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

Abstract

We examined whether the α_1L-adrenoceptor (AR), which shows low affinity (pA₂ < 9) for prazosin (an α₁-AR antagonist) and high affinity (pA₂ ≈ 10) for tamsulosin/silodosin (α_1A-AR antagonists), is involved in phenylephrine-induced contractions in the guinea pig (GP) thoracic aorta (TA). Intracellular signaling induced by α_1L-AR activation was also examined by focusing on Ca²⁺ influx pathways. Tension changes of endothelium-denuded TAs were isometrically recorded and mRNA encoding α-ARs/Ca²⁺ channels and their related molecules were measured using RT-quantitative PCR. Phenylephrine-induced contractions were competitively inhibited by prazosin/tamsulosin, and their pA₂ value were calculated to be 8.53/9.74, respectively. These contractions were also inhibited by silodosin concentration-dependently. However, the inhibition was not competitive fashion with the apparent pA₂ value being 9.48. In contrast, phenylephrine-induced contractions were not substantially suppressed by L-765314 (an α_1B-AR antagonist), BMY 7378 (an α_1D-AR antagonist), yohimbine, and idazoxan (α₂-AR antagonists). Phenylephrine-induced contractions were markedly inhibited by YM-254890 (a Gq protein inhibitor) or removal of extracellular Ca²⁺, and partially inhibited by verapamil (a voltage-dependent Ca²⁺ channel (VDCC) inhibitor). The residual contractions in the presence of verapamil were slightly inhibited by LOE 908 (a receptor-operated Ca²⁺ channel (ROCC) inhibitor) and strongly inhibited by SKF-96365 (a store-operated Ca²⁺ channel (SOCC) and ROCC inhibitor). Among the mRNA encoding α-ARs/SOCC-related molecules, α_1A-AR (Adra1a)/Orai3, Orai1, and Stim2 were abundant in this tissue. In conclusion, phenylephrine-induced contractions in the GP TA can be triggered by stimulation of Gq protein-coupled α_1L-AR, followed by activation of SOCCs and VDCCs.

INTRODUCTION

α₁-Adrenoceptors (ARs), representative G protein-coupled receptors,¹⁾ are classified into three genetically distinct subtypes (α_1A, α_1B, and α_1D).²⁾ Stimulation of these three α₁-AR subtypes with AR agonists has been reported to produce contractions in various smooth muscle tissues^3–42) (Table 1). Cell lines overexpressing each α₁-AR subtype show high affinity for the α₁-AR antagonist prazosin (pK_B > 9).^43–46) However, in native smooth muscle (SM) tissues, the presence of an α₁-AR showing low affinity for prazosin (pA₂ < 9) was suggested, and this α₁-AR type was proposed to be classified as α_1L-AR, as opposed to the α_1H-AR that shows high affinity for prazosin (pA₂ > 9).^43–46) Although many approaches have been used to identify the α_1L-AR-encoding gene, it has not yet been found. Currently, α_1L-AR is regarded as an α_1A-AR-derived phenotype that is modified specifically in SM tissues. The evidence supporting this hypothesis is as follows: 1) α_1L-AR shows high affinity for α_1A-AR antagonists such as tamsulosin and silodosin (pA₂ ≈ 10); 2) while α_1L-AR characteristic binding sites can be detected using native SMs, the binding site characteristics change to the α_1A-AR type using membrane fractions prepared by homogenization; and 3) in α_1A-AR knockout mice, both α_1L- and α_1A-AR characteristics are not detected, while α_1L-AR characteristics remain in α_1B- and α_1D-AR knockout mice.^43–46) α_1L-AR is abundant in the lower urinary tract and genital SMs and is substantially present in some vascular SM tissues^{19,28,32,38,39,47–65)} (Table 1).

Table 1. α-Adrenoceptor (AR) Subtypes Involved in Various Smooth Muscle Contractions

α1-AR Subtypes	Species	Smooth muscle tissues	References
α1A-AR	Human	Vas deferens	3,4)
		Prostate	5)
		Corpus cavernosum	6)
	Marmoset	Urethra	7)
	Dog	Superior tarsal muscle	8)
		Mesenteric artery	9)
	Pig	Urethra	10)
		Coronary artery	11)
	Rabbit	Thoracic aorta	12)
		Common iliac artery	12)
		Bronchus	13)
	Rat	Vas deferens	14–17)
		Prostate	18)
		Urinary bladder	19)
		Urethra	20)
		Cauda epididymis	21)
		Caudal artery	22)
		Renal artery	22)
		Renal interlobar arteries	23)
	Hamster	Ureter	24)
	Mouse	Urethra	7)
α1B-AR	Human	Iliac artery branches	25)
		Internal mammary artery	26)
	Rabbit	Corpus cavernosum	27)
		Thoracic aorta	12)
		Common iliac artery	12)
		Cutaneous resistance arteries	28)
	Guinea pig	Spleen	29)
	Rat	Spleen	14,15)
		Tail artery	30,31)
		Mesenteric resistance artery	22)
		Portal vein	32)
	Mouse	Spleen	33)
α1D-AR	Rabbit	Renal artery	34)
		Iliac artery	34)
	Rat	Vas deferens	17)
		Thoracic/Abdominal aorta	22,31,35–38)
		Superior mesenteric artery	22,39)
		Mesenteric artery	31,37)
		Pulmonary artery	37)
		Iliac artery	22,40)
		Carotid artery	31)
		Femoral artery	22)
	Mouse	Vas deferens	41)
		Thoracic aorta	42)
α1L-AR	Human	Bladder neck	47)
		Prostate	47)
		Urethra	47)
		Vas deferens	48)
		Erectile tissue	49)
		Iris dilator muscle	50)
	Dog	Prostate	51)
		Urethra	52)
		Subcutaneous resistance arteries	53)
	Pig	Urethra	54)
	Rabbit	Bladder trigone	55,56)
		Urethra	55,56)
		Prostate	56,57)
		Mesenteric artery	56)
		Ear artery	58)
		Cutaneous resistance arteries	28)
		Iris dilator muscle	50)
	Guinea pig	Prostate	59)
		Aorta	60)
		Nasal mucosa vasculature	61)
	Rat	Urinary bladder	19)
		Superior mesenteric artery	39)
		Small mesenteric artery	62)
		Tail artery	38)
		Femoral artery	63)
		Portal vein	32)
	Mouse	Vas deferens	64)
		Prostate	64,65)

Regarding SM contractions mediated through α_1H-ARs (α_1A, α_1B, and α_1D), extracellular Ca²⁺ influx through Ca²⁺ channels plays a primary role similar to the contractions mediated through other drug receptors, and this is evidenced by the following findings: 1) α_1A-AR-mediated, noradrenaline (NA)-induced contractions in rat vas deferens were elicited by Ca²⁺ influxes through voltage-dependent Ca²⁺ channels (VDCCs) as well as intracellular Ca²⁺ release from ryanodine-sensitive stores⁶⁶⁾; 2) α_1A-AR-mediated, phenylephrine-induced contractions in rat tail and iliac arteries involved VDCC-dependent Ca²⁺ influxes, the activation of which was triggered by cation influxes sensitive to LOE 908, an inhibitor of receptor-operated Ca²⁺ channels (ROCCs)⁶⁷⁾; 3) α_1B-AR-mediated, phenylephrine-induced contractions in rat spleen involved Ca²⁺ influxes sensitive to SKF-96365, an inhibitor of ROCC and store-operated Ca²⁺ channels (SOCCs), and intracellular Ca²⁺ release⁶⁸⁾; and 4) α_1D-AR-mediated, NA-induced contractions in the rat aorta involved nifedipine-sensitive and SKF-96365-sensitive Ca²⁺ influxes.⁶⁹⁾ These findings suggest that VDCC-independent and -dependent Ca²⁺ entry routes trigger α_1H-AR-mediated SM contractions. However, to the best of our knowledge, the dependency on extracellular Ca²⁺ and its entry routes responsible for α_1L-AR-mediated SM contractions have not been studied to date.

In this study, to clarify the extracellular Ca²⁺ entry routes responsible for α_1L-AR-mediated SM contractions, we focused on the guinea pig (GP) thoracic aorta (TA), in which NA is reported to induce α_1L-AR-mediated contractions.⁶⁰⁾ GP TA is a commonly used tissue for pharmacological studies and could be more suitable for studying α_1L-AR than resistant arteries and lower urinary tract tissues, in which α_1H-AR subtypes may be present in addition to α_1L-AR (Table 1). Here, we report that α_1L-AR-mediated, phenylephrine-induced contractions in the GP TA are triggered by Ca²⁺ influxes through both SOCCs and VDCCs. We also suggest that the principal SOCC candidates are ORAI3/ORAI1 and stromal interaction molecule 2 (STIM2) based on their mRNA expression in GP TA.

MATERIALS AND METHODS

Animals

This study was approved by the Toho University Animal Care and User Committee (Approval Nos. 20-51-444, accredited on May 19, 2020; 21-52-444, accredited on April 21, 2021; 22-53-444, accredited on April 9, 2022). The experiments were conducted in accordance with the guidelines of the Laboratory Animal Center of the Faculty of Pharmaceutical Sciences, Toho University. For this study, we purchased male GPs (age, 5–11 weeks; weight, 287–550 g) from Kyudo, Co., Ltd. (Saga, Japan). The GPs were housed under controlled conditions (21–22 °C, relative air humidity 50 ± 5%) and a fixed 12/12 h light–dark cycle (08:00–20:00), with food and water available ad libitum.

Preparation of GP TA

The GPs were anesthetized using isoflurane inhalation and euthanized by exsanguination via the carotid artery. The isolated TAs were cleaned of connective tissue and fat and cut into ring preparations (approximately 2 mm in length) in normal Tyrode’s solution containing (mM): NaCl, 158.3; KCl, 4.0; CaCl₂, 2.0; MgCl₂, 1.05; NaH₂PO₄, 0.42; NaHCO₃, 10.0; and D-(+)-glucose, 5.6. The intimal surface of ring preparations was rubbed to remove the endothelium.

Measurement of Tension Changes

The ring preparations were suspended under an optimal resting tension of 1.0 g using stainless steel hooks (outer diameter, 200 µm) in a 5-mL organ bath (UC-5, UFER Medical Instrument, Kyoto, Japan) containing normal Tyrode’s solution. The resting tension was determined according to methods previously described.⁶⁰⁾ The solution was maintained at 35.0 ± 1.0 °C (pH = 7.4) and continuously bubbled with a mixture of 95% O₂ and 5% CO₂. TA tension changes were isometrically recorded using force-displacement transducers (T7-8-240, Orientec, Tokyo, Japan; ULA-10GR, MinebeaMitsumi Inc., Nagano, Japan) connected to amplifiers (AM20AZ, Unipulse Corporation, Tokyo, Japan; AP-621G, Nihon Kohden, Tokyo, Japan) and PowerLab™/LabChart™ software (ADInstruments Pty., Ltd., Bella Vista, NSW, Australia). After equilibrated for 60 min in normal Tyrode’s solution, the TA segments were contracted with phenylephrine (10⁻⁵ M). After then, to verify the functional absence of endothelium, the TA segments were challenged with acetylcholine (ACh, 10⁻⁵ M). TA preparations hardly affected by ACh were regarded as endothelium-denuded TA preparations. The TA preparations were then contracted four times with phenylephrine (3 × 10⁻⁴ M). To prevent any possible effects of endogenous prostaglandins, all experiments were performed in the presence of indomethacin (3 × 10⁻⁶ M).

Effects of α-AR Antagonists and Ca²⁺ Channel Inhibitors on Concentration–Response Curves (CRCs) for Phenylephrine

After the procedures described in “Measurement of Tension Changes,” to obtain a CRC for the control, phenylephrine was cumulatively added to the bath solution. For some experiments, the vehicle (0.2% ethanol (EtOH) for silodosin; 6 × 10⁻⁴, 0.02, and 0.3% dimethyl sulfoxide (DMSO) for L-765314, YM-254890, and LOE 908, respectively), and/or verapamil (a VDCC inhibitor, 10⁻⁵ M) was administered 20 min before control CRC determination. After washing out, prazosin (an α₁-AR antagonist, 3 × 10⁻⁹–3 × 10⁻⁸ M), tamsulosin (an α_1A-AR antagonist, 3 × 10⁻¹⁰–3 × 10⁻⁹ M), silodosin (an α_1A-AR antagonist, 10⁻⁹–3 × 10⁻⁸ M), L-765314 (an α_1B-AR antagonist, 3 × 10⁻⁸ M), BMY 7378 (an α_1D-AR antagonist, 10⁻⁸ M), yohimbine (an α₂-AR antagonist, 10⁻⁸ M), idazoxan (an α₂-AR antagonist, 10⁻⁸ M), YM-254890 (a Gq protein inhibitor, 10⁻⁶ M), verapamil (10⁻⁵ M), verapamil (10⁻⁵ M) + LOE 908 (3 × 10⁻⁵ M), or verapamil (10⁻⁵ M) + SKF-96365 (3 × 10⁻⁵ M) was applied in the bath medium and incubated for 20 min. The concentrations and incubation time of the antagonists and inhibitors used in this study were sufficient to inhibit the targeted receptors and channels based on previous reports.^{20,43–46,60,70–75)} After then, to obtain the CRC in the presence of drugs, phenylephrine was cumulatively added again.

To examine the effects of the Ca²⁺-free solution, the bath solution was replaced 20 min before CRC with the following solution (mM): NaCl, 158.3; KCl, 4.0; MgCl₂, 1.05; NaH₂PO₄, 0.42; NaHCO₃, 10.0; D-(+)-glucose, 5.6; and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N'-tetraacetic acid (EGTA), 0.2.

RT-Quantitative (q)PCR of α-AR, SOCC-Related, and ROCC-Related mRNA Expression

In the mRNA experiment, the endothelium of the TA preparations was carefully denuded. RT-qPCR was performed according to the methods described in a previous report.⁷⁶⁾ Briefly, total RNA was extracted from the endothelium-denuded TA preparations by the acid guanidinium thiocyanate-phenol-chloroform method. After treated with deoxyribonuclease (Nippon Gene Co., Ltd., Tokyo, Japan) for 30 min at 37 °C, total RNA pellets were obtained by phenol-chloroform extraction/ethanol precipitation and dissolved in diethyl pyrocarbonate-treated water. We used ReverTra Ace^® qPCR RT Master Mix with gDNA Remover (TOYOBO Co., Ltd., Osaka, Japan) to synthesize first-strand cDNA by reverse transcription. RT-qPCR was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, U.S.A.) using THUNDERBIRD^® Next SYBR^® qPCR Mix (TOYOBO Co., Ltd.). The primers used in this study are shown in Supplementary Table 1. The thermal cycler parameters were set to 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 35 s. Fluorescence intensity was measured at each 72 °C step. The mRNA expression level of each gene was analyzed by Sequence Detection Software (Applied Biosystems) and calculated as a relative value to that of β-actin gene (Actb), which was set to 1. We considered the mRNA expression level as 0 for samples that did not reach the fluorescence intensity threshold after 40 cycles.

Drugs

The following drugs were used in this study: l-phenylephrine hydrochloride, idazoxan hydrochloride, (±)-verapamil hydrochloride, and indomethacin (Sigma-Aldrich Co., St. Louis, MO, U.S.A.); prazosin hydrochloride, silodosin, yohimbine hydrochloride, and YM-254890 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); tamsulosin hydrochloride (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan); L-765314 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.); BMY 7378 dihydrochloride (Research Biochemicals Inc., MA, U.S.A.); LOE 908 (Nippon Boehringer Ingelheim Co., Ltd., Hyogo, Japan); SKF-96365 (Cayman Chemical Co., Ann Arbor, MI, U.S.A. or Tokyo Chemical Industry Co., Ltd.); and Ach chloride (Daiichi Sankyo Co., Ltd., Tokyo, Japan).

Silodosin was dissolved in EtOH to prepare stock solutions of 5 × 10⁻³ M and diluted with EtOH to the desired concentrations. Indomethacin was dissolved in EtOH to prepare a stock solution of 10⁻² M. L-765314/YM-254890 and LOE 908 were dissolved in DMSO to prepare stock solutions of 5 × 10⁻³ M and 10⁻² M. All the other drugs were dissolved/diluted with distilled water.

Data and Statistical Analyses

The extent of phenylephrine-induced contractions was calculated relative to the tension level before the phenylephrine administration (0% contraction) and to the 3 × 10⁻⁴ M phenylephrine-induced contractions in the control CRC (100% contraction). The data were plotted as a function of phenylephrine concentration and fitted to the following equation:

  

where E is % contraction at a given phenylephrine concentration, E_max is the maximum contraction, A is the phenylephrine concentration, n_H is the Hill coefficient, and EC₅₀ is the phenylephrine concentration that produces a half-maximal response. Curve fitting was performed using GraphPad Prism™ (GraphPad Software Inc., San Diego, CA, U.S.A.). α-AR antagonist potencies were expressed as pA₂ values, which were calculated from a Schild plot analysis.⁷⁷⁾

Data are expressed as the mean ± standard error of the mean (S.E.M.) or the mean with 95% confidence interval (95% CI), where n refers to the number of experiments. Statistical analyses were performed by Šidák’s test after two-way ANOVA using GraphPad Prism™. All statistical analyses were conducted with a significance level of α = 0.05 (p < 0.05).

RESULTS

Effects of Prazosin, Tamsulosin, and Silodosin on Phenylephrine-Induced Contractions

Prazosin (3 × 10⁻⁹–3 × 10⁻⁸ M) inhibited the phenylephrine-induced contractions and shifted the CRCs for phenylephrine to the right (Fig. 1Aa). The slope of the regression line for the Schild plot of prazosin vs. phenylephrine was 1.06 (95% CI: 0.85–1.27), which was not significantly different from unity (Fig. 1Ab). The pA₂ value of prazosin vs. phenylephrine was 8.53 (95% CI: 8.42–8.69).

Fig. 1. Effects of Prazosin on Phenylephrine-Induced Contractions in Guinea Pig (GP) Thoracic Aorta (TA)

a: Effects of prazosin (3 × 10⁻⁹–3 × 10⁻⁸ M, Aa), tamsulosin (3 × 10⁻¹⁰–3 × 10⁻⁹ M, Ba), and silodosin (10⁻⁹–3 × 10⁻⁸ M, Ca) on the concentration-response curves (CRCs) for phenylephrine-induced contractions (Aa: n = 16 for control, n = 6 for 10⁻⁸ M prazosin, and n = 5 for 3 × 10⁻⁹ M and 3 × 10⁻⁸ M prazosin; Ba: n = 18 for control and n = 6 for 3 × 10⁻¹⁰–3 × 10⁻⁹ M tamsulosin; Ca: n = 20 for control and n = 5 for 10⁻⁹–3 × 10⁻⁸ M silodosin). b: Schild plot for prazosin (Ab), tamsulosin (Bb), and silodosin (Cb) vs. phenylephrine (Ab: n = 6 for 10⁻⁸ M prazosin and n = 5 for 3 × 10⁻⁹ M and 3 × 10⁻⁸ M prazosin; Bb: n = 6 for each; Cb: n = 5 for each). Data are presented as the mean ± standard error of the mean (S.E.M.). Slope and pA₂ values (b) are presented as the mean with 95% confidence interval (95% CI).

Tamsulosin (3 × 10⁻¹⁰–3 × 10⁻⁹ M) inhibited the phenylephrine-induced contractions and shifted the CRCs for phenylephrine to the right (Fig. 1Ba). The slope of the regression line for the Schild plot of tamsulosin vs. phenylephrine was 1.14 (95% CI: 0.88–1.41), which was not significantly different from unity (Fig. 1Bb). The pA₂ value of tamsulosin vs. phenylephrine was 9.74 (95% CI: 9.58–9.99).

Silodosin (10⁻⁹–3 × 10⁻⁸ M) inhibited the phenylephrine-induced contractions and shifted the CRCs for phenylephrine to the right (Fig. 1Ca). However, the slope of the regression line for the Schild plot of silodosin vs. phenylephrine was 0.58 (95% CI: 0.37–0.78), which was significantly less than unity (Fig. 1Cb). The apparent pA₂ value of silodosin vs. phenylephrine was 9.48 (95% CI: 9.12–10.19).

Effects of L-765314, BMY 7378, Yohimbine, and Idazoxan on Phenylephrine-Induced Contractions

Neither L-765314 (3 × 10⁻⁸ M, Fig. 2A) nor BMY 7378 (10⁻⁸ M, Fig. 2B) significantly affected the phenylephrine-induced contractions (vs. DMSO/control).

Fig. 2. Effects of L-765314 (3 × 10⁻⁸ M, A), BMY 7378 (10⁻⁸ M, B), Yohimbine (10⁻⁸ M, C), and Idazoxan (10⁻⁸ M, D) on the CRCs for Phenylephrine-Induced Contractions in GP TA

Data are presented as the mean ± S.E.M. (n = 5 for each). * p < 0.05, ** p < 0.01 vs. control (two-way ANOVA followed by Šidák’s test).

Both yohimbine (10⁻⁸ M, Fig. 2C) and idazoxan (10⁻⁸ M, Fig. 2D) significantly, but only slightly, inhibited or augmented the phenylephrine-induced contractions (vs. control). We concluded that the effects of yohimbine and idazoxan were negligible.

Comparison of mRNA Expression Levels of α-ARs Assessed by RT-qPCR

The relative mRNA expression levels of α-ARs (α_1A-AR (Adra1a), α_1B-AR (Adra1b), α_1D-AR (Adra1d), α_2A-AR (Adra2a), α_2B-AR (Adra2b), and α_2C-AR (Adra2c)) in endothelium-denuded TA are shown in Fig. 3. The most abundant α-AR mRNA was Adra1a, followed by Adra1d, Adra1b, and Adra2c. In contrast, mRNA expression levels of Adra2a and Adra2b were the lowest.

Fig. 3. Comparison of mRNA Expression Levels of α_1A-Adrenoceptor (AR) (Adra1a), α_1B-AR (Adra1b), α_1d-AR (Adra1d), α_2A-AR (Adra2a), α_2B-AR (Adra2b), and α_2C-AR (Adra2c) in GP TA Measured by RT-qPCR

The expression level of each mRNA is shown relative to the mRNA expression level of Actb, which is set as 1. Data are expressed as the mean ± S.E.M. (n = 12 for Adra1a, Adra1b, and Adra2b; n = 11 for Adra2a and Adra2c; and n = 10 for Adra1d).

Effects of YM-254890 on Phenylephrine-Induced Contractions

YM-254890 (10⁻⁶ M) completely inhibited the phenylephrine-induced contractions (Fig. 4).

Fig. 4. Effects of YM-254890 (10⁻⁶ M) on the CRCs for Phenylephrine-Induced Contractions in GP TA

Data are presented as the mean ± S.E.M. (n = 5). ** p < 0.01 vs. DMSO (YM-254890 vehicle) (two-way ANOVA followed by Šidák’s test). DMSO: dimethyl sulfoxide.

Effects of Verapamil and Removal of Ca²⁺ on Phenylephrine-Induced Contractions

The CRCs for phenylephrine-induced contractions in normal Tyrode’s solution and Ca²⁺-free solution containing 0.2 mM EGTA are shown in Fig. 5A. The phenylephrine-induced contractions were strongly inhibited by the Ca²⁺-free solution; at 3 × 10⁻⁴ M phenylephrine, the contraction was inhibited by approx. 95% (Fig. 5A).

Fig. 5. Effects of 0.2 mM EGTA-Containing Ca²⁺-Free Solution (A) and Verapamil (10⁻⁵ M, B), LOE 908 (3 × 10⁻⁵ M, C), and SKF-96365 (3 × 10⁻⁵ M, D) on the CRCs for Phenylephrine-Induced Contractions in the Absence (A, B) or Presence (C, D) of Verapamil (10⁻⁵ M) in GP TA

Data are presented as the mean ± S.E.M. (n = 5 for each). * p < 0.05, ** p < 0.01 vs. control/verapamil/verapamil + DMSO (LOE 908 vehicle) (two-way ANOVA followed by Šidák's test).

Verapamil (10⁻⁵ M) significantly inhibited the contractions at low concentrations (10⁻⁶–3 × 10⁻⁵ M) (vs. control) (Fig. 5B). In contrast, the contractions were not affected by verapamil (10⁻⁵ M) at higher concentrations (≥10⁻⁴ M).

Effects of LOE 908 and SKF-96365 on Phenylephrine-Induced Contractions in the Presence of Verapamil

LOE 908 (3 × 10⁻⁵ M) slightly but significantly inhibited the CRCs for phenylephrine-induced contractions in the presence of verapamil (10⁻⁵ M) (vs. verapamil + DMSO) (Fig. 5C).

SKF-96365 (3 × 10⁻⁵ M) strongly inhibited the CRCs for phenylephrine-induced contractions in the presence of verapamil (10⁻⁵ M); at 3 × 10⁻⁴ M, the contraction was inhibited by approx. 60% (Fig. 5D).

Comparison of mRNA Expression Levels of SOCC-Related Molecules (Orai and Stim) Assessed by RT-qPCR

The relative mRNA expression levels of SOCC-related molecules (Orai (Orai1–3) and Stim (Stim1 and Stim2)) in endothelium-denuded TA are shown in Fig. 6. The most abundant SOCC-related molecule mRNA was Orai3 and Stim2, followed by Orai1. In contrast, the mRNA expression levels of Orai2 and Stim1 were the lowest.

Fig. 6. Comparison of mRNA Expression Levels of Orai (Orai1, Orai2, and Orai3) and Stim (Stim1 and Stim2) in GP TA Measured by RT-qPCR

The expression level of each mRNA is shown relative to the mRNA expression level of Actb, which is set as 1. Data are expressed as the mean ± S.E.M. (n = 5 for each).

Comparison of mRNA Expression Levels of ROCC-Related Molecules (Transient Receptor Potential (TRP) Channels) Assessed by RT-qPCR

The relative mRNA expression levels of ROCC-related molecules (TRP channels (Trpc1, Trpc3, Trpc4, Trpc5, Trpc6, Trpc7, Trpm8, and Trpv4)) in endothelium-denuded TA are shown in Supplementary Fig. 1. We tested the presence of TRPC channels which are considered to play a major role in receptor-operated Ca²⁺ entry in SMs⁷⁸⁾ and TRPM8/TRPV4 channels which are reported to involve rat aortic SM contractility.⁷⁹⁾ The most abundant TRP channel mRNA was Trpc6, followed by Trpc1 and Trpc3. The mRNA expression levels of the other TRP channels were very low compared with the Trpc6 mRNA level, in the following order of expression: Trpc5 ≈ Trpv4 > Trpc4 ≈ Trpc7 ≫ Trpm8.

DISCUSSION

We showed that phenylephrine-induced contractions in GP TA SMs were triggered by the stimulation of Gq protein-coupled α_1L-AR, which was followed by Ca²⁺ influx through SOCCs (ORAI1/3 and STIM2) and VDCCs. GP TA could be more suitable for studying α_1L-AR than resistant arteries and lower urinary tract tissues, in which α_1H-AR subtypes may be present in addition to α_1L-AR (Table 1). Furthermore, its easy-to-handle properties will contribute to the progress of α_1L-AR research.

First, we discuss the effect of prazosin on phenylephrine-induced contractions in GP TA. These contractions were competitively suppressed by prazosin, and its pA₂ value was calculated to be 8.53 (Fig. 1A). Since this pA₂ value was <9, we concluded that the α₁-AR responsible for phenylephrine-induced contractions was α_1L-AR, as α_1H-AR has a pA₂ > 9.^43–46) Yamamoto and Koike also reported that the α₁-AR mediating NA-induced contractions in GP TA was α_1L-AR.⁶⁰⁾

Second, we discuss the effects of α_1A-AR antagonists (tamsulosin/silodosin) on phenylephrine-induced contractions. α_1L-AR is defined as α₁-AR showing low affinity for prazosin and high affinity for α_1A-AR antagonists such as tamsulosin/silodosin (pA₂ ≈ 10).^43–46) In the present study, the phenylephrine-induced contractions of GP TA were competitively suppressed by tamsulosin, with a pA₂ value of 9.74 (Fig. 1B). This value is consistent with the pA₂ or pK_B values of tamsulosin for α_1L-AR: 9.57 (pA₂ vs. NA/GP TA)⁶⁰⁾ and 9.99 (pK_B vs. phenylephrine/rabbit prostate).⁸⁰⁾

We also tested the effects of silodosin, another specific inhibitor of α_1A-AR. Silodosin is reported to show a competitive antagonistic action on α_1A-AR/α_1L-AR in the following SM tissues based on the regression line slopes of the Schild plots for silodosin vs. the α₁-AR agonists being unity: hamster ureter (pA₂ = 9.44 vs. phenylephrine), rat tail artery (pA₂ = 10.0 vs. NS-49 (an α_1A-AR agonist)), dog mesenteric artery (pA₂ = 9.9 vs. NS-49), and rabbit ureter (pA₂ = 8.71 vs. NA).^24,81,82) Although the phenylephrine-induced contractions were suppressed by silodosin in a concentration-dependent manner (Fig. 1Ca), the slope of the regression line for the Schild plot of silodosin vs. phenylephrine was less than unity (0.58 (95% CI: 0.37–0.78)) (Fig. 1Cb), which does not support a competitive antagonist action vs. phenylephrine. Similar results have been reported in human and rabbit prostates, in which α_1L-AR mediates NA-/phenylephrine-induced contractions. In both SM tissues, Schild plot analysis was not performed for silodosin-induced inhibition, possibly because of its lack of apparent competitive antagonistic action. In these studies, instead of pA₂ values, apparent pK_B values were calculated for silodosin (9.45 vs. NA, human prostate; 10.05/9.6 vs. phenylephrine/NA, rabbit prostate).^80,83,84) Furthermore, in human prostate tissue, silodosin was suggested to be prone to bind to non-α₁-AR sites as well as α₁-AR. Namely, the specific binding of [³H]silodosin (500 pM) was shown to be almost equal to the non-specific binding of [³H]silodosin (500 pM).⁸⁵⁾ Therefore, in GP TA and in prostate tissues, the apparent non-competitive antagonistic action of silodosin vs. phenylephrine might be partly explained by its tendency to bind to non-α₁-AR sites. Although competitive antagonism of silodosin vs. phenylephrine was not observed in GP TA, its apparent pA₂ value was tentatively calculated to be 9.48, which is consistent with the values for α_1A-AR/α_1L-AR. Therefore, we conclude that the pharmacological characteristics of α_1L-AR, which mediates phenylephrine-induced contractions in GP TA, are almost the same as those in other SM tissues.

Third, we discuss the effects of other α-AR (α_1B-, α_1D-, and α₂-AR) antagonists on the phenylephrine-induced contractions. These contractions were not substantially suppressed by L-765314 (an α_1B-AR antagonist, 3 × 10⁻⁸ M), BMY 7378 (an α_1D-AR antagonist, 10⁻⁸ M), yohimbine (an α₂-AR antagonist, 10⁻⁸ M), and idazoxan (an α₂-AR antagonist, 10⁻⁸ M) (Fig. 2). Therefore, α_1B-AR/α_1D-AR/α₂-AR are unlikely to be involved in the phenylephrine-induced contractions and this is supported for the following reasons: 1) the concentration of L-765314 used in this study (30 nM) could sufficiently antagonize α_1B-AR since its K_i value for α_1B-AR is 5.4 nM⁷⁰⁾; 2) the concentration of BMY 7378 used in this study (10 nM) could sufficiently antagonize α_1D-AR since its K_i value for α_1D-AR is 0.43 nM²⁰⁾; and 3) the concentrations of yohimbine and idazoxan used in this study (10⁻⁸ M, negative logarithm of 8.0) could sufficiently antagonize α₂-AR since the pA₂ values of yohimbine and idazoxan for α₂-AR calculated using rat vas deferens are reported to be 8.24 and 8.70, respectively.⁷¹⁾

The pharmacological characteristics of the α-AR responsible for phenylephrine-induced contractions in GP TA are consistent with those of α_1L-AR, which is proposed to be derived from the α_1A-AR mRNA (Adra1a).^43–46) This is also supported by our present mRNA expression results, showing that Adra1a is the most abundant α-AR (Fig. 3).

Sex differences in α-AR have been reported as follows: 1) Administration of phenylephrine dose-dependently reduced finger blood flow in men, but not in women.⁸⁶⁾ 2) The number of α-ARs in platelets of women who are hypertensive is 1.5 times higher than in those of women who are normotensive, but the affinity of [³H]dihydroergocryptine for α-AR did not differ between these two groups, or between men and women.⁸⁷⁾ 3) The binding properties of [³H]prazosin to the rabbit urethra are the same in males and females.⁸⁸⁾ In this study, we determined that α_1L-AR is predominant in GP TA using only male GPs. Further studies using female GPs are required to clarify whether there are sex differences in the function and subtype of α₁-ARs in the GP TA.

The pathophysiological role of α_1L-AR in GP TA is currently unclear. However, in rat femoral arteries in which α_1H-AR and α_1L-AR are present, the α₁-AR subtypes mediating contraction in the spontaneously hypertensive rat femoral artery were identical with those in the normotensive rat tissue.⁶³⁾ In addition, a study using young adult and aged rat urinary bladder SM suggests that the promotion of contractile responses mediated by α_1A-AR, but not α_1L-AR, may be the cause of unstable bladder in those who are elderly.¹⁹⁾ Therefore, α_1L-AR is unlikely to actively be involved in hypertension and unstable bladder in older individuals. Moreover, in human benign prostatic hyperplasia tissue in which α_1H-AR and α_1L-AR are present, treatment with α₁-AR antagonists may not change the conditions of α₁-ARs in this tissue.⁸⁹⁾ Thus, α₁-AR antagonists that strongly suppress α_1L-AR are needed for the treatment of benign prostatic hyperplasia.

Fourth, we discuss the post-receptor mechanisms of phenylephrine-induced contractions. These contractions were completely suppressed by a Gq-selective inhibitor YM-254890⁹⁰⁾ (Fig. 4), suggesting that they were mediated by Gq protein activation. In addition, the phenylephrine-induced contractions were potently suppressed by the removal of extracellular Ca²⁺ (Fig. 5A). This finding indicates that these contractions strongly depend on extracellular Ca²⁺ influx. One extracellular Ca²⁺ influx route was indicated to be VDCC-mediated because verapamil (a VDCC inhibitor) partly but significantly suppressed the contractions induced by low concentrations (10⁻⁶–3 × 10⁻⁵ M) of phenylephrine (vs. control) (Fig. 5B). The concentration of verapamil (10⁻⁵ M) used in this study was sufficient to inhibit VDCC because it completely suppressed the binding of [³H]D888 (4.2 nM) to rat cardiac membranes in a previous study.⁷²⁾ Therefore, VDCC plays an important role in the Ca²⁺ influx pathway when α_1L-AR is weakly activated. However, VDCC is not the only Ca²⁺ influx pathway, and the contribution of VDCC-independent Ca²⁺ influx is speculated to be substantial because the contractions induced by high concentrations of phenylephrine (≥10⁻⁴ M) were not affected by verapamil (10⁻⁵ M) (Fig. 5B). VDCC-independent Ca²⁺ influx is the main pathway when α_1L-AR is strongly activated. In support of our results, Tanaka et al. also reported that the contractile responses of GP TA induced by a high concentration (3 × 10⁻⁵ M) of NA were not suppressed by various VDCC inhibitors (nicardipine/diltiazem/verapamil).⁷³⁾ In the mouse prostate, a VDCC inhibitor (nifedipine) has been reported to suppress α_1L-AR-mediated contractions induced by electrical field stimulation, but the suppression rate differs depending on the frequency of electrical stimulation.⁹¹⁾

To clarify the possible involvement of ROCCs and SOCCs in VDCC-independent Ca²⁺ influx pathways, the effects of LOE 908 and SKF-96365 on the phenylephrine-induced contractions were investigated. LOE 908 inhibits ROCCs, although it also inhibits VDCCs.⁹²⁾ SKF-96365 is an inhibitor of SOCCs/ROCCs and shows inhibitory effects on VDCCs.^92,93) Therefore, in this study, the inhibitory effects of LOE 908 for ROCC were evaluated in the presence of verapamil, and those of SKF-96365 for SOCC were evaluated in the presence of verapamil but in the absence of LOE 908 since the effects of LOE 908 were very limited. Our findings showed that the inhibitory effects of LOE 908 (3 × 10⁻⁵ M) vs. phenylephrine were significant, but only slight (Fig. 5C), and those of SKF-96365 (3 × 10⁻⁵ M) were very strong (Fig. 5D). Therefore, SOCCs are suggested to represent the principal VDCC-independent Ca²⁺ influx pathway, although ROCCs may be involved. In GP TA, SKF-96365-inhibitable characteristics have also been reported for NA-induced contractions.⁷³⁾ An important role of SOCCs in α₁-AR-mediated contraction has also been reported in non-vascular SM tissues. Specifically, in mouse urethral SM, phenylephrine-induced contractions were strongly attenuated by SOCC inhibitors,⁹⁴⁾ although it is unclear whether α_1A-AR/α_1L-AR is predominant in this tissue.⁷⁾ To the best of our knowledge, our study is the first to show that α_1L-AR-mediated SM contraction is triggered by SOCCs/VDCCs through Gq protein activation. However, to clarify whether functional coupling of α_1L-AR with SOCCs/VDCCs is conserved among SM type, further studies are required using tissues in which α_1L-AR functionally predominates, such as genital and lower urinary tract SMs.

Finally, we discuss the molecular candidates for SOCC/ROCC that are activated by the stimulation of α_1L-AR. ORAI channels are molecular candidates for the SOCC, and ORAI channel activation is regulated by STIMs, which are Ca²⁺ sensors of the endoplasmic reticulum.⁹⁵⁾ To identify the SOCC/STIM molecular candidates responsible for phenylephrine-induced α_1L-AR-mediated contractions, the Orai and Stim mRNA expression levels were assessed by RT-qPCR. Among the tested Orai and Stim mRNA, Orai3 and Stim2 mRNA expression levels were the highest in GP TA, followed by Orai1 (Fig. 6). Therefore, stimulation of α_1L-AR in the GP TA is speculated to activate ORAI1 and/or ORAI3 through STIM2. In addition, TRP channels are molecular candidates for the ROCC.⁹⁶⁾ The relative mRNA expression of the tested TRP channels in GP TA was Trpc6 > Trpc1 > Trpc3 (Supplementary Fig. 1). Therefore, in addition to SOCCs, these TRP channels may function as VDCC-independent Ca²⁺ influx pathways in α_1L-AR-mediated GP TA contractions, although their contributions are very small compared to those of SOCCs/VDCCs. Another role of ROCCs in SM contraction is to trigger the activation of VDCCs.⁹⁷⁾ Therefore, the verapamil-sensitive component of the phenylephrine-induced α_1L-AR-mediated contractions may be generated by being triggered by LOE 908-sensitive ROCCs. By using ROCC inhibitors without VDCC inhibition, this role of ROCCs may be clarified, and this issue should be studied in the future.

Acknowledgments

The authors would like to thank Dr. Fumiko Yamaki for her expert technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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