Contractile Effects of Serotonin (5-HT) in the Rat Cauda Epididymis: Expression and Functional Characterization of 5-HT Receptors.

Serotonin [5-hydroxytryptamine (5-HT)] exerts multiple central and peripheral functions. High concentrations of 5-HT have been found in the epididymis, a ductal organ that plays pivotal roles in sperm transport and maturation. The contraction of the epididymal smooth muscle is essential for sperm transport and emission during ejaculation. The contributions of the epididymal 5-HT system to these events are poorly understood. Here, we assessed the contractile function of 5-HT in the rat cauda epididymis (CE), pharmacologically targeting the receptor(s) and the reuptake mechanism involved in this system. Segments of CE duct from adult Wistar rats were set up in an organ bath system for isometric tension recordings, and concentration-response curves to 5-HT and norepinephrine were obtained. 5-HT elicited concentration-dependent contractions of the CE duct (pEC50 = 6.5 ± 0.1) that were potentiated with high potency by the norepinephrine transporter (NET) inhibitor desipramine and with low potency by the highly selective serotonin transporter inhibitor paroxetine, indicating that the NET is the major mediator of 5-HT reuptake in vitro. CE contractions to 5-HT were antagonized by the α 1-adrenoceptor (α 1-AR) antagonist prazosin (pA 2 ≅ 8.9), 5-HT2A/2C antagonists ketanserin (pA 2 ≅ 9.4) and fluoxetine (pA 2 ≅ 7.4), and 5-HT1A ligands WAY 100635 (pA 2 ≅ 8.9) and buspirone (pA 2 ≅ 7.3). Reverse transcriptase polymerase chain reaction analysis demonstrated that 5-HT1A and 5-HT2A transcripts are highly abundant in the cauda epididymis, whereas 5-HT2C transcript was not found. Altogether, our results reveal that contractions of the CE duct to 5-HT encompasses at least activation of α 1-ARs and 5-HT1A and 5-HT2A receptors, providing new insights into the roles of 5-HT on the epididymal function.

Noradrenaline, oxymetazoline and phorbol myristate acetate induce distinct functional actions and phosphorylation patterns of α1A-adrenergic receptors.

In LNCaP cells that stably express α1A-adrenergic receptors, oxymetazoline increased intracellular calcium and receptor phosphorylation, however, this agonist was a weak partial agonist, as compared to noradrenaline, for calcium signaling. Interestingly, oxymetazoline-induced receptor internalization and desensitization displayed greater effects than those induced by noradrenaline. Phorbol myristate acetate induced modest receptor internalization and minimal desensitization. α1A-Adrenergic receptor interaction with ß-arrestins (colocalization/coimmunoprecipitation) was induced by noradrenaline and oxymetazoline and, to a lesser extent, by phorbol myristate acetate. Oxymetazoline was more potent and effective than noradrenaline in inducing ERK 1/2 phosphorylation. Mass spectrometric analysis of immunopurified α1A-adrenergic receptors from cells treated with adrenergic agonists and the phorbol ester clearly showed that phosphorylated residues were present both at the third intracellular loop and at the carboxyl tail. Distinct phosphorylation patterns were observed under the different conditions. The phosphorylated residues were: a) Baseline and all treatments: T233; b) noradrenaline: S220, S227, S229, S246, S250, S389; c) oxymetazoline: S227, S246, S381, T384, S389; and d) phorbol myristate acetate: S246, S250, S258, S351, S352, S401, S402, S407, T411, S413, T451. Our novel data, describing the α1A-AR phosphorylation sites, suggest that the observed different phosphorylation patterns may participate in defining adrenoceptor localization and action, under the different conditions examined.

Contraction of Rat Cauda Epididymis Smooth Muscle to α1-Adrenoceptor Activation Is Mediated by α1A-Adrenoceptors.

The cauda epididymis (CE), the site of sperm storage until the ejaculation, is densely innervated by the sympathetic nervous system. Contraction of CE smooth muscle via α1-adrenoceptors (α1-ARs) plays a key role during the seminal emission phase of ejaculation and α1-AR antagonism has been suggested as a nonhormonal and reversible male contraceptive target. Since the α1-AR subtype mediating contraction of rat CE is not known, this study investigates the expression and role of α1-AR subtypes on the proximal and distal rat CE duct contraction to norepinephrine in vitro. Alpha1a, α1b, and α1d transcripts were detected by real-time quantitative polymerase chain reaction in proximal and distal CE segments and α1a and α1d were shown to predominate over α1b The inhibition of [3H]prazosin specific binding to intact CE segments from proximal and distal CE by RS 100329 and 5-methylurapidil (α1A-selective) and BMY 7378 (α1D-selective) showed that α1A- and α1D-ARs are expressed at similar densities. Norepinephrine-induced contractions of CE were competitively antagonized with high affinity by RS 100329 (pKB ≈ 9.50) and 5-methylurapidil (pKB ≈ 9.0) and with low affinity by BMY 7378 (pKB ≈ 7.0) and the α1B-selective L-765,314 (pA2 < 7.0), suggesting contractions are mediated by α1A-ARs. The clinically used α1A/D-ARs antagonist tamsulosin potently (pA2 ≈ 10.0) inhibited the norepinephrine-induced CE contractions. Altogether, our results show that α1A- and α1D-ARs are expressed in the CE duct and α1A-AR is the main subtype mediating contraction to norepinephrine. Our results highlight the importance of α1A-AR in the peripheral control of ejaculation and strengthen the α1A-AR as a target for a nonhormonal approach to male contraception.

Cardiac hyporesponsiveness in severe sepsis is associated with nitric oxide-dependent activation of G protein receptor kinase.

G protein-coupled receptor kinase isoform 2 (GRK2) has a critical role in physiological and pharmacological responses to endogenous and exogenous substances. Sepsis causes an important cardiovascular dysfunction in which nitric oxide (NO) has a relevant role. The present study aimed to assess the putative effect of inducible NO synthase (NOS2)-derived NO on the activity of GRK2 in the context of septic cardiac dysfunction. C57BL/6 mice were submitted to severe septic injury by cecal ligation and puncture (CLP). Heart function was assessed by isolated and perfused heart, echocardiography, and ß-adrenergic receptor binding. GRK2 was determined by immunofluorescence and Western blot analysis in the heart and isolated cardiac myocytes. Sepsis increased NOS2 expression in the heart, increased plasma nitrite + nitrate levels, and reduced isoproterenol-induced isolated ventricle contraction, whole heart tension development, and ß-adrenergic receptor density. Treatment with 1400W or with GRK2 inhibitor prevented CLP-induced cardiac hyporesponsiveness 12 and 24 h after CLP. Increased labeling of total and phosphorylated GRK2 was detected in hearts after CLP. With treatment of 1400W or in hearts taken from septic NOS2 knockout mice, the activation of GRK2 was reduced. 1400W or GRK2 inhibitor reduced mortality, improved echocardiographic cardiac parameters, and prevented organ damage. Therefore, during sepsis, NOS2-derived NO increases GRK2, which leads to a reduction in ß-adrenergic receptor density, contributing to the heart dysfunction. Isolated cardiac myocyte data indicate that NO acts through the soluble guanylyl cyclase/cGMP/PKG pathway. GRK2 inhibition may be a potential therapeutic target in sepsis-induced cardiac dysfunction.NEW & NOTEWORTHY The main novelty presented here is to show that septic shock induces cardiac hyporesponsiveness to isoproterenol by a mechanism dependent on nitric oxide and mediated by G protein-coupled receptor kinase isoform 2. Therefore, G protein-coupled receptor kinase isoform 2 inhibition may be a potential therapeutic target in sepsis-induced cardiac dysfunction.

Implication of Rho-kinase and soluble guanylyl cyclase enzymes in prostate smooth muscle dysfunction in middle-aged rats.

AIMS: Aging is highly associated with benign prostate hyperplasia (BPH). We investigated here the alterations of the contractile and relaxant machinery in prostates of middle-aged rats, focusing on the Rho-kinase, nitric oxide (NO)-soluble guanylyl cyclase (sGC), α1- and ß-adrenoceptor pathways. METHODS: Male Wistar young (3.5-month old) and middle-aged rats (10-month old) were used. Quantitative image analysis of prostates and functional assays evaluating the prostate contractions and relaxations were employed. Measurement of [3 H]-noradrenaline efflux, western blotting for α1 and ß1 sGC subunits, and cyclic nucleotide levels were carried out. RESULTS: Prostates of middle-aged rats showed significant increases in lumen and smooth muscle cells, but no alterations in the relative prostate weight were observed. In vivo, noradrenaline (10-7 -10-4 g/kg) produced greater prostatic contractions in middle-aged compared with control rats. Likewise, the in vitro contractions to phenylephrine (1 nM-100 µM) and α,ß-methylene ATP (1-10 µM) were greater in middle-aged rats. Electrical-field stimulation (EFS, 1-32 Hz) promoted higher [3 H]-noradrenaline efflux and prostate contractions in middle-aged rats. Reduced expressions of α1 and ß1 sGC subunits and diminished NO-mediated prostate relaxations in middle-age were observed. Isoproterenol-induced relaxations and cAMP levels were reduced in prostates of middle-aged rats. The Rho-kinase inhibitor fasudil (50 mg/kg, 2 weeks) normalized the prostate hypercontractility in middle-age rats. CONCLUSIONS: Prostate hypercontractility in middle-aging is associated with increased release of noradrenaline and Rho-kinase pathway, as well as with impairments of NO-sGC and ß-adrenoceptor pathways. Middle-aged rats are suitable to explore the enhanced prostatic tone in the absence of prostate overgrowth. Neurourol. Urodynam. 36:589-596, 2017. © 2016 Wiley Periodicals, Inc.

Reproductive disorders in female rats after prenatal exposure to betamethasone.

Betamethasone is the drug of choice for antenatal treatment, promoting fetal lung maturation and decreasing mortality. Previous studies in rats reported male programming and alteration in sperm parameters and sexual behavior following intrauterine betamethasone exposure. The impact on the female reproductive development is not known. In this study, rat female offspring was assessed for sexual development, morphophysiology of the reproductive tract and fertility after maternal exposure to 0.1 mg kg-1 of betamethasone or vehicle on gestational days 12, 13, 18 and 19. The treatment promoted reduction of litter weight on postnatal day 1, morphological masculinization in females, delay in the age of puberty onset, reduction in estrus number, increase in estrous cycle length and increase in luteinizing hormone serum levels and uterus weight. The females from the betamethasone group showed an increase of myometrial uterine area and decrease in endometrial uterine area. These animals also performed less lordosis during the sexual behavior test and showed impaired reproductive performance. The uterus showed higher contraction in the treated group as shown by a pharmacological assay. In conclusion, prenatal betamethasone exposure in rats promoted female masculinization, altered sexual development and reproductive parameters. Copyright © 2017 John Wiley & Sons, Ltd.

A Latin American Perspective on G Protein-Coupled Receptors.

G protein-coupled receptors are sensors that interact with a large variety of elements, including photons, ions, and large proteins. Not surprisingly, these receptors participate in the numerous normal physiologic processes that we refer to as health and in its perturbations that constitute disease. It has been estimated that a large percentage of drugs currently used in therapeutics target these proteins, and this percentage is larger when illegal drugs are included. The state of the art in this field can be defined with the oxymoron "constant change," and enormous progress has been made in recent years. A group of scientists working in Latin America were invited to contribute minireviews for this special section to present some of the work performed in this geographical region and foster further international collaboration.

Recent updates on GPCR biased agonism.

G protein-coupled receptors (GPCRs) are the most important targets for drug discovery and not surprisingly ∼40% of all drugs currently in the market act on these receptors. Currently, one of the most active areas in GPCRs signaling is biased agonism, a phenomenon that occurs when a given ligand is able to preferentially activate one (or some) of the possible signaling pathways. In this review, we highlight the most recent findings about biased agonism, including an extension of this concept to intracellular signaling, allosterism, strategies for assessment and interpretation, and perspectives of therapeutic applications for biased agonists.

In Vitro Contraction of Isolated Cauda Epididymal Duct Smooth Muscle as a Complimentary Approach to Physiological, Pathological, Toxicological, and Pharmacological Studies on Epididymal Function.

Contraction of cauda epididymal duct (CE) smooth muscle is one of the very first events of the seminal emission phase of ejaculation. The contraction of CE smooth muscle is governed by a complex interaction of hormones, autacoids, and by the neurotransmitters released from the epididymal intramural nerve endings, and any impairment in the CE smooth muscle contraction has the potential to impair male fertility. Apart the obvious pathophysiological and toxicological importance of CE smooth muscle contraction, modulation of CE contraction has pharmaceutical interest offering a druggable target to development of drugs to improve/impair male fertility. The in vitro contraction experiments constitute a valuable approach to an in-depth evaluation of functional and molecular changes resulting from pathologies or drug exposure. Therefore, this chapter consists in a description of in vitro pharmacological reactivity contractility of the epididymal duct in a controlled medium, maintained at 30 °C of temperature and continuously bubbled with 95% O2 and 5% CO2 to obtain cumulative concentration-response curves that has been fundamental to some of our investigations on epididymal physiology, toxicology, and pharmacology.

The affinity and selectivity of α-adrenoceptor antagonists, antidepressants, and antipsychotics for the human α1A, α1B, and α1D-adrenoceptors.

α1-adrenoceptor antagonists are widely used for hypertension (eg, doxazosin) and benign prostatic hypertrophy (BPH, eg, tamsulosin). Some antidepressants and antipsychotics have been reported to have α1 affinity. This study examined 101 clinical drugs and laboratory compounds to build a comprehensive understanding of α1-adrenoceptor subtype affinity and selectivity. [3H]prazosin whole-cell binding was conducted in CHO cells stably expressing either the full-length human α1A, α1B, or α1D-adrenoceptor. As expected, doxazosin was a high-affinity nonselective α1-antagonist although other compounds (eg, cyclazosin, 3-MPPI, and ARC239) had higher affinities. Several highly α1A-selective antagonists were confirmed (SNAP5089 had over 1700-fold α1A selectivity). Despite all compounds demonstrating α1 affinity, only BMY7378 had α1D selectivity and no α1B-selective compounds were identified. Phenoxybenzamine (used in pheochromocytoma) and dibenamine had two-component-binding inhibition curves at all three receptors. Incubation with sodium thiosulfate abolished the high-affinity component suggesting this part is receptor mediated. Drugs used for hypertension and BPH had very similar α1A/α1B/α1D-adrenoceptor pharmacological profiles. Selective serotonin reuptake inhibitors (antidepressants) had poor α1-adrenoceptor affinity. Several tricyclic antidepressants (eg, amitriptyline) and antipsychotics (eg, chlorpromazine and risperidone) had high α1-adrenoceptor affinities, similar to, or higher than, α blockers prescribed for hypertension and BPH, whereas others had poor α1 affinity (eg, protriptyline, sulpiride, amisulpiride, and olanzapine). The addition of α blockers for the management of hypertension or BPH in people already taking tricyclic antidepressants and certain antipsychotics may not be beneficial. Awareness of the α-blocking potential of different antipsychotics may affect the choice of drug for those with delirium where additional hypotension (eg, in sepsis) may be detrimental.

Multiple effects of sibutramine on ejaculation and on vas deferens and seminal vesicle contractility.

Sibutramine is an inhibitor of norepinephrine and 5-HT reuptake largely used in the management of obesity. Although a fairly safe drug, postmarketing adverse effects of sibutramine were reported including abnormal ejaculation in men. This study investigates the effects of sibutramine on ejaculation and vas deferens and seminal vesicle contractility. Adult male rats received sibutramine (5; 20; or 50 mg kg(-1), ip) and after 60 min were exposed to receptive females for determination of ejaculation parameters. The vasa deferentia and seminal vesicles of untreated rats were mounted in isolated organ baths for recording of isometric contractions and HEK293 cells loaded with fluorescent calcium indicator were used to measure intracellular Ca(2+) transients. Sibutramine 5 and 20 mg kg(-1) reduced ejaculation latency whereas 50 mg kg(-1) increased ejaculation latency. Sibutramine 3 to 30 microM greatly increased the sensitivity of the seminal vesicle and vas deferens to norepinephrine, but at concentrations higher than 10 microM there were striking depressions of maximal contractions induced by norepinephrine, carbachol and CaCl(2). In HEK293 cells, sibutramine 10 to 100 microM inhibited intracellular Ca(2+) transients induced by carbachol. Depending on the doses, sibutramine either facilitates or inhibits ejaculation. Apart from its actions in the central nervous system, facilitation of ejaculation may result from augmented sensitivity of smooth muscles to norepinephrine while reductions of intracellular Ca(2+) may be involved in the delayed ejaculation observed with high doses of sibutramine.

Mirabegron elicits rat corpus cavernosum relaxation and increases in vivo erectile response.

Mirabegron is the first ß3-adrenoceptor agonist approved on the market and may offer beneficial pharmacological action in patients with overactive bladder and erectile dysfunction. Here, we further investigate the mechanisms by which mirabegron induces rat corpus cavernosum (CC) relaxation. Adult male Wistar rats were used. The CC were isolated for in vitro functional assays and ß-adrenoceptors subtypes mRNA expression evaluation. Animals were treated orally with mirabegron (30 mg/kg, 3 h), tadalafil (10 mg/kg, 3 h) or both for intracavernous pressure (ICP). Intracellular levels of cAMP and cGMP were also determined. The ß1-, ß2- and ß3-adrenoceptors subtypes were expressed in rat CC. Mirabegron produced concentration-dependent CC relaxations that were unaffected by the ß1-, ß2- or ß3-adrenoceptor antagonists atenolol (1 µM), ICI-118,551 (1 µM) and L748,337 (10 µM), respectively. Mirabegron-induced relaxations were not affected by the phosphodiesterase type 4 inhibitor, rolipram, or the adenylyl cyclase selective inhibitor, SQ 22,536. Potassium channel- or calcium influx-blockade are not involved in mirabegron-induced relaxations. In contrast, mirabegron produced rightward shifts in the contractile response induced by the α1-adrenoceptor agonist, phenylephrine. Finally, cavernous nerve stimulation caused frequency-dependent ICP increases, which were significantly increased in rats treated with mirabegron in a similar degree of tadalafil-treated rat, without promoting a significant cAMP or cGMP accumulation. Together, our results demonstrate that mirabegron induced CC relaxation through α1-adrenoceptor blockade. Care should be taken to translate the effect of mirabegron into the clinic, especially when using rat as an animal model of erectile dysfunction.

alpha1-Adrenoceptors in proximal segments of tail arteries from control and reserpinised rats.

It has been recently shown that the supersensitivity of distal segments of the rat tail artery to phenylephrine after chemical sympathectomy with reserpine results from the appearance of alpha(1D)-adrenoceptors. It is known that both alpha(1A)- and alpha(1D)-adrenoceptors are involved in the contractions of proximal portions of the rat tail artery. Therefore, this study investigated whether sympathectomy with reserpine would induce supersensitivity in proximal segments of the rat tail artery, a tissue in which alpha(1D)-adrenoceptors are already functional. Proximal segments of tail arteries from reserpinised rats were three- to sixfold more sensitive to phenylephrine and methoxamine than were arteries from control rats (n = 6-2; p < 0.05). The imidazolines N-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide hydrobromide (A-61603) and oxymetazoline, which activate selectively alpha(1A)-adrenoceptors, were equipotent in tail arteries from control and reserpinised rats (n = 4-2; p < 0.05), whereas buspirone, which activates selectively alpha(1D)-adrenoceptor, was approximately 4-fold more potent in tail arteries from reserpinised rats (n = 4-6; p < 0.05). Prazosin (nonselective) and 5-methylurapidil (alpha(1A)-selective), were competitive antagonists of contractions induced by phenylephrine and were equipotent in tail arteries from control and reserpinised rats (n = 4-6). The selective alpha(1D)-adrenoceptor antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) presented similar complex antagonism in tail arteries from control and reserpinised rats, with Schild slopes much lower than 1.0 (p < 0.05, n = 4-6). Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) revealed that mRNA encoding alpha(1A)-and alpha(1B)-adrenoceptors are similarly distributed in tail arteries from control and reserpinised rats, whereas mRNA for alpha(1D)-adrenoceptors is twice more abundant in the tail artery from reserpinised rats. In conclusion, the supersensitivity induced by reserpine is related only to alpha(1D)-adrenoceptors, even in tissues where this receptor subtype is already present and functional. Only the use of subtype-selective alpha(1)-adrenoceptor agonists detected the increased alpha(1D)-adrenoceptor component after reserpinisation, as the antagonists behaved similarly in tail arteries from control and reserpinised rats.

Long-term adverse effects on reproductive function in male rats exposed prenatally to the glucocorticoid betamethasone.

Betamethasone is the drug of choice for antenatal treatment, promoting fetal lung maturation, decreasing the incidence of respiratory distress syndrome and neonatal mortality. Previous studies reported that prenatal treatment with this drug reduced testosterone levels, sperm quality and fertility in adult rats. We aimed to further evaluate the reproductive consequences of prenatal betamethasone exposure in male rats. Pregnant Wistar rats (n=13/group) were separated into two groups: control (vehicle) and betamethasone- treated (0.1mg/kg IM) and rats were injected on gestational days 12, 13, 18 and 19. Body weight, sexual behavior, reproductive organ weights, serum hormone levels, accessory glands contractility, sperm parameters, and fertility after in utero artificial insemination were evaluated. Our results showed that prenatal betamethasone exposure provoked a significant reduction in body weight at PND 01 and, at adulthood, decrease in FSH levels, sperm motility and production. Furthermore, seminal vesicle weight was decreased while testicular and ventral prostate weights were increased. Serum LH levels and the percentage of abnormal sperm were significantly increased. Although sexual behavior was not altered, a significant reduction in fertility in the adult rats exposed prenatally to betamethasone was noted. We concluded that prenatal betamethasone exposure leads to long-term reproductive impairment in male rats. These results may have important implications for humans, considering the use of this glucocorticoid in pregnant women.

Differential antagonism by conotoxin rho-TIA of contractions mediated by distinct alpha1-adrenoceptor subtypes in rat vas deferens, spleen and aorta.

The ability of the conotoxin rho-TIA, a 19-amino acid peptide isolated from the marine snail Conus tulipa, to antagonize contractions induced by noradrenaline through activation of alpha1A-adrenoceptors in rat vas deferens, alpha1B-adrenoceptors in rat spleen and alpha1D-adrenoceptors in rat aorta, and to inhibit the binding of [125I]HEAT (2-[[beta-(4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) to membranes of human embryonic kidney (HEK) 293 cells expressing each of the recombinant rat alpha1-adrenoceptors was investigated. rho-TIA (100 nM to 1 microM) antagonized the contractions of vas deferens and aorta in response to noradrenaline without affecting maximal effects and with similar potencies (pA2 approximately 7.2, n=4). This suggests that rho-TIA is a competitive antagonist of alpha1A- and alpha1D-adrenoceptors with no selectivity between these subtypes. Incubation of rho-TIA (30 to 300 nM) with rat spleen caused a significant reduction of the maximal response to noradrenaline, suggesting that rho-TIA is a non-competitive antagonist at alpha1B-adrenoceptors. After receptor inactivation with phenoxybenzamine, the potency of rho-TIA in inhibiting contractions was examined with similar occupancies (approximately 25%) at each subtype. Its potency (pIC50) was 12 times higher in spleen (8.3+/-0.1, n=4) than in vas deferens (7.2+/-0.1, n=4) or aorta (7.2+/-0.1, n=4). In radioligand binding assays, rho-TIA decreased the number of binding sites (B(max)) in membranes from HEK293 cells expressing the rat alpha1B-adrenoceptors without affecting affinity (K(D)). In contrast, in HEK293 cells expressing rat alpha1A- or alpha1D-adrenoceptors, rho-TIA decreased the K(D) without affecting the B(max). It is concluded that rho-TIA will be useful for distinguishing the role of particular alpha1-adrenoceptor subtypes in native tissues.

Involvement of α1B-adrenoceptors in the anti-immobility effect of imipramine in the tail suspension test.

Imipramine is a tricyclic antidepressant inhibitor of norepinephrine and serotonin neuronal reuptake. The roles of specific α1-adrenoceptor subtypes that might be targeted by the increased synaptic levels of noradrenaline induced by imipramine are not well understood. This study investigates the α1-adrenoceptor subtypes involved in the anti-immobility effect of imipramine in the mouse tail suspension test. The anti-immobility effect of imipramine (32mg/kg, i.p.) was significantly antagonised by the non-subtype-selective α1-adrenoceptor antagonist prazosin (0.5 and 1.0mg/kg, i.p.). Neither the selective α1A-adrenoceptor antagonist 5-methyl-3-[3-[3-[4-[2-(2,2,2,-trifluroethoxy)phenyl]-1-piperazinyl]propyl]-2,4-(1H,3H)-pyrimidinedione (RS-100329, 0.5 and 1.0mg/kg) nor the selective α1D-adrenoceptor antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride, (BMY-7378, up to 1.0mg/kg, i.p.) affected the anti-immobility effect of imipramine. However, the anti-immobility effect of imipramine was significantly antagonised by the selective α1B-adrenoceptor antagonist (2S)-4-(4-amino-6,7-dimethoxy-2-quinazolinyl)-2-[[(1,1-dimethylethyl)amino]carbonyl]-1-piperazinecarboxylate (L-765,314). In addition, mice treated only with RS-100329 or BMY-7378, but not with L-765,314, showed reduced immobility times in comparison to mice treated with vehicle. These results indicate that the selective antagonism of α1A- and α1D-adrenoceptors results in antidepressant-like effects and that the α1B-subtype is the main target for the increased levels of noradrenaline caused by imipramine.

N-terminal truncation of human alpha1D-adrenoceptors increases expression of binding sites but not protein.

The role of the N-terminus of human alpha(1D)-adrenoceptors was examined by deleting the first 79 amino acids (Delta(1-79)) and epitope-tagging to facilitate immunoprecipitation and detection. Following transfection into HEK293 cells, 6- to 13-fold increases in the density of specific [125I]BE 2254 binding sites were observed for both tagged and untagged Delta(1-79)alpha(1D)- compared to full-length alpha(1D)-adrenoceptors, while agonist and antagonist affinities remained unchanged. In contrast, immunoprecipitation of tagged receptors showed that full-length alpha(1D)-adrenoceptor protein was at least twice as abundant as Delta(1-79)alpha(1D)-adrenoceptor protein. Photoaffinity labelling with [125I]arylazidoprazosin showed much more intense labelling of tagged Delta(1-79)alpha(1D)- than of full-length alpha(1D)-adrenoceptors. Substantial N-linked glycosylation of tagged Delta(1-79)alpha(1D)-adrenoceptors was observed, although full-length alpha(1D)-adrenoceptors contain two consensus glycosylation sites but are not glycosylated. These results suggest that N-terminal truncation of alpha(1D)-adrenoceptors enhances processing of a binding competent form in HEK293 cells; and show a clear dissociation between abundance of receptor protein and density of receptor binding sites.

Effects of castration and of testosterone replacement on alpha(1)-adrenoceptor subtypes in the rat vas deferens.

The contractions of the rat vas deferens in response to noradrenaline are mediated through alpha(1A)-adrenoceptors. We observed participation of alpha(1B)-adrenoceptors in these contractions after castration. We now investigated the time course of this plasticity and the effects of testosterone by determining the actions of competitive antagonists on noradrenaline-induced contractions after 7, 14, 21 and 30 days of castration. BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride) antagonised noradrenaline-induced contractions in control and castrated rats with low pA(2) values (approximately = 6.8). In control vas deferens, WB 4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) had a slope in the Schild plot no different from 1.0, while slopes lower than 1.0 (approximately 0.6) were observed for vas deferens from castrated rats. Chloroethylclonidine was ineffective in the control vas while it inhibited noradrenaline-induced contractions in vasa from castrated rats and converted the complex antagonism by WB 4101 into simple competitive antagonism. Treatment of castrated rats with testosterone prevented the effects of castration. The results suggest that alpha(1B)-adrenoceptors are detectable in vas deferens from at least the 7th through the 30th day after castration and that testosterone prevents this plasticity.

Functional characterisation of alpha(1)-adrenoceptors in denervated rat vas deferens.

We investigated whether or not surgical denervation of the rat vas deferens changes the alpha(1)-adrenoceptor subtypes involved in the contractions to noradrenaline. Denervated vas deferens was approximately 22 times more sensitive to noradrenaline (pD(2)=7.35+/-0.04) than control vas (pD(2)=6.01+/-0.03). This difference in noradrenaline potency was eliminated when cocaine (6 microM) was added to control vas (pD(2)=7.22+/-0.04). The noradrenaline-induced contractions of control and denervated vas deferens were insensitive to the alpha(1B)/alpha(1D)-adrenoceptor alkylating agent chloroethylclonidine (100 microM, 45 min). The concentration-response curves to noradrenaline in control and denervated vas deferens were competitively antagonised by prazosin (pA(2) approximately equal to 9.6), WB-4101 (pA(2) approximately equal to 9.5), 5-methyl urapidil (pA(2) approximately equal to 8.4), phentolamine (pA(2) approximately equal to 8.7), yohimbine (pA(2) approximately equal to 6.9), BMY 7378 (pA(2) approximately equal to 6.9) and indoramin (pA(2) approximately equal to 8.7). After the treatment of control and denervated vas deferens with phenoxybenzamine, the partial agonist oxymetazoline antagonised competitively the concentration-response curves to noradrenaline showing pA(2) values approximately 7.4 in both groups. We conclude that noradrenaline-induced contractions in control and denervated rat vas deferens are mediated by alpha(1A)-adrenoceptors and that surgical denervation of the rat vas deferens is not able to change the alpha(1)-adrenoceptor subtypes involved in the contractions to noradrenaline.

Effects of castration on alpha 1-adrenoceptor subtypes in the rat aorta.

The expression of alpha 1-adrenoceptor subtypes in several tissues is regulated by gonadal hormones. In this study, we investigated whether castration regulates the alpha 1-adrenoceptor subtypes mediating the contractions of the aorta from male rats to noradrenaline. Noradrenaline induced similar concentration-dependent contractions in the aorta from control and castrated rats. Treatment of the aorta from both control and castrated rats with the alpha 1B/alpha 1D-adrenoceptor alkylating agent chloroethylclonidine resulted in approximately 1600-fold rightward shift in the concentration-response curves to noradrenaline. The pA2 values found for WB 4101, benoxathian (alpha 1A-selective) and BMY 7378 (alpha 1D-selective) indicate that alpha 1D-adrenoceptors are involved in the contractions of the aorta from control and castrated rats to noradrenaline. However, there was a 15-fold difference between the pKB estimated through the lowest effective concentrations of the alpha 1A-adrenoceptor selective antagonist 5-methyl-urapidil in the aorta from control and castrated rats. The pKB estimated in aorta from control rats is consistent with the interaction with alpha 1D-adrenoceptors (7.58 +/- 0.06), while that calculated in organs from control rats is consistent with alpha 1A-adrenoceptors (8.76 +/- 0.09). These results suggest that castration induces plasticity in the alpha 1-adrenoceptor subtypes involved in the contractions of the aorta to noradrenaline.