Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2004 Sep 27;143(6):745–752. doi: 10.1038/sj.bjp.0705987

Sympathectomy reveals α1A- and α1D-adrenoceptor components to contractions to noradrenaline in rat vas deferens

Linda Cleary 1, James Slattery 1, Sotiria Bexis 1, James R Docherty 1,*
PMCID: PMC1575931  PMID: 15451776

Abstract

  1. We have previously demonstrated that contractions of rat vas deferens to exogenous noradrenaline involve predominantly α1A-adrenoceptors, but that contractions to endogenous noradrenaline involve predominantly α1D-adrenoceptors. In this study, we have examined the effects of sympathectomy on the subtypes of α1-adrenoceptor in rat vas deferens in radioligand binding and functional studies.

  2. In vehicle-treated tissues, antagonist displacement of [3H]prazosin binding to α1-adrenoceptors was consistent with a single population of α1-adrenoceptors. Binding affinities for a range of α1-adrenoceptor antagonists were expressed as pKi values and correlated with known affinities for α1-adrenoceptor subtypes. The correlation was significant only with α1A-adrenoceptors.

  3. In tissues from rats sympathectomised with 6-hydroxy-dopamine (2 × 100 mg kg−1 i.p.), binding affinity for the α1D-adrenoceptor antagonist BMY 7378 fitted best with a two-site model.

  4. In functional studies, the potency of noradrenaline at producing total (phasic plus tonic) but not tonic contractions was increased in tissues from sympathectomised rats.

  5. Results obtained from sympathectomised rats suggest that phasic contractions are mainly α1D-adrenoceptor mediated, whereas tonic contractions are mainly α1A-adrenoceptor mediated, based on the effects of BMY 7378 and the α1A-adrenoceptor antagonist RS 100329.

  6. It is concluded that the predominant α1-adrenoceptor in vehicle-treated rat vas deferens is the α1A-adrenoceptor, both in terms of ligand binding and contractions to exogenous agonists. The α1D-adrenoceptor is only detectable by ligand binding following chemical sympathectomy, but is involved in noradrenaline-evoked contractions, particularly phasic contractions, of rat vas deferens.

Keywords: Rat vas deferens, α1A-adrenoceptors, α1D-adrenoceptors, prazosin, BMY 7378, RS100329

Introduction

α1-Adrenoceptors were initially subdivided into α1A- and α1B-subtypes in ligand binding studies (Morrow & Creese, 1986; Han et al., 1987). Molecular cloning techniques revealed initially four subtypes of α1-adrenoceptor: α1a, α1b, α1c and α1d (Cotecchia et al., 1988; Schwinn et al., 1990; Lomasney et al., 1991; Perez et al., 1991). The α1a/α1d clone represented a novel subtype of α1-adrenoceptor (α1D), whereas the α1c is now identified with the α1A-ligand binding site. These clones have now been renamed to match the functional receptors: α1A (formerly α1c), α1B (formerly α1b), and α1D (formerly α1a/α1d) (Heible et al., 1995). Selective agonists such as A61603 are available for α1A-adrenoceptors, with selective antagonists such as RS 100329 for α1A-adrenoceptors, and BMY 7378 for α1D-adrenoceptors (see Docherty, 1998; Guimares & Moura, 2001).

It has been variously suggested that contractions of rat vas deferens to exogenous noradrenaline or adrenaline are mediated predominantly by α1A-adrenoceptors (Han et al., 1987; Hanft & Gross, 1989; Aboud et al., 1993), or the postulated α1L-adrenoceptor in addition to α1A-adrenoceptors (Ohmura et al., 1992). In a previous study of rat vas deferens, we found that contractions to exogenous noradrenaline were mediated predominantly by α1A-adrenoceptors, and contractions to endogenous noradrenaline by α1D-adrenoceptors (Honner & Docherty, 1999). In this study, we have examined the subtypes of α1-adrenoceptor present in rat vas deferens in normal rat and how these are changed following sympathectomy. Some of these results have been published in abstract form (Slattery et al., 2003).

Methods

Male Wistar rats (250–350 g) were obtained from Trinity College Dublin, and vas deferens was employed as outlined below. Some animals were injected with 6-Hydroxy-Dopamine (100 mg kg−1 i.p.) on days 1 and 4 to produce a chemical sympathectomy, or with vehicle. Tissues were investigated on day 5 or 6. Animals were killed by CO2 overdose. The studies were carried out in accordance with the Declaration of Helsinki and have been approved by the Department of Health and by the RCSI Research Ethics Committee.

Rat vas deferens: membrane preparation

Membrane radioligand binding was performed using a method adapted from those of Cheung et al. (1982), Neylon & Summers (1985), Michel et al. (1989) and Connaughton & Docherty (1990). The vasa deferentia were removed and carefully cleaned of any adherent connective tissue. They were then weighed and chopped finely using small sharp scissors.

The tissue was diluted in 10 volumes of ice-cold initial wash buffer (Tris-HCl 50 mM, EDTA 5 mM: pH 7.4 at 4°C). The soluble components of the tissue were removed by a series of homogenisation and centrifugation steps. The tissue was homogenised in an Ultra-Turrax homogeniser for 30 s and centrifuged using an Eppendorf Micro-Centrifuge 5415C at 16,000 × g for 12 min at 4°C. Following this, the supernatant was discarded and the pellet fraction was resuspended in 10 volumes of ice-cold initial wash buffer. The homogenisation and centrifugation steps were repeated. The supernatant was again discarded and the pellet suspended in 10 volumes of ice-cold incubation buffer (Tris-HCl 50 mM, EDTA 5 mM: pH 7.4 at 25°C) and homogenised again. The homogenate was filtered through two layers of nylon 43 T-mesh gauze to remove connective tissue. Prepared membranes were used immediately in the radioligand binding assays.

Competition radioligand binding

Competition binding assays were carried out in duplicate in 5 ml polypropylene test tubes. A measure of 100 μl membrane aliquots were incubated with 100 μl of radioligand ([3H]prazosin, 2 nM) and 100 μl of unlabelled ligand, incubation buffer or phentolamine (10 μM). Assays were performed at 25°C for 30 min.

Specific binding was determined by subtracting the radioligand binding to nonspecific sites from the total radioligand binding for all sites. Nonspecific binding was determined in the presence of 10 μM phentolamine.

Following the 30-min incubation period, bound and free radioligand were separated by vacuum filtration. The assays were terminated by the addition of 5-ml ice-cold wash buffer (Tris-HCl 50 mM, EDTA 5 mM: pH 7.4 at 4°C) to all tubes. This was followed by rapid filtration through Whatman GF/C glass fibre filters using a Brandell Call Harvester. Filters and tubes were then washed four times with 5 ml of ice-cold wash buffer.

Each filter was placed in a standard polypropylene scintillation vial and 5 ml of organic liquid scintillation medium (Pico-Fluor 40, Packard) was added to each vial. The vials were placed on a shaker for 20 min before counting on a LKB 1214 Rack Beta counter.

Saturation radioligand binding

Saturation binding experiments were carried out as described above for competition studies, but employing increasing concentrations of [3H]prazosin (70–87 Ci mmol−1, New England Nuclear) (0.1–20 nM). Prazosin KD was 0.33±0.12 nM (n=6) and Bmax was 740±155 fmol mg−1 protein in vehicle-treated tissues.

Rat vas deferens: contractile studies

Rats were killed by CO2 overdose, and whole vas deferens was dissected out. Tissues were placed between platinum electrodes and attached to myograph transducers, orientated with the epididymal portion uppermost, under 1.0 g tension in organ baths at 37°C in Krebs–Henseleit solution of the following composition: (mM): NaCl 119; NaHCO3 25; D-glucose 11.1; KCl 4.7; CaCl2 2.5; KH2PO4 1.2; MgSO4 1.0. The solution was bubbled with 5%CO2/95%O2. Cocaine (3 μM) was additionally present prior to and during noradrenaline concentration–response curves. Data acquisition was carried out using a MacLab system.

Contractile responses to electrical stimulation and to exogenous agonists

Tissues were equilibrated for 30 min then stimulated with a single pulse, followed 5 min later by 40 pulses at 10 Hz (supramaximal voltage, 0.5 ms duration. Tissues were then contracted with noradrenaline (10 μM). Cocaine was then added and bathing fluid was changed every 15 min for the next hour. Following 45 min exposure to BMY 7378 (0.3 μM) or RS 100329 (0.03 μM) or vehicle, a single noradrenaline concentration–response curve was obtained per tissue. Tissues were contracted with noradrenaline cumulatively in 1.0 log unit increments at approximately 2-min intervals beginning with 100 nM. Responses to noradrenaline consisted of phasic and tonic contractions. Phasic contractions were measured as the total contraction (phasic plus tonic) and called total contraction. Tonic contractions were measured as the underlying baseline on which phasic contractions were superimposed. These tonic contractions were more difficult to measure in tissues from sympathectomised animals due to the high level of phasic contractions.

Drugs

Benoxathian hydrochloride (Research Biochemicals, U.S.A.); BMY 7378 (8-[2-(4-(2-methoxyphenyl) piperazin-1-yl)ethyl]-8-azaspiro[4,5]decane-7,9-dione; Research Biochemicals); HV 723 (α-ethyl-3,4,5-trimethoxy-α-(3-((2-(2-methoxyphenoxy)ethyl)-amino)-propyl)-benzene acetonitrile fumarate; gift: Hokurika, Japan); 6-hydroxy-dopamine hydrobromide (Sigma, Ireland); 5-methyl-urapidil (gift: Byk, Germany); phentolamine hydrochloride (Research Biochemicals); prazosin hydrochloride (gift; Pfizer, Sandwich, U.K.); RS 17053 (N-[2(2-cyclopropylmethoxy)ethyl]-5-chloro-α, α-dimethyl-1H-indole-3-ethylamine hydrochloride; gift: Roche Bioscience, U.S.A.); RS 100329 (5-methyl-3-[3-[4-[2-(2,2,2,-trifluoroethoxy)phenyl]-1-piperazinyl]propyl]-2,4-(1H)-pyrimidinedione;Tocris); spiperone (Research Biochemicals, U.S.A.); WB 4101 (2-(2′,6′-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxan; Research Biochemicals, U.S.A.).

Drugs were dissolved in distilled water, except for 6-hydroxy-dopamine (ascorbic acid 1 mg ml−1), spiperone (100% ethanol), and RS 17053 (DMSO), and dilutions were made up in distilled water.

Statistics

Values are mean±s.e.m. from n experiments. Statistical and graphical analysis was carried out using Instat for Macintosh and GraphPad Prism for PC. Antagonist Ki values were obtained from the equation Ki=IC50/(1+[3H]/KD), where Ki is the ligand affinity constant, IC50 is the concentration of ligand that produces 50% displacement of binding, [3H] is the concentration of radioligand employed (2 nM) and KD is the equilibrium dissociation constant for [3H]prazosin (0.33 nM). Correlations were made between functional antagonist potency and pooled binding affinity data from a number of studies. Two- and one-site models of ligand binding were compared by an F test based on increase in sum of squares divided by increase in degrees of freedom in going from two site to one. Hill slopes were considered significantly different from 1 when 95% confidence limits did not include 1.0.

Results

In vehicle-treated rats, displacement of [3H]prazosin binding to membranes of rat vas deferens fitted best with a one-site model for all 10 antagonists investigated, with Hill slopes not significantly different from 1, although Hill slopes were relatively low for several ligands including the α1A-adrenoceptor selective antagonists RS 17053 and RS 100329 and the α1D-adrenoceptor selective antagonist BMY 7378 (Table 1).

Table 1.

Ligand Ki values (−log M) and Hill slopes (mean±s.e.m.) for inhibition of [3H]prazosin binding at α1-adrenoceptor sites in membranes of vehicle-treated and sympathectomised rat whole vas deferens

  pKi Hill slope n
Vehicle-treated (all one-site models)      
 Prazosin 9.51±0.06 −0.99±0.05 6
 5-methyl-urapidil 8.05±0.16 −0.99±0.12 5
 RS 17053 7.46±0.19 −0.80±0.09 6
 Benoxathian 8.25±0.10 −1.10±0.09 4
 WB 4101 9.89±0.30 −0.86±0.15 4
 HV 723 8.50±0.29 −0.90±0.13 4
 BMY 7378 7.10±0.12 −0.84±0.11 6
 Phentolamine 7.70±0.20 −0.95±0.02 6
 Spiperone 7.56±0.17 −0.89±0.05 4
 RS 100329 9.61±0.19 −0.79±0.12 4
Sympathectomised      
 BMY 7378 (one-site model) 7.70±0.24 −0.46±0.03* 5
(two-site model) 9.20±0.24    
  7.17±0.14    
 RS 100329 (one-site model) 8.70±0.23 −0.64±0.12* 7
Open in a new tab
*

Hill slopes significantly different from 1.

In sympathectomised tissues, binding affinity for the α1D-adrenoceptor selective antagonist BMY 7378 fitted best with a two-site model (significant in five from five individual experiments and for all data combined), with pKi values of 9.59±0.30 (46±10% of specific binding) and 7.18±0.14 for high- and low-affinity sites respectively (n=5) (values quoted are the means from individual experiments and differ from those in Table 1). For the α1A-adrenoceptor selective antagonist RS 100329, binding affinity still fitted best with a one-site model in individual experiments (two-site model was best in only one from seven experiments), although the Hill slopes for both BMY 7378 and RS 100329 were significantly different from 1 (Table 1).

For BMY 7378, all data points obtained in five to six separate experiments for both vehicle-treated (n=6) and sympathectomised (n=5) tissues are shown in Figure 1. From these data, a Ki value for BMY 7378 of 7.10±0.12 was obtained for a one-site model in vehicle-treated tissue, and high- and low-affinity pKi values of 9.20±0.24 (42% of specific binding) and 7.17±0.14 were obtained for a two-site model in sympathectomised tissues. These affinities closely fit the published affinities of BMY 7378 for α1D- and α1A-adrenoceptors, respectively, and were used for calculation of correlations in Table 2.

Figure 1.

Figure 1

Open in a new tab

Displacement by BMY 7378 of [3H]prazosin binding to membranes of vas deferens from normal (vehicle-treated) (top) and sympathectomised rats (bottom). Values are expressed as % of specific binding of [3H]prazosin (2 nM). For sympathectomised tissues, the two-site model was significantly better than a one-site model, and both are shown. For normal tissues, the two-site model was not significantly different from the one-site model so that only the onr site model is shown. Data points shown are the mean of duplicates in five (sympathectomised) or six experiments (vehicle treated).

Table 2.

Correlation between published antagonist affinities for α1A-, α1B- and α1D- adrenoceptor ligand binding sites (see Honner & Docherty for affinity values of nine antagonists; additional values for RS 100329 taken from Williams et al., 1999) and antagonist affinities for ligand binding in rat vas deferens (Table 1), using both high- and low-affinity values obtained for BMY 7378

  α1A α1B α1D
BMY 7378 high-affinity site (9.20)
 r 0.23 0.17 0.78
 Significance NS NS P<0.01
BMY 7378 low-affinity site (7.17)
 r 0.78 0.47 0.57
 Significance P<0.01 NS NS
Open in a new tab

NS=not significant.

Correlation analysis between ligand affinities for α1-adrenoceptors in rat vas deferens (Table 1) and published data for their affinities at subtypes of α1 was carried out (Table 2). Correlation of rat vas deferens binding using the low-affinity site for BMY 7378 was significant only with the α1A-adrenoceptor (r=0.78, P<0.01), and was poor with the α1B-adrenoceptor (r=0.47, NS) and the α1D-adrenoceptor (r=0.57, NS). If the value of BMY 7378 at the high-affinity sites obtained in sympathectomised rats were used in correlations with α1A- and α1D-adrenoceptors, respectively, the correlation with the α1D-adrenoceptor (r=0.78, P<0.01) became significant, and that with the α1A-adrenoceptor (r=0.23, NS) became nonsignificant (Table 2).

Rat vas deferens; contractile studies

Single pulse stimulation produced contractions of 1.22±0. 22 g (n=16) and 0.42±0.06 g (n=22) in tissues from vehicle-treated and sympathectomised animals (P<0.001). Stimulation at 10 Hz for 4 s produced contractions of 3.85±0.43 g (n=16) and 1.68±0.15 g (n=22) in tissues from vehicle-treated and sympathectomised animals (P<0.001). Responses to 10 Hz stimulation in sympathectomised rats were prolonged in terms of time to maximum response and time from end of stimulation to baseline.

Noradrenaline produced contractions of rat vas deferens consisting of phasic and tonic components (Figures 2 and 3), but the relative importance of the phasic response was greater in tissues from sympathectomised animals, so that responses were analysed as total response (phasic and tonic combined) and tonic (maintained) response (Figures 4 and 5).

Figure 2.

Figure 2

Open in a new tab

Traces obtained from typical experiments showing response to increasing concentrations of noradrenaline in vas deferens from vehicle-treated (top) and sympathectomised (bottom) rat. Note the predominance of phasic spikes to low concentrations of noradrenaline in tissues from sympathectomised animals.

Figure 3.

Figure 3

Open in a new tab

Traces obtained from typical experiments showing the effects of RS 100329 (0.03 μM) on responses to increasing concentrations of noradrenaline in vas deferens from vehicle-treated (top) and sympathectomised (bottom) rat. Note the predominance of phasic spikes in both tissues.

Figure 4.

Figure 4

Open in a new tab

Concentration–response curves obtained to noradrenaline following vehicle, RS 100329 (0.03 μM) or BMY 7378 (0.3 μM) in vas deferens from vehicle-treated (top) and sympathectomised rats (bottom). The graphs shows total contractions (spike plus tonic), Vertical bars represent s.e. of mean from five to seven experiments.

Figure 5.

Figure 5

Open in a new tab

Concentration–response curves obtained to noradrenaline following vehicle, RS 100329 (0.03 μM) or BMY 7378 (0.3 μM) in vas deferens from vehicle-treated (top) and sympathectomised rats (bottom). The graphs show tonic contractions. Vertical bars represent s.e. of mean from five to seven experiments.

In control experiments (in the absence of receptor antagonists), there were no significant differences between vehicle-treated and sympathectomised rats in the maximum total or tonic responses to noradrenaline (Figures 4 and 5). There was no difference between vehicle-treated and sympathectomised animals in the potency of noradrenaline in producing tonic contractions (Figure 5). However, noradrenaline was significantly more potent at producing phasic contractions in tissues from sympathectomised animals (Figure 4 and Table 3).

Table 3.

Potency of noradrenaline (pD2, −log M) in the presence of vehicle (control), BMY 7378 (0.3 μM) or RS 100329 (0.03 μM) at producing total and phasic contractions in t whole vas deferens from vehicle-treated and sympathectomised rats

  Vehicle treated Sympathectomised
NA pD2: total contractions
 Control 5.98±0.18 6.68±0.23**
 BMY 7378 5.11±0.19* 5.88±0.23*
 RS 100329 4.03±0.22* 6.52±0.23**
NA pD2: tonic contractions
 Control 5.93±0.21 6.03±0.14
 BMY 7378 5.24±0.12* 5.56±0.33
 RS 100329 <3.50* 4.08±0.14*
Open in a new tab

Values are mean and s.e. of mean from five to seven experiments.

*

P<0.05 from potency of noradrenaline in relevant control experiments.

**

P<0.05 from potency of noradrenaline in vehicle-treated animals.

The total contraction to noradrenaline was unaffected by RS 100329 (0.03 μM) in tissues from sympathectomised rats (Figures 3 and 4 and Table 3), whereas this concentration of RS 100329 produced a very marked shift in the total contaction to noradrenaIine in tissues from vehicle-treated animals (Figures 3 and 4 and Table 3). RS 100329 produced large shifts in the potency of noradrenaline at producing tonic contractions in tissues from both vehicle-treated and sympathectomised animals (Figures 3 and 5).

BMY 7378 (0.3 μM) significantly shifted the potency of noradrenaline in terms of total contraction in both groups of rat, but significantly shifted tonic contractions only in tissues from vehicle-treated animals (Figures 4 and 5 and Table 3).

Discussion

In this study we have found that the ligand binding affinity of BMY 7378 is altered by sympathectomy in rat vas deferens. Firstly, sympathectomy using the current protocol was not complete in that some response to nerve stimulation remained. Despite this, there were marked changes in ligand binding and in responses to exogenous noradrenaline.

BMY 7378 is especially interesting as an α1D-adrenoceptor selective antagonist (Goetz et al., 1995) that has high potency as an antagonist in rat aorta, but low potency in vas deferens (Deng et al., 1996). In a previous study, taking the published data for α1-adrenoceptor ligand binding sites, we correlated with contractile data and found that the predominant receptor mediating contractions to exogenous noradrenaline in rat vas deferens was an α1A-adrenoceptor (Honner & Docherty, 1999), in agreement with previous findings (Teng et al., 1994; Burt et al., 1995). For responses to endogenous noradrenaline (nerve-mediated responses) in rat vas deferens, there was a significant correlation only with α1D-adrenoceptors (Honner & Docherty, 1999), in agreement with evidence for expression of α1D-adrenoceptors in rat vas deferens (Perez et al., 1991; Rokosh et al., 1994).

In the present study, we initially investigated whether these two subtypes of α1-adrenoceptor can be identified in ligand binding studies of rat vas deferens. In vehicle-treated tissues, antagonist binding was consistent with a single population of α1-adrenoceptors, with Hill slopes not significantly different from unity. Binding affinities for a range of α1-adrenoceptor antagonists were expressed as pKi values and correlated with known affinities for α1-adrenoceptors. There was a significant correlation with published data for ligand binding affinities at α1A-adrenoceptors but not at α1B- or α1D-adrenoceptors. Furthermore, the pKi values obtained in the present study correlated significantly with the potency of antagonists at inhibiting contractions to exogenous noradrenaline (an α1A-adrenoceptor mediated response: r=0.81, P<0.01), but not with the potency of antagonists at inhibiting the nifedipine-insensitive α1-adrenoceptor mediated electrically evoked contraction (α1D-adrenoceptor mediated; r=0.49, NS) (functional data taken from Honner & Docherty, 1999). Hence, in vas deferens from vehicle-treated rats, ligand binding reveals only the predominant α1A-adrenoceptor involved in contractions to exogenous noradrenaline, which is presumably located widely on the smooth muscle cells. The α1D-adrenoceptor involved in nerve-stimulation evoked contractions is presumably localised to areas postjunctional to nerve terminals, and, although making a major contribution to contractile responses to endogenous noradrenaline, is relatively unimportant in terms of receptor number. We next hypothesised that it might be possible to identify these α1D-adrenoceptors following chemical sympathectomy, which should result in denervation supersensitivity. Hence, experiments were carried out employing 6-hydroxy-dopamine to cause chemical sympathectomy (Thoenen & Tranzer, 1968).

Following chemical sympathectomy, binding of BMY 7378 to rat vas deferens membranes fitted best with a two-site model, yielding affinities which fitted with its known affinity for α1D-adrenoceptors (high-affinity site) and with α1A- or α1B-adrenoceptors (low-affinity site). However, since the predominant site in untreated rat vas deferens is an α1A-adrenoceptor, it can be assumed that the low-affinity site in sympathectomised tissue is an α1A-adrenoceptor. For the α1A-adrenoceptor selective antagonist RS 100329, binding to vas deferens from sympathectomised rats fitted best with a one-site model, although the Hill slope was significantly less than 1 (0.64), a two-site model was best in one experiment and the affinity value (8.43) was significantly lower that that in untreated vessels (9.61), but still higher than its affinity for α1B- or α1D-adrenoceptors. This may suggest that the presence of an increased number of α1D-adrenoceptors affected the affinity of RS 100329, but not enough to produce a two-site model. Presumably, since BMY 7378 shows 300-fold selectivity for α1D over α1A but RS 100329 shows only 30-fold selectivity for α1A over α1D (see Table 2), a two-site model is more easy to obtain with BMY 7378.

It has long been known that, following chemical or surgical denervation of adrenergic nerves, a postjunctional supersensitivity to agonists occurs. Part of the supersensitivity to exogenous noradrenaline is prejunctional by loss of nerve terminals, and so loss of noradrenaline re-uptake, similar to a cocaine supersensitivity (Trendelenburg, 1966; Iversen, 1967). A postjunctional component has also been postulated. Reserpine increases the potency of phenylephrine (Nasseri et al., 1985), and surgical denervation increases the potency of noradrenaline (Campos et al., 2003) in rat vas deferens Surgical or chemical sympathectomy produces supersensitivity in other tissues (Amark & Olson, 1992; Chou et al., 2001). The enhanced contractile response of rat vas deferens to noradrenaline following surgical denervation has been linked to protein kinase C and to decreased involvement of nifedipine-sensitive calcium entry (Abraham et al., 2003). In our neurotransmission studies, the α1D-adrenoceptor mediated contaction of rat vas deferens is indeed insensitive to nifedipine (Honner & Docherty, 1999), suggesting that the changes seen by Abraham et al. (2003) may involve α1D-adrenoceptors.

In terms of receptor number, the number of α1D-adrenoceptors increases from undetectable in vehicle-treated tissue (say, less than 10%) to 46% in sympathectomised tissues. We did not look at maximum number of binding sites in sympathectomised tissues (given the large variation in Bmax values obtained in vehicle-treated tissues, we would only have been able to detect large changes in Bmax), so that we cannot say whether this represents an increase in total number of receptors or substitution of α1D for α1A. Previous studies of the effects of chemical sympathectomy in rat or guinea-pig vas deferens with 6-hydroxydopamine or reserpine reported no change in α1-adrenoceptor density (Watanabe et al., 1982; Cowan et al., 1985; Nasseri et al., 1985). If there is no increase in receptor number following chemical sympathectomy, then supersensitivity to agonists must be due to change in receptor subtype. Indeed, noradrenaline and phenylephrine are more potent in rat aorta (α1D) than in rat mesenteric artery (α1A) (e.g. from our group: Cawley et al., 1995; Connolly et al., 1999), so that it is likely that such a change in receptor subtype would result in supersensitivity to agonists.

The second part of our study looked at whether changes in ligand binding sites resulted in changes in functional responses to noradrenaline. To eliminate a prejunctional component to supersensitivity (due to loss of nerve terminals and noradrenaline transporters), experiments were carried out in the presence of cocaine to block noradrenaline uptake. As always with rat vas deferens, the results were complicated, but interesting. The contractile response to noradrenaline changed from a largely tonic response with small phasic contractions superimposed in tissues from vehicle-treated animals, to a predominantly phasic contraction in tissues from sympathectomised animals. Potency of noradrenaline was significantly increased in tissues from sympathectomised animals in terms of total contraction but not tonic contraction. We next asked whether phasic contractions differed from tonic contractions in the receptor subtype involved. We have previously investigated this problem but found no clear differences (Honner & Docherty, 1999). The results from vehicle-treated animals in the present study might suggest that tonic and phasic contractions involve both α1A- and α1D-adrenoceptors, although α1A-adrenoceptors may predominate in tonic contractions. The results from sympathectomised animals give a much more clear answer: phasic contractions are predominatly α1D-adrenoceptor mediated (antagonised by BMY 7378 but not RS 100329) and tonic contractions are predominantly α1A-adrenoceptor mediated (antagonised by RS 100329 but not BMY 7378). Given that it is less easy to separate the two components of contraction in vehicle-treated animals, we would like to suggest that the same is true for vehicle-treated animals: α1D-adrenoceptor mediate phasic and α1A-adrenoceptor mediate tonic contractions.

α1-Adrenoceptor agonists have been used with some limited success in the treatment of male infertility (Kamischke & Nieschlag, 2002). They act to contract the vas deferens, to treat particularly anejaculation (AE). Phasic contractions are more likely to produce propulsive movements than tonic contractions. This study suggests that development of an α1D-adrenoceptor agonist may lead to more effective therapy for AE, and possibly in retrograde ejaculation (RE), with fewer side effects. α1D-Adrenoceptor knockout mice are apparently fertile, so that this receptor does not seem to be essential for sperm propulsion (Tanoue et al., 2003). However, in combination with, for instance a P2X1 receptor antagonist (Mulryan et al., 2000), an α1D-adrenoceptor antagonist may potentially have a role also as a male contraceptive.

It is concluded that the predominant α1-adrenoceptor in vehicle-treated rat vas deferens is the α1A-adrenoceptor, both in terms of ligand binding and contractions to exogenous agonists. The α1D-adrenoceptor, although involved in nerve-evoked contractions of rat vas deferens, is only detectable by ligand binding following chemical sympathectomy. This suggests that sympathectomy increases the number of receptors in the junctional region of the postjunctional membrane, or that these spread to other areas of the smooth muscle. The evidence obtained from sympathectomised rats suggests that phasic contractions are mainly α1D-adrenoceptor mediated, whereas tonic contractions are mainly α1A-adrenoceptor mediated.

Acknowledgments

This work was supported by The Health Research Board (Ireland), RCSI and the Irish Heart Foundation.

Abbreviations

BMY 7378

(8-[2-(4-(2-methoxyphenyl) piperazin-1-yl)ethyl]-8-azaspiro[4,5]decane-7,9-dione)

HV 723

(α-ethyl-3,4,5-trimethoxy-α-(3-((2-(2-methoxyphenoxy)ethyl)-amino)-propyl)-benzene acetonitrile fumarate)

RS 17053

(N-[2(2-cyclopropylmethoxy)ethyl]-5-chloro-α, α-dimethyl-1H-indole-3-ethylamine hydrochloride)

RS 100329

(5-methyl-3-[3-[4-[2-(2,2,2,-trifluoroethoxy)phenyl]-1-piperazinyl]propyl]-2,4-(1H)-pyrimidinedione)

WB 4101

(2-(2′,6′-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxan)

References

  1. ABOUD R.W., SHAFII M., DOCHERTY J.R. Investigation of the subtypes of α1-adrenoceptor mediating contraction of rat aorta, vas deferens and spleen. Br. J. Pharmacol. 1993;109:80–87. doi: 10.1111/j.1476-5381.1993.tb13534.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. ABRAHAM S.T., ROBINSON M., RICE P.J. A role for protein kinase C in the supersensitivity of the rat vas deferens following chronic surgical denervation. Pharmacology. 2003;67:32–40. doi: 10.1159/000066784. [DOI] [PubMed] [Google Scholar]
  3. AMARK P., OLSON L. Alpha-adrenoceptor function before and after chemical sympathectomy in human and feline detrusor muscles. Urol. Res. 1992;20:265–269. doi: 10.1007/BF00300256. [DOI] [PubMed] [Google Scholar]
  4. BURT R.P., CHAPPLE C.R., MARSHALL I. Evidence for a functional α1A- (α1c-) adrenoceptor mediating contraction of the rat epididymal vas deferens and an α1B-adrenoceptor mediating contraction of the rat spleen. Br. J. Pharmacol. 1995;115:467–475. doi: 10.1111/j.1476-5381.1995.tb16356.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. CAMPOS M., DE LUCENA MORAIS P., PUPO A.S. Functional characterization of alpha(1)-adrenoceptors in denervated rat vas deferens. Naunyn-Schmeideberg's Arch. Pharmacol. 2003;368:72–80. doi: 10.1007/s00210-003-0770-z. [DOI] [PubMed] [Google Scholar]
  6. CAWLEY T., GERAGHTY J., OSBORNE H., DOCHERTY J.R. Effects of portal hypertension on responsiveness of rat mesenteric artery and aorta. Br. J. Pharmacol. 1995;114:791–796. doi: 10.1111/j.1476-5381.1995.tb13274.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. CHEUNG T.D., BARNETT D.B., NAHORSKI S.R. [3H]-rauwolscine and [3H]-yohimbine binding to rat cerebral cortex and human platelet membranes: possible heterogeneity of alpha2-adrenoceptors. Eur. J. Pharmacol. 1982;84:79–85. doi: 10.1016/0014-2999(82)90159-5. [DOI] [PubMed] [Google Scholar]
  8. CHOU P.I., LU D.W., CHEN J.T. Adrenergic supersensitivity of rabbit choroidal blood vessels after sympathetic denervation. Curr. Eye Res. 2001;23:352–356. doi: 10.1076/ceyr.23.5.352.5447. [DOI] [PubMed] [Google Scholar]
  9. CONNAUGHTON S., DOCHERTY J.R. No evidence for differences between pre- and postjunctional alpha2-adrenoceptors in the periphery. Br. J. Pharmacol. 1990;99:97–102. doi: 10.1111/j.1476-5381.1990.tb14660.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. CONNOLLY C., CAWLEY T., MCCORMICK P.A., DOCHERTY J.R. Portal hypertension increased vasoconstrictor responsiveness of rat aorta. Clin. Sci. 1999;96:41–47. [PubMed] [Google Scholar]
  11. COTECCHIA S., SCHWINN D.A., RANDALL R.R., LEFKOWITZ R.J., CARON M.G., KOBILKA B.K. Molecular cloning and expression of the cDNA for the hamster α1-adrenergic receptor. Proc. Natl. Acad. Sci. U.S.A. 1988;85:7159–7163. doi: 10.1073/pnas.85.19.7159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. COWAN F.F., WONG S.K., WESTFALL D.P., FLEMING W.W. Effect of postganglionic denervation and pretreatment with reserpine on alpha-adrenoceptors of the guinea-pig vas deferens. Pharmacology. 1985;30:289–295. doi: 10.1159/000138080. [DOI] [PubMed] [Google Scholar]
  13. DENG X.F., CHEMTOB S., VARMA D.R. Characterization of α1D-adrenoceptor subtype in rat myocardium, aorta and other tissues. Br. J. Pharmacol. 1996;119:269–276. doi: 10.1111/j.1476-5381.1996.tb15981.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. DOCHERTY J.R. Subtypes of functional alpha1- and alpha2-adrenoceptor adrenoceptors. Eur. J. Pharmacol. 1998;361:1–15. doi: 10.1016/s0014-2999(98)00682-7. [DOI] [PubMed] [Google Scholar]
  15. GOETZ A.S., KING H.K., WARD S.D.C., TRUE T.A., RIMELE T.J., SAUSSY D.L. BMY 7378 is a selective antagonist of the D subtype of α1-adrenoceptors. Eur. J. Pharmacol. 1995;272:R5–R6. doi: 10.1016/0014-2999(94)00751-r. [DOI] [PubMed] [Google Scholar]
  16. GUIMARES S., MOURA D. Vascular adrenoceptors: an update. Pharmacol. Rev. 2001;53:319–356. [PubMed] [Google Scholar]
  17. HAN C., ABEL P.W., MINNEMAN K.P. α1-Adrenoceptor subtypes linked to different mechanisms for increasing intracellular Ca2+ in smooth muscle. Nature. 1987;329:333–335. doi: 10.1038/329333a0. [DOI] [PubMed] [Google Scholar]
  18. HANFT G., GROSS G. Subclassification of α1-adrenoceptor recognition sites by urapidil derivatives and other selective antagonists. Br. J. Pharmacol. 1989;97:691–700. doi: 10.1111/j.1476-5381.1989.tb12005.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. HEIBLE J.P., BYLUND D.B., CLARKE D.E., EIKENBURG D.C., LANGER S.Z., LEFKOWITZ R.J., MINNEMAN K.P., RUFFOLO R.R. International Union of Pharmacology recommendation or nomenclature of α1-adrenoceptors: consensus update. Pharmacol. Rev. 1995;47:267–270. [PubMed] [Google Scholar]
  20. HONNER V., DOCHERTY J.R. Investigation of the subtypes of alpha1-adrenoceptor mediating contractions of rat vas deferens. Br. J. Pharmacol. 1999;128:1323–1331. doi: 10.1038/sj.bjp.0702913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. IVERSEN L.L. The Uptake and Storage of Noradrenaline in Sympathetic Nerves. Cambridge: Cambridge University Press; 1967. [Google Scholar]
  22. KAMISCHKE A., NIESCHLAG E. Update on medical treatment of ejaculatory disorders. Int. J. Androl. 2002;25:333–344. doi: 10.1046/j.1365-2605.2002.00379.x. [DOI] [PubMed] [Google Scholar]
  23. LOMASNEY J.W., COTECCHIA S., LORENZ W., LEUNG W.-Y., SCHWINN D.A., YANG-FENG T.L., BROWNSTEIN M., LEFKOWITZ R.J., CARON M.G. Molecular cloning and expression of the cDNA for the α1A-adrenergic receptor. J. Biol. Chem. 1991;266:6365–6369. [PubMed] [Google Scholar]
  24. MICHEL A.D., LOURY D.N., WHITING R.L. Identification of a single alpha1-adrenoceptor corresponding to the alpha1A-subtype in rat submaxillary gland. Br. J. Pharmacol. 1989;98:883–889. doi: 10.1111/j.1476-5381.1989.tb14617.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. MORROW A.L., CREESE I. Characterization of α1-adrenergic subtypes in rat brain: a reevaluation of [3H] WB 4101 and [3H] prazosin binding. Mol. Pharmacol. 1986;29:321–330. [PubMed] [Google Scholar]
  26. MULRYAN K., GITTERMAN D.P., LEWIS C.J., VIAL C., LECKIE B.J., COBB A.L., BROWN J.E., CONLEY E.C., BUELL G., PRITCHARD C.A., EVANS R.J. Reduced vas deferens contraction and male infertility in mice lacking P2X1 receptors. Nature. 2000;403:86–89. doi: 10.1038/47495. [DOI] [PubMed] [Google Scholar]
  27. NASSERI A, BARAKEH J.F., ABEL P.W., MINNEMAN K.P. Reserpine-induced postjunctional supersensitivity in rat vas deferens and caudal artery without changes in alpha adrenergic receptors. J. Pharmacol. Exp. Ther. 1985;234:350–357. [PubMed] [Google Scholar]
  28. NEYLON C.B., SUMMERS R.J. [3H]-rauwolscine binding to alpha2-adrenoceptors in the mammalian kidney: apparent receptor hetyerogeneity between species. Br. J. Pharmacol. 1985;85:349–359. doi: 10.1111/j.1476-5381.1985.tb08868.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. OHMURA T., OSHITA M., KIGOSHI S., MURAMATSU I. Identification of α1-adrenoceptor subtypes in the rat vas deferens: binding and functional studies. Br. J. Pharmacol. 1992;107:697–704. doi: 10.1111/j.1476-5381.1992.tb14509.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. PEREZ D.M., PIASCIK M.T., GRAHAM R.M. Solution-phase library screening for the identification of rare clones: Isolation of an α1D-adrenergic receptor cDNA. Mol. Pharmacol. 1991;40:876–883. [PubMed] [Google Scholar]
  31. ROKOSH D.G., BAILEY B.A., STEWART A.F.R., KARNS L.R., LONG C.S., SIMPSON P.C. Distribution of α1c-adrenergic receptor mRNA in adult rat tissues by RNase protection and comparison with α1B and α1D. Biochem. Biophys. Res. Commun. 1994;200:1177–1184. doi: 10.1006/bbrc.1994.1575. [DOI] [PubMed] [Google Scholar]
  32. SCHWINN D.A., LOMASNEY J.W., LORENZ W., SZKLUT P.J., FREMEAU R.T., YANG-FENG T.L., CARON M.G., LEFKOWITZ R.J., COTECCHIA S. Molecular cloning and expression of the cDNA for a novel α1-adrenergic receptor subtype. J. Biol. Chem. 1990;265:8183–8189. [PubMed] [Google Scholar]
  33. SLATTERY J., HONNER V., CLEARY L., DOCHERTY J.R. Correlation between ligand binding sites and subtypes of alpha1-adrenoceptor mediating contractions of rat vas deferens. Br. J. Pharmacol. 2003;140:51P. doi: 10.1038/sj.bjp.0702913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. TANOUE A., KOSHIMIZU T.A., SHIBATA K., NASA Y., TAKEO S., TSUJIMOTO G. Insights into alpha1 adrenoceptor function in health and disease from transgenic animal studies. Trends Endocrinol. Metab. 2003;14:107–113. doi: 10.1016/s1043-2760(03)00026-2. [DOI] [PubMed] [Google Scholar]
  35. TENG C.-M., GUH J.-H., KO F.-N. Functional identification of α1-adrenoceptor subtypes in human prostate: comparison with those in rat vas deferens and spleen. Eur. J. Pharmacol. 1994;265:61–66. doi: 10.1016/0014-2999(94)90223-2. [DOI] [PubMed] [Google Scholar]
  36. THOENEN H., TRANZER J.P. Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine. Naunyn-Schmiedeberg's Arch. Pharmacol. 1968;261:271–288. doi: 10.1007/BF00536990. [DOI] [PubMed] [Google Scholar]
  37. TRENDELENBURG U. Mechanisms of supersensitivity and subsensitivity to sympatmomimetic amines. Pharmacol. Rev. 1966;18:629–640. [PubMed] [Google Scholar]
  38. WATANABE Y., LAI R.T., MAEDA H., YOSHIDA H. Reserpine and sympathetic denervation cause an increase of postsynaptic alpha2-adrenoceptors. Eur. J. Pharmacol. 1982;80:105–108. doi: 10.1016/0014-2999(82)90183-2. [DOI] [PubMed] [Google Scholar]
  39. WILLIAMS T.J., BLUE D.R., DANIELS D.V., DAVIS B., ELWORTHY T., GEVER J.R., KAVAS M.S., MORGANS D., PADILLA F, TASSA S., VIMONT R.L., CHAPPLE C.R., CHESS-WILLIAMS R., EGLEN R.M., CLARKE D.E., FORD A.P. In vitro alpha1-adrenoceptor pharmacology of Ro 70-0004 and RS-100329, novel alpha1A-adrenoceptor selective antagonists. Br. J. Pharmacol. 1999;127:252–254. doi: 10.1038/sj.bjp.0702541. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES