Effects of nociceptin and endomorphin 1 on the electrically stimulated human vas deferens

Abstract

Aims

To examine the effects of nociceptin (NC) and endomorphin 1 (EM1) on electrical field stimulation (EFS)-induced contractions of the human vas deferens (hVD).

Methods

Concentration-response curves to NC and EM1 were constructed in the absence and in presence of peptidase inhibitors (PI). In some experiments a NC receptor antagonist, [Phe¹ψ(CH₂-NH)Gly²]NC(1–13)NH₂ [F/G], 10 µm) or naloxone (1 µm) were included.

Results

All data are mean(95%CI). In the presence of PI, NC inhibited twitches (E_max = 67(44,90)%; pEC₅₀ = 7.28(6.95,7.61)). NC inhibition was sensitive to [F/G]. EM1 also inhibited twitches both in the absence (E_max = 82(73,91)% pEC₅₀ = 7.07(6.92,7.22)) and presence (E_max = 83(76,90)%; pEC₅₀ = 7.00(6.91, 7.09)) of PI. EM1 inhibition was sensitive to naloxone.

Conclusions

These data suggest that hVD express NC and opioid receptors that inhibit neurogenic contractions.

Introduction

Nociceptin (NC) has recently been identified as the endogenous ligand of the opioid receptor like-1 receptor (ORL1, here referred to as NCR). Actions mediated by the NC/NCR system have been investigated in different animal models, in cell lines expressing native receptors or in cells transfected with recombinant receptors (see [1] for a review).

The aim of this work was to investigate the expression and function of native NCR receptors using a pharmacological preparation of human origin, namely the vas deferens. The effects elicited by NC were compared with those evoked by EM1, the recently identified endogenous ligand of the µ-opioid receptor [2].

Methods

Following local ethics committee approval and with written informed consent, 35 patients presenting for vasectomy under local anaesthesia were recruited. Immediately after removal, the tissues were placed into Krebs solution (composition in mm: NaCl 118.5, KCl 4.7, KH₂PO₄ 1.2, NaHCO₃ 25, CaCl₂ 2.5, glucose 10), oxygenated with 95% O₂ and 5% CO₂, packed into ice and transported to the laboratory.

Within 1 h of collection a segment of each vas deferens (∼1 cm in length without tears) was suspended in a 5 ml organ bath containing Krebs buffer at 37 °C and a resting tension of 1 g applied. Once equilibrated tissue responsiveness was tested with KCl 60 mm. Tissue was then subjected to electrical field stimulation (EFS) through two platinum electrodes, with 40 V square wave pulses of 0.5ms duration, at a frequency of 10Hz for 5 s, every 3 min. Electrically evoked contractions (twitches) were measured isotonically.

Concentration-response curves to NC and EM1 were performed in the absence and in the presence of the peptidase inhibitors (PI, 30 µm) amastatin, bestatin, captopril (Sigma, UK), and phosporamidon (Peptide Institute Inc, Japan). NC and EM1 were administered at 6 min intervals immediately after EFS. In a further series of experiments, 1 µm naloxone and 10 µm of the NCR antagonist [Phe¹ψ(CH₂-NH)Gly²]NC(1–13)NH₂ ([F/G]), concentrations which had been shown to be selective for opioid and NCR receptors, respectively, were preincubated for 15min prior to the application of NC and EM1 (1 µm). At the end of the experiment, 1 µm tetrodotoxin (Sigma, UK) was applied 15 min before the final EFS. All peptides used were prepared and purified as previously described [3, 4].

Graphical data are expressed as means±s.e.mean of n experiments whilst derived values are mean(95%CI). The pharmacological terminology used in this paper follows recent IUPHAR recommendations [5]. Apparent agonist affinities are given as pEC₅₀, which is –log₁₀ of the agonist molar concentration that produces 50% of the maximal possible agonist effect. E_max is the maximal effect that an agonist can elicit in a given tissue. pEC₅₀ values were obtained by computer-assisted curve fitting (Logistic function, GRAPHPAD-PRISM).

Results

Tissues from the first 12 patients were used to optimize the experimental conditions. Of the remaining 23 patients samples a further two were excluded because of stimulating electrode failure. Data presented are from 21 patients (mean age 36.3; range 25–51 years), although not all agents were tested in all specimens.

EFS of hVD produced a reproducible contractile effect amounting to 86 ± 14% (mean±s.e.mean) of the contraction induced by KCl (60 mm) and was blocked (data not shown) by 1 µm tetrodotoxin or 1 µm prazosin. A full concentration-response curve to NC was performed in the absence of PI in nine specimens, and in the presence of PI in 13 specimens. In the presence of PI, NC inhibited the twitch response to EFS in six specimens whereas in seven specimens there was no appreciable depression. The data presented in Figure 1a and Table 1 are from the six NC sensitive specimens.

Figure 1.

Inhibitory effects of NC (a), mean±s.e. mean, (n = 6–9) and EM1 (b), mean±s.e. mean, (n = 11–16) in the absence and presence of peptidase inhibitors (PI) on electrically stimulated hVD. In panel (c) the effect of [F/G] (10 µm) and naloxone (1 µm) on the inhibitory effect of NC (▪) and EM1 (□) (both 1 µm) are shown. Data in (c) are from n ≥ 3 (mean±s.e. mean) except for naloxone vs NC where the mean of 2 determinations showing no inhibition are given.

Table 1.

Comparision of potency (pEC₅₀) and efficacy (E_max) of nociceptin and endomorphin-1 inhibition of electrically evoked contractions of the hVD.

	Without peptidase inhibitors		With peptidase inhibitors
Peptide	pEC50	Emax (%)	pEC50	Emax (%)
Nociceptin	< 5	ND*	7.28 (6.95, 7.61)	67 (44, 90)
	(n = 9)		(n = 6)
Endomorphin-1	7.07 (6.92, 7.22)	82 (73, 91)	7.00 (6.91, 7.09)	83 (76, 90)
	(n = 16)		(n = 11)

Data are mean (95%CI)

^*concentration–response curve incomplete at 10 µm therefore the E_max could not be determined (ND).

A full concentration-response curve to EM1 was performed in the absence of PI in 16 specimens and in the presence of PI in 11 specimens. EM1 induced a concentration-dependent inhibition of control twitch (Figure 1b, Table 1) in the absence and in the presence of PI.

Naloxone (1 µm) failed to antagonize the inhibitory action of 1 µm NC, but blocked the inhibitory effect of 1 µm EM1. In contrast, [F/G] (10 µm) antagonized the inhibitory action of 1 µm NC, but not 1 µm EM1 (Figure 1c). Neither [F/G] nor naloxone alone showed any significant direct effects.

In two experiments, NC and EM1 did not modify resting tone of the hVD or phasic contractions induced by 10 µm phenylephrine (data not shown).

Discussion

To the best of our knowledge, the present report describes the first use of hVD to examine the effects of NC and EM1, where both inhibit electrically evoked twitches via an interaction at NCR and opioid receptors, respectively. In addition, the human vas deferens displays high peptidase activity as the NC response is only observed in the presence of PI.

In agreement with animal studies [6–8], contraction of the hVD elicited by EFS was tetrodotoxin (a voltage-sensitive Na⁺ channel blocker) sensitive and hence neurogenic. The mechanism by which EFS results in contraction is thought to be via catecholamine release and subsequent activation of α₁-adrenoceptors. Indeed, the α₁-antagonist prazosin (1 µm) prevents both the contractile effect of exogenously applied phenylephrine and that elicited by EFS. Both EM1 and NC inhibit the contractions elicited by EFS but not those evoked by exogenously applied phenylephrine indicating that the receptors involved in this inhibition are located presynaptically where they inhibit the release of neurotransmitter, as seen in animal studies [6–8].

It is worthy of mention that all the examined tissues were sensitive to EM1 while only 50% responded to NC even in the presence of PI. The reason for this variability in NC sensitivity is unknown. However, in the rat and mouse vas deferens the maximal effect elicited by NC (70–80%) is lower than that induced by opioid receptor agonists (100%) [5, 6, 9]. Therefore, it is possible that in some tissues expressing a low number of sites the effects of NC disappear before those of opioids.

In the absence of peptidase inhibitors, NC was inactive. However, in their presence, NC produced a concentration-dependent inhibition, with relatively high potency and efficacy. On the basis of these results we suggest that NC is rapidly metabolized to inactive fragments. In contrast, EM1 produced a concentration-dependent inhibition with similar potency and efficacy both in the absence and presence of peptidase inhibitors indicating that the short sequence (Tyr-Pro-Trp-Phe-NH₂) and the amide functional group at the C-terminus may confer metabolic stability.

That NC and EM1 actions are mediated by NCR and µ-opioid receptor were confirmed by the observation that [F/G], a peripheral NCR antagonist [10], blocks the effect of NC but not EM1. Moreover, naloxone blocks the effects of EM1 but not NC.

The clinical effects of opioids with activity at the µ-receptor (e.g. morphine) are well known [11]. In marked contrast, the effects of NC in man are currently unknown. However, some therapeutic indications of NCR agonists and antagonists can be predicted from their actions in animals. NC produces antinociceptive actions when administered intrathecally (i.t) or pronociceptive actions when administered intracerebroventricularly (i.c.v.). In addition, i.c.v. NC stimulates food intake and produces anxiolysis. Moreover i.c.v. and i.v NC produce bradycardia, hypotension, and diuresis with antinatriuresis (see for reviews [1, 12]).

In conclusion, we suggest that the human vas deferens represents a suitable pharmacological preparation to assess activity of novel agents active at native NC and µ-opioid receptors.