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Am J Physiol Heart Circ Physiol 294: H1923-H1932, 2008. First published January 25, 2008; doi:10.1152/ajpheart.01221.2007
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Rho kinase is involved in Ca2+ entry of rat penile small arteries

Nuria Villalba,1,2 Edgaras Stankevicius,2 Ulf Simonsen,2 and Dolores Prieto1

1Departamento de Fisiología, Facultad de Farmacia, Universidad Complutense, Madrid, Spain; and 2Department of Pharmacology, Faculty of Health Sciences, University of Aarhus, Aarhus C, Denmark

Submitted 22 October 2007 ; accepted in final form 25 January 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tonic physiological activity of RhoA/Rho kinase contributes to the maintenance of penile flaccidity through its involvement in the Ca2+ sensitization of erectile tissue smooth muscle. The present study hypothesized that Rho kinase is also involved in the modulation of Ca2+ entry induced by {alpha}1-adrenoceptor stimulation of penile arteries. Rat penile arteries were mounted in microvascular myographs for simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and force. The Rho-kinase inhibitor Y-27632 markedly reduced norepinephrine-mediated electrically induced contractions and the increases in both [Ca2+]i and tension elicited by the {alpha}1-adrenoceptor agonist phenylephrine (Phe). In contrast, the protein kinase C (PKC) inhibitor Ro-31-8220 reduced tension without altering the Phe-induced increase in [Ca2+]i. In the presence of nifedipine, Y-27632 still inhibited the non-L-type Ca2+ signal and blunted Phe contraction. Y-27632 did not impair the capacitative Ca2+ entry evoked by store depletion with cyclopiazonic acid but largely reduced the Ba2+ influx stimulated by Phe in fura-2 AM-loaded arteries. The addition of Y-27632 to arteries depolarized with high KCl markedly reduced tension without changing [Ca2+]i. In {alpha}-toxin-permeabilized penile arteries stimulated with threshold Ca2+ concentrations, Y-27632 inhibited the sensitization induced by either guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S) or Phe in the presence of GTP{gamma}S. However, Y-27632 failed to alter contractions induced by a maximal concentration of free Ca2+. These results suggest that Rho kinase, besides its contribution to the Ca2+ sensitization of the contractile proteins, is also involved in the regulation of Ca2+ entry through a nonselective cation channel activated by {alpha}1-adenoceptor stimulation in rat penile arteries.

penile arteries; calcium entry; nonselective cation channels; calcium sensitization

PENILE ERECTION OCCURS when nitric oxide (NO) released from nerves and endothelium upon sexual stimulation relaxes smooth muscle of the corpus cavernosum (CC) and penile arteries, leading to blood filling of the sinuses and restriction of venous outflow (2, 25). During the flaccid state, erectile tissue is contracted by the release of neural and local factors, such as norepinephrine, neuropeptide Y, endothelin-1, and prostanoids that increase smooth muscle cytosolic Ca2+ ([Ca2+]i) and/or Ca2+ sensitization through activation G protein-coupled receptors (2, 20, 25). Erectile dysfunction (ED), considered as a sign of early endothelial dysfunction and cardiovascular disease, is three times more prevalent in men with Types 1 and 2 diabetes than in men without diabetes (3, 33). About 50% of these patients exhibit suboptimal responses to oral phophodiestarase 5 inhibitors, such as sildenafil (33), that enhance the NO-mediated vasodilatation leading to penile erection. As an alternative, inhibition of the Rho/Rho-kinase signaling pathway has been proposed as a potential molecular target for the development of novel therapies for ED since Rho-kinase inhibitors can stimulate penile erection in a NO-independent manner (6) and can increase intracavernous pressure in both normal and diabetic animals (4).

RhoA is a member of the Ras superfamily of small GTP-binding proteins, and its effector Rho kinase regulates Ca2+ sensitivity of vascular smooth muscle and vasoconstriction (28). Tonic physiological activity of RhoA/Rho kinase is also involved in the regulation of erectile function and the maintenance of penile flaccidity. Thus the inhibition of Rho kinase in a rat in vivo model induces a marked increase in CC pressure and erection, and adeno-associated viral gene transfer of dominant-negative RhoA mutant enhances erectile function (5, 6). Moreover, RhoA and Rho kinase are expressed in penile smooth muscle, the content of RhoA in both human and rabbit CC being much higher than that in ileum smooth muscle (35).

Whereas Rho kinase is involved in the Ca2+ sensitization of the contractile proteins in CC smooth muscle (36), it is unclear whether this kinase plays a role in the Ca2+ handling of penile arteries. In small arteries, Rho kinase has recently been suggested to regulate smooth muscle Ca2+ entry (9). Penile arterial vasoconstriction is largely dependent on extracellular Ca2+ influx (34), and, therefore, in the present study, we hypothesized that Rho kinase, in addition to Ca2+ sensitization, also modulates Ca2+ entry in penile small arteries. To address the hypothesis, simultaneous measurements of smooth muscle [Ca2+]i and force were performed to investigate the effect of Rho-kinase inhibition on Ca2+ entry and force development in rat penile arteries. The role of Rho kinase in Ca2+ sensitization was examined in {alpha}-toxin-permeabilized arteries. The present study suggests that Rho kinase modulates Ca2+ entry besides contributing to Ca2+ sensitization in rat penile small arteries.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Dissection, mounting, and force measurement. All the protocols were approved by the Institutional Animal Care and Use Committee at Complutense University (Madrid, Spain). Adult male Wistar rats (12–14 wk) were euthanized by decapitation and subsequent exsanguination. The penis was excised and transferred into cold physiological saline solution (PSS) of the following composition: (in mM) 119 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4, 1.5 CaCl2, 24.9 NaHCO3, 0.027 EDTA, and 11 glucose (pH 7.4). The penile arteries, first- or second-order branches of the rat dorsal penile artery, were carefully dissected by removing the connective and fat tissue and mounted in a microvascular myograph (Danish Myotechnology) as ring preparations by inserting two 40-µm tungsten wires into the vessel lumen. The arteries were equilibrated for 30 min in Krebs solution at 37°C, continuously gassed with 95% O2-5% CO2, and the relationship between passive wall tension and internal circumference was then determined for each individual artery. From this, the internal circumference L100 corresponding to a transmural pressure of 100 mmHg for a relaxed vessel in situ was calculated. The arteries were set to an internal circumference L1 equal to 0.9 times L100 (L1 = 0.9 x L100), at which tension development is maximal in small arteries (16, 26). The normalized internal diameter of the penile arteries used in the present study was 120–210 µm. After the normalization procedure, the arteries were exposed to the various experimental conditions.

Simultaneous measurements of [Ca2+]i and tension. Simultaneous measurements of the [Ca2+]i and tension were performed in intact arterial segments by fura-2 AM fluorescence as described previously (34). Briefly, the arteries were loaded in the dark in PSS containing 8 µM fura-2 AM and 0.05% Cremophor EL for 3 h at 37°C. They were washed three times in PSS, and the solution was changed to PSS with fresh fura-2 AM after 90 min. The myograph chamber was mounted on a Zeiss-inverted microscope equipped for dual-excitation wavelength fluorimetry (Deltascan, Photon Technology International). The arteries were illuminated with alternating 340- and 380-nm light, and the intensity of the emitted fluorescence was collected at a wavelength of 510 nm using a photomultiplier and monitored together with the tension (19, 34). At the end of each experiment, Ca2+-insensitive signals were determined after quenching with Mn2+, and the values obtained were subtracted from those obtained during the experiment. The ratio of fluorescence at 340 and 380 nM (F340/F380) corrected for autofluorescence was taken as a measure of [Ca2+]i. In some experiments, in particular when Ba2+ was used instead of Ca2+, the same procedure was used.

Permeabilization of the small penile arteries with {alpha}-toxin. Permeabilization of penile small arteries was achieved by using {alpha}-toxin from Staphylococcus aureus, as described previously (34). The arterial rings were first equilibrated for 20 min in relaxing solution containing (in mM) 2 EGTA, 130 potassium methanesulphonate, 4 MgCl2·6H2O, 20 Tris maleate, 4 Na2ATP·3H2O, and 10 phosphocreatine and 0.1 mg/ml creatine phosphokinase (pH titrated to 7.1 with KOH). The arteries were then permeabilized by immersion in relaxing solution with 4 mM EGTA and 5,000 U/ml {alpha}-toxin for 2 h at room temperature. After permeabilization, the arteries were mounted in vascular myographs and passively stretched to their L1 as described above. Contraction experiments were made at room temperature (20–22°C). For contraction experiments, the EGTA concentration was 2 mM and the relaxing solution was gassed with 100% O2. The effectiveness of the permeabilization procedure was assessed by a stimulation of arteries with a high concentration of free Ca2+ (pCa 4.9), which elicited a gradual development in tension. The calculation of free Ca2+ concentration was made with a computer program based on association constants from Tsien and Pozzan (30).

Experimental procedure for the functional studies. After normalization, the contractile ability of each arterial preparation was tested by stimulating it with a high-K+ solution (equivalent to PSS, except that NaCl was exchanged for KCl on an equimolar basis, giving a final concentration of 123.7 mM K+). The effects of the selective inhibitor of Rho kinase, Y-27632, were evaluated on the contraction elicited by electrical field stimulation (EFS) and on the changes in isometric tension and [Ca2+]i induced by the {alpha}1-adrenoceptor agonist phenylephrine (Phe) and high-K+ depolarization in rat penile small arteries.

EFS was performed with a pair of thick platinum square electrodes fixed in the mounting jaws of the myograph and connected to an electrical stimulator (Cibertec, CS20, Barcelona, Spain). EFS was conducted at 35 mA, 0.3 ms pulse width, and 20-s trains at varying frequencies (1–32 Hz) (26). These parameters have earlier been shown to evoke specific neurogenic contractile responses abolished by tetrodotoxin in penile small arteries (26). To test the influence of Rho-kinase inhibition on the contractions elicited by electrical stimulation of perivascular nerves in the arterial wall, frequency-response curves were performed in the absence and the presence of a submaximal concentration of Y-27632 (10 µM) (9). These experiments were carried out in the presence of NG-nitro-L-arginine (L-NNA, 100 µM) (22) to avoid the effects of neural-released NO. The effects of Y-27632 were also tested on the contractions induced by the {alpha}1-adrenoceptor agonist Phe. To confirm the adrenergic origin of the responses to EFS, chemical sympathectomy was performed in vitro by treatment with guanethidine (10 µM) for 1 h, changing the solution every 15 min (19). Frequency-responses curves for EFS were then performed after denervation with guanethidine.

In a second set of experiments, the effects of Rho-kinase inhibition on the changes in isometric [Ca2+]i and tension induced by Phe and by high-K+ depolarization were evaluated. Increasing concentrations of Y-27632 (0.1–100 µM) were cummulatively added on arteries precontracted with a submaximal dose of Phe (3 µM). To discard a possible nonspecific action of Y-27632 on PKC, the effects of increasing concentrations of the selective inhibitor of PKC, Ro-31-8220, were also evaluated on the changes in [Ca2+]i and tension induced by Phe. When the effects of Rho-kinase inhibition were evaluated in arteries depolarized with a high-K+ solution (KPSS), phentolamine (0.3 µM) was used to avoid the effects of neural-released norepinephrine.

Furthermore, we determined the effects of Y-27632 on the Phe-induced changes in [Ca2+]i/tension under conditions of L-type Ca2+ channel blockade with nifedipine (1 µM) and on the capacitative Ca2+ entry evoked after emptying the sarcoplasmic reticulum Ca2+ store with cyclopiazonic acid (CPA, 30 µM) (34). In some experiments, Ba2+ (1 mM BaCl2) was used instead of Ca2+ to evaluate the influx of divalent cations activated by Phe (9, 12). The extent of Ba2+ influx was assessed as the ratio of fluorescence intensity at 340 and 380 nm after correction for autofluorescence in fura-2 AM-loaded penile arteries. As a positive control, the nonselective inhibitor of cation channels SKF-96365 (100 µM) was added at the end of each experiment in the arteries stimulated by Phe, to confirm the Ba2+ influx through nonselective cations channels.

In {alpha}-toxin permeabilized arteries, the effects of Y-27632 (0.1 to 10 µM) were evaluated on the sensitization induced by both nonselective and {alpha}1-adrenoceptor activation of G protein-coupled receptors, with 3 µM guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S) and 10 µM Phe plus 1 µM GTP{gamma}S, respectively, in arteries stimulated with threshold concentrations of free Ca2+ (pCa < 6.71) (34).

Drugs. Creatinine phosphokinase, GTP{gamma}S, guanethidine, L-phenylephrine, L-NNA, nifedipine, phentolamine, phosphocreatine disodium salt, and SKF-96365 were obtained from Sigma. The Staphylococcus aureus {alpha}-toxin was also purchased from Sigma. CPA and (R)-(+)-trans-N-4-(pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) were from Tocris (Bristol, UK). Adenosine 5'-triphosphate disodium (Na2ATP) and Ro-31-8220 were from Calbiochem. Stock solutions of CPA and nifedipine were made in dimethyl sulfoxide (DMSO) and ethanol, respectively, and further diluted in distilled water. Fura-2 AM (Molecular Probes) was prepared in DMSO.

Statistical analysis and data presentation. Mechanical responses of the arteries were measured as force and expressed as active wall tension, {Delta}T, which is the increase in force, {Delta}F, divided by twice the segment length. The results are expressed as either absolute values [Nm–1 of tension or units of F340/F380] or as a percentage of the responses induced by KPSS or Phe. Sensitivity to the agonists is expressed in terms of pEC50, defined as –logEC50. The concentration of the Rho-kinase inhibitor Y-27632 producing 50% inhibition (IC50) of the responses elicited by either Phe or KPSS was calculated by nonlinear curve fitting of the concentration-response curves for the inhibitor to the classical Hill equation by using the computer program GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). The IC50 value for each individual curve was first obtained, and thereafter the average value for a given set of experiments was calculated.

Data are expressed as means ± SE (n = number of arteries, 1 to 2 from each animal). In the experiments for simultaneous measurements of Ca2+ and tension, one artery per animal was studied. The differences between means were analyzed using paired or unpaired Student's t-test when appropriate. If more than two means of multiple groups were compared, a one-way ANOVA was used, followed by a Bonferroni's as a posterior test. Probability levels of <5% were considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of Rho-kinase inhibition on EFS- and {alpha}1-adrenoceptor agonist-induced contractions. In the continuous presence of L-NNA (100 µM) to inhibit nitrergic neurotransmission, EFS (1–32 Hz) performed at resting tension elicited frequency-dependent contractions of penile small arteries with an internal lumen diameter of 134 ± 11 µm (n = 7, Fig. 1A). The selective Rho-kinase inhibitor Y-27632 (10 µM) markedly reduced the electrically induced contractions, maximal responses at 32 Hz being 1.16 ± 0.16 and 0.21 ± 0.06 Nm–1 (P < 0.0001, n = 7) in the absence and presence of Y-27632, respectively (Fig. 1, A and C). The EFS-evoked contractions were reproducible and restored to control values when removing the inhibitor from the bath (data not shown) and reduced to 17 ± 10% of the KPSS response by guanethidine (10 µM), thus confirming its adrenergic origin (26).


Figure 1
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  		Fig. 1. The Rho-kinase inhibitor Y-27632 reduces the electrically and agonist-induced contractions in rat penile arteries. Isometric force recording showing the electrical field stimulation (EFS)-induced contractions (A) and the contractile response (B) elicited by the {alpha}1-adrenoceptor agonist phenylephrine (Phe) in 2 penile small arteries in the absence (top) and in the presence (bottom) of 10 µM Y-27632. Average effects of Y-27632 on the EFS-induced contractions (C) and Phe-induced contractions (D) in dorsal penile arteries. Values are means ± SE of 5–7 arteries, 1 to 2 arteries/animal. L-NNA, NG-nitro-L-arginine; KPSS, high-K+ solution. *P < 0.01, **P < 0.01, and ***P < 0.001, significantly different from Phe responses in the absence of Y-27632.

 
A cumulative addition to the bath of the {alpha}1-adrenoceptor agonist Phe (0.01–30 µM) evoked concentration-dependent contractions of penile arteries with a maximum response and pEC50 value of 1.50 ± 0.12 and 6.54 ± 0.13 Nm–1 (n = 4), respectively (Fig. 1, B and D). Y-27632 (10 µM) caused a profound inhibition of the contractions elicited by Phe, maximum response and pEC50 value being 0.47 ± 0.05 and 5.36 ± 0.21 Nm–1, respectively (n = 4, P < 0.001), after Y-27632 treatment (Fig. 1).

Effects of Rho-kinase inhibition on the changes in [Ca2+]i and tension elicited by {alpha}1-adrenoceptor activation. Figure 2 shows simultaneous measurements of [Ca2+]i and contraction and the effects of Y-27632 (0.1–100 µM) on the steady-state responses induced by a submaximal concentration of Phe (3 µM). The Rho-kinase inhibitor markedly reduced both the increases in [Ca2+]i and the contractions elicited by Phe in a concentration-dependent manner (Fig. 2A). The pIC50 values for the inhibitory effect of Y-27632 on [Ca2+]i and tension were 5.44 ± 0.10 and 6.14 ± 0.06 (n = 5), respectively. These results initially suggest that Rho kinase is involved in both Ca2+ entry and Ca2+ sensitization induced by Phe in penile small arteries (Fig. 2).


Figure 2
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  		Fig. 2. The Rho-kinase inhibitor Y-27632 reduces the increases in both intracellular Ca2+ concentration ([Ca2+]i) and tension elicited by Phe. A: representative traces of the changes in both [Ca2+]i (top) and force (bottom) induced by cumulative addition of Y-27632 to arteries submaximally precontracted with 3 µM Phe. Summarized data showing the changes in [Ca2+]i (B) and force (C) in response to Y-27632. Responses are percentage of the rise in 340- and 380-nm fluorescence ratio (F340/F380) and contraction elicited by Phe before the addition of Y-27632. Values are means ± SE of 4 arteries, 1 artery/animal. *P < 0.05 and ***P < 0.001, significantly different from Phe responses in the absence of Y-27632.

 
To rule out the possibility that the effects of Y-27632 could be mediated via other kinases than the Rho kinase and particularly PKC, the effects of a selective inhibitor of PKC, Ro-31-8220, were evaluated on the responses to Phe. Figure 3 illustrates the inhibitory effect of Ro-31-8220 in arteries precontracted with Phe (3 µM). Unlike the Rho-kinase inhibitor that maximally reduced both [Ca2+]i and tension (Fig. 2), the PKC inhibitor produced a concentration-dependent inhibition of the Phe-evoked contraction without changing the increase in [Ca2+]i (Fig. 3). The pIC50 value for the inhibitory effect of Ro-31-8322 on tension was 7.40 ± 0.14 (n = 4).


Figure 3
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  		Fig. 3. The PKC inhibitor Ro-31-8220 inhibits the vasoconstriction but not the increase in [Ca2+]i induced by Phe in rat penile arteries. Concentration-inhibition curve for effects of Ro-31-8220 on the Ca2+ signal and the contraction evoked by Phe in rat dorsal penile arteries. Responses are percentages of the rise in F340/F380 and contraction elicited by Phe before the addition of Ro-31-8220. Values are means ± SE of 4 arteries, 1 artery/animal.

 
Effect of Rho-kinase inhibition on non-L-type Ca2+ entry activated by Phe. In rat penile small arteries, the vasoconstriction elicited by {alpha}1-adrenoceptor activation is largely dependent on extracellular Ca2+ entry through both voltage-operated (VOC) L-type and non-L-type Ca2+ channels (34). Therefore, to investigate the Rho-kinase interactions with a specific Ca2+ entry pathway, the effects of Rho-kinase inhibition were evaluated on the changes in [Ca2+]i and tension under conditions of L-type Ca2+ channel blockade. In the presence of nifedipine (1 µM), Y-27632 (10 µM) partially reduced the non-L-type Ca2+ signal (33 ± 7% inhibition, n = 4) and blunted the contraction (91 ± 1% inhibition, n = 4) elicited by Phe (10 µM) (Fig. 4, A and B).


Figure 4
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  		Fig. 4. Y-27632 reduces the non-L-type Ca2+ entry activated by the {alpha}1-adrenoceptor agonist Phe. A: representative traces of the changes in both [Ca2+]i (top) and force (bottom) induced by 3 µM Phe alone (left) and Y-27632 (10 µM; right) on the responses elicited by 3 µM Phe in the presence of 1 µM nifedipine. C: traces showing the effect of 10 µM Y-27632 on the changes in [Ca2+]i (top) and force (bottom) induced by Ca2+ readdition (1.5 mM) on arteries kept in Ca2+-free in the presence of 1 µM nifedipine and further stimulated with 3 µM Phe. B and D: summarized data showing the effects of 10 µM Y-27632 on the non-L-type increases in [Ca2+]i (top) and contraction (bottom) induced by Phe. Contraction is expressed as percentage of the control responses induced by Phe (3 µM) in physiological saline solution (PSS) (1.5 mM Ca2+). Values are means ± SE of 4 to 5 arteries, 1 artery/animal. *P < 0.05 and **P < 0.01, significantly different from Phe responses in the absence of Y-27632.

 
The interactions of the Rho-kinase pathway with the non- L-type Ca2+ entry evoked by {alpha}1-adrenoceptor activation were further investigated in penile arteries kept in a Ca2+-free solution in the presence of 1 µM nifedipine (Fig. 4, C and D). In the absence of extracellular Ca2+, Phe did not affect either [Ca2+]i or tension, but this agonist elicited sustained increases in [Ca2+]i and tension (83 ± 6% and 40 ± 13% of Phe-induced responses in control conditions, respectively), when Ca2+ (1.5 mM) was restored (Fig. 4C). Y-27632 (10 µM) reduced by 31 ± 2% and 88 ± 10% (n = 5) the non-L-type Ca2+ entry and contraction, respectively, elicited by Phe (Fig. 4, C and D).

Effect of Rho-kinase inhibition on the Ba2+ entry activated by Phe. The contribution of nonselective cation channels to the Ca2+ entry stimulated by {alpha}1-adrenoreceptor activation was specifically evaluated using Ba2+ fluorescence instead of Ca2+ to investigate the influx of divalent cations activated by Phe without any intervention of Ca2+ extrusion or Ca2+ storage mechanisms (12). Dorsal penile arteries were treated with nifedipine (1 µM) to block VOC channels in a nominally Ca2+-free solution and stimulated with Phe (3 µM). As shown in Fig. 5A and summarized in Fig. 5C, the addition of Ba2+ (1 mM) to the Ca2+-free solution increased the fura-2 AM ratio by 0.28 ± 0.018 (69.6 ± 5% of KPSS-evoked response, n = 7). Treatment with Y-27632 (10 µM) significantly reduced the increase in the Ba2+ fluorescence ratio in response to the {alpha}1-adrenoceptor activation to 0.13 ± 0.01 (39.5 ± 6% of KPSS-evoked response, P < 0.0001, n = 7) (Fig. 5B). The nonselective inhibitor of cation channels, SKF-96365 (100 µM), abolished the influx of Ba2+ activated by Phe in control and Y-27632-treated arteries (fluorescence ratio of 0.01 ± 0.01 and 0.02 ± 0.01, respectively, n = 3, after SKF-96365 treatment).


Figure 5
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  		Fig. 5. Inhibitory effect of Y-27632 on the Ba2+ influx stimulated by Phe. Representative traces showing the Phe-stimulated Ba2+ influx in control conditions (A) and in the presence of Y-27632 (10 µM; B) in dorsal penile arteries loaded with fura-2 AM. Arteries were first stimulated with 120 mM KCl-PSS (KPSS) (left). After application of nifedipine (1 µM) in a nominally Ca2+-free solution, they were stimulated with Phe (3 µM), and afterward Ba2+ (1 mM) was added to the bath solution (right). The Ba2+ influx was assessed by the increase in F340/F380 after correction for autofluorescence by quenching with Mn2+. C: histograms showing the mean values of the change in F340/F380 evoked by addition of Ba2+ (1 mM) in the presence and in the absence of Y-27632 (10 µM). SKF-96365 (100 µM) was used as positive control to block nonselective cation channels. Values are means ± SE of 4–7 arteries, 1 artery/animal. ***P < 0.001, significantly different from Phe responses in the absence of Y-27632.

 
Effect of Rho-kinase inhibition on capacitative Ca2+ entry. The role of Rho kinase in the Ca2+ entry through store-operated Ca2+ (SOC) channels of penile small arteries was assessed by evaluating the effects of Y-27632 on the Ca2+ influx evoked by store depletion with the sarcoendoplasmic reticulum Ca2+-ATPase inhibitor CPA, under conditions of L-type channel blockade. Ca2+ readmission in arteries treated with CPA (30 µM) in a Ca2+-free medium in the presence of nifedipine (1 µM) evoked a sustained increase in [Ca2+]i not coupled to significant contractions (3 ± 1% of KPSS, n = 4) (Fig. 6, A and B). The application of Y-27632 (10 µM) did not affect the Ca2+ entry evoked by store depletion with CPA, the Ca2+ signal in the absence and the presence of the inhibitor being 37 ± 5% and 37 ± 4% (n = 5) of the KPSS-induced increase in [Ca2+]i, respectively (Fig. 6, B and C).


Figure 6
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  		Fig. 6. The capacitative Ca2+ entry evoked by store depletion with cyclopiazonic acid (CPA) is unaltered by Y-27632. A and B: representative traces showing the changes in both [Ca2+]i (top) and tension (bottom) induced by 120 mM KCl-PSS (A) and Y-27632 (10 µM; B) on the Ca2+ entry evoked by 1.5 mM Ca2+ readdition after store depletion with CPA (30 µM) in the presence of nifedipine (1 µM). C: summarized data showing the changes in [Ca2+]i (top) and contraction (bottom) in response to Y-27632. Force is expressed as percentage of that induced by KPSS. Values are means ± SE of 4 arteries, 1 artery/animal.

 
Effect of Rho-kinase inhibition on the changes in [Ca2+]i and tension elicited by KCl depolarization. In the presence of phentolamine (0.3 µM) to avoid the effects of neural-released norepinephrine, a change of the bathing medium from PSS to a KPSS evoked a rapid rise in both [Ca2+]i and tension followed by sustained levels of slower rise (Fig. 7, A and B). A cumulative addition of the Rho-kinase inhibitor Y-27632 (0.1–10 µM) on the steady-state increases in [Ca2+]i and force induced by KCl concentration dependently reduced tension, this inhibition at the highest concentration of Y-27632 (10 µM) being 80 ± 7% (n = 5) and pIC50 5.71 ± 0.41. However, the relaxation produced by Y-27632 was not associated with significant decreases in [Ca2+]i (Fig. 7, B and C), although the L-type Ca2+ channel blocker nifedipine (0.3 µM) inhibited by 74 ± 4% (n = 5) the KCl-induced tonic increase in [Ca2+]i and further reduced tension to basal levels (Fig. 7B). These data suggest that KCl depolarization induces Ca2+ sensitization by activation of Rho kinase in penile arterial tissue.


Figure 7
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  		Fig. 7. The Rho-kinase inhibitor Y-27632 reduces tension but not [Ca2+]i in rat penile arteries depolarized with KCl. A and B: representative traces showing the responses in both [Ca2+]i (top) and force (bottom) induced by a KPSS (A) and cumulative addition of Y-27632 (B) to arteries precontracted with KPSS in the presence of phentolamine (0.3 µM). C: summarized data showing the changes in [Ca2+]i (top) and force (bottom) in response to Y-27632. Responses are percentage of the rise in F340/F380 and contraction elicited by KPSS before the addition of Y-27632. Values are means ± SE of 5 arteries, 1 artery/animal. **P < 0.01 and ***P < 0.001, significantly different from KPSS responses in the absence of Y-27632.

 
Effect of Rho-kinase inhibition on {alpha}-toxin permeabilized arteries. The participation of Rho kinase in the Ca2+ sensitization of the contractile machinery was further investigated by using {alpha}-toxin-permeabilized penile small arteries. Rho-kinase inhibition reduced Ca2+ sensitization elicited by both nonselective G protein-coupled receptor and {alpha}1-adrenoceptor activation with Phe (Fig. 8). Thus Y-27632 concentration dependently inhibited the potentiation induced by either the nonhydrolizable GTP analogous GTP{gamma}S (3 µM) (Fig. 8, A and D) or Phe (10 µM) in the presence of GTP{gamma}S (1 µM) (Fig. 8. B and D) on the contractions elicited by threshold concentrations of Ca2+ (pCa ≤ 6.71). These contractions averaged 137 ± 18% (n = 4) and 96 ± 7% (n = 5), respectively, of the responses induced by a maximal concentration of free Ca2+ (pCa 4.9). pIC50 values and maximal effects for the inhibitory action of Y-27632 were 5.77 ± 0.14% and 72 ± 6% (n = 4) and 5.96 ± 0.01% (n = 7) and 72 ± 5% (n = 7) in GTP{gamma}S and Phe plus GTP{gamma}S-activated arteries, respectively. However, Y-27632 (10 µM) only inhibited by 9 ± 2% (n = 6) the contractions induced by a maximal free Ca2+ concentration (pCa 4.9) (Fig. 8, C and D).


Figure 8
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  		Fig. 8. Y-27632 inhibits Ca2+ sensitization in {alpha}-toxin-permeabilized smooth muscle of rat penile arteries. A: representative traces showing the responses to a maximal stimulation with 10 µM Ca2+ free (left) and cumulative addition to Y-27632 (right) on a permeabilized artery stimulated with threshold free Ca2+ (pCa ≤ 6.71) in the presence of 3 µM 5'-O-(3-thiotriphosphate) (GTP{gamma}S). B: Y-27632 dose-response curve for Y-27632 in an artery stimulated with threshold free Ca2+ in the presence of 10 µM Phe and 1 µM GTP{gamma}S. C: effect of 10 µM Y-27632 on an artery contracted with maximal free Ca2+ (10 µM). D: histograms showing the mean inhibitory effects of Y-27632. Measurements were made after the sustained contraction had reached constant tension. Experiments were done at 20–22°C and pH 7.1. Values are means ± SE of 4–7 arteries, 1 to 2 arteries/animal. ***P < 0.001, significantly different from contractions in the absence of Y-27632. W, wash.

 

   DISCUSSION
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 ABSTRACT
 METHODS
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 DISCUSSION
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Physiological activity of RhoA/RhoK is involved in the maintenance of penile flaccidity, but its role in penile arterial vasoconstriction is poorly understood. The present study demonstrates that Rho kinase not only plays an important role in the Ca2+ sensitization of the erectile tissue but also regulates Ca2+ entry through nonselective cation channels activated by {alpha}1-adrenoceptors in penile small arteries.

Sympathetic noradrenergic nerves are responsible for the detumescence of the erect penis through the local release of norepinephrine that contracts penile arterial and trabecular smooth muscle (2, 26). RhoA and Rho kinase are highly expressed in cavernosal smooth muscle, and the inhibition of Rho kinase stimulates rat penile erection by blocking endogenous vasoconstriction (6). In the present study, the inhibition of Rho kinase reduced both norepinephrine-mediated neurally evoked contractions and those induced by selective activation of {alpha}1-adrenoceptors with Phe in rat penile arteries. These findings are consistent with previous work in human penile CC showing that Rho kinase is involved in the noradrenergic contractile pathway (23) and suggest that the in vivo erectogenic effect elicited by Rho-kinase inhibitors (6) is not only due to its relaxant action on cavernosal smooth muscle but also to the strong vasodilator effect on penile arteries.

Contraction of penile CC involves potent Ca2+ sensitization mechanisms that have been ascribed to its greater content of RhoA compared with other smooth muscles (35). This, along with the fact that Rho-kinase inhibitors reverse CC Ca2+ sensitization in vitro (29, 35) and cause a dramatic increase of intracavernous pressure and erection in vivo (6), has led to the suggestion that Rho kinase-mediated Ca2+ sensitization makes a remarkable contribution to the maintenance of flaccid-contracted state of the penis. However, the results of the present study demonstrate that in penile arterial tissue, Rho kinase is involved not only in Ca2+ sensitization of the contractile proteins but also in the regulation of Ca2+ entry across the plasma membrane. Thus Y-27632 markedly inhibited the increase in [Ca2+]i evoked by Phe with an inhibitory potency slightly lower than that found for the reduction of Ca2+ sensitization but within the micromolar range reported for the in vitro inhibition of the Rho-associated protein kinase p160 Rho kinase (31). The lack of effect of a selective PKC inhibitor on the increase in [Ca2+]i evoked by {alpha}1-adrenoceptor activation initially rules out that the effects of Y-27632 are mediated by PKC and confirms the selectivity of this compound on Rho kinase. In rabbit CC, no significant inhibition by Y-27632 of the Ca2+ entry evoked by Phe was found (29). Therefore, the present results first showing a role for Rho kinase in the modulation of Ca2+ handling in penile small arteries confirm the differences in the intracellular Ca2+ signaling mechanisms between trabecular and arterial penile smooth muscle (29, 34, 36).

In penile small arteries, {alpha}1-adrenoceptor vasoconstriction is largely dependent on Ca2+ entry through both L-type VOC and receptor-operated Ca2+ (ROC) channels (34). The Rho-kinase inhibitor Y-27632 reduced the nifedipine-resistant Ca2+ influx induced by Phe but did not affect the increase in [Ca2+]i induced by KCl depolarization, a classical stimulus for activation of L-type VOC channels. These data initially suggest that the inhibition of Rho kinase suppresses Ca2+ entry through cation channels distinct from VOC. The role of Rho kinase in the regulation of cation influx was further investigated by measuring Ba2+ fluorescence, which provides an advantage over Ca2+, measuring the genuine divalent cations influx because Ba2+ is not extruded or taken up into internal stores by Ca2+-ATPases (14) and it is almost equally as permeable as Ca2+ through nonselective cation channels (18). The marked inhibitory effect of Y-27632 on the nifedipine-resistant Ba2+ influx stimulated by {alpha}1-adrenoceptor activation confirms that Rho-kinase inhibition specifically affects Ca2+ influx through nonselective cation channels rather than any mechanisms of Ca2+ release, Ca2+ sequestration, or Ca2+ efflux. Since {alpha}1-adrenoceptor activation does not mobilize intracellular Ca2+ in penile arterial tissue (34), it seems unlikely that Y-27632 has an inhibitory effect on the Ca2+ influx through SOC channels. Moreover, a Ca2+ readdition under conditions of store depletion with CPA and L-type channel blockade induces a capacitative Ca2+ entry in penile small arteries that was unaltered by the Rho-kinase inhibitor Y-27632. Taken together, the present results suggest that Rho kinase is likely regulating Ca2+ influx through a ROC channel different from SOC or VOC. This is consistent with findings in tracheal smooth muscle (13) and in rat aorta and small mesenteric arteries (9) where Rho kinase regulates the Ca2+ entry upon activation of muscarinic and {alpha}1-adrenergic receptors, respectively. However, this involvement is probably dependent on the type of receptor and/or vascular bed since inhibition of Rho kinase did not significantly alter the Ca2+ influx evoked by thromboxane A2 in either porcine coronary artery (17) or pulmonary arteries (7).

Several members of the canonical transient receptor potential channel (TRPC) family act as Ca2+-permeable cation channels that can be activated in response to stimulation of G protein (Gq/11)-coupled receptors (8, 12). TRPC6 is highly expressed in vascular smooth muscle cells and has been suggested to be the molecular correlate of the {alpha}1-adrenoceptor-activated nonselective cation channel in rabbit portal vein (1, 12) and mesenteric artery (11) myocytes. Moreover, TRPC6 and one of its splicing variant (TRPC6{alpha}) are expressed in corporal myocytes from human CC (27), and, therefore, this TRPC subtype could be a molecular candidate for the nonselective cation channel activated by {alpha}1-adrenoceptors and regulated by Rho kinase in penile arteries. However, TRPC proteins can form heteromeric and homomeric channels (8), and, therefore, specific molecular and functional studies are needed to clarify the expression of the various TRPC subunits, its possible contribution to ROC entry, and its specific regulation by Rho kinase in penile arteries.

Rho Kinase is a major determinant for Ca2+ sensitization in erectile tissue smooth muscle (35), and, therefore, we evaluated the contribution of this kinase to the Ca2+-independent changes in force of penile arteries by clamping Ca2+ concentrations in {alpha}-toxin-permeabilized arteries. Both direct activation of G proteins with GTP{gamma}S and of {alpha}1-adrenoceptors with Phe plus GTP{gamma}S in the presence of threshold Ca2+ concentrations evoked marked contractions similar or even larger than those induced by a maximum free Ca2+ concentration. This observation is in agreement with the unusually large Ca2+ sensitization observed in human and rabbit CC and consistent with the greater expression of RhoA and Rho kinase in erectile tissue compared with other smooth muscles (35). Accordingly, the RhoK-inhibitor Y-27632 completely reversed the Ca2+ sensitization induced by both nonspecific and {alpha}1-adrenergic receptor activation in permeabilized penile arteries. Moreover, the scarce inhibitory effect of Y-27632 on the contractions induced by Ca2+ is consistent with its high selectivity on Rho kinase compared with MLC kinase.

Although Rho kinase is activated by excitatory agonists via receptors coupled to the G{alpha}12/13 family of heterotrimeric G proteins (10), recent studies have demonstrated that Ca2+ sensitization involving Rho-kinase activation can also be brought about by membrane depolarization (15, 24, 32). In the present study, the Rho-kinase inhibitor Y-27632 dose dependently reduced the contraction but not the increase in [Ca2+]i induced by KCl, which suggests that Rho kinase is involved in Ca2+ sensitization evoked by depolarization and confirms previous reports in arterial smooth muscle from several vascular beds (15, 24, 32). The inhibitory effect of Y-27632 on the contractions of both [Ca2+]-clamped {alpha}-toxin-permeabilized arteries and on those induced by K+ depolarization in intact arteries suggests that both Ca2+-independent and -dependent mechanisms, respectively, are involved in the Rho kinase-mediated Ca2+ sensitization. This in turn may explain the involvement of Ca2+ sensitization in the vasoconstriction evoked by {alpha}1-adrenoceptor activation of penile arteries that is largely dependent on extracellular Ca2+ influx (34). It has been suggested that physiological vasoconstrictors acting on G protein-coupled receptors use a dual signaling pathway, i.e., the G{alpha}12/13 pathway and the Gq/Ca2+-dependent pathway for RhoA activation-mediated Ca2+ sensitization (24). The present results support the involvement of both Ca2+-independent and -dependent mechanisms in the Rho-mediated regulation of Ca2+ sensitivity of penile small arteries. Moreover, a role for Rho kinase in the regulation of Ca2+ entry through nonselective cation channels coupled to the {alpha}1-adrenoceptor is also demonstrated, which likely contributes to the Ca2+-dependent regulation of Ca2+ sensitivity by Rho kinase.

ED is highly prevalent in men with diabetes in whom endothelial dysfunction and reduced NO bioavailability may be related to the lower response of these patients to the currently used oral phosphodiesterase 5 inhibitors (3, 33). These compounds amplify the relaxing effects of endothelial NO on penile arteries (21) and, therefore, the present data showing that Rho-kinase inhibitors can reduce both Ca2+ influx and Ca2+ sensitization underlying penile arterial vasoconstriction, support a role for the Rho/Rho-kinase signaling pathway as an alternative therapeutic target for ED. The novel finding that Rho kinase regulates Ca2+ entry through nonselective Ca2+ channels, along with a future molecular identification of the Rho-kinase isoform and the characterization of the TRP subfamily of the channel protein in penile arteries, would allow the development of more selective Rho-kinase inhibitors while minimizing unwanted effects on other vascular beds.


   GRANTS
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 ABSTRACT
 METHODS
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REFERENCES
 
This work was supported by the Comisión Internacional de Ciencia y Tecnología, Ministerio de Educación y Ciencia (Spain) Grant n° SAF2006-09191.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Prieto, Dept. de Fisiología, Facultad de Farmacia, Univ. Complutense, 28040-Madrid, Spain (e-mail: dprieto{at}farm.ucm.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 DISCUSSION
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