiles between transgenic and normal brains. The results indicated changes in expression of apoptotic, calcium regulatory, neurodegenerative and genes involved in neurotransmission. Defects in regulation of intracellular calcium are known to play a role in cell death; thus, these genes may provide clues as to the molecular basis of alpha1BAR-induced neurodegeneration. Epilepsy is a disorder that can be caused by an imbalance between excitatory (e.g. glutamate) and inhibitory (e.g. GABA) signals. Microarray analysis of transgenic brains showed increased N-methyl-d-aspartate (NMDA) receptors and decreased GABAA, which were confirmed with immunohistochemistry, western blot and radioligand binding studies. The alpha1BAR also co-localized with the glutamatergic distribution, suggesting a glutamate imbalance as a molecular rationale for the epileptic seizures.

Allosteric α1-Adrenoreceptor Antagonism by the Conopeptide ρ-TIA

A peptide contained in the venom of the predatory marine snail Conus tulipa, rho-TIA, has previously been shown to possess alpha1-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, rho-TIA inhibited alpha1-adrenoreceptor-mediated increases in cytosolic Ca2+ concentration that were triggered by norepinephrine, but did not affect presynaptic alpha2-adrenoreceptor-mediated responses. In radioligand binding assays using [125I]HEAT, rho-TIA displayed slightly greater potency at the alpha 1B than at the alpha 1A or alpha 1D subtypes. Moreover, although it did not affect the rate of association for [3H]prazosin binding to the alpha 1B-adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by rho-TIA. N-terminally truncated analogs of rho-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of rho-TIA (Arg4). An alanine walk of rho-TIA confirmed the importance of Arg4 for activity and revealed a number of other residues clustered around Arg4 that contribute to the potency of rho-TIA. The unique allosteric antagonism of rho-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive alpha1-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.

Role of adrenergic receptor subtypes in the control of human placental blood vessels.

The use of beta2-Adrenergic Receptor (AR) agonists and the potential use of alpha1-AR blockers as tocolytics raise the question of how they influence placental circulation. The receptor profile was characterized via the amounts of the mRNA of alpha1-AR subtypes and beta2-ARs. The mRNAs were detected by reverse transcription-polymerase chain reaction. Electric field stimulation (EFS) was applied to test the pharmacological reactivity of the placental vessels. Expressions of beta2- and all subtypes of alpha1-AR mRNA were demonstrated in human placental vessels, and were significantly higher in the arteries. A significant difference was not found between the veins and the arteries as concerns the amount of alpha1D-AR mRNA. There was a preponderance of alpha1A- and alpha1B-AR mRNA as compared to alpha1D-AR mRNA both in the arteries and in the veins. beta2-AR agonists and alpha1-AR antagonists antagonized the EFS-induced contractions of the placental arteries in a dose-dependent manner. These effects were significantly less marked on veins at all applied doses. Urapidil antagonized the EFS-induced contractions of both the placental arterial and vein rings in a dose-dependent manner. The beta2-, alpha1A-, and alpha1B-AR are the important subtypes involved in the regulation of the contractility of the human term placental vessels. The possible increase in placental blood flow mediated by these ARs can even be beneficial during pregnancy. Accordingly, the use of beta2-AR agonists and the potential use of alpha1-blockers as tocolytics seem safe on the basis of in vitro examinations as concerns the placental circulation.

Spinophilin Stabilizes Cell Surface Expression of α2B-Adrenergic Receptors

The third intracellular (3i) loops of the alpha 2A- and alpha 2B-adrenergic receptor (AR) subtypes are critical for retention of these receptors at the basolateral surface of polarized Madin-Darby canine kidney (MDCKII) cells at steady state. The third intracellular loops of the alpha 2A, alpha 2B, and alpha 2C-AR subtypes interact with spinophilin, a multidomain protein that, like the three alpha 2-AR subtypes, is enriched at the basolateral surface of MDCKII cells. The present studies provide evidence that alpha 2-AR interaction with spinophilin contributes to cell surface stabilization of the receptor. We exploited the unique targeting profile of the alpha 2B-AR subtype in MDCKII cells: random delivery to apical and basolateral surfaces with rapid (t(1/2) < or = 60 min) apical versus slower (t(1/2) = 10-12 h) basolateral turnover. Apical delivery of a spinophilin subdomain containing the alpha 2-AR-interacting region (Sp151-483) by fusion with apically targeted p75NTR extended the half-life of alpha 2B-AR at the apical surface to approximately 3.6 h and eliminated the rapid phase (0-60 min) of alpha 2B-AR turnover on that surface. Furthermore, we examined alpha 2B-AR turnover at the surface of mouse embryo fibroblasts derived from wild type (Sp+/+) or spinophilin knock-out (Sp-/-) mice. Two independent experimental approaches demonstrated that agonist-evoked internalization of HA-alpha 2B-AR was accelerated in mouse embryo fibroblasts derived from Sp-/- mice. These findings are consistent with the interpretation that endogenous spinophilin contributes to the stabilization of alpha 2B-AR and presumably all three alpha2-AR subtypes at the surface of target cells and may act as a scaffold that could link alpha 2-ARs to proteins interacting with spinophilin via other domains.

2‐(Anilinomethyl)imidazolines as α1 Adrenergic Receptor Agonists: α1a Subtype Selective 2′‐Heteroaryl Compounds.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Alpha1-adrenergic receptors activate Ca(2+)-permeable cationic channels in prostate cancer epithelial cells.

The prostate gland is a rich source of alpha1-adrenergic receptors (alpha1-ARs). alpha1-AR antagonists are commonly used in the treatment of benign prostatic hyperplasia symptoms, due to their action on smooth muscle cells. However, virtually nothing is known about the role of alpha1-ARs in epithelial cells. Here, by using two human prostate cancer epithelial (hPCE) cell models - primary cells from resection specimens (primary hPCE cells) and an LNCaP (lymph node carcinoma of the prostate) cell line - we identify an alpha1A subtype of adrenergic receptor (alpha1A-AR) and show its functional coupling to plasmalemmal cationic channels via direct diacylglycerol (DAG) gating. In both cell types, agonist-mediated stimulation of alpha1A-ARs and DAG analogues activated similar cationic membrane currents and Ca(2+) influx. These currents were sensitive to the alpha1A-AR antagonists, prazosin and WB4101, and to transient receptor potential (TRP) channel blockers, 2-aminophenyl borate and SK&F 96365. Chronic activation of alpha1A-ARs enhanced LNCaP cell proliferation, which could be antagonized by alpha1A-AR and TRP inhibitors. Collectively, our results suggest that alpha1-ARs play a role in promoting hPCE cell proliferation via TRP channels.

Molecular biological researches of the lower urinary tract function

Adrenergic alpha1 and beta receptors are present in the target organs of sympathetic nerve and they participate in the signal transduction mechanism of the lower urinary tract. Adrenergic alpha1 receptors are present in urethral and prostatic smooth muscles, and contract these muscles. Among these receptor subtypes, the alpha1-A receptor has the most important role, and mRNA expression of the corresponding alpha1-a subtype is predominant. In the human urinary bladder detrusor smooth muscle, the expression of adrenergic beta3 receptor subtype mRNA is predominant, and relaxation of detrusor smooth muscle is mediated mainly via beta3 receptor. Afferent nerve with lower threshold can easily transmit bladder sensation and takes an important role in the pathophysiology of urge urinary incontinence. Successful molecular cloning of vanilloid receptors, which are present in these afferent nerves, revealed that vanilloid receptors are ion-channels, sensitive for heat and pH, and termed VR1 and VRL1. Among purinergic receptors, ion channel type P2X3 receptor is found in afferent nerve fibers and plays some roles in the signal transduction of bladder sensation. In the near future, agonist for the adrenergic beta3 receptor and selective antagonists for VR1, VRL1, or P2X3 will possibly become drugs for pollakisuria and urge urinary incontinence.

Variation in the alpha2B-adrenoceptorgene as a risk factor for prehospitalfatal myocardial infarction and sudden cardiac death

ObjectivesOur aim was to corroborate the observed association between the deletion/deletion (DD) genotype of the insertion/deletion polymorphism in the alpha2B-adrenoceptor (AR) and increased risk for acute myocardial infarction (AMI), and to study whether this genotype also confers an increased risk for sudden cardiac death (SCD). BackgroundVasospasm has been suggested to play a role in AMI. Alpha2-AR mediate coronary vasoconstriction in humans, and studies on mice suggest the involvement of the alpha2-AR subtype B in vasoconstriction. A deletion variant of the human alpha2B-AR has been associated with impaired receptor desensitization in vitro. In a population-based prospective study of 912 middle-aged men, the DD genotype of the alpha2B-AR conferred an increased risk for AMI. MethodsA series of 700 unselected sudden out-of-hospital deaths of middle-aged white men subjected to medico-legal autopsy was analyzed. ResultsGenotype information was obtained for 683 men (DD = 22%, insertion/deletion = 51%, insertion/insertion = 27%). Carriers of the DD genotype had an increased risk for SCD (n = 278, odds ratio [OR] = 2.0, p = 0.01) and fatal AMI (n = 84, OR = 2.1, p = 0.04) compared with the other two genotypes combined. The risks for SCD and fatal AMI were higher in carriers of the DD genotype who died before the age of 55 years (OR = 4.5 and 5.0, p < 0.001 for both). ConclusionsMiddle-aged white men carrying the DD genotype of the alpha2B-AR have a significantly increased risk for SCD and AMI, especially before the age of 55 years.

Subtype-specific neuronal differentiation of PC12 cells transfected with α 2-adrenergic receptors

Cells of the PC12 rat pheochromocytoma cell line acquire characteristics of sympathetic neurons under appropriate treatment. Stably transfected PC12 cells expressing individual alpha2-adrenergic receptor (alpha2-AR) subtypes were used to assess the role of alpha2-ARs in neuronal differentiation and to characterise the signalling pathways activated by the alpha2-AR agonist epinephrine in these cells. The effects of alpha2-AR activation were compared with the differentiating action and the signalling mechanisms of nerve growth factor (NGF). Epinephrine induced neuronal differentiation of PC12alpha2 cells through alpha2-AR activation in a subtype-dependent manner, internalization of all human alpha2-AR subtypes, and activation of mitogen-activated protein kinase (MAPK) and the serine-threonine protein kinase Akt. Epinephrine and NGF showed synergism in their differentiating effects. The MAPK kinase (MEK-1) inhibitor PD 98059 abolished the differentiating effect of epinephrine indicating that the differentiation is dependent on MAPK activation. Activating protein-1 (AP-1) DNA-binding activity was increased after epinephrine treatment in all three PC12alpha2 subtype clones. Evaluation of the potential physiological consequences of these findings requires further studies on endogenously expressed alpha2-ARs in neuronal cells.

2-(Anilinomethyl)imidazolines as alpha1 adrenergic receptor agonists: the discovery of alpha1a subtype selective 2'-alkylsulfonyl-substituted analogues.

A series of 2'-alkylthio-2-(anilinomethyl)imidazolines were prepared to examine the effect of the alkyl group size, sulfur oxidation state, and phenyl ring substitution on ligand binding and agonism of alpha-adrenergic receptor subtypes alpha1a, alpha1b, alpha1d, alpha2a, and alpha2c. Binding at all receptor subtypes decreased for compounds in the sulfone oxidation state as compared to their sulfide analogues. While sulfides were generally potent, nonselective agonists, sulfones exhibited alpha1a subtype selectivity in a cell-based functional assay. Sulfone (32) was 250-7000-fold selective for alpha1a vs all other subtypes.

Signalling components involved in the coupling of alpha 1-adrenoceptors to phospholipase D in neonatal rat cardiac myocytes.

Activation of phospholipase D (PLD) is assumed to be one major pathway by which alpha(1)-adrenoceptors (alpha(1)ARs) induce hypertrophic responses in cardiac myocytes. Heterotrimeric G proteins, protein kinase C (PKC) isoforms, protein tyrosine kinases, monomeric GTPases of the ADP-ribosylation factor (ARF) and Rho families, and as important cofactor phosphatidylinositol 4,5-bisphosphate (PIP(2)) seem to participate in the G protein-coupled receptor dependent regulation of PLD. We therefore studied the role of these components in the coupling of alpha(1)ARs to PLD in neonatal rat cardiac myocytes (NRCM). Stimulation of alpha(1)ARs, most likely of the alpha(1A) subtype, by noradrenaline increased PLD activity three- to fourfold concomitant with the stimulation of phospholipase C (PLC). In contrast, the partial agonist phenylephrine stimulated PLC, but failed to increase PLD activity. The PLC and PLD responses were pertussis toxin insensitive and treatment of the cells with the G(q)-activating toxin of Pasteurella multocida stimulated both phospholipases about fourfold. Over-expression of the G(q)-and G(i)-type-specific regulator of G protein signalling RGS4 blunted alpha(1)AR-induced PLC and PLD stimulation. Ro 31-8220, known to inhibit Ca(2+)-dependent and -independent PKC isoforms, strongly inhibited PLD activity, whereas Gö 6976, known to inhibit preferentially Ca(2+)-dependent PKC isozymes, was without effect. The ARF signalling inhibitor brefeldin A, protein tyrosine kinase inhibitors and the Rho-inactivating toxin B of Clostridium difficile blunted alpha(1)AR-induced PLD stimulation and largely reduced the cellular PIP(2) content. In membranes of toxin B-treated NCRM, PLD activity was similarly reduced, but was fully restored by addition of exogenous PIP(2). We conclude, that alpha(1A)ARs stimulate PLD activity via a G(q/11)-PLCbeta-novel PKC isoform-dependent pathway in NRCM. ARF and Rho GTPases as well as protein tyrosine kinases contribute to PLD stimulation in NRCM, most likely by regulating the supply of PIP(2).

Alpha-1 adrenoceptor up-regulation induced by prazosin but not KMD-3213 or reserpine in rats.

1. We have investigated the effects of chronic administration of prazosin (a subtype-nonspecific alpha-1 AR antagonist), KMD-3213 (an alpha-1A AR subtype-specific antagonist) and reserpine (a catecholamine depletor) on the density of alpha-1 AR subtypes in various rat tissues (liver, kidney, submaxillary gland, heart and spleen). 2. Administration of prazosin (2 mg kg(-1) day(-1), i.p.) for 2 weeks did not affect K(D) values for [(3)H]-prazosin or [(3)H]-KMD-3213 of alpha-1 ARs in five rat tissues tested. However, it caused 52% up-regulation of alpha-1B AR in the spleen, and 84% and 107% up-regulation of alpha-1A- and alpha-1B ARs, respectively, in the heart. Although major subtypes of alpha-1 AR are alpha-1A AR in the submaxillary gland, alpha-1B AR in the liver, and alpha-1A and alpha-1B ARs in the kidney, these tissues showed no up-regulation. The mRNA levels of alpha-1 AR subtypes were not affected by prazosin administration in any tissue tested. 3. Neither administration of KMD-3213 (2 mg kg(-1) day(-1), i.p.) nor reserpine (0.5 - 1 mg kg(-1) day(-1), i.p.) for 2 weeks caused any change in either the binding affinity for [(3)H]-prazosin or [(3)H]-KMD-3213 or the density of the alpha-1 AR subtypes in the five rat tissues. 4. Neither prazosin nor KMD-3213 treatment reduced the noradrenaline content in the five rat tissues, in contrast to reserpine treatment, which markedly reduced it. 5. The findings of the present study demonstrated that up-regulation of alpha-1 AR is selectively caused by prazosin treatment in some tissues but neither by KMD-3213 treatment nor by chemical denervation with reserpine. These results suggest that up-regulation of alpha-1 ARs is not caused by a simple blockade of sympathetic tone.

MODULATION OF BLADDER α1-ADRENERGIC RECEPTOR SUBTYPE EXPRESSION BY BLADDER OUTLET OBSTRUCTION

alpha1-Adrenergic receptor (alpha1AR) antagonists are effective for relieving obstructive and irritative symptoms in patients with bladder outlet obstruction. While the alpha1aAR 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 alpha1AR subtypes may be involved in this process. 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. alpha1AR subtype messenger (m)RNA was quantitated using quantitative competitive reverse transcriptase-polymerase chain reaction and protein expression was determined using radioligand binding with the alpha1AR antagonist [125iodine]2-(-[4-hydroxyphenyl]-ethyl-aminomethyl)tetralone (saturation analysis for total alpha1AR density and competition analysis with BMY7378 and 5-methylurapidel to determine alpha1AR subtypes). In this model 6-week surgical obstruction produced a 6.3-fold increase in bladder weight versus sham operation (p <0.001), concurrent with increased voiding frequency versus before obstruction (p <0.004). Although bladder alpha1AR density did not increase overall with obstruction, striking changes in alpha1AR subtype expression occurred. In control animals 70% of alpha1AR mRNA was the alpha1a subtype, 5% were alpha1b and 25% were alpha1d, whereas in obstructed animals bladder alpha1AR expression changed to 23% alpha1a, 2% alpha1b and 75% alpha1d. Changes in alpha1AR mRNA expression were of similar magnitude throughout the bladder dome, mid body and base. Parallel changes were also evident at the protein level with 100% alpha1aAR expression in control animals changing to new onset alpha1dAR expression (mean plus or minus standard error of mean 36% +/- 7%) in animals with a 5-fold or greater increase in bladder weight. Our findings indicate a remarkable increase in bladder alpha1dAR mRNA and protein expression after 6 weeks of obstruction and resultant detrusor hypertrophy. This finding is potentially important since alpha1dARs have 10 to 100-fold higher affinity for the endogenous neurotransmitter norepinephrine than the alpha1a or alpha1bAR subtypes. These findings imply that targeting alpha1d may provide a new therapeutic approach for controlling bladder irritative symptoms and possibly detrusor overactivity associated with bladder outlet obstruction.