Evidence Showing Bombesin-Like Peptides Contract Guinea Pig Vas Deferens Smooth Muscle

Abstract

In this study, we investigated (1) the functional role of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in the regulation of guinea pig vas deferens smooth muscle (VDSM) contractions and (2) the potential contractile effects of 33 physiologically active substances and related chemicals that have not been previously reported to contract VDSM. Iberiotoxin (an inhibitor of BK_Ca channels, 10^–7 M) was the most potent enhancer of both noradrenaline (10^–5 M)- and ATP (10^–6 M)-induced contractions among the 6 types of K⁺ channel inhibitors. In addition, BK_Ca channel mRNA expression was the highest among the 32 types of K⁺ channel mRNAs. Iberiotoxin also enhanced the contractions induced by acetylcholine (10^–6 M), histamine (5 × 10^–5 M), bradykinin (10^–6 M), neurokinin A (10^–6 M), neurokinin B (10^–6 M), and substance P (10^–6 M). In the 33 tested physiologically active substances and related chemicals (15 peptides, 5 amino acids and their derivatives, 11 prostanoid- and isoprostane-related drugs, 1 endocannabinoid, and 1 phospholipid), we found that 3 bombesin-like peptides, neuromedin B (NMB) (10^–6 M), gastrin-releasing peptide (GRP, 10^–8 M), and NMC (10^–6 M), contracted VDSM in the absence of iberiotoxin, and these contractions were strongly enhanced in the presence of iberiotoxin. Among the 3 bombesin receptor subtypes, the mRNA expression level of Grpr (BB₂ receptor) was the highest. These findings suggest that (1) BK_Ca channels are the most powerful negative regulator of VDSM contractility and (2) NMB, GRP, and NMC are physiologically active substances that contract VDSM.

INTRODUCTION

The vas deferens (VD) is a long and thin tube composed of smooth muscle (SM), the primary function of which is the transport of sperm. The VD is densely innervated by adrenergic nerves and has therefore been widely used in pharmacological studies as a model organ for adrenergic neurotransmission.¹⁾ In fact, noradrenaline (NA) induces strong contractions in VDSM through the stimulation of α₁-adreneroceptors.²⁾ Subsequent research using VDSM has revealed that ATP, as well as NA, acts as a sympathetic neurotransmitter in VDSM.²⁾ Furthermore, studies using knockout mice demonstrated that the predominant ATP receptor subtype in VDSM is the purine P2X1 receptor.³⁾

In addition to NA and ATP, acetylcholine,⁴⁾ dopamine,⁵⁾ and histamine⁶⁾ have been reported as physiologically active substances that induce contractions in VDSM. In the late 1980s, several new peptides were discovered, some of which were reported to contract VDSM. These included bradykinin⁷⁾ and tachykinins (neurokinin A,⁸⁾ neurokinin B,⁸⁾ and substance P⁹⁾).

Although many new peptides have been discovered since the 1990s, their contractile effects on VDSM have not been reported, possibly because the contractile tension changes induced by the assessed substances were too small to have been considered active mechanical changes. This is plausible because, as in other SMs, various K⁺ channels are abundantly expressed in the VDSM and suppress its contractile activity. In fact, contractions of human VDSM induced by ATP and ATP analogs are reported to be potently enhanced by iberiotoxin, an inhibitor of large-conductance Ca2+-activated K⁺ (BK_Ca) channels.10) Therefore, detection of the contractile effects of physiologically active substances on VDSM should be performed under conditions in which the influence of K⁺ channels is excluded by the presence of K⁺ channel inhibitors.

In this study, we used guinea pig (GP) VDSM strips to investigate the potential contractile effects of 33 physiologically active substances and related chemicals that, to the best of our knowledge, have not been previously reported to contract VDSM. The contractile effects were tested in the presence of iberiotoxin because mRNA expression of the BK_Ca channel was the highest among the K⁺ channels tested in this study. In addition, iberiotoxin was the most potent enhancer of both NA- and ATP-induced contractions among the tested K⁺ channel inhibitors. Three types of peptides, neuromedin B (NMB), gastrin-releasing peptide (GRP), and neuromedin C (NMC), were newly found to contract VDSM.

MATERIALS AND METHODS

Drugs

The tested substances in this study were as follows: (R)-(−)-NA hydrogen tartrate monohydrate (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan or Tokyo Chemical Industry Co., Ltd., Tokyo, Japan); ATP sodium salt hydrate, dopamine hydrochloride, and histamine dihydrochloride (Sigma-Aldrich Co., LLC, St. Louis, MO, U.S.A.); acetylcholine chloride (Daiichi Sankyo Co., Ltd., Tokyo, Japan); glycine, serotonin creatinine sulfate monohydrate, and melatonin (FUJIFILM Wako Pure Chemical Corporation); l-glutamic acid (Tokyo Chemical Industry Co., Ltd.); bradykinin, neurokinin A, neurokinin B, substance P, NMB, GRP, NMC, NMU, neurotensin, orexin-A, glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, ghrelin (human), motilin, somatostatin, angiotensin II, atrial natriuretic peptide, and endothelin-1 (human) (Peptide Institute Inc., Osaka, Japan); urotensin II, prostaglandin A₂ (PGA₂), PGB₂, PGD₂, PGE₂, PGI₂, U46619, 8-iso PGA₂, 8-iso PGE₂, 8-iso PGF_2α, unoprostone, 2-arachidonoyl glycerol (dissolved in acetonitrile), and platelet-activating factor (C16) (Cayman Chemical Co., Ann Arbor, MI, U.S.A.); and PGF_2α (Fuji Pharma Co., Ltd., Tokyo, Japan). The tested K⁺ channel inhibitors in this study were as follows: iberiotoxin and apamin (Peptide Institute Inc.); TRAM-34 (Cayman Chemical Co.); and Ba²⁺ chloride, 4-aminopyridine (4-AP), and glibenclamide (Sigma-Aldrich Co., LLC). Indomethacin was purchased from Sigma-Aldrich Co., LLC.

ATP was dissolved in 25 mM Tris–HCl (pH = 8.0) to prepare a stock solution of 10^–2 M and diluted with 25 mM Tris–HCl (pH = 7.4). PGA₂, PGB₂, PGD₂, PGE₂, PGF_2α, PGI₂, U46619, 8-iso-PGA₂, 8-iso-PGE₂, 8-iso PGF_2α, and unoprostone were dissolved in ethanol to prepare stock solutions of 2 × 10^–2 M. Indomethacin was dissolved in ethanol to prepare a stock solution of 10^–2 M. Platelet-activating factor was dissolved in ethanol to prepare a stock solution of 2 × 10^–3 M and stored at –80°C; to prepare a 2 × 10^–4 M working solution, the ethanol solvent was evaporated, and the platelet-activating factor was redissolved with 0.25% bovine serum albumin. Neurokinin B/TRAM-34/glibenclamide were dissolved in dimethyl sulfoxide to prepare 2.5 × 10^–4 M/10^–3 M/5 × 10^–3 M solutions. All the other drugs were prepared as aqueous solutions and diluted with distilled water.

Animals

This study was approved by the Toho University Animal Care and Use Committee (Approval Numbers: 19-55-294 and 20-444) and was performed in compliance with the guidelines of the Laboratory Animal Center of the Faculty of Pharmaceutical Sciences, Toho University. Male GPs (weight: 255–680 g, age: 5–16 weeks; Kyudo Co., Ltd., Saga, Japan) were housed under a fixed 12/12 h light/dark cycle (08 : 00–20 : 00) and controlled conditions (20–22°C, relative air humidity: 50 ± 5%), with food and water available ad libitum.

GP VDSM Strips

The GPs were anesthetized by isoflurane inhalation and exsanguinated from the carotid artery. The VD was rapidly removed and placed in a Locke–Ringer solution containing (in mM) NaCl, 154; KCl, 5.6; CaCl₂, 2.2; MgCl₂, 2.1; NaHCO₃, 5.9; and d-(+)-glucose, 2.8. After the VD interior was irrigated with the Locke–Ringer solution, VDSM strips of approximately 20 mm in length were prepared.

The VDSM strips were equilibrated under a 1 g resting tension for 60 min in a 5- or 20-mL organ bath containing the Locke–Ringer solution equilibrated with 95% O₂ and 5% CO₂ at 32 ± 1°C. Tension changes were isometrically recorded using force–displacement transducers (TB-612T; Nihon Kohden Co., Tokyo, Japan) connected to amplifiers (MSC-1 [Lab Support. Co., Osaka, Japan] and AD-601G [Nihon Kohden Co.]) and PowerLab™/LabChart™ software (ADInstruments Pty. Ltd., Bella Vista, NSW, Australia).

The VDSM strips were contracted using NA (3 × 10^–5 M) at least 3 times at 5 min intervals and then incubated for 30 min (preliminary procedures). All experiments were performed in the presence of indomethacin (3 × 10^–6 M).

Effects of Various K⁺ Channel Inhibitors on NA- and ATP-Induced Contractions

After preliminary procedures, the VDSM strips were contracted using NA (10^–5 M) or ATP (10^–6 M) for 5 min at least twice at 30 min intervals. After washing out, the following K⁺ channel inhibitors were added to the solution and incubated for 60 min: iberiotoxin (a BK_Ca channel inhibitor, 10^–7 M), apamin (a small-conductance Ca²⁺-activated K⁺ (SK_Ca) channel inhibitor, 10^–7 M), TRAM-34 (an intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channel inhibitor, 10^–6 M), Ba²⁺ (an inward-rectifier K⁺ (K_ir) channel inhibitor, 10^–4 M), 4-AP (a voltage-gated K⁺ (K_v) channel inhibitor, 3 × 10^–3 M), or glibenclamide (an ATP-sensitive K⁺ (K_ATP) channel inhibitor, 10^–6 M). After a 60-min incubation, the VDSM strips were contracted using NA (10^–5 M) or ATP (10^–6 M) for 5 min.

Contractile Effects of Various Substances in the Absence or Presence of Iberiotoxin

After preliminary procedures, the substances listed in Table 1 were added to the solution and incubated for 5 min. Concentrations near the half-maximal effective concentration were used for ATP (10^–6 M), acetylcholine (10^–5 M), and histamine (5 × 10^–5 M). For NA and bradykinin, we used 10^–5 and 10^–6 M, respectively, which allowed sufficient contractile responses. Based on these results, we estimated that 10^–4 M for inexpensive amino acids and their derivatives and 10^–6 M for other substances would be sufficient to observe contractile responses. We used 10^–5 M dopamine because the solution in the bath discolored when using 10^–4 M. Contractions induced by 10^–6 M GRP remained even after peptide removal; thus, we used 10^–8 M GRP, which could be washed out. After washout, iberiotoxin (10^–7 M) was added to the solution and incubated for 60 min. After this 60-min incubation, the substances shown in Table 1 were again added to the solution and incubated for 5 min. As repeated administration of platelet-activating factor is known to cause desensitization, its effects in the absence and presence of iberiotoxin were examined using separate strips.

Table 1. Contractile Effects of the 41 Substances Tested on Guinea Pig Vas Deferens in the Absence and Presence of Iberiotoxin (IbTX, 10^–7 M)

Category/substance	Without IbTX	With IbTX
Sympathetic neurotransmitters
Noradrenaline (10–5 M)	+	++
ATP (10–6 M)	+	++
Peptides
Bradykinin (10–6 M)	+	++
Neurokinin A (10–6 M)	+	++
Neurokinin B (10–6 M)	+	++
Substance P (10–6 M)	+	++
Neuromedin B (10–6 M)	+	++
Gastrin-releasing peptide (10–8 M)	+	++
Neuromedin C (10–6 M)	+	++
Neuromedin U (10–6 M)	–	–
Neurotensin (10–6 M)	–	–
Orexin-A (10–6 M)	–	–
Glucagon-like peptide-1 (10–6 M)	–	–
Glucose-dependent insulinotropic polypeptide (10–6 M)	–	–
Ghrelin (10–6 M)	–	–
Motilin (10–6 M)	–	–
Somatostatin (10–6 M)	–	–
Angiotensin II (10–6 M)	–	–
Atrial natriuretic peptide (10–6 M)	–	–
Endothelin-1 (10–6 M)	–	–
Urotensin II (10–6 M)	–	–
Amino acids and their derivatives
Acetylcholine (10–6 M)	+	++
Histamine (5 × 10–5 M)	+	++
Dopamine (10–5 M)	–	–
Glycine (10–4 M)	–	–
Glutamic acid (10–4 M)	–	–
Serotonin (10–4 M)	–	–
Melatonin (10–4 M)	–	–
Prostanoid- and isoprostane-related drugs
Prostaglandin A2 (10–6 M)	–	–
Prostaglandin B2 (10–6 M)	–	–
Prostaglandin D2 (10–6 M)	–	–
Prostaglandin E2 (10–6 M)	–	–
Prostaglandin F2α (10–6 M)	–	–
Prostaglandin I2 (10–6 M)	–	–
U46619 (Prostanoid TP receptor agonist) (10–6 M)	–	–
8-iso-Prostaglandin A2 (10–6 M)	–	–
8-iso-Prostaglandin E2 (10–6 M)	–	–
8-iso-Prostaglandin F2α (10–6 M)	–	–
Unoprostone (10–6 M)	–	–
Endocannabinoid
2-Arachidonoyl glycerol (10–6 M)	–	–
Phospholipid
Platelet-activating factor (10–6 M)	–	–

+: contraction; ++: enhanced contraction compared to that in the absence of IbTX; –: no contraction. The effect of platelet-activating factor was examined in the presence of 0.25% bovine serum albumin.

Measurement of mRNA Expression Levels

RT-quantitative (q)PCR was performed as previously described.¹¹⁾ Briefly, total RNA was isolated from the GP VD. First-strand cDNA was synthesized using ReverTra Ace^® qPCR RT Master Mix with gDNA Remover (TOYOBO Co., Ltd., Osaka, Japan). RT-qPCR was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, U.S.A.)/CronoSTAR™ 96 Real-Time PCR System (TaKaRa Bio Inc., Shiga, Japan) using THUNDERBIRD^® Next SYBR^® qPCR Mix (TOYOBO Co., Ltd.). Supplementary Table 1 shows the primers used in this study. The thermal cycler parameters were set at 95°C for 2 min/30 s, followed by 40 cycles of 95°C for 15/5 s, 60°C for 30/10 s, and 72°C for 35/15 s. The fluorescence intensities were measured at each 72°C step to confirm DNA amplification. Sequence Detection Software Version 1.4 (Applied Biosystems)/CronoSTAR™ 96 Software (TaKaRa Bio Inc.) was used to analyze the mRNA expression level of each gene, which was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) that was set to 1. Samples that did not produce a Ct value after 40 cycles were considered to have no expression.

Data Analysis

The VDSM contractile responses induced by various substances were evaluated as the area under the contraction curve (AUC) for 5 min (AUC_{5 min}) using LabChart™ software. AUC_{5 min} was defined as “% of 3 × 10^–5 M NA-induced contraction × 5 min” by dividing by the maximum contraction (g) induced by NA (3 × 10^–5 M) (100%) in the final preliminary procedures (Supplementary Fig. 1). The numbers of contractile responses over a 5-min period were visually counted (Supplementary Fig. 1). Individual data points are plotted with mean ± standard error of the mean, where n is the number of experiments. Statistical analyses were performed using GraphPad Prism (version 6) (GraphPad Software, Inc., San Diego, CA, U.S.A.) using t-tests. Statistical significance was set at p < 0.05.

RESULTS

Effects of Various K⁺ Channel Inhibitors on NA-Induced Contractile Responses

Figure 1A depicts representative traces showing the effects of various K⁺ channel inhibitors on NA-induced contractions in GP VDSM, and Fig. 1B depicts summarized data of the AUCs of the NA-induced contractions shown in Fig. 1A. The contractions induced by NA (10^–5 M) were potently enhanced by the BK_Ca channel inhibitor iberiotoxin (10^–7 M) (Figs. 1Aa, 1Ba). In addition to iberiotoxin, the AUC of the NA-induced contractions was significantly enhanced by the K_ir channel inhibitor Ba²⁺ (10^–4 M) (Figs. 1Ad, 1Bd) and the K_v channel inhibitor 4-AP (3 × 10^–3 M) (Figs. 1Ae, 1Be). In contrast, the AUC was not significantly enhanced by the SK_Ca channel inhibitor apamin (10^–7 M) (Figs. 1Ab, 1Bb), the IK_Ca channel inhibitor TRAM-34 (10^–6 M) (Figs. 1Ac, 1Bc), or the K_ATP channel inhibitor glibenclamide (10^–6 M) (Figs. 1Af, 1Bf).

Fig. 1. Representative Traces (A) and Summarized Data (B) Showing the Effects of K⁺ Channel Inhibitors on Noradrenaline (NA, 10^-5 M)-Induced Contractions in Guinea Pig Vas Deferens

Tested K⁺ channel inhibitors are iberiotoxin (IbTX, 10^–7 M, a), apamin (10^–7 M, b), TRAM-34 (10^–6 M, c), Ba²⁺ (10^–4 M, d), 4-aminopyridine (4-AP, 3 × 10^–3 M, e), and glibenclamide (Gli, 10^–6 M, f). Data are expressed as the means ± standard error of the mean, with each data point plotted (n = 6 [Ba] and n = 5 [Bb–Bf]). **p < 0.01 vs. control (paired t-test). w: wash out; AUC: area under the contraction curve.

Supplementary Figure 2A depicts summarized data of the contractile response counts of the NA-induced contractions shown in Fig. 1, which were not significantly affected by any of the tested K⁺ channel inhibitors except Ba²⁺ (10^–4 M).

Effects of Various K⁺ Channel Inhibitors on ATP-Induced Contractile Responses

Figure 2A depicts representative traces showing the effects of various K⁺ channel inhibitors on ATP-induced contractions in GP VDSM, and Fig. 2B depicts summarized data of the AUCs of the ATP-induced contractions shown in Fig. 2A. The contractions induced by ATP (10^–6 M) were potently enhanced by iberiotoxin (10^–7 M) (Figs. 2Aa, 2Ba). In addition to iberiotoxin, the AUC of the ATP-induced contractions was significantly enhanced by Ba²⁺ (10^–4 M) (Figs. 2Ad, 2Bd). In contrast, the AUC was not significantly enhanced by apamin (10^–7 M) (Figs. 2Ab, 2Bb), TRAM-34 (10^–6 M) (Figs. 2Ac, 2Bc), 4-AP (3 × 10^–3 M) (Figs. 2Ae, 2Be), or glibenclamide (10^–6 M) (Figs. 2Af, 2Bf).

Fig. 2. Representative Traces (A) and Summarized Data (B) Showing the Effects of K⁺ Channel Inhibitors on ATP (10^–6 M)-Induced Contractions in Guinea Pig Vas Deferens

Tested K⁺ channel inhibitors are iberiotoxin (IbTX, 10^–7 M, a), apamin (10^–7 M, b), TRAM-34 (10^–6 M, c), Ba²⁺ (10⁻⁴ M, d), 4-aminopyridine (4-AP, 3 × 10^–3 M, e), and glibenclamide (Gli, 10^–6 M, f). Data are expressed as the means ± standard error of the mean, with each data point plotted (n = 6 [Bd] and n = 5 [Ba–Bc, Be, and Bf]). *p < 0.05, **p < 0.01 vs. control (paired t-test). w: wash out; AUC: area under the contraction curve; NA: noradrenaline.

Supplementary Figure 2B depicts summarized data of the contractile response counts of the ATP-induced contractions shown in Fig. 2, which were not significantly affected by any of the tested K⁺ channel inhibitors.

mRNA Expression Levels of Various K⁺ Channels

Figure 3 shows the mRNA expression levels of various K⁺ channels (α subunits) in GP VD tissues, as assessed by RT-qPCR. Among the tested K⁺ channels, the mRNA expression of K_Ca1.1 (a BK_Ca channel inhibited by iberiotoxin) was the highest. In addition, the mRNA expression levels of K_Ca2.3 (an SK_Ca channel inhibited by apamin), K_ir2.4 (a K_ir channel inhibited by Ba²⁺), and K_v1.6/K_v2.1 (K_v channels inhibited by 4-AP) were relatively higher than those of the other K⁺ channels.

Fig. 3. mRNA Expression Levels of K⁺ Channels (K_Ca 1.1–3.1, K_ir 1.1–6.2, and K_v 1.1–4.2) in Guinea Pig Vas Deferens Tissues as Assessed by RT-qPCR

The expression level of each mRNA is shown relative to that of glyceraldehyde-3-phosphate dehydrogenase (Gapdh), which is arbitrarily set as 1. Data are expressed as the means ± standard error of the mean, with each data point plotted (n = 4 each).

Contractile Effects of Various Physiologically Active Substances and Related Chemicals in the Presence of Iberiotoxin

Table 1 summarizes the contractile effects of various physiologically active substances and related chemicals in the absence and presence of iberiotoxin in GP VDSM. In addition to NA and ATP, acetylcholine (10^–6 M), histamine (5 × 10^–5 M), bradykinin (10^–6 M), neurokinin A (10^–6 M), neurokinin B (10⁻⁶ M), and substance P (10^–6 M) contracted VDSM in the absence of iberiotoxin, and these contractions were strongly enhanced in the presence of iberiotoxin (Supplementary Fig. 3). In this study, we also found that 3 peptide substances, NMB (10^–6 M), GRP (10^–8 M), and NMC (10^–6 M), contracted VDSM in the absence of iberiotoxin, and these contractions were strongly enhanced in the presence of iberiotoxin (Fig. 4). Iberiotoxin (10^–7 M) did not significantly affect the contractile response counts induced by NMB (10^–6 M; Supplementary Fig. 4A); however, it significantly decreased those induced by GRP (10^–8 M) and NMC (10^–6 M) (Supplementary Figs. 4B and 4C, respectively).

Fig. 4. Representative Traces (A) and Summarized Data (B) Showing the Contractile Effects of Neuromedin B (NMB, 10^-6 M, a), Gastrin-Releasing Peptide (GRP, 10^-8 M, b), and Neuromedin C (NMC, 10^–6 M, c) in the Absence and Presence of Iberiotoxin (IbTX, 10^-7 M) in Guinea Pig Vas Deferens

Data are expressed as the means ± standard error of the mean, with each data point plotted (n = 11 [Ba], n = 6 [Bb], and n = 5 [Bc]). *p < 0.05, **p < 0.01 vs. control (paired t-test). w: wash out; AUC: area under the contraction curve; NA: noradrenaline.

In contrast, the other physiologically active substances and their derivatives tested in this study (12 peptides, 5 amino acids and their derivatives, 11 prostanoid- and isoprostane-related drugs, 1 endocannabinoid, and 1 phospholipid) did not contract VDSM either in the absence or presence of iberiotoxin (Table 1).

mRNA Expression Levels of Bombesin Receptor Subtypes

Figure 5 shows the mRNA expression levels of Nmbr (BB₁ receptor), Grpr (BB₂ receptor), and Brs3 (BB₃ receptor) in GP VD tissues, as assessed by RT-qPCR. Among the 3 subtypes, the mRNA expression level of Grpr (BB₂ receptor) was the highest, that of Brs3 (BB₃ receptor) was clearly detected, and that of Nmbr (BB₁ receptor) was extremely low.

Fig. 5. mRNA Expression Levels of Bombesin Receptors (Nmbr, Grpr, and Brs3) in Guinea Pig Vas Deferens Tissues as Assessed by RT-qPCR

The expression level of each mRNA is shown relative to that of glyceraldehyde-3-phosphate dehydrogenase (Gapdh), which is arbitrarily set as 1. Data are expressed as the means ± standard error of the mean, with each data point plotted (n = 5 each).

DISCUSSION

In this study, we used GP VDSM strips and found that contractions induced by the sympathetic neurotransmitters NA and ATP were potently enhanced by the BK_Ca channel inhibitor iberiotoxin. In addition, we investigated the potential contractile effects of 33 physiologically active substances in the presence of iberiotoxin and identified the contractile activity of the 3 peptides: NMB, GRP, and NMC.

VDSM contractility is known to be suppressed by K⁺ channels, especially BK_Ca channels, similar to that observed in other SMs.^12–14) For example, NA-induced contractions in rat VDSM were inhibited by the BK_Ca channel activator NS 1619,¹⁴⁾ and electrical field stimulation-induced contractions in GP VDSM in the presence of a purinergic receptor antagonist were enhanced by iberiotoxin,¹⁵⁾ suggesting that the activation of BK_Ca channels inhibits VDSM contractility. In this study, we found that iberiotoxin significantly enhanced NA-induced VDSM contraction (Fig. 1), demonstrating the importance of BK_Ca channels in the negative control of VDSM contractility. We also found that VDSM contractions induced by the selective α₁-adrenoceptor agonist phenylephrine were strongly enhanced by iberiotoxin (Supplementary Fig. 5). Furthermore, we found that the expression of BK_Ca channel mRNA (K_Ca1.1) in GP VD was the highest among the 32 types of K⁺ channel mRNAs (5 types of Ca²⁺-activated K⁺ channels, 11 types of K_ir, and 16 types of K_v) (Fig. 3), strongly supporting the experimental results that NA-induced contractions were potently enhanced by iberiotoxin. NA-induced VDSM contractions were induced by α₁-adrenoceptor stimulation followed by extracellular Ca²⁺ influx through voltage-dependent calcium channels (VDCCs).¹⁵⁾ The intracellular Ca²⁺ increases induced by the extracellular Ca²⁺ influx could activate BK_Ca channels, which would inhibit VDCC activation and thereby suppress NA-induced contractions. Indeed, the enhancement of NA-induced VDSM contractions by iberiotoxin was strongly inhibited by an L-type VDCC inhibitor (verapamil, 10^–5 M) (Supplementary Fig. 6A). Regarding the α-adrenoceptor subtypes in GP VD tissues, the expression of mRNA encoding α_1A-adrenoceptor (Adra1a) was the highest among the tested mRNAs (Supplementary Fig. 7A). This finding suggests that the α-adrenoceptor, which mediates NA-induced contractions in GP VDSM, is the α_1A-adrenoceptor or its phenotype (α_1L-adrenoceptor).¹⁶⁾

ATP-induced VDSM contractions were significantly enhanced by iberiotoxin (Fig. 2). These results are consistent with a previous report that ATP-induced contractions in human VDSM are strongly enhanced by iberiotoxin.¹⁰⁾ ATP-induced VDSM contractions are induced by the stimulation of P2X1 ionotropic receptors,¹⁷⁾ followed by extracellular Ca²⁺ influx through both P2X1 receptors and VDCCs.¹⁸⁾ Therefore, as in the case of NA-induced contractions, the intracellular Ca²⁺ increases induced by these Ca²⁺ influxes activate BK_Ca channels, which then inhibit VDCC activation, and thus inhibit ATP-induced contractions. In fact, in GP VD, (1) the expression of mRNA encoding the P2X1 receptor (P2rx1) was the highest (Supplementary Fig. 7B), and (2) the enhancement of ATP-induced contractions by iberiotoxin was strongly inhibited by an L-type VDCC inhibitor (verapamil, 10^–5 M) (Supplementary Fig. 6B). Therefore, during contractions induced by both NA and ATP, the activation of BK_Ca channels may suppress extracellular Ca²⁺ influx through L-type VDCCs. However, the iberiotoxin-enhanced contractions due to both NA and ATP were not completely suppressed by verapamil (Supplementary Fig. 6), suggesting that in addition to L-type VDCCs, non-L-type VDCCs are involved in the enhancement by iberiotoxin. Regarding the potential involvement of non-L-type VDCCs, T-type as well as L-type VDCC has been reported to substantially contribute to VDSM contractions in GPs and rats.^15,19) Therefore, the iberiotoxin-induced enhancement that remains in the presence of verapamil may be mediated through T-type VDCCs. To clarify this issue, further studies are needed.

In addition to the BK_Ca channel inhibitor iberiotoxin, we examined the effects of 5 types of K⁺ channel inhibitors on NA- and ATP-induced contractions in GP VDSM. The tested K⁺ channel inhibitors were apamin (10^–7 M), an SK_Ca channel inhibitor; TRAM-34 (10^–6 M), an IK_Ca channel inhibitor; Ba²⁺ (10^–4 M), a K_ir channel inhibitor; 4-AP (3 × 10^–3 M), a K_v channel inhibitor; and glibenclamide (10^–6 M), a K_ATP channel inhibitor. Of these, TRAM-34 and glibenclamide did not show any significant enhancing effects on NA- or ATP-induced contractions. Although apamin slightly enhanced both contractions, this effect was not statistically significant. In contrast, Ba²⁺ significantly enhanced both types of contractions, and 4-AP significantly enhanced NA-induced contractions and slightly but insignificantly enhanced ATP-induced contractions. In RT-qPCR experiments, we detected the mRNA expression of SK_Ca channels (K_Ca2.2/K_Ca2.3) inhibited by apamin, K_ir channels (K_ir2.4) inhibited by Ba²⁺, and K_v channels (K_v1.6/K_v2.1) inhibited by 4-AP, in addition to that of the BK_Ca channel (K_Ca1.1) (Fig. 3). These findings suggest that these K⁺ channels also function as inhibitory regulatory mechanisms in NA- and ATP-induced VDSM contractions. However, since the degree of enhancement by these 5 types of K⁺ channel inhibitors was not as remarkable as that by iberiotoxin, we concluded that iberiotoxin was optimal for detecting the VDSM contractile effects of physiologically active substances whose effects on VDSM are still unknown. The use of iberiotoxin can be justified by the finding that this K⁺ channel inhibitor also strongly enhanced VDSM contractions in response to the tested reagents other than NA and ATP: 2 amines (acetylcholine and histamine) and 4 peptides (bradykinin, neurokinin A, neurokinin B, and substance P) that have been previously reported to contract VDSM (Supplementary Fig. 3). Our conclusion, deduced from the findings using iberiotoxin, is that this BK_Ca channel inhibitor is one of the best pharmacological tools for detecting VDSM contractions induced by physiologically active substances, even when the contractions are so small that they are undetectable in its absence.

In this study, we also investigated the potential contractile effects of 33 physiologically active substances and related drugs on VDSM and found that the 3 bombesin-related peptides, NMB, GRP, and NMC, possess contractile activity (Table 1 and Fig. 4). The contractions induced by NMB (10^–6 M), GRP (10^–7 M), and NMC (10^–6 M) in the presence of iberiotoxin were not significantly affected by the α-adrenoreceptor antagonist phentolamine (10^–6 M) and the purine P2 receptor antagonist suramin (10^–4 M) (Supplementary Fig. 8). These results indicate that the contractile responses induced by these bombesin-like peptides are not mediated by NA or ATP but are exerted by the direct action of these peptides on VDSM. For the other 30 substances, no substantial contractions were detected either in the absence or presence of iberiotoxin (Table 1).

NMB, GRP, and NMC are known as bombesin-related peptides because their amino acid sequences are similar to that of bombesin isolated from frog skin.^20,21) There are 2 types of endogenous bombesin-related peptides in mammals, GRP and NMB.^20,21) NMC belongs to the GRP group and is an active fragment of GRP (GRP_18–27).^20,21) NMB, GRP, and NMC have been reported to cause a wide variety of physiological effects,^21–23) including contractility of gastrointestinal and urinary bladder SMs. Specifically, NMB and GRP contract esophagus, gastric fundus, and duodenum SMs in cats²⁴⁾; NMB, GRP, and NMC contract rat gastric fundus and GP gallbladder SMs²⁵⁾; and NMB, GRP, and NMC contract rat and GP urinary bladder SMs.²⁵⁾ However, to the best of our knowledge, ours is the first report on the contractile effects of NMB, GRP, and NMC on VDSM. These VDSM contractile effects may have gone unnoticed because the magnitude of the contractility is so small. In fact, a previous study indicated that GRP did not exhibit contractile effects on GP VDSM.²⁶⁾ In the present study, in the absence of iberiotoxin, the magnitude of VDSM contractions induced by NMB and GRP were less than 50% of that induced by NA (3 × 10^–5 M) and were relatively small. Especially in the case of GRP, it was sometimes difficult to distinguish the contractile responses from baseline fluctuations, and careful observation was required. However, in the presence of iberiotoxin, obvious contractile responses to bombesin-related peptides were observed in all experimental cases, confirming that these peptides have VDSM contractile activity.

Currently, 3 subtypes of bombesin-related peptide receptors have been identified.²⁰⁾ They are BB₁ (NMB-preferring receptor), BB₂ (GRP-preferring receptor), and BB₃ (bombesin receptor subtype-3; unknown endogenous ligand).^20,27) However, functional experiments do not allow identification of the receptor subtypes for bombesin-related peptides because selective antagonists against these receptor subtypes are not currently available. Therefore, in this study, we investigated the mRNA expression levels of the 3 types of bombesin-related peptide receptors (BB₁, BB₂, and BB₃) in GP VD tissues using RT-qPCR to identify the receptor subtypes that mediate contractions induced by NMB, GRP, and NMC. Among the 3 subtypes, the mRNA expression of BB₂ (Grpr) was the highest, followed by that of BB₃ (Brs3), whereas that of BB₁ (Nmbr) was extremely low (Fig. 5). However, BB₃ is an orphan receptor, and its endogenous ligand is currently unknown.²⁷⁾ Therefore, in GP VDSM, the primary target of NMB, GRP, and NMC is suggested to be BB₂ (Grpr). NMB has a higher affinity for BB₁ than BB₂ (pK_i values vs. BB₁/BB₂: 8.1–10.3 [human]/6.4–8.1 [human]),²⁸⁾ but the mRNA expression level of BB₁ is extremely low, suggesting that BB₁ is an unlikely NMB target. Since the concentration of NMB used in our experiment, 10^–6 M (–log M of 6), is larger than the concentrations corresponding to its pK_i value vs. BB₂, it is quite possible that NMB targets BB₂. GRP has a higher affinity for BB₂ than BB₁ (pK_i values vs. BB₁/BB₂: 6.4 [rat]/6.3–8.2 [human]),²⁹⁾ and the concentration of GRP used in our experiment, 10^–8 M (–log M of 8), is consistent with the concentrations corresponding to its pK_i value vs. BB₂. Regarding NMC, since the concentration used in the experiment, 10^–6 M (–log M of 6), is larger than the concentrations corresponding to its pK_i value vs. BB₂ (7.7 [rat]),³⁰⁾ it is quite possible that NMC targets BB₂.

Iberiotoxin decreased the frequency of contractile responses induced by GRP (10^–8 M) and NMC (10^–6 M), but not NMB (10^–6 M) as shown in Supplementary Fig. 4. To determine the mechanisms underlying this phenomenon, we examined the effects of other K⁺ channel inhibitors on the contractile responses induced by NMC, an active fragment of GRP, and these results are shown in Supplementary Fig. 9. K⁺ channel inhibitors other than iberiotoxin did not significantly affect the frequency of contractile responses induced by NMC (Supplementary Fig. 9C). Therefore, the decrease in NMC-induced contractions in the presence of iberiotoxin is likely due to inhibition of BK channels rather than desensitization caused by repeated administration of NMC. This decrease is predicted to be caused by the strong contractions elicited in the presence of iberiotoxin. In the absence of iberiotoxin, GRP and NMC often induced rhythmic contractile responses. In contrast, in the presence of iberiotoxin, the duration as well as the magnitude of each rhythmic contractile response was increased, which delayed the occurrence of subsequent rhythmic contractions and thus decreased the contraction frequency. Regardless of the absence or presence of iberiotoxin, rhythmic contractions hardly occurred in response to NMB. One explanation for this observation could be the lower affinity of NMB for the BB₂ receptor compared to that of GRP and NMC.

Regarding the clinical significance of this study, infertility is one of the major challenges in reproductive medicine, affecting an estimated 70 million people worldwide.³¹⁾ It is estimated that 9% of couples worldwide suffer from infertility problems, and 50% of these cases involve the male.³¹⁾ GRP-related peptides have been reported to be associated with female reproductive functions as follows: (1) GRP-related peptides, including GRP and NMC, are produced in the ovine uterus during the estrous cycle and pregnancy³²⁾; (2) GRP receptors are expressed in human uterine SMs obtained from women who have undergone a cesarean section, and in some strips, GRP increases spontaneous uterine activity³³⁾; and (3) NMB may induce labor.³⁴⁾ GRP may also be involved in male reproductive functions as follows: (1) In rabbits, GPs, and rats, bundles of nerves or fine varicose nerve fibers containing GRP are found in the VD and, to a lesser extent, in the seminal vesicles²⁶⁾; (2) Leydig cells in rat testes produce GRP³⁵⁾; (3) GRP promotes testosterone secretion from and Grpr mRNA synthesis in pig Leydig cells³⁶⁾; and (4) GRP does not affect normal sperm motility, but the proportion of sperm that have undergone an acrosome reaction increases when GRP is used in combination with calcium ionophore.³⁷⁾ In this study, we found VDSM contractile activity of 3 types of bombesin-related peptides and propose that these peptides can control VDSM contractility. Abnormalities in VDSM contractility can reportedly cause male infertility. For example, mice lacking P2X1 receptors show a 90% decrease in fertility in their ejaculate due to reduced VD motility.³⁾ Therefore, excessive or decreased production of bombesin-related peptides could impair normal VD motility and lead to male infertility. Further investigations into the roles of bombesin-related peptides in the male reproductive system may elucidate one of the causes of male infertility.

This study had some limitations. (1) The tissues used for RT-qPCR may have contained nerve tissue in addition to SM, and this should be considered when interpreting the mRNA expression results. (2) We used epithelium-intact VDSM strips, and thus the VD epithelium could have influenced the VDSM contractions. In fact, the VD epithelium has been reported to regulate VDSM contractions in rats.³⁸⁾ Specifically, the stimulation of the VD epithelium by ATP inhibits VDSM contractions through the production of PGE₂ but not nitric oxide. Our experiments were performed in the presence of indomethacin to eliminate the influence of endogenous prostanoids. Therefore, even if the tested substances stimulated the VD epithelium, the epithelial influence could be eliminated under conditions where prostanoid production was suppressed. (3) We used GPs from 5 to 16 weeks old. Therefore, our study results might contain some age-related variability. To examine whether there were age-related differences in mRNA expression, we compared the expression of K_Ca1.1 and 3 bombesin receptor mRNAs (Nmbr, Grpr, and Brs3) between 5–7- and 16-week-old GPs (Supplementary Fig. 10). Although a small, significant difference was observed for Nmbr, no substantial age-related differences were observed in the expression of these mRNAs. Next, we examined whether potentiation by iberiotoxin was observed in both 5- and 16-week-old GPs, and the results are shown in Supplementary Fig. 11. The results clearly show that both NA- and ATP-induced contractions were significantly potentiated in the presence of iberiotoxin in both 5- and 16-week-old animals. Therefore, regardless of age or body weight, responses to physiologically active substances and related chemicals can be detected as potentiated contractions in the presence of iberiotoxin, which validates the methodology of this study.

In conclusion, (1) GP VDSM contractility is suppressed by BK channels, which inhibit VDCCs; and (2) 3 bombesin-like peptides (NMB, GRP, and NMC) contribute to the regulation of VDSM contraction through stimulation of BB₂ receptors (Fig. 6).

Fig. 6. Study Summary

NA: noradrenaline; NMB: neuromedin B; GRP: gastrin-releasing peptide; NMC: neuromedin C; VDCCs: voltage-dependent Ca²⁺ channels; BK_Ca channels: large-conductance Ca²⁺-activated K⁺ channels.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

The data that support the findings of this study are available from the corresponding author, K.O., upon reasonable request.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1)  Burnstock G, Verkhratsky A. Vas deferens—a model used to establish sympathetic cotransmission. Trends Pharmacol. Sci., 31, 131–139 (2010).
• 2)  Sneddon P, Westfall DP. Pharmacological evidence that adenosine triphosphate and noradrenaline are co-transmitters in the guinea-pig vas deferens. J. Physiol., 347, 561–580 (1984).
• 3)  Mulryan K, Gitterman DP, Lewis CJ, Vial C, Leckie BJ, Cobb AL, Brown JE, Conley EC, Buell G, Pritchard CA, Evans RJ. Reduced vas deferens contraction and male infertility in mice lacking P2X₁ receptors. Nature, 403, 86–89 (2000).
• 4)  Ozawa H, Aihara K, Abe F, Sugawara K. Pharmacological analysis of acetylcholine-induced contraction in mouse vas deferens. Jpn. J. Pharmacol., 24, 814–816 (1974).
• 5)  Tayo FM. Occurrence of excitatory dopaminoceptors in the rat and guinea-pig vas deferens. Clin. Exp. Pharmacol. Physiol., 6, 275–279 (1979).
• 6)  Lau LC, Adaikan PG, Ratnam SS. Effect of histamine on human vas deferens in vitro. Br. J. Urol., 64, 423–427 (1989).
• 7)  Rifo J, Pourrat M, Vavrek RJ, Stewart JM, Huidobro-Toro JP. Bradykinin receptor antagonists used to characterize the heterogeneity of bradykinin-induced responses in rat vas deferens. Eur. J. Pharmacol., 142, 305–312 (1987).
• 8)  Osakada F, Kubo K, Goto K, Kanazawa I, Munekata E. The contractile activities of neurokinin A, B and related peptides on smooth muscles. Eur. J. Pharmacol., 120, 201–208 (1986).
• 9)  Miranda HF, Silva WI, Wolstenholme WW, Cuevas N, Ucros A, Bustamante D, Fernández EH. Pharmacological actions of substance P in the rat vas deferens. Neuropeptides, 18, 209–213 (1991).
• 10)  Amobi NI, Guillebaud J, Smith IC. Perspective on the role of P2X-purinoceptor activation in human vas deferens contractility. Exp. Physiol., 97, 583–602 (2012).
• 11)  Liu G, Kaneko M, Yoshioka K, Obara K, Tanaka Y. Platelet-activating factor (PAF) strongly enhances contractile mechanical activities in guinea pig and mouse urinary bladder. Sci. Rep., 12, 2783 (2022).
• 12)  Ohi Y, Yamamura H, Nagano N, Ohya S, Muraki K, Watanabe M, Imaizumi Y. Local Ca²⁺ transients and distribution of BK channels and ryanodine receptors in smooth muscle cells of guinea-pig vas deferens and urinary bladder. J. Physiol., 534, 313–326 (2001).
• 13)  Ohya S, Yamamura H, Muraki K, Watanabe M, Imaizumi Y. Comparative study of the molecular and functional expression of L-type Ca²⁺ channels and large-conductance, Ca²⁺-activated K⁺ channels in rabbit aorta and vas deferens smooth muscle. Pflugers Arch., 441, 611–620 (2001).
• 14)  Huang Y, Lau CW, Ho IHNS. 1619 activates Ca²⁺-activated K⁺ currents in rat vas deferens. Eur. J. Pharmacol., 325, 21–27 (1997).
• 15)  Shishido T, Sakai S, Tosaka T. T- and L-type calcium channels mediate α₁-adrenoceptor-evoked contraction in the guinea-pig vas deferens. Neurourol. Urodyn., 28, 447–454 (2009).
• 16)  Obara K, Yoshioka K, De Dios Regadera M, Matsuyama Y, Yashiro A, Miyokawa M, Iura R, Tanaka Y. Phenylephrine-induced contraction in guinea pig thoracic aorta is triggered by stimulation of α_1L-adrenoceptors functionally coupled with store-operated Ca²⁺ channels and voltage-dependent Ca²⁺ channels. Biol. Pharm. Bull., 46, 309–319 (2023).
• 17)  Kennedy C, Tasker PN, Gallacher G, Westfall TD. Identification of atropine- and P2X₁ receptor antagonist-resistant, neurogenic contractions of the urinary bladder. J. Neurosci., 27, 845–851 (2007).
• 18)  Inomata H, Ji JJ, Mimata T, Yamagata H. Concentration-related effects of extracellular application of ATP on the action potential and membrane currents of the guinea-pig vas deferens. Eur. J. Pharmacol., 202, 245–252 (1991).
• 19)  Seto SW, Docherty JR. Effects of T-type calcium channel blockers and thalidomide on contractions of rat vas deferens. Br. J. Pharmacol., 159, 1211–1216 (2010).
• 20)  Hirooka A, Hamada M, Fujiyama D, Takanami K, Kobayashi Y, Oti T, Katayama Y, Sakamoto T, Sakamoto H. The gastrin-releasing peptide/bombesin system revisited by a reverse-evolutionary study considering Xenopus. Sci. Rep., 11, 13315 (2021).
• 21)  Gajjar S, Patel BM. Neuromedin: an insight into its types, receptors and therapeutic opportunities. Pharmacol. Rep., 69, 438–447 (2017).
• 22)  Ohki-Hamazaki H. Neuromedin B. Prog. Neurobiol., 62, 297–312 (2000).
• 23)  Ischia J, Patel O, Shulkes A, Baldwin GS. Gastrin-releasing peptide: different forms, different functions. Biofactors, 35, 69–75 (2009).
• 24)  Milusheva EA, Kortezova NI, Mizhorkova ZN, Papasova M, Coy DH, Bálint A, Vizi ES, Varga G. Role of different bombesin receptor subtypes mediating contractile activity in cat upper gastrointestinal tract. Peptides, 19, 549–556 (1998).
• 25)  Rouissi N, Rhaleb NE, Nantel F, Dion S, Drapeau G, Regoli D. Characterization of bombesin receptors in peripheral contractile organs. Br. J. Pharmacol., 103, 1141–1147 (1991).
• 26)  Stjernquist M, Håkanson R, Leander S, Owman C, Sundler F, Uddman R. Immunohistochemical localization of substance P, vasoactive intestinal polypeptide and gastrin-releasing peptide in vas deferens and seminal vesicle, and the effect of these and eight other neuropeptides on resting tension and neurally evoked contractile activity. Regul. Pept., 7, 67–86 (1983).
• 27)  Tang H, Shu C, Chen H, Zhang X, Zang Z, Deng C. Constitutively active BRS3 is a genuinely orphan GPCR in placental mammals. PLoS Biol., 17, e3000175 (2019).
• 28)  The IUPHAR/BPS Guide to PHARMACOLOGY. “neuromedin B.”: <https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=613>, accessed 30 September, 2024.
• 29)  The IUPHAR/BPS Guide to PHARMACOLOGY. “gastrin-releasing peptide.”: <https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=612>, accessed 30 September, 2024.
• 30)  The IUPHAR/BPS Guide to PHARMACOLOGY. “neuromedin C.”: <https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6178>, accessed 30 September, 2024.
• 31)  Fainberg J, Kashanian JA. Recent advances in understanding and managing male infertility. F1000Res., 8, 670 (2019).
• 32)  Whitley JC, Shulkes A, Salamonsen LA, Vogiagis D, Familari M, Giraud AS. Temporal expression and cellular localization of a gastrin-releasing peptide-related gene in ovine uterus during the oestrous cycle and pregnancy. J. Endocrinol., 157, 139–148 (1998).
• 33)  Tattersall M, Cordeaux Y, Charnock-Jones DS, Smith GC. Expression of gastrin-releasing peptide is increased by prolonged stretch of human myometrium, and antagonists of its receptor inhibit contractility. J. Physiol., 590, 2081–2093 (2012).
• 34)  Zhang WS, Xie QS, Wu XH, Liang QH. Neuromedin B and its receptor induce labor onset and are associated with the RELA (NFKB P65)/IL6 pathway in pregnant mice. Biol. Reprod., 84, 113–117 (2011).
• 35)  Gnessi L, Isidori A, Bolotti M, Altamura S, Ulisse S, Jannini EA, Fabbri A, Spera G. Identification of immunoreactive gastrin-releasing peptide related substances in adult rat Leydig cells. Endocrinology, 124, 558–560 (1989).
• 36)  Ma Z, Zhang Y, Su J, Li X, Yang S, Qiao W, Suo C, Lei Z. Distribution of the pig gastrin-releasing peptide receptor and the effect of GRP on porcine Leydig cells. Peptides, 99, 142–152 (2018).
• 37)  Levy R, Eustache F, Pilikian S, Clavel C, Cordonier H, Benchaib M, Lornage J, Pinatel MC, Guerin JF. Regulators of sperm function: effect Effect of gastrin-releasing peptide on sperm functions. Mol. Hum. Reprod., 2, 867–872 (1996).
• 38)  Ruan YC, Wang Z, Du JY, Zuo WL, Guo JH, Zhang J, Wu ZL, Wong HY, Chung YW, Chan HC, Zhou WL. Regulation of smooth muscle contractility by the epithelium in rat vas deferens: role of ATP-induced release of PGE₂. J. Physiol., 586, 4843–4857 (2008).