Postsynaptic alpha adrenoceptors on vascular smooth muscle

A heterogeneous population of alpha adrenoceptors mediates vasoconstriction in the canine saphenous vein (CSV). Studies with isolated strips of venous smooth muscle incubated with selective alpha-adrenoceptor agonists and antagonists revealed that both alpha 1 and alpha 2 adrenoceptors exist independently in this tissue and both subtypes mediate a contractile response. Measurement of contractile responses in reduced or zero external calcium conditions indicates that stimulation of alpha 1 adrenoceptors induces contractions by influx of extracellular calcium and release of calcium from internal stores. In contrast, 45Ca uptake studies suggest that activation of the postsynaptic alpha 2 adrenoceptor produces vasoconstriction dependent only on influx of extracellular calcium. The influx of calcium produced by the selective alpha 2-adrenoceptor agonist BHT-920 is inhibited by calcium entry blockers. Measurements of transmembrane potentials from smooth muscle cells of the CSV suggest that alpha 1-adrenoceptor activation produces depolarization and contraction (electromechanical coupling) whereas alpha 2-adrenoceptor stimulation does not result in concentration-dependent depolarization of the smooth muscle cells (pharmacomechanical coupling).     

Ligand design for alpha1 adrenoceptor subtype selective antagonists

Alpha1 adrenoceptors have three subtypes and drugs interacting selectively with these subtypes could be useful in the treatment of a variety of diseases. In order to gain an insight into the structural principles governing subtype selectivity, ligand based drug design (pharmacophore development) methods have been used to design a novel 1,2,3-thiadiazole ring D analogue of the aporphine system. Synthesis and testing of this compound as a ligand on cloned and expressed human alpha1 adrenoceptors is described. Low binding affinity was found, possibly due to an unfavourable electrostatic potential distribution. Pharmacophore models for antagonists at the three adrenoceptor sites (alpha1A, alpha1B, alpha1D) were generated from a number of different training sets and their value for the design of new selective antagonists discussed. The first preliminary antagonist pharmacophore model for the alpha1D adrenoceptor subtype is also reported.     

Design, synthesis and biological evaluation of pyridine-phenylpiperazines: a novel series of potent and selective alpha1a-adrenergic receptor antagonist

Beginning from the screening hit and literature alpha1-adrenergic compounds, a hybridized basic skeleton A was proposed as the pharmacophore for potent and selective alpha1a-AR antagonists. Introduction of a hydroxy group to increase the flexibility afforded B which served as the screening model and resulted in the identification of the second-generation lead 1. Using the Topliss approach, a number of potent and selective alpha1a-AR antagonists were discovered. In all cases, binding affinity and selectivity at the alpha1a-AR of S-hydroxy enantiomers were higher than the R-hydroxy enantiomers. As compared to the des-hydroxy analogues, the S-hydroxy enantiomers displayed comparable potency and better selectivity at alpha1a-AR. The S-hydroxy enantiomer 17 (Ki = 0.79 nM; alpha1b/alpha1a = 800; alpha1d/alpha1a = 104) was slightly less potent but much more selective at alpha1a-AR than tamsulosin (Ki = 0.13 nM, alpha1b/alpha1a = 15, alpha1d/alpha1a = 1.4). Compound 17 displayed higher selectivity in inhibiting rat prostate contraction over rat aorta contraction and also exhibited a higher degree of uroselectivity than tamsulosin in the anesthetized dog model.     

Aminocyclohexylsulfonamides: discovery of metabolically stable alpha(1a/1d)-selective adrenergic receptor antagonists for the treatment of benign prostatic hyperplasia/lower urinary tract symptoms (BPH/LUTS)

Benign prostatic hyperplasia/lower urinary tract symptoms (BPH/LUTS) can be effectively treated by alpha(1) adrenergic receptor antagonists, but these drugs also produce side effects that are related to their subtype non-selective nature. To overcome this limitation, it was hypothesized that an alpha(1a/1d) subtype-selective antagonist would be efficacious while keeping side effects to a minimum. To discover alpha(1a/1d)-selective antagonists and improve metabolic stability of our previously reported compounds, we have designed and synthesized a series of (phenylpiperazinyl)- or (phenylpiperidinyl)-cyclohexylsulfonamides. By incorporating the information obtained from metabolism studies, we were able to discover several compounds that are both alpha(1a/1d) adrenoceptor subtype selective and show increased stability toward human liver microsomal metabolism. The selectivity profile of these compounds provides great improvement over the commercial drug tamsulosin, hence may pave the way to the development of new and efficacious therapeutic agents with reduced side effects.     

Adrenergic pharmacology of human and canine peripheral veins

A comparison has been made of the factors concerned with the response of canine and human saphenous veins to adrenergic stimulation. Both vessels have prejunctional muscarinic and beta-adrenergic receptors. When activated by appropriate agonists these receptors decrease and increase the output, respectively, of norepinephrine from the nerve endings. Both vessels have postjunctional alpha 1 and alpha 2 adrenoceptors and postjunctional beta adrenoceptors. Activation of the former two receptors leads to contraction of the smooth muscle, and of the latter to relaxation. There are, however, qualitative differences. In the human veins the responsiveness of the prejunctional beta adrenoceptors exceeds that of the postjunctional, whereas the reverse is true in the dog. As a consequence, in the human vein beta-adrenergic agonists augment, and in the canine veins they depress, the contractile response to sympathetic nerve stimulation.     

Functional role of PKC in contraction of cultured human prostatic stromal cells

The contractile activity of prostatic stromal cells contributes to symptoms of benign prostatic hyperplasia (BPH). However, the mechanisms for this contraction have not yet been fully elucidated. In this study, we investigated the role of protein kinase C (PKC) in prostatic contraction by measuring the isometric tension development of cultured human prostatic stromal cells (CHPSCs) derived from BPH patients. Fresh human BPH tissue was used only in a Western blot analysis. A ring preparation made of CHPSCs and collagen gel could develop an isometric tension during activation with various agonists. Phorbol 12,13 dibutyrate (PDBu), a PKC activator, induced a relaxation. A Western blot analysis revealed the expression of PKC-potentiated protein phosphatase-1 inhibitory protein (CPI-17) in both CHPSCs and fresh human BPH tissue to be much lower than that in the rabbit aorta. When CPI-17 was over-expressed, PDBu induced a large contraction, but the agonist-induced contraction did not become larger than expected. In alpha-toxin permeabilized preparations, PDBu induced a relaxation in control CHPSCs, while it induced a contraction at a constant [Ca2+]i in CPI-17 over-expressing CHPSCs. These results indicated that the activation of PKC in CHPSCs induces a relaxation probably due to low expression level of CPI-17 and also that the PKC-CPI-17 pathway does not appear to play a major role in the agonist-induced contraction even when CPI-17 was over-expressed.     

Noradrenergic agonists and antagonists: effects on avoidance behaviour in rats

The effects of various agonists and antagonists of both alpha and beta adrenoceptors on the acquisition of avoidance behaviour were investigated in the rat. Clonidine, a selective agonist of alpha 2 adrenoceptors depressed avoidance acquisition whilst yohimbine, an antagonist of these receptors produced an opposite effect. Prazosin which showed postsynaptic alpha 1 adrenoceptor blocking activity reduced avoidance behaviour. A similar effect was produced by propranolol, a non-selective antagonist of beta adrenoceptors. On the other hand, salbutamol preferentially stimulating beta 2 adrenoceptors facilitated avoidance behaviour. In general, the results show a fairly good correlation between avoidance acquisition and efficacy of noradrenergic neurotransmission.     

Neurogenic dilatation and constriction of rat superior mesenteric artery in vitro: mechanisms and mediators

In the rat superior mesenteric arteries, the mechanical responses to perivascular nerve stimulation were characterized. The predominant response was contraction mediated by the release of norepinephrine, acting postjunctionally on alpha 1-adrenoceptors. These frequency-dependent contractions were unaffected by the alpha 2-selective adrenoceptor antagonist yohimbine, but were markedly attenuated by clonidine, the alpha 2-selective adrenoceptor agonist. In the presence of prazosin, the alpha 1-selective antagonist, a significant component of the nerve-mediated contraction was still present. At the concentrations used, prazosin, yohimbine, as well as clonidine acted as competitive antagonists of response to exogenous norepinephrine. This differential inhibition of norepinephrine- and nerve-mediated responses suggested the presence of distinct postjunctional adrenoceptors. The effects of clonidine and yohimbine are interpreted to arise from prejunctional modulation of norepinephrine release. In 30 of the 100 vessels studied, there was spontaneous myogenic tone. In these arteries, field stimulation caused frequency- and voltage-dependent relaxations. These responses were neural in origin, dependent on sympathetic nerve activity, but were nonadrenergic and noncholinergic in nature. Naloxone, indomethacin, and substance P inhibited these relaxations with no significant effect on the tone. The opioid agonist, 1-13 dynorphin relaxed these vessels and only naloxone inhibited this response. The effects of these agents were selective against field-stimulated responses since they did not alter the relaxation to the nonspecific agent sodium nitroprusside. These results provide circumstantial evidence for opioid-mediated vascular relaxation that is presynaptically modulated by prostanoids and substance P.     

Binding of [3H] methyltrienolone (R 1881) in rat prostate and human benign prostatic hypertrophy (BPH).

Methyltrienolone (R 1881 - 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) binding to rat ventral prostate cytosol has a specificity typical of an androgen receptor. In human benign prostatic hypertrophy (BPH) tissue, the specificity of [3H] R 1881 binding is different from that measured in rat prostate: progesterone and R 5020 (17, 21-dimethyl-19-nor-4, 9-pregnadiene-3, 20-dione) being more potent while 19-nortestosterone is less potent competitor. Moreover, the synthetic progestin [3H] R 5020 binds to BPH tissue with a similar specificity. These data suggest the presence of progestin binding components or of an atypical androgen receptor in human BPH cytosol.     

Adrenergic regulation of adipocyte metabolism

Adipocytes can be readily isolated from intact adipose tissue. In adipocytes from hamster and human white adipose tissue it is possible to demonstrate beta, alpha 1, and alpha 2 adrenoceptors. Alpha 2 adrenoceptor activation inhibits while beta adrenoceptor activation stimulates cyclic AMP accumulation and lipolysis. The effects of catecholamines on cyclic AMP accumulation are mediated through regulation of adenylate cyclase activity, which is activated through beta adrenoceptors and inhibited through alpha 2 adrenoceptors. Activation of alpha 1 adrenergic receptors has been shown to be associated with elevations of cytosol calcium and increased turnover of phosphatidylinositol. In white adipocytes, the only known alpha 1 adrenergic effects are inhibition of glycogen synthase and stimulation of glycogen phosphorylase via mechanisms distinct from those by which cyclic AMP produces similar end effects. In brown adipocytes, alpha 1 adrenoceptor activation stimulates respiration. Thyroid hormones primarily regulate the sensitivity of adipocytes to beta-adrenergic amines while having little effect on alpha adrenoceptor sensitivity.     

Synthesis,pharmacology and pharmacokinetics of 3-(4-aryl-piperazin-1-ylalkyl)-uracils as uroselective alpha1A-antagonists

Predominance in the urethra and prostate of the alpha(1A)-adrenoceptor subtype, which is believed to be the receptor mediating noradrenaline induced smooth muscle contraction in these tissues, led to the preparation of alpha(1A)-selective antagonists to be tested as uroselective compounds for the treatment of benign prostatic hyperplasia. Thus, a number of selective alpha(1A)-adrenoceptor antagonists were synthesized and assayed in vitro for potency and selectivity. Dog pharmacokinetic parameters of 12 (RO700004) and its metabolite 40 (RO1104253) were established. The relative selectivity of intravenously administered 12, 40 and standard prazosin to inhibit hypogastric nerve stimulation-induced increases in intraurethral prostatic pressure versus phenylephrine-induced increases in diastolic blood pressure in anesthetized dogs was 76, 71 and 0.6, respectively.     

Pharmacological properties of A-204176, a novel and selective alpha1A adrenergic agonist, in in vitro and in vivo models of urethral function.

A-204176 (N-[5-(1H-imidazol-4-y1)-5,6,7,8-tetrahydro-1-naphthalenyl]methanesulfonamide) is a potent and selective alpha1A adrenoceptor agonist that binds with 17-fold and 9-fold greater affinity to the alpha1A (Ki=176 nM) than the alpha1b and alpha1d subtypes, respectively. In functional studies A-204176 is potent (pD2=6.4) and efficacious (83% of maximum control phenylephrine response) at rabbit urethra alpha1A receptors, with weaker potency and greatly reduced efficacy at rat spleen alpha1B (pD2=5.3, 11%) and rat aorta alpha1D (pD2=4.4, 10%) subtypes. In anesthetized female dogs, A-204176 is more potent than the non-selective alpha1 adrenoceptor agonist phenylpropanolamine (PPA) to increase measures of urethral tone and is more efficacious to increase pressure in the proximal region of the urethra. Significant increases on parameters of the urethral pressure profilometry were induced at 100 and 300 nmol/kg, i.v., by A-204176 and PPA, respectively. A-204176 was more potent than PPA to increase the abdominal pressure required to produce leakage. In the simultaneous measurement of intraurethral pressure and mean arterial blood pressure, A-204176 displays enhanced urethral selectivity relative to PPA. However, despite its selectivity for alpha1A versus alpha1B and alpha1D adrenoceptors in vitro, A-204176 did not display the degree of urethral selectivity in vivo that would have been expected. The observed effect of A-204176 on blood pressure may be due to the presence of extra-synaptic alpha1A adrenoceptors in the vasculature or to activation of spinal and supraspinal alpha1A adrenoceptors. These data indicate that A-204176 may represent a useful pharmacological tool to investigate the functional role of the alpha1A adrenoceptor in the urethra and to elucidate the lack of uroselectivity observed in vivo.     

Binding characteristics of naftopidil and alpha 1-adrenoceptor antagonists to prostatic alpha-adrenoceptors in benign prostatic hypertrophy.

Binding properties of naftopidil and alpha 1-adrenoceptor antagonists to alpha-adrenoceptors in prostates from benign prostatic hypertrophy (BPH) were characterized by radioreceptor assays using [3H]prazosin and [3H]rauwolscine. Specific binding of [3H]prazosin and [3H]rauwolscine in human prostatic membranes was saturable and of high affinity, and it showed a pharmacological specificity which characterized alpha 1 and alpha 2-adrenoceptors, respectively. Naftopidil and several alpha 1 antagonists competed for prostatic [3H]prazosin binding in order: R-(-)-YM-12617 greater than prazosin greater than bunazosin greater than terazosin greater than naftopidil greater than urapidil, and the inhibitory effect (Ki = 11.6 nM) of naftopidil was 10 to 45 times less potent than quinazoline derivatives such as prazosin, bunazosin and terazosin. The potencies of these antagonists in competing for [3H]prazosin binding sites in human prostates correlated well with their pharmacological potencies (pA2). Scatchard analysis indicated that the decrease of prostatic [3H]prazosin binding by naftopidil was due to a marked increase in the Kd value without a change in the Bmax value. The inhibition of prostatic [3H]prazosin binding by naftopidil was reversible. Naftopidil also inhibited prostatic [3H]rauwolscine binding (Ki = 70.0 nM). Thus, it is suggested that naftopidil antagonizes alpha 1-adrenoceptors in human prostates in a competitive and reversible manner.     

Existence of two types of postjunctional alpha-adrenoceptors in the isolated canine internal carotid artery

The pharmacological characteristics of postjunctional alpha-adrenoceptors in isolated canine internal carotid arteries were investigated by the use of selective agonists and antagonists for alpha 1- and alpha 2-adrenoceptors. Norepinephrine, phenylephrine, and xylazine caused concentration-dependent contractions in the helical strips. The contraction induced by 10(-4)M xylazine was significantly smaller than that produced by 10(-4)M norepinephrine or 10(-4)M phenylephrine. The contraction induced by 10(-4)M phenylephrine was almost the same value as that induced by 10(-4)M norepinephrine. Phentolamine (10(-8) and 10(-7)M) caused a parallel shift to the right of the concentration-response curve to norepinephrine. The contractile responses to low concentrations of norepinephrine were significantly suppressed by pretreatment with an alpha 2-antagonist such as yohimbine (10(-9) and 10(-8)M) or DG5128 (10(-7) and 10(-6)M). On the other hand, the responses to higher concentrations of norepinephrine were mainly reduced by low concentrations of an alpha 1-antagonist, prazosin (3 x 10(-10) and 3 x 10(-9)M). These results suggest that both alpha 1- and alpha 2-adrenoceptors are located on the plasma membrane of smooth muscle cells in canine internal carotid arteries and that the norepinephrine-induced contractions at low and high concentrations are mainly mediated by activation of alpha 2- and alpha 1-adrenoceptors, respectively.     

Effect of 2 new derivatives of imidazoline and their optical isomers on postsynaptic alpha adrenergic receptors in pithed rats

The effects of the stereoisomers of two alpha adrenoceptor antagonists [S 10089 (Imidazolinyl-2)-2 benzocyclobutane and S 9871 (Imidazolinyl-2)-2 dihydro 2,3 benzofuran] were studied in pithed Rats. Vasoconstriction elicited via stimulation of alpha 1 adrenoceptors by cirazoline was antagonized, stimulation of alpha 2 adrenoceptors by azepexole was also antagonized by all these derivatives except (-) S 9871 which was ineffective on the pressor response of azepexole.     

(Phenylpiperazinyl)cyclohexylureas: discovery of alpha1a/1d-selective adrenergic receptor antagonists for the treatment of benign prostatic hyperplasia/lower urinary tract symptoms (BPH/LUTS)

Benign prostatic hyperplasia/lower urinary tract symptoms (BPH/LUTS) can be effectively treated with alpha(1) adrenergic receptor antagonists. Unfortunately, currently marketed alpha(1) blockers produce CV-related side effects that are caused by the subtype non-selective nature of the drugs. To overcome this problem, it was postulated that an alpha(1a/1d) subtype-selective antagonist would bring more benefit for the treatment of BPH/LUTS. As a continuation of our effort to develop selective alpha(1a/1d) ligands, a series of (phenylpiperazinyl)cyclohexylureas was synthesized and evaluated for the ability to bind to three cloned human alpha(1)-adrenergic receptor subtypes. Several trans isomers were shown to have equal affinity for both alpha(1a), and alpha(1d) subtypes, with 14- to 47-fold selectivity versus the alpha(1b) subtype and >15-fold selectivity versus dopamine D(2).     

Actions of prostaglandins E1 and F2alpha on isolated intrapulmonary vascular smooth muscle.

The effects of PGE1 and PGF2alpha were studied on isolated strips of intrapulmonary arteries and veins from dog, sheep, swine and man. PGF2alpha contracted human arterial strips in a dose-dependent fashion, relaxed slightly sheep arteries and had no effect on dog arteries. Canine, sheep and human venous strips were contracted by PGF2alpha. PGE1 relaxed slightly both veins and arteries from dog and sheep. Human arteries usually contracted slightly and human veins usually relaxed slightly to PGE1. In a limited number of experiments, swine arteries and veins failed to respond to PGF2alpha or PGE1. All the vascular strips contracted well when exposed to NE. These results suggest that the responses of intrapulmonary vessels to PGF2alpha and PGE1 are species-dependent. PGF2alpha generally exhibits a contractile action, especially on veins. PGE1 usually relaxes intrapulmonary vessels. With regard to vessels from man, PGF2alpha is a powerful stimulant while PGE1 produces only small, variable effects.     

Androgens and estrogens in benign prostatic hyperplasia: past, present and future

Benign prostatic hyperplasia (BPH) and associated lower urinary tract symptoms (LUTS) are common clinical problems in urology. While the precise molecular etiology remains unclear, sex steroids have been implicated in the development and maintenance of BPH. Sufficient data exists linking androgens and androgen receptor pathways to BPH and use of androgen reducing compounds, such as 5α-reductase inhibitors which block the conversion of testosterone into dihydrotestosterone, are a component of the standard of care for men with LUTS attributed to an enlarged prostate. However, BPH is a multifactorial disease and not all men respond well to currently available treatments, suggesting factors other than androgens are involved. Testosterone, the primary circulating androgen in men, can also be metabolized via CYP19/aromatase into the potent estrogen, estradiol-17β. The prostate is an estrogen target tissue and estrogens directly and indirectly affect growth and differentiation of prostate. The precise role of endogenous and exogenous estrogens in directly affecting prostate growth and differentiation in the context of BPH is an understudied area. Estrogens and selective estrogen receptor modulators (SERMs) have been shown to promote or inhibit prostate proliferation signifying potential roles in BPH. Recent research has demonstrated that estrogen receptor signaling pathways may be important in the development and maintenance of BPH and LUTS; however, new models are needed to genetically dissect estrogen regulated molecular mechanisms involved in BPH. More work is needed to identify estrogens and associated signaling pathways in BPH in order to target BPH with dietary and therapeutic SERMs.