- Top of page
This study was intended to quantify the amounts of the α1-adrenoceptor subtype mRNAs in human vas deferens, and demonstrate the receptor subtype responsible for the vas contraction.
The RNase protection assay showed that the mean total amount of α1a mRNA was 7.4±2.2 pg/5 μg of poly (A)+ RNA (97.0% of the total α1 mRNA) in the epididymal portion (E-vas) and 4.9±0.8 pg/5 μg of poly (A)+ RNA (96.3% of the total) in the pelvic portion (P-vas). The E-vas showed a tendency to have a greater α1a mRNA abundance than the P-vas (P=0.11). The α1b and α1d mRNAs were absent or of extremely low abundance.
By an in situ hybridization, the α1a and α1d mRNAs were recognized in the smooth muscle cells of the E-vas and the P-vas, and the distribution pattern the same in both tissues. The α1b mRNA positive site was scarcely detectable in both vas portions.
In a functional study, l-phenylephrine produced concentration-dependent contraction in the E-vas (Emax=2.24±0.70 g; pD2=5.32±0.09) and the P-vas (Emax=2.46±0.46 g; pD2=5.07±0.12). KMD-3213, a novel α1A-adrenoceptor-selective antagonist, caused parallel rightward shifts of the concentration–response curves for l-phenylephrine. Apparent pKB values were 9.90±0.16 for the E-vas and 9.71±0.17 for the P-vas. There was no significant difference in Emax, pD2 or pKB estimates between the two portions.
We have found that α1a mRNA is predominant in the human vas deferens, and confirmed that contraction of this organ is mediated by the α1A-adrenoceptor.
- Top of page
Using antisense RNA probes prepared from the cloned genes, various investigators have detected mRNAs of these subtypes in many organs and tissues (Price et al., 1993; 1994; Weinberg et al., 1994; Moriyama et al., 1996; Nasu et al., 1996). The vas deferens has a muscular coat (tunica muscularis) that is made up of a middle circular layer surrounded by inner and outer longitudinal muscle layers (Neaves, 1975). The thickness of the muscle layers gradually decreases along the length of the ductus deferens (Paniagua et al., 1982). Adrenergic nerves are observed in all three layers of the tunica muscularis, but their density is the greatest in the outer longitudinal layer (McConnell et al., 1982), and noradrenaline appears to be the main mediator of contractile responses via adrenoceptors (Holmquist et al., 1990).
Many mechanisms contribute to the transport of spermatozoa in the ductus deferens (Guha et al., 1975; Neaves, 1975). Among these, muscle peristaltic contraction induced by adrenergic neurotransmitters is important in propelling the ductal contents (Bruschini et al., 1977; Lipshultz et al., 1981). A defect in the contractility of the vas deferens is thought to account for some cases of infertility. Recently, Furukawa et al. (1995) reported that the contractile response to l-phenylephrine is mediated by the α1A-adrenoceptor subtype in human vas deferens, and pointed out that this tissue is readily available for functional studies of this receptor.
The vas deferens may be divided into the following five portions: (1) the epididymal portion within the tunica vaginalis, (2) the scrotal portion, (3) the inguinal division, (4) the retroperioneal or pelvic portion, and (5) the ampulla (Lich et al., 1978). The regional variation in purinergic and adrenergic responses has been examined in the vas deferens of various experimental animals, and noradrenaline was found to contribute more to contraction in the epididymal portion (E-vas) than in the pelvic portion (P-vas) (Sneddon & Machaly, 1992). However, there is no such report for human vas deferens.
The purpose of this study was to quantify the amounts of the α1-adrenoceptor subtype mRNAs and to estimate the ratio of these mRNAs in the two main parts of the human vas deferens (E-vas and P-vas), and demonstrate the subtype responsible for the vas contraction. This information is relevant to the design of pharmacological stimulators of the contraction of vas deferens.
- Top of page
The existence of three α1-adrenoceptor subtypes has recently been demonstrated by use of molecular biological techniques, and the expression of these subtype mRNAs has previously been investigated in various human tissues (Price et al., 1994; Weinberg et al., 1994).
Functional studies of human prostate samples obtained from prostatectomy have concluded that the predominant functional receptor in this tissue has the pharmacological characteristics of the native α1A-adrenoceptor, which appears to be identical to the cloned α1a-adrenoceptor (Lepor et al., 1993; Chapple et al., 1994). RNase protection assay also showed that the cloned α1a-adrenoceptor is the predominant receptor in the human prostate (Price et al., 1993; Nasu et al., 1996).
Investigations of the regional distribution of functional α1-adrenoceptors in the rat vas deferens, using pharmacological and molecular pharmacological techniques, revealed that the α1A-adrenoceptor subtype is predominant in the E-vas (Faure et al., 1994; Rokosh et al., 1994). Regional variation in purinergic and adrenergic responses of the vas deferens was examined in various experimental animals, and noradrenaline was reported to be more responsible for contraction in the E-vas than in the P-vas (Sneddon & Machaly, 1992). Furthermore, Furukawa et al. (1995) recently reported that the contractile responses of human vas deferens (E-vas) to l-phenylephrine were mediated through the α1A-adrenoceptor subtype.
However, the expression and distribution of α1-adrenoceptor subtype mRNAs have not been confirmed yet in human vas deferens. Additionally, regional distribution of these receptor has not been studied. The aim of the present study was to characterise the α1 subtype responsible for mediating contractile responses of the human vas deferens as well as the subtype variation in its two main portions (E-vas and P-vas). The three α1-adrenoceptor subtypes mRNAs (α1a, α1b and α1d) were quantified with RNase protection assay, and the distribution of each subtype was confirmed by in situ hybridization. Functional studies were also performed using KMD-3213, a novel α1A-adrenoceptor-selective antagonist (Shibata et al., 1995).
We have quantified each α1-adrenoceptor subtype mRNA expressed in the E-vas and the P-vas by using an RNase protection assay. The predominant subtype mRNA was the α1a subtype in both portions of the vas deferens, where it accounted for almost 100% of the total α1 mRNA, and the α1b and the α1d subtypes were absent or extremely low. These findings are supported by our in situ hybridization study (Figure 2). Furthermore, our functional study (Figure 3), as well as the previous work (Furukawa et al., 1995), also indicate the view that the α1A-adrenoceptor mediates the contraction of the human vas deferens. This low abundance of α1d mRNA is in contrast to its relatively high levels in human prostate (Moriyama et al., 1996; Nasu et al., 1996), which showed about 15% being α1d in benign prostatic hypertrophy (BPH) and 30% being α1d in non-BPH. We also found no regional variation in ratios of α1-adrenoceptor subtype mRNAs in human vas deferens. This lack of regional variation, and the extremely low abundance of α1b and α1d may be characteristic of human vas deferens. In the contractile experiments, however, KMD-3213 (1 nm), an α1A-adrenoceptor-selective antagonist, reduced the Emax values to l-phenylephrine by 16.4% only in the E-vas. This finding suggests that the insurmountability of KMD-3213 in the E-vas may be slightly higher than that in the P-vas.
Through in situ hybridization, we revealed that the α1a and the α1d mRNAs were primarily localised in all of the muscle layers in both E-vas and P-vas, in which there was no clear difference in localisation of either subtype. The scarce α1b-adrenoceptor mRNA could be demonstrated. The staining intensities of α1a and α1d mRNA were not proportional to the mRNA levels determined by RNase protection assay. However, in situ hybridization is primarily a qualitative technique that demonstrates the location of the mRNA but not its exact amount. Thus, the findings on in situ hybridization are consistent with the results of the RNase protein assay and functional study.
In the experiments with human recombinant adrenoceptors, KMD-3213 showed a Ki value of 0.036 nm for the α1A-adrenoceptor, but has 580- and 56-fold lower affinity for α1B and α1D-adrenoceptors. KMD-3213 also has a potent affinity for the native α1A-adrenoceptor with a pKB value of 10.0 in rabbit prostate and a pKi value of 9.82 in the rat submaxillary gland (Shibata et al., 1995; Yamagishi et al., 1996), and has a lower affinity for the α1D-adrenoceptor with a pA2 value of 8.13 (Yamagishi et al., 1996). In the present study, the pKB values for KMD-3213 were 9.90 in the E-vas and 9.71 in the P-vas, values that show good agreement with the pKB value and pKi value in the recombinant or native α1A-adrenoceptor. They are also comparable to the value of tamsulosin (pKB = 10.0) for the human vas deferens (Furukawa et al., 1995). Furakawa et al. (1995) concluded that the α1B-adrenoceptor is not the predominant subtype mediating responses of human vas deferens, because l-phenylephrine produced dose-dependent contractions, the treatment with CEC failed to alter responses significantly, and WB4101, 5-methylurapidil and oxymetazoline had relatively high affinities (Furukawa et al., 1995). These data suggest that the α1A-adrenoceptor mediates the contractile response in the human vas deferens. In the vas deferens of various experimental animals, α1-adrenoceptor agonists induced rapid and transient contraction of the P-vas, while they induced slower and sustained contraction of the E-vas (Sneddon & Machaly, 1992). In human vas deferens, pKB estimates for KMD-3213 were not significantly different between the E-vas and the P-vas, but there was a tendency for the E-vas to have a greater total α1, namely, α1a mRNA abundance than the P-vas. This result may be related to the observation that the thickness of the entire muscular layer gradually decreases along the length of the vas deferens (Paniagua et al., 1982).
In conclusion, we have found molecular biologically that α1a mRNA is predominant in at least two major portions (E-vas and P-vas) of the human vas deferens. We have also confirmed that contraction of the human vas deferens is pharmacologically mediated by the α1A-adrenoceptor.
This investigation was supported in part by the Human Science Foundation and a Grant-in-Aid for Scientific Research (B), No 08457421, from the Ministry of Education, Science, and Culture, Japan.