Role of Alpha Adrenergic Receptors in Erectile Function

Home , Adrenergic, Adrenergic agonist, Adrenergic receptor, Agonist, Alpha blocker, Aphrodisiac

Role of alpha adrenergic receptors in erectile function

A Traish1,2*, NN Kim1, RB Moreland1 and I Goldstein1

1Department of Urology, Boston University School of Medicine, Boston, MA 02118, USA; 2Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA

Penile erection is a complex physiological process in which the regulation of penile hemodynamics depends on signal input from central and peripheral nervous systems, and on the balance and interplay between several local physiological mediators (neurotransmitters, vasoactive agents and endocrine factors). A role for the sympathetic nervous system in attaining or maintaining penile ¯accidity and detumescence is well recognized. However, the exact mechanisms of alpha-adrenergic receptor mediated erectile function remain poorly de®ned. The objective of this review is to summarize data presented in the literature and from our laboratory on alpha-adrenergic receptors, and to discuss the conceptual framework by which the alpha- adrenergic receptor pathway modulates penile corpus cavernosum smooth muscle contractility. We will integrate the current state of knowledge of penile erection into one framework encompassing the biochemical and physiological pathways responsible for trabecular smooth muscle relaxation (erection) and contraction (detumescence). We will focus our discussion on local control mechanisms of alpha-adrenergic receptors and explore the following topics: (1) functional activity of alpha-1 and alpha-2 adrenergic receptors in the physiology of human penile erection; (2) identi®cation, classi®cation and characterization of alpha-1 and alpha-2 adrenergic receptor subtypes in human penile erectile tissue; (3) molecular mechanism of action of alpha-1 and alpha-2 adrenergic receptors in human penile erectile tissue; (4) blockade of alpha-1 and alpha-2 adrenergic receptor action by selective and non-selective alpha-1 and alpha-2 adrenergic receptor antagonists (blockers); (5) physiologic (functional) antagonism of alpha-1 and alpha-2 adrenergic receptor activity by vasodilators mediating smooth muscle relaxation; and (6) key areas of consensus, as well as critical gaps in the existing scienti®c knowledge concerning the rationale and the potential use of alpha-blockers in erectile function. International Journal of Impotence Research (2000) 12, Suppl 1, S48±S63

Keywords: alpha adrenergic receptor; vascular smooth muscle; corpus cavernosum; erectile dysfunction

Introduction trol of trabecular smooth muscle contractility. In the ¯accid penis, the smooth muscle of the trabeculae and penile cavernosal arteries is maintained in the Penile erectile function is a complex physiological contracted state by contractile agonists. These process involving integration of multiple biochemical messengers interact with speci®c mem- chemical signals elicited in response to several brane receptors and ion channels modulating in- neurotransmitters and vasoactive agents involved tracellular calcium and=or altering calcium in regulation of penile erection and ¯accidity.1±5 sensitivity to contractile proteins producing smooth Physiological and biochemical studies over the past muscle contraction.2,9 three decades have demonstrated that corpus caver- One of the key pathways modulating penile nosum trabecular smooth muscle is an important ¯accidity is the release of norepinephrine from the structure in the penis and contributes to control of adrenergic nerves and binding to post-junctional penile erection and ¯accidity.6±8 Adrenergic nerves, alpha-1 and alpha-2 adrenergic receptors localized via release of the neurotransmitter norepinephrine to the smooth muscle of cavernosal arteries and and synthesis and release of vasoconstrictor sub- trabeculae.1,2,9±18 This reaction causes activation of stances from the endothelium such as endothelins G-protein coupled alpha-adrenergic receptors and and contractile prostaglandins, mediate local con- activation of several signal transduction pathways leading to smooth muscle contraction. Upon sexual stimulation, activation of non-adrenergic non-choli- *Correspondence: A Traish, Boston University, School of nergic nerves results in the synthesis and release of Medicine, Department of Urology, 700 Albany Street, Room W 607A, Boston MA, 02118. nitric oxide (NO), among other substances, which E-mail: [email protected] diffuses into the arterial and trabecular smooth Received 1 October 1999; accepted 23 November 1999 muscle of corpus cavernosum.19±22 NO interacts Alpha adrenergic receptor blockade in erectile dysfunction A Traish et al S49 with guanylyl cyclase resulting in its activation, Currently, the ®rst step in management of male increasing cyclic guanosine monophosphate (cGMP) erectile dysfunction involves the use of oral phar- synthesis. The NO=cGMP signal transduction path- macological agents, vacuum constrictive devices or ways, integrates into the overall regulation of sex therapy. Research on nitric oxide mediated myosin light chain kinase, and myosin light chain relaxation of trabecular smooth muscle and en- phosphatase and intracellular calcium concentra- hancement of penile erection has led to the devel- tion. The net effect is reduced intracellular calcium, opment of a new, safe and effective oral agent, silde- via subcellular sequestration into intracellular cal- na®l citrate (ViagraTM) for treatment of male erectile cium stores or by extrusion, reduced smooth muscle dysfunction.25 27 Since alpha-adrenergic receptors tone and enhanced smooth muscle relaxation and play a critical role in initiating and=or maintaining penile erection.23 penile ¯accidity, understanding the mechanism of Several physiological and biochemical mechan- the alpha adrenergic receptor pathway in modulat- isms involved in the integration and ®ne regulation ing erectile function may provide new insights into of multiple signal transduction pathways maintain- the use of alpha adrenergic receptor antagonists ing smooth muscle contractility have been de- (blockers), alone or in combination with other scribed.1±5,24 These mechanisms are critical for agents, in the treatment of erectile dysfunction. penile erection. Erectile dysfunction may be a result of an imbalance in the integration of bio- Role of alpha-1 and alpha-2 adrenergic receptors in chemical messengers and signals in this complex human penile erectile function physiological process. Thus, biochemical changes in neurotransmitter synthesis or release, receptor expression, cellular distribution or function, and=or The alpha-adrenergic neuroeffector system plays modi®cations in any of the multiple downstream a critical physiological role in erectile func- events along the signal transduction cascade re- tion.2; 9; 10; 12; 14; 15; 28 31 Evidence derived from in sponsible for vasoconstriction over vasodilation vitro and in vivo studies has indicated that adrener- may result in erectile dysfunction.3,4,24 Thus, gic nerves, a major source of physiologically active fundamental knowledge of the biochemical and norepinephrine, innervate human penile corpus physiological mechanisms of neurotransmitters cavernosum.2,32,33 Release of norepinephrine from and vasoactive substances and their receptor func- the sympathetic nerve ®bers of the human corpus tion modulating smooth muscle contraction and cavernosum is modulated by presynaptic alpha-2 relaxation is critical to our understanding of erectile adrenergic receptors and cholinergic nerves via pre- function, and for the development of new pharma- junctional muscarinic acetylcholine receptors (see cotherapeutic strategies to manage patients with Figure 1).6,12,34,35 Detailed analyses of these interac- erectile dysfunction. tions are provided in the accompanying article by

A B

Cholinergic Cholinergic nerve Adrenergic nerve NANC nerve Ach nerve Ach α α A 2 A - - M Ach 2 receptor M Ach + - receptor receptor receptor

NOS NO

α α 1 & 2 adrenergic receptors Contraction of Relaxation of smooth muscle smooth muscle

Figure 1 (A) Prejunctional modulation of the adrenergic nerve by alpha-2 and muscarinic acetylcholine receptors. Norepinephrine (NE) released from the nerve binds to prejunctional alpha-2 adrenergic receptors and inhibits further norepinephrine release. Norepinephrine binds to post-junctional alpha-1 and alpha-2 adrenergic receptors on the trabecular smooth muscle cells resulting in contraction. Acetylcholine released from the cholinergic nerve binds to muscarinic acetylcholine receptors (MACh) on the adrenergic nerves and inhibits norepinephrine release. (B) Nitric oxide (NO) synthesis and release from the nonadrenergic, noncholinergic nerves (NANC) is negatively modulated by NE binding to prejunctional alpha-2 adrenergic receptors. Acetylcholine positively modulates NO synthesis and release from the NANC nerves.

International Journal of Impotence Research Alpha adrenergic receptor blockade in erectile dysfunction A Traish et al S50 Saenz de Tejada et al in this issue. Contraction of Classi®cation of alpha-adrenergic receptor subtypes trabecular smooth muscle by norepinephrine is in human corpus cavernosum dependent on expression of post-junctional alpha- 1 and alpha-2 adrenergic receptors.2,9,15 Alpha adrenergic receptor antagonists (blockers), adminis- Classi®cation and nomenclature of the alpha 1 and tered systemically, facilitate penile erection and in alpha 2 adrenergic receptor subtypes has been some cases produce prolonged erection or priap- recently reported.43±49 The alpha-1 adrenergic re- ism.36±42 In in vitro studies with tissue strips of ceptors were classi®ed into alpha-1a (formerly corpus cavernosum the alpha adrenergic receptor alpha-1c), alpha-1b and alpha-1d (formerly alpha- antagonists prazosin (alpha-1) and yohimbine (alpha- 1a) based on cDNA sequences and agonist and 2) produced right-ward parallel shifts in the pheny- antagonists binding characteristics.46,50±51 Capital lephrine concentration-response curve.11; 13; 15 17; 31 letters denote the pharmacological classi®cation and The af®nity of the receptor for prazosin was greater lower case letters denote the cloned receptor than that for yohimbine, suggesting predominance subtypes (Tables 1±3). Throughout this review we of alpha-1 adrenergic receptors over alpha-2 adre- will adhere to this convention of classi®cation and nergic receptors in human erectile tissue.13,15 These nomenclature. studies demonstrated the important role of alpha-1 Pharmacological and ligand binding studies with adrenergic receptors in erectile function. selective agonists and antagonists lead to classi®ca-

Table 1 Nomenclature and characteristics of human alpha adrenergic receptors

Pharmacologic Receptor Cloned Receptor Other names Predicted MW Glycosylation Acylation Disul®de Bridges

96 a1AAR a1aAR a1CAR 47,482±54,964 Asn60, Asn76 Cys413, Cys415 Cys163±240 97 a1BAR a1bAR - 56,524 Asn10, Asn14, Asn34 Cys375 Cys377 Cys118±195 98 a1DAR a1dAR a1AAR 54,646 Asn7, Asn13, Asn22 None Cys99±176 99 a2AAR a2aAR - 49,016 Asn10, Asn14 Cys442 Cys106±188 100 a2BAR a2bAR - 49,948 None Cys442 Cys85±164 101 a2CAR a2cAR a2c2AR 49,513 Asn19, Asn33 None Cys124±202 [Taken from refs 96±102].

Table 2 Pharmacologic characteristics of alpha-1 adrenergic receptors

Receptor Agonist Antagonist Signalling

a1A Oxymetazoline WB4101 5-Methylurapidil S-Niguldipine a1B None Chloroethylclonidine a1D None Chloroethylclonidine All Subtypes Norepinephrine Prazosin Active Gq/11, voltage-gated Phenylephrine Corynanthine Ca2 channels, 2 Methoxamine :IP3, :Ca [Adapted from refs 44,46,47,49].

Table 3 Pharmacologic characteristics of alpha-2 adrenergic receptors

Receptor Agonist Antagonist Signalling

a2A Oxymetazoline BRL44408 BRL48962 a2B None Imiloxan ARC238 a2C None MK912 2 All Subtypes Norepinephrine Delequamine (RS15385) Active Gi,Ca -dependent Clonidine Yohimbine K channels, UK14,304 Rauwolscine ;adenylate cyclase, Prazosin ;cAMP

[Adapted from references 45,46,48,49,51].

International Journal of Impotence Research Alpha adrenergic receptor blockade in erectile dysfunction A Traish et al S51 tion of alpha-1 adrenergic receptors into three sub- characterized.46; 47; 50; 52; 53 These clones were ex- types (alpha-1A,-1B and-1D; Table 2). Receptors pressed in mammalian cell systems and their ligand which displayed high af®nity for the antagonists WB binding characteristics were determined (reviewed 4101, 5-methylurapidil and prazosin were de®ned as in refs 46, 47). A summary of the selective agonists alpha-1A and alpha-1D. Receptor subtypes with low and antagonists for these three subtypes is shown in af®nity for WB 4101, 5-methylurapidil were desig- Table 2. A fourth receptor subtype referred to as nated as alpha-1B subtype. Furthermore, alpha-1A alpha-1L (low af®nity to prazosin) has been reported subtype is insensitive to the alkylating agent cholor- (reviewed in ref. 44). However, the cDNA for this ethylclonidine (CEC), whereas the alpha-1B is sensi- subtype is yet to be described. Because of insuf®- tive to CEC.46,47 The alpha-1D subtype is only partially cient molecular and pharmacological information sensitive to the alkylating agent CEC and exhibits on this subtype it will not be discussed further in lower af®nity to the agonist oxymetazoline.46,47 this review. Three alpha-1 adrenergic receptor cDNAs repre- Biochemical studies employing pharmacological senting three distinct genes have been cloned and agents clearly suggested that, while discrimination

Norepinephrine

α α α 1a 1b? 1d Smooth muscle cell

αq γ γ αq + β β GTP GDP

PLC PIP2 IP3 + DAG

Norepinephrine

α α α 2a 2b? 2c Smooth muscle cell

αi γ γ αi + β β GTP GDP

AC - cAMP

© L. Messenger Figure 2 (A) adrenergic receptor subtypes identi®ed in human corpus cavernosum smooth muscle cells by molecular and pharmacological approaches. (B) Alpha-2 adrenergic receptor subtypes identi®ed in human corpus cavernosum smooth muscle cells by molecular and pharmacological approaches.

International Journal of Impotence Research Alpha adrenergic receptor blockade in erectile dysfunction A Traish et al S52 between alpha-1 and alpha-2 adrenergic receptors is tile response by these subtypes is yet to be fully possible, it is dif®cult to unequivocally distinguish determined in human corpus cavernosum. the alpha-1 or alpha-2 adrenergic receptor subtypes in human corpus cavernosum using a single bio- Identi®cation of alpha-1 adrenergic receptor chemical or pharmacological approach. Alpha adre- subtypes in human penile corpus cavernosum nergic selective agonists and antagonists bind to the various receptor subtypes over a wide range of concentrations with varying af®nities and selectiv- Several pharmacological and biochemical studies ities but little de®ned speci®city.16±18,51,54 This lack have con®rmed the presence of alpha-adrenergic of receptor subtype `speci®c' ligands for the alpha 1 receptors in penile tissue. However, these studies adrenergic receptor has hampered the unequivocal did not identify the nature of receptor subtypes assignment of the receptor subtypes responsible expressed in human penile corpus cavernosum. In for the contractile response to norepinephrine. The a series of studies, we demonstrated that human availability of speci®c molecular biological probes to erectile corpus cavernosum tissue expresses mRNA identify and characterize the various subtypes, to- transcripts for alpha-1a, alpha-1b and alpha-1d gether with biochemical approaches had facilitated adrenergic receptor subtypes (Figure 2A).16 The identi®cation of these subtypes.50 However, assigning abundance of mRNA transcripts for these subtypes biological functions for each of these subtypes and in human corpus cavernosum, suggested that alpha- determining the relative contribution to the contrac- 1a and alpha-1d adrenergic receptors are predominant

Norepinephrine OH OH

NH2 CH2 CH OH

Extracellular

Cell membrane α γ β Intracellular Gq/11 protein α 1 adrenergic receptor

Extracellular

Cell membrane γ β Intracellular α α 1 adrenergic Gq/11 receptor activated protein activated © L. Messenger

Figure 3 Interaction of norepinephrine with alpha-1 adrenergic receptor and activation of the Gq=11 signal transduction pathway.

International Journal of Impotence Research Alpha adrenergic receptor blockade in erectile dysfunction A Traish et al S53 over the alpha-1b adrenergic receptor subtype. Using usefulness of targeted alpha-blockers as drugs for different molecular approaches, the expression of therapeutic purposes? Clearly, investigation of the three distinct alpha-1 adrenergic receptor subtypes (a, role of the various subtypes in regulating corpus b and d) in human corpus cavernosum, has also been cavernosum function is warranted. reported by Dausse et al,55 corroborating our studies. Expression of three alpha-1 adrenergic receptor sub- Identi®cation and characterization of alpha-2 type mRNAs (a, b, and d) in the rat corpus cavernosum adrenergic receptor subtypes in penile corpus has recently been reported.56 These studies document cavernosum the expression of three alpha-1 adrenergic receptors in erectile tissue. Based on pharmacological studies, Tong and Pharmacological studies in human corpus caverno- Cheng57 have suggested that the alpha-1A receptor sum tissue have suggested the expression of post- subtype was responsible for inducing contraction in junctional alpha-2 adrenergic receptors. However, it the rat corpus cavernosum. In contrast, Furukawa et was concluded that alpha-2 adrenergic receptors do al58 have suggested that the alpha-1B adrenergic not contribute signi®cantly to the contractile activity receptor subtype mediates the contractile response of trabecular smooth muscle.2,15,31 This conclusion in the rabbit corpus cavernosum. This conclusion was is based on the observations that the constrictor based on pharmacological studies using WB 4101, 5- response to electrical ®eld stimulation is blocked methylurapidil, chloroethylchlonidine, oxymetazo- mainly with alpha-1 adrenergic receptor antagonists line and tamsulosin. Although it is becoming clear but not with alpha-2 adrenergic receptor antagonists that at least three alpha-1 adrenergic receptor sub- (Tables 2 and 3).15,31 Since high concentrations of types are expressed in erectile tissue, their functional antagonists were used in these studies, the selec- and physiological signi®cance remains unknown. tivity of the antagonists for alpha-1 and alpha-2 Several questions remain to be answered. What role adrenergic receptors is compromised at high drug does each receptor subtype play in mediating smooth concentrations due to lack of speci®city.46,51,54 muscle contractility? What is the relative contribution Therefore, the assignment of a speci®c functional of each of these receptor subtypes to the signalling activity to a speci®c receptor class or subclass based pathway modulating intracellular calcium levels? only on pharmacological analyses is suggestive, at How does the selectivity of these receptor subtypes best. Further, although yohimbine and rauwolscine to various alpha antagonists determine the potential (highly selective alpha-2 adrenergic receptor anta-

Figure 4 Proposed molecular mechanism of action of alpha-1 adrenergic receptors in penile corpus cavernosum smooth muscle cells.

International Journal of Impotence Research Alpha adrenergic receptor blockade in erectile dysfunction A Traish et al S54 gonists) were shown to inhibit norepinephrine-in- observations strongly suggest that alpha-1 adrener- duced contraction in corpus cavernosum, no physio- gic receptor subtypes modulate trabecular smooth logical role was assigned for alpha-2 adrenergic muscle contractility in human erectile tissue. receptors in human penile erectile tissue.15,28,31,59 Functional studies with human and rabbit erec- In the absence of detailed biochemical and molec- tile tissue using UK14304, a selective alpha-2 ular biological analyses of the alpha-2 adrenergic adrenergic agonist demonstrated dose-dependent receptors expressed in corpus cavernosum tissue contractions.11,17 UK14304-dose response curve and their functional activities, it remains unclear if was shifted to the right by selective alpha-2 alpha-2 adrenergic receptors are expressed in the adrenergic receptor antagonists, rauwolscine and corpus cavernosum tissue and if these receptors are delequamine (RS15385-197).11,17 Furthermore, physiologically functional. phentolamine caused a dose-dependent relaxation We have demonstrated mRNA expression for of corpus cavernosum tissue strips pre-contracted alpha-2a, alpha-2b and the alpha-2c adrenergic with UK14304.18 UK14304 inhibited PGE1 and receptor subtypes in human corpus cavernosum forskolin induced-cAMP synthesis in cultured tissue (Figure 2B).17 Ligand binding studies with human and rabbit corpus cavernosum smooth [3H]RX 821002 and [3H]rauwolscine, selective lig- muscle cells.11,17 The UK14304 mediated inhibition ands for alpha-2 adrenergic receptors, in isolated of forskolin-induced cAMP synthesis and the shift membranes of cultured human corpus cavernosum of the contractile dose response curve of UK14304 smooth muscle cells, demonstrated speci®c, high by delequamine (RS15385-197) suggest that post- af®nity (Kd ~ 0.63 nM) binding with limited capacity junctional alpha-2 adrenergic receptors in human (25±30 fmol=mg protein). Binding of [3H]RX821002 penile erectile tissue are biochemically and physio- was displaced with unlabeled rauwolscine or norepi- logically functional. nephrine but not with phenylephrine, an alpha-1 The alpha-1 adrenergic receptor agonists elicit selective ligand. Competitive binding studies with greater contractile responses than those obtained [3H]RX821002 and phentolamine (a non-selective with the alpha-2 adrenergic receptor agonists.11 This alpha antagonist) demonstrated speci®city of bind- suggests that alpha 1 adrenergic receptors contribute ing to alpha-2 adrenergic receptors.17 Physio- more to the contractility of human corpus caver- logical studies with selective alpha-2 antagonists nosum smooth muscle.2,12,14±16,18,31,60 This physio- have clearly supported the expression of biochemi- logical difference may be attributed to a lower cally functional alpha-2 adrenergic receptors.11,17 The density of the alpha-2 adrenergic receptors on penile data from the molecular biology, biochemistry and smooth muscle. Also, post-synaptic alpha-2 adre- physiological studies suggest that human corpus nergic receptors may be localized on the smooth cavernosum expresses physiological functional al- muscle distal to the adrenergic nerve terminals. pha-2 adrenergic receptors. Furthermore, the distribution of alpha-2 adrenergic receptors on the different cell types of penile erectile tissue may be different from that of the alpha-1 Functional (physiological) studies of alpha-1 and adrenergic receptors. alpha-2 adrenergic receptors in erectile tissue Molecular mechanism of alpha-1 adrenergic receptors action in erectile tissue corpus The blockade of electric ®eld stimulation induced- cavernosum contraction by prazosin in human corpus cavernosum supports a role for functional post-junctional alpha-1 adrenergic receptors in penile erectile Norepinephrine released from the adrenergic nerve tissue.15 Norepinephrine causes dose-dependent terminals binds to alpha-1 adrenergic receptor contraction in human corpus cavernosum smooth subtypes localized on the smooth muscle of corpus muscle.2,12,15,16,18,31,60 Norepinephrine dose re- cavernosum and cavernosal arteries. This binding sponse curve was shifted to the right with increasing reaction results in conformational changes in the concentrations of the alpha-1A=1D adrenergic re- 7-transmembrane receptor molecule leading to acti- ceptors antagonist WB4101 and the alpha-1B recep- vation of the heterotrimeric Gq=11 protein and tor antagonist chloroethylchlonidine.60 Phento- increased rate of GDP dissociation (Figures 3 and lamine, an alpha-1 and alpha-2 antagonist, shifted 4). The exchange of GDP with GTP in the binding the phenylephrine contractile dose-response curve site of the a-subunit of the G-protein results in its to the right.18 Phentolamine produced dose-depen- dissociation into alpha and beta=gamma subunits. dent relaxation response in corpus cavernosum The Ga=q subunit interacts with phospholipase Cb1 tissue strips pre-contracted with norepinephrine, and stimulates its activity producing hydrolysis of oxymetazoline or phenylephrine. Similar relaxation phosphatidylinositol 4,5-bis-phosphate (PIP2) into responses, in a dose-dependent fashion, were 1,4,5-inositol trisphosphate (IP3) and diacylglycerol obtained with doxazosin, terazosin, prazosin and (DAG). IP3 diffuses into the cytoplasm where it tamsulosin (N. Kim, unpublished data). These binds to a speci®c receptor on the sarcoplasmic

International Journal of Impotence Research Alpha adrenergic receptor blockade in erectile dysfunction A Traish et al S55 reticulum (SR) causing release of Ca2 from intra- contraction in the absence of increased intracellular cellular stores. DAG, in the presence of Ca2, Ca2 (Figure 4). Other mechanisms of smooth activates protein kinase C (PKC), which causes muscle contraction have implicated G protein further increases in intracellular Ca2. Further, activating Ca2-sensitizing cascade via monomeric norepinephrine binding to the receptor may GTpase RhoA. The downstream Ca sensitizing increase Ca2 in¯ux through L-type Ca2 channels effector of RhoA is mediated by Rho kinase, which by as yet unknown mechanism. Both of these mech- phosphorylates the regulatory subunit of myosin anisms result in increased intracellular calcium light chain phosphatase and inhibits its catalytic 2 2 63 ([Ca i]). Binding of Ca to calmodulin activates activity. myosin light chain kinase (MLCK) and may inhibit myosin light chain phosphatase (MLCP). The biochemical mechanisms mediating smooth Molecular mechanism of action of alpha-2 muscle contraction involves phosphorylation of the adrenergic receptors in erectile tissue corpus myosin regulatory light chain (MLC20) by myosin cavernosum light chain kinase (MLCK). Phosphorylation of myosin light chain induces actin to activate myosin ATPase, causing smooth muscle contraction. De- The mechanism of alpha-2 adrenergic receptor phosphorylation of MLC20 by heterotrimeric smooth action in human corpus cavernosum is yet to be muscle myosin phosphatase (SMPP-1M)61 produces fully understood. Studies in other vascular systems smooth muscle relaxation. It is not known if have shown that activation of the alpha-2 adrenergic contractile agonists inhibit MLCP activity in addi- receptor inhibits adenylate cyclase activity via tion to activating the MLCK, and bring about heterotrimeric Gi-proteins (reviewed in ref. 45) and contraction. In addition to these primary mechan- reduce intracellular cAMP. We have demonstrated isms other mechanisms are proposed in which that UK14304 decreased the forskolin or PGE1- phosphorylation of MLC20 takes place in the induced cAMP synthesis in cultured human and absence of increased intracellular Ca2-calmodu- rabbit corpus cavernosum smooth muscle cells lin.62 The altered balance between the activities of (Figure 5).11,17 This reduction in cAMP synthesis Ca2-independent kinase and inhibition of phos- leads to reduced protein kinase A (PKA) activation phatase increases MLC20 phosphorylation causing and increased intracellular calcium or calcium=

Alpha2 Adrenergic Receptors in Smooth Muscle Function N

I II III IV V VI VII

α γ γ β β

GDP C

αi GTP

• Activation of Ion channels • Inhibition of cAMP • Activation of phospholipases • Activation of phosphodiesterases

Figure 5 Proposed molecular mechanism of action of alpha-2 adrenergic receptors in penile corpus cavernosum smooth muscle cells.

International Journal of Impotence Research Alpha adrenergic receptor blockade in erectile dysfunction A Traish et al S56 calmodulin sensitivity of myosin light chain kinase. concentration±response curves produced by nor- Also, alpha-2 adrenergic receptor have been shown epinephrine (NE) are displaced to the left in the to modulate Ca2 in¯ux either by G-protein coupled absence of the endothelium, without a change in the mechanisms or by G-protein-independent mechan- magnitude of the maximal response.64 This suggests isms.45 These receptors are also implicated in that NE-mediated contraction is antagonized by activation of phospholipase C, phospholipase endothelium-derived vasoactive substances, at the D, potassium channels and Na=H exchange.45 receptor level and=or at a step in the biochemical Whether these mechanisms are integrated in in- pathway leading to contraction. Contractile agonists creasing corpus cavernosum smooth muscle tone is may modulate smooth muscle relaxation by binding unknown at present. Nevertheless, the demonstra- to a speci®c receptor on the endothelium and tion of functional post-junctional alpha-2 adrenergic releasing vasorelaxatroy substances. For instance, receptor in human and rabbit corpus cavernosum methoxamine, phenylephrine, clonidine and B-HT suggests that these receptors contribute to the 920 (agonists that stimulate contractility in smooth contractility of corpus cavernosum smooth mus- muscle) increased intracellular cGMP only in the cle.11,17 presence but not in the absence of the endothelium, Modulation of agonist induced contraction of suggesting release of nitric oxide (NO), presumably smooth muscle by the endothelium has been via alpha-2 adrenergic receptors on the endo- reported in other vascular beds.64 In aortic rings, thelium.65 Microvascular constriction caused by

α antagonist

X Norepinephrine unable to bind Extracellular

Cell membrane α γ β Intracellular Gq/11 protein α 1 adrenergic receptor

Extracellular

Figure 6 Inhibition of norepinephrine binding to alpha adrenergic receptors by an alpha antagonist and blockade of activation of the signal transduction pathways.

International Journal of Impotence Research Alpha adrenergic receptor blockade in erectile dysfunction A Traish et al S57 alpha-1 and alpha-2 adrenergic receptors activation actions, at the alpha-adrenergic receptors, has been is signi®cantly modulated by endothelium in vas- the basis of drug therapy in urogenital disorders.66 cular beds. Thus, it remains to be determined if As summarized in Figure 6, competitive inhibition human corpus cavernosum endothelium modulates or displacement of norepinephrine binding to the smooth muscle contractility via activation of NO by alpha-receptor by the antagonist inhibits receptor binding of NE to alpha-2 adrenergic receptors on the activation and, in turn, attenuates the signal trans- endothelium. duction pathway. Alpha-adrenergic blockade was ®rst used in the early 1980s in treatment of erectile dysfunction.36,37,39 Nevertheless, this approach has Blockade of alpha-1 and alpha-2 adrenergic yet to be fully exploited, in part, because of limited receptor activity by selective and non-selective understanding of the mechanism of adrenergic receptor function in erectile tissue. In this section, alpha-1 and -2 receptor antagonists we summarize studies from our laboratory and work by others on the functional antagonism of alpha- Blockade of the sympathetic neurotransmitter nor- adrenergic receptors by selective antagonists in epinephrine and the neurohormone epinephrine erectile tissue. Injection of phenoxybenzamine has

Yohimbine: Alpha2 Selective Antagonist Terazosin: Alpha1D Selective Antagonist

N N N H H N CH3O N O H

N H CH3O

CH3O2C NH2

Doxazosin: Alpha1A and Alpha1D Antagonist Tamsulosin: Alpha1A and Alpha1D Selective Antagonist O

O N

N CH O N O H 3 H N O 2 N S O N O CH3O CH3 O CH3

CH3O NH2

Delaquamine: Alpha2 Selective Antagonist

CH3O

H H

H O N S H C 3 O Figure 7 Chemical structures of selected alpha-1 and alpha-2 adrenergic receptor-antagonists.

International Journal of Impotence Research Alpha adrenergic receptor blockade in erectile dysfunction A Traish et al S58 been demonstrated to cause full erection in six out alpha-2 adrenergic receptor selective antagonist, of 11 impotent men.36 Further studies utilizing the from alpha-2 adrenergic receptors in corpus caver- alpha-blocker, phentolamine as an injectable agent nosum membranes. Delequamine (RS 15385-197) produced mixed results.37 These observations and rauwolscine, selective alpha-2 adrenergic suggested potential usefulness of alpha blockade in receptor antagonists, competed effectively for bind- treatment of erectile dysfunction, however, basic ing of [3H]rauwolscine to alpha-2 adrenergic recep- science and clinical research must be carried out to tors. Although prazosin and 5-methylurapidil were investigate this fully. Buccal and oral phentolamine thought to be alpha-1 selective agents, we observed produced satisfactory erections for penetration in cross-reactivity of these agents with alpha-2 adren- impotent men.42 Oral phentolamine, similar to ergic receptors indicating a lack of discrete receptor intracavernosal injection of phentolamine, pro- speci®city, in corpus cavernosum smooth muscle. In duced penile tumescence and enhanced responsive- experiments utilizing corpus cavernosum smooth ness to sexual stimulation which in turn produced muscle cells in culture, phentolamine mesylate was rigidity.39 Phentolamine (RegitineTM) has been effective in displacing the alpha-2 adrenergic an- used alone and in combination with a variety of tagonists [3H]rauwolscine and [3H]RX821002 in a vasodilators (such as papaverine and PGE1) as an concentration-dependent manner. Norepinephrine intracavernosal injectable treatment for erectile also displaced [3H]RX821002 but was less effec- dysfunction (reviewed in ref. 67). Combinations of tive than phentolamine. The selective alpha-1 vasoactive intestinal peptide (VIP) and phentola- adrenergic receptor agonist phenylephrine did mine have been tested in pilot studies as intra- not compete with either [3H]rauwolscine or cavernosal therapeutics.68,69 In a recent clinical [3H]RX821002.17 These data clearly indicate that trial, Becker et al.70 evaluated the ef®cacy of oral phentolamine is an alpha-1 and alpha-2 antagonist phentolamine mesylate in treatment of men with in penile smooth muscle. erectile dysfunction. The authors concluded that Phentolamine mesylate, at concentrations ranging oral phentolamine may be of bene®t for the treat- from 1nM to 10mM, displaced the phenylephrine ment of erectile dysfunction. Unpublished studies contractile dose-response curve to the right in organ have demonstrated safety and ef®cacy of phento- bath studies with corpus cavernosum strips.18 In lamine in treatment of erectile dysfunction (I. human and rabbit corpus cavernosum erectile Goldstein, personal communication). Although tissues, phentolamine caused dose-dependent re- phentolamine has been used as an intracavernosal laxation in tissue strips pre-contracted with 3 mM and oral agent in treatment of erectile dysfunction phenylephrine. The half-maximal relaxation re- (reviewed in refs 27, 67), a detailed analysis of its sponse (EC50) occurred at 10±30nM phentolamine mechanism of action in erectile tissue was not and the maximal relaxation response was reached at reported. Several classes of alpha adrenergic block- 1 mM. Phentolamine caused dose-dependent relaxa- ers have been synthesized and screened for their tion in strips pre-contacted with 1 mM phenylephr- potential usefulness in inhibiting receptor activity ine, 5 mM norepinephrine or 1 mM oxymetazoline. (reviewed in refs 46, 51). Different alpha-adrenergic Phentolamine mesylate, in concentrations ranging receptor blockers possess various structural deter- from 10 nM to 1 mM, also displaced the UK14304 minants that confer selectivity, af®nity and ef®cacy contractile response to the right. Phentolamine (Figure 7). Thus, it is expected that different alpha- caused dose-dependent relaxation in strips pre- blockers will have different physiological responses contracted with 3 or 10 mM UK14304. In human in vivo. Below is a summary of the action of several erectile tissues pre-contracted with 3 mM UK14304, alpha-blockers, which have been employed, in the half-maximal relaxation response (EC50)oc- physiological studies and in clinical trials in erectile curred at 30±50 nM phentolamine, and the maximal dysfunction. relaxation response occurred at 1mM. These results suggest that phentolamine mesylate is an effective alpha-1 and alpha-2 antagonist and inhibits norepi- Alpha-1 and alpha-2 selective antagonists nephrine mediated responses in vitro. Phentolamine mesylate (VasomaxTM). In a recent In rabbit corpus cavernosum strips pre-contracted study, we demonstrated that phentolamine compe- with 80 mM KCl or 30 nM endothelin-1, phentola- titively displaces binding of the alpha-1 antagonists mine mesylate caused concentration-dependent [125I]HEAT and [3H]prazosin from alpha-1 adrener- relaxation.18 Since the mechanisms of contraction gic receptors isolated from human and rabbit corpus induced by KCl or endothelin-1 are not mediated by cavernosum.18 Phentolamine mesylate also exhib- alpha adrenergic receptors, an additional non- ited similar binding af®nity to that of 5-methylur- adrenergic mechanism for phentolamine-induced apidil, an alpha-1A selective adrenergic receptor corpus cavernosum relaxation was suspected. Phen- antagonist. Prazosin was more effective in displac- tolamine mesylate did not inhibit the binding of the ing bound [125I] HEAT than either phentolamine or L-type calcium channel-blocker, [3H]PN 200-110, a 5-methylurapidil. Phentolamine mesylate competi- nitripidine analogue, in human corpus cavernosum tively displaced binding of [3H] rauwolscine, an membranes, suggesting that phentolamine may not be

International Journal of Impotence Research Alpha adrenergic receptor blockade in erectile dysfunction A Traish et al S59 acting on this type of calcium channel (N. Kim and A. sin is clinically used for the treatment of benign Traish, unpublished data). Phentolamine mesylate- prostatic hyperplasia, but not for the treatment of induced relaxation in corpus cavernosum smooth hypertension.83 muscle pre-contracted with 80 mM KCl and this Trazodone hydrochloride (DesyrelTM) is an oral response was shifted to the right in the presence serotonergic antidepressant which was noted to of the reversible nitric oxide synthase inhibitor, L improve erectile function in impotent men and nitroarginine (LNNA, 30mM). Similarly, the relaxation cause prolonged erections and priapism in potent response of corpus cavernosum strips pre-contracted men.40,41 Injection of trazodone intracavenosally in with 30nM endothelin-1 was reduced in the presence the rabbit penis produced full penile erection in of LNNA (30 mM). These observations suggest that 76±84% of animals. Injection of trazodone into phentolamine, by blocking the action of alpha-1 and volunteers produced tumescence but not full penile alpha-2 activity by endogenously released norepi- erection. It was concluded that trazodone acts as an nephrine, enhances the effectiveness of the NO alpha adrenergic receptor antagonist in enhancing released constitutively by the corpus cavernosum penile erection but not initiating penile endothelium producing relaxation of tissue pre- erection.40 Intracavernosal trazadone was found to contracted with K or with endothelins. be associated with priapism in a canine model.38 Saenz de Tejada et al reported on the pathophysiology of prolonged erections associated with Alpha-1 selective antagonists trazodone use.41 Trazodone signi®cantly improved Prazosin hydrochloride (MinipressTM) is an alpha- nocturnal penile tumescence. Trazadone displaced 1A, -1B and -1D selective antagonist.51 While NE-induced dose dependent contractile response introduced primarily as an antihypertensive agent,71 curve to the right and caused dose dependent it has been used orally to treat psychogenic erectile relaxation of tissue strips pre-contracted with NE. dysfunction.72 Prazosin has also been used, in Trazodone also reduced the contractile response to combination with alprostadil (PGE1), as an intraur- electrical ®eld stimulation.41 These observations ethral agent for treatment of erectile dysfunction.73 suggest that trazodone hydrochloride acts periph- Although prazosin is thought to be alpha-1 selective, erally via a mechanism mimicking alpha blocking it does cross-react with alpha-2 adrenergic recep- properties.38,40,41 It should be noted that metabolites tors, as suggested from biochemical binding studies. of trazadone can activate hydroxytryptamine recep- Doxazosin mesylate (CarduraTM) is an alpha-1A tors (HT1c) in the spinal cord, and these metabolites and-1D selective blocker and possesses no af®nity are believed to contribute to the erectogenic effect of for alpha-2 adrenergic receptors.51 Doxazosin has this drug (reviewed in ref. 27). A recent clinical trial been utilized clinically in the treatment of hyperten- showed some ef®cacy of trazadone in treatment of sion and benign prostatic hyperplasia.74,75 Kaplan et psychogenic erectile dysfunction.84 al76 showed that oral doxazosin increased the ef®cacy of alprostadil injected intracavernosally. It was noted that patients taking doxazosin experience Alpha-2 selective antagonists lower incidence of erectile dysfunction (2.8%) than Yohimbine is an indole alkaloid that has been other hypotensive agents. The authors concluded widely used for treatment of psychogenic impo- that their data do not support the use of doxazosin tence.85±88 Yohimbine's aphrodisiac activity may be alone in the management of erectile dysfunction.76 mediated through a combination of central nervous However, it is interesting to note that in the system effects and peripheral effects including Treatment of Mild Hypertension Study, patients the blockade of pre-synaptic alpha-2 adrenergic taking doxazosin reported a lower incidence of receptors, resulting in enhanced blood ¯ow and erectile dysfunction.77 decreased blood out¯ow from erectile tissue and Terazosin hydrochloride (HytrinTM) is an alpha-1 enhanced libido. Although yohimbine, a selective adrenergic selective antagonist with high af®nity for alpha-2 adrenergic receptor antagonist, has been alpha-1D receptor subtype.78 It is clinically utilized widely used in treatments of psychogenic male for the treatment of benign prostatic hyperplasia but erectile dysfunction,86±89 with some success; its not hypertension.79 It has been reported in a single exact mechanism of action remains unknown. A case study that terazosin caused prolonged erection recent study has shown that yohimbine is ineffec- in a spinal cord injury patient.80 tive in treatment of organic erectile dysfunction.90 Tamsulosin hydrochloride (FlomaxTM) is an al- Inhibition of presynaptic alpha-2 adrenergic recep- pha-1 adrenergic receptor blocker, which possesses tors regulate the exocytotic norepinephrine release higher af®nity for alpha-1A and alpha-1D than (Figure 1).15,88,91 This would suggest increased alpha-1B.78,81,82 A detailed pharmacological analy- smooth muscle contractility due to increased cate- sis of interaction of tamsulosin with cloned alpha-1 cholamine release. However, yohimbine may facil- adrenergic receptor subtypes revealed that tamsulo- itate trabecular smooth muscle relaxation by binding sin is a high af®nity antagonist with selectivity to post-junctional alpha-2 adrenergic receptors on for alpha-1D alpha-1A > alpha-1B.81,82 Tamsulo- the smooth muscle or on the cavernosal arteries, and

International Journal of Impotence Research Alpha adrenergic receptor blockade in erectile dysfunction A Traish et al S60 inhibiting contraction. A possible mechanism of Modulation by the endothelium of agonist-induced yohimbine action in regulation of erectile function, contraction of smooth muscle by vasodilators has especially in the patients with psychogenic erectile been recognized.64,65 In other vascular tissues, con- dysfunction, is a combined effect on the central centration±response curves produced by norepi- nervous system by increased systemic blood pressure nephrine are displaced to the left in the absence of and peripheral effects at penile arterial and trabecular the endothelium, without a change in the magnitude smooth muscle by attenuating contraction. of the maximal response.64,65 This suggests that Delequamine is a selective alpha-2 adrenergic norepinephrine-mediated contraction is antagonized receptor antagonist which was shown to shift the by endothelium-derived vasoactive substances, either UK 14,304 dose±response curve to the right in at the receptor level and=or at a step in the biochemical human corpus cavernosum17 and in rabbit corpus pathway leading to contraction. Contractile agonists cavernosum.11 In an elegant and detailed study, may modulate smooth muscle relaxation by binding to Munoz et al92,93 demonstrated that delequamine a speci®c receptor on the endothelium. For instance, when administered to healthy volunteers, had methoxamine, phenylephrine, clonidine and B-HT several effects. An increase in spontaneous erection, 920 (agonists that stimulate contractility in smooth increased subjective rating of sexual arousal, in- muscle) increased intracellular cGMP only in the creased duration of erectile response to erotic presence but not in the absence of the endothelium, stimuli and increases in systolic blood pressure suggesting release of NO, presumably via alpha-2 and heart rate before and during erotic stimulation. adrenergic receptors on the endothelium.64 Micro- It was noted that the adverse effects of the drug were vascular constriction caused by alpha-1 and alpha-2 minimal. In a subsequent study, Munoz et al92,93 adrenergic receptor activation is signi®cantly modu- further investigated the effects of delequamine in lated by endothelium. young and old men with suspected psychogenic Increased activation of endothelial or neural nitric erectile dysfunction. It was suggested that delequa- oxide synthase physiologically antagonizes alpha mine may reverse the inhibition of the general adrenergic receptors action by synthesis and release arousal response in young men with psychogenic of NO. In addition to activation of guanylyl cyclase, impotence and that this response declines with age. NO may also activate K channels or Na=K ATPase 2 94,95 and reduces [Ca ]i. Further, the endothelium releases vasoactive substances such as prostaglan- Physiological (functional) antagonism of alpha-1 dins, which activate speci®c Gs-protein-coupled and alpha-2 adrenergic receptors activity by receptors on the smooth muscle and increase cAMP vasodilators: the balance between contraction synthesis. VIP released from the VIPergic nerves and relaxation interacts with Gs-protein coupled receptors and results in activation of adenylyl cyclases and increased intracellular cAMP synthesis. The signal Physiological antagonism refers to the action of an transduction pathway of the NO=cGMP via PKG agonist (e.g. PGE1, VIP or NO), which produces the counteract and overcomes the signal transduction opposite biological effect (relaxation) to the substance pathway of norepinephrine mediated increase in IP3 being antagonized (norepinephrine) which produces turnover and increased intracellular calcium. This contraction. In this case, the antagonist (PGE1, VIP or shift in the balance towards reduced intracellular NO) acts via its own receptor or enzyme and activates calcium results in reduced contractility and enhanced a signal transduction cascade that will attenuate or relaxation. Increased intracellular levels of cAMP and oppose the action of the agonist, resulting in the ®nal cGMP result in activation of cGMP and cAMP- response observed. This concept, while de®ned in the dependent protein kinases, activation of K channels pharmacological literature, is often not understood and hyperpolarization. Hyperpolarization decreases when several mechanisms or pathways are integrated Ca2 in¯ux through L-type Ca2 channels thus to produce ®nal response. decreasing intracellular calcium and causing smooth Non-adrenergic non-cholinergic nerves and the muscle relaxation. vascular endothelium lining the lacunar spaces and Since the alpha adrenergic pathway exerts a the penile arteries release vasoactive factors that relax dominant regulatory effect on trabecular smooth the trabecular smooth muscle. Nitric oxide released muscle tone, any physiological perturbations in this from the non-adrenergic non-cholinergic nerves dif- pathway, regardless of the nature of the mediator, fuses into the smooth muscle and activates guanylyl may have profound in¯uence on smooth muscle cyclase. This results in increased synthesis of intra- contractility. An important concept to emphasize cellular cGMP. Intracellular concentration of cGMP is regarding the interaction of multiple and overlap- regulated by cGMP-speci®c phosphodiesterases. ping regulatory mechanisms is that no single path- Binding of cGMP to speci®c protein kinase G (PKG) way is completely inactive at any given time and is results in its activation and phosphorylation of always operative at some basal level. This is protein substrates leading to decreased intracellular illustrated by the ability of phosphodiesterase calcium by sequestration and extrusion. inhibitors to elevate intracellular levels of cyclic

International Journal of Impotence Research Alpha adrenergic receptor blockade in erectile dysfunction A Traish et al S61 nucleotides and induce relaxation in organ bath standing of the role of alpha adrenergic receptor studies in the absence of exogenously added agonist function in modulating trabecular smooth muscle or nerve stimulation (N Kim and A Traish, unpub- tone and penile erection. With the advent of lished data). Similarly, contraction is enhanced biochemical and molecular biological approaches when basal release of NO synthesis is inhibited by coupled with pharmacological and histological arginine analogs (L-NMMA) in the presence of methods, it is possible to address these questions exogenous adrenergic agonist, suggesting that such and delineate the molecular mechanism of alpha pathways are always active at some basal level. adrenergic function in penile erection. Investigation Therefore, it is important to appreciate the contribu- of the signal transduction pathways that maintain tions of the contractile and vasodilator effector the delicate balance between vaosactive factors systems on smooth muscle contractility. This re- regulating trabecular smooth muscle contraction quires a comprehensive approach to understand the and relaxation, in normal erectile function, is potential interactions (synergistic or antagonistic) necessary. Such research should provide better of these vasodilator and vasoconstrictor systems in understanding of the pathophysiological mechan- the physiology of erectile function. isms disrupting this delicate balance in aging and in diseases causing erectile dysfunction.

Future research directions Acknowledgements

The current conceptual framework of penile erection, is that the state of penile trabecular smooth muscle We are grateful to Ms. Jerie McGrath-Cerqua for her tone depends on the balance and integration of signals administrative assistance. This work was supported derived from the biochemical pathways regulating by NIH grants DK 02696, DK 39080, DK 40025 and smooth muscle contraction and relaxation. In the DK 47950. ¯accid state, functional synergism between the adrenergic system and other vasoconstrictors (such as endothelins, angiotension, and prostanoids), main- References tains contractility. Subsequent to sexual stimulation, activation of the physiological mechanisms mediating 1 Saenz de Tejada I. In the physiology of erection, signposts to relaxation antagonizes alpha adrenergic receptor impotence. Contemporary Urol 1992; 7: 52±68. function and modi®es alpha-adrenergic receptor 2 Andersson K-E, Wagner G. Physiology of penile erection. signaling pathway either at the receptor level and=or Physiol Rev 1995; 75: 191±236. 2 3 Lue TF, Dahiya R. Molecular biology of erectile function and at one or more key steps in the cascade of [Ca ]i homeostasis. Better understanding of the biochemical dysfunction. Mol Urol 1997; 1: 55±64. 4 Adams MA, et al. Vascular control mechanisms in penile and physiological mechanisms modulating this func- erection: phylogeny and the inevitability of multiple and tional antagonism should lead to the development of overlapping systems. Int J Impot Res 1997; 9: 69±84. better strategies for the management of erectile 5 Stief CG, Noack T, Andersson K-E. Signal transduction in dysfunction with potential use of alpha-blockers. cavernous smooth muscle. World J Urol 1997; 15: 27±31. 6 Saenz de Tejada I, et al. Trabecular smooth muscle While several studies have reported on the modulates the capacitor function of the penis. Studies on a expression, identi®cation and function of alpha rabbit model. Am J Physiol 1991; 260: H1590±H1595. adrenergic receptors and their subtypes in erectile 7 Hatzichristou DG, et al. In vivo assessment of trabecular tissue,16,17,55,56 no studies have addressed the smooth muscle tone, it's application in pharmacocaverno- following questions. What are the cellular distribu- sometry and analysis of intracavernosal pressure determi- nants. J Urol 1995; 153: 1126±1131. tion of the alpha-1 and alpha-2 adrenergic receptor 8 Udelson, D et al. Engineering analysis of penile hemody- subtypes on the trabecular and penile arterial namic and structural dynamic relationships: Part I.- Clinical smooth muscle in erectile tissue? Are alpha-1 and implications of penile tissue mechanical properties. Int J Imp alpha-2 adrenergic receptor subtypes expressed Res 1998; 10: 15±24. with similar densities on the trabecular smooth 9 Diederichs W, Stief CG, Lue, TF, Tanagho EA. Norepinephr- ine involvement in penile detumescence. J Urol 1990; 143: muscle? What is the relative contribution of the 1264±1266. various alpha adrenergic receptor subtypes to the 10 Adaikan PG, Karim SMM. Adrenoceptors in the human contractile response? What are the key biochemical penis. J Auton Pharmacol 1991; 1: 199±203. reactions by which the vasodilators functionally 11 Gupta S, et al. Corpus cavernosum smooth muscle expresses functional post-synaptic alpha-2-adrenergic receptors. Br J antagonize the activity of the alpha adrenergic Pharmacol 1998; 123: 1237±1245. receptor at the second messenger level? What are 12 Hedlund H, Andersson KE, Mattiasson A. Pre- and post- the basis of functional selectivity and how such junctional adreno- and muscarinic receptor function in the information may be utilized to improve the effec- isolated corpus spongiosum urethrae. J Auton Pharmacol tiveness of alpha-blockers? 1984; 4: 241±246. 13 Kirkeby HJ, Forman A, Sorensen S, Andersson KE. Alpha Future research will have to address many of adrenoceptor function in isolated penile circum¯ex veins these questions in order to broaden our under- from potent and impotent men. J Urol 1989; 142: 1369±1371.

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