Introduction

In the flaccid state, there is a high resistance to blood flow into the penis due to contraction of smooth muscle cells located in penile vessels and in the stroma of the corpora cavernosa.¹ The constricted state of penile vasculature is considered to be mediated by the release of norepinephrine, endothelin-1, and other vasoactive agents.² These agents bring about vasoconstriction by elevating intracellular calcium and activating myosin light chain kinase resulting in myosin phosphorylation and cross-bridge activation. Additionally, a calcium sensitization process is activated through agonist stimulation of heterotrimeric G-protein-coupled receptors, activation of RhoA through exchange of GTP for GDP and dissociation from an inhibitor (GDI).^{3, 4} The activated RhoA then activates Rho kinase, which inhibits myosin light chain phosphatase activity, resulting in a net increase in myosin phosphorylation and force at low calcium levels.^{5, 6}

In other vascular smooth muscle, it has been shown that norepinephrine (through 1-adrenoreceptors) activates phospholipase C (PLC). PLC catalyzes the hydrolysis of phosphatidyl inositol 4,5-bisphosphate and leads to the formation of 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP₃), which respectively activate protein kinase C (PKC) and release Ca²⁺ from the sarcoplasmic reticulum.^{7, 8} In many tonic smooth muscles, it is believed that IP₃ is involved in the phasic component of smooth muscle contraction,⁵ and there is experimental evidence that indicates that DAG is involved in the tonic phase of the contractile response.^{9, 10, 11} The potential role of PKC in the regulation of smooth muscle was originally suggested by the finding that tumor-promoting phorbol esters, which specifically activate PKC, induce slowly developing and sustained contractions.^{9, 10} More recently, it is believed that PKC's contractile effect can be associated or linked with a Ca²⁺-sensitization process.^{5, 6} The PKC family is comprised of at least 11 isoforms encoded by different genes and have been identified by molecular cloning and biochemical means.^{12, 13} The isoforms have been found to differ in enzymological properties, amino-acid sequence, specific intracellular localization, tissue expression, mode of activation, and cofactor requirements. The conventional PKC isoforms (, _I, _II, ) are activated by Ca²⁺, phosphatidylserine, and DAG. The novel PKC isoforms (, , , ) are also activated by phosphatidylserine and DAG, but Ca²⁺ is not required for their activation. Atypical PKCs (, , and ) are DAG- and Ca²⁺-independent, but are activated by phosphatidylserine.

Because the penile vasculature is maintained in a vasoconstricted state for long periods of time, we postulated that mechanisms that aid this process are active in corpora cavernosa. We hypothesized that PKC isoforms may act as regulators of penile vascular tone. To test this hypothesis, studies were designed to identify PKC isoforms, to determine their subcellular distribution, and to investigate their roles in 1-adrenergic receptor agonist (phenylephrine)-induced muscle contraction in the isolated rat corpus cavernosum.

Materials and methods

Materials

Monoclonal antibodies to PKC isoforms (, , , , , , ) were obtained from BD Transduction Laboratories (San Diego, CA). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Nitrocellulose membranes, enhanced chemiluminescent (ECL) reagents, and ECL hyperfilms were obtained from Amersham Pharmacia (Piscataway, NJ). Phenylephrine and phorbol 12-myristate13-acetate (PMA) were obtained from Sigma Chemical Co. (St Louis, MO). Chelerythrine chloride was obtained from Calbiochem (La Jolla, CA).

Methods

Animals

Intact male Sprague–Dawley rats (275–300 g, Harlan Laboratories, Indianapolis, IN) were anesthetized with ketamine (87 mg/kg body weight) and xylazine (13 mg/kg body weight), and cavernosal tissue removed as described earlier.¹⁴ All procedures were performed in accordance with the Guiding Principles in the Care and Use of Animals established by the National Institute of Health and approved by the Institutional Committee on the use of Animals in Research and Education.

Preparation of subcellular fractions

Cavernosal smooth muscle strips (cleaned of the corpus spongiosum and dorsal vein) were allowed to equilibrate for 45 min in 1 ml of physiological salt solution (PSS) at pH 7.4 at 37°C bubbled with breathing air. The PSS was composed of the following: 140 mM NaCl, 5 mM KCl, 1.2 mM MgCl₂, 1.2 mM Na₂HPO₄, 1.4 mM CaCl₂, and 5.6 mM D-glucose. The tissues were then incubated in the absence or presence of agonists (3 M phenylephrine for 10 min or 100 nM PMA for 30 min). The reaction was stopped by immersing the tissue in liquid nitrogen at -80°C. The frozen tissues were homogenized in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonate fluoride, 10% (v/v) glycerol, 4 g/ml aprotinin, 4 g/ml pepstatin, and 4 g/ml leupeptin using a glass homogenizer. The homogenates were centrifuged at 600 g for 10 min at 4°C. We did not find any PKC isoform immunoreactivity in the 600 g pellets. The supernatant was again centrifuged at 100 000 g for 45 min at 4°C. The supernatant was saved as the cytosolic fraction. The pellet was resuspended in homogenization buffer containing 0.1% (v/v) Triton X-100. The resultant cytosolic and particulate fractions were used for Western blotting or frozen in aliquots at -80°C. The protein concentration of each fraction was determined using Micro BCA protein analysis system (Pierce Lab., Rockford, IL).

Western blotting

PKC isoforms were detected in cytosolic and particulate fractions using the procedure described elsewhere.¹⁵ Briefly, equal amounts of proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk (Biorad, Hercules, CA) followed by incubation with anti-PKC isoform antibodies (1:1000 dilution) for 3 h at 20°C with gentle shaking. After washing, the membranes were incubated with secondary antibodies (HRP-conjugated goat anti-mouse IgG at 1:3000 dilution) for 1 h at 20°C. Prestained multimark (Invitrogen Life Technologies, Carlsbad, CA) was run in parallel as protein molecular weight markers. Rat brain extract was run as a positive control for immunodetection of PKC isoforms. Specificity of PKC isoform antisera was confirmed by the use of inhibitory peptides (ie an immunogenic peptide that was used to generate the antibodies, data not shown). In order to selectively block the binding of antibodies to an antigen, an inhibitory peptide (1–2 g/ml) was added to the primary antibodies prior to incubation with the blots. For chemiluminescent detection, the membranes were treated with ECL reagent (Amersham Pharmacia, Piscataway, NJ) for 1 min and subsequently exposed to ECL hyperfilm for 1–2 min.

To ascertain that quantitative comparisons could be made for each isoform of PKC, 5–30 g cytosolic and particulate total protein levels were loaded on the SDS-PAGE followed by immunoblotting. The blots were scanned and densitometrically analyzed to assure a proportional response between sample loaded and signal obtained. We also determined the concentration of each PKC isoform antibodies necessary for optimal signal.

Quantification of PKC isoforms

The densities of the PKC isoform bands were determined by scanning with densitometer (Alpha imager TM 2200 documentation and Analysis System, Alpha Innotech Corp., San Leandro, CA). Specific immunoreactive bands were expressed as arbitrary units (AU), which were calculated from the area under the curve of the selected band scanned by the densitometer. The arbitrary values were then normalized to the area under the curve for -actin staining and then expressed as percent of the unstimulated (PSS) condition. -Actin detection was accomplished using a monoclonal anti--actin antibody (Sigma Chemical Co., St Louis, MO) at a dilution of 1:5000.

Force measurement in isolated cavernosal muscle strips

Cavernosal strips were prepared by removal of the corpus spongiosum and dorsal vein. Strips were bathed in PSS at pH 7.4, 37°C, bubbled with breathing air. Tissue resting force was set to 500 mg as described earlier.¹⁴ Setting the tissue to a preset resting force of 500 mg was found to correlate to an optimal length force generation in response to maximal K⁺ depolarization (data not shown). All tissues were contracted by the addition of 109 mM K⁺ physiological saline solution (KPSS). KPSS was prepared by stoichiometric substitution of KCl for NaCl in PSS. Tissues were depolarized for 10 min and then relaxed with repeated washes of PSS at 10 min intervals before the start of any experimental protocol. Strips were then used to construct cumulative concentration–response curves (0.1–10 M) for the 1-adrenergic agonist phenylephrine in the absence or presence of PKC activator PMA (0.1 M) or inhibitor chelerythrine chloride (30 M).

Data analysis and statistics

Force responses were recorded digitally with POLYview data acquisition (Astro-Med, Inc., West Warwick, RI). Postacquisition analysis included conversion of force (in grams) to stress (mN/mm²). Force was converted to stress values (force/cross-sectional area) by the equation ((force (mg) 98.07)/area)/1.055 (density conversion), where area is calculated from wet weight (mg)/length (mm). Concentration–response profiles were constructed for the individual traces for each experimental condition. Normalized stress was calculated by dividing the measured stress level by the maximal stress level measured during the construction of the concentration–response profile. The profiles were fit by Sigma Plot (SPSS Science, Chicago, IL), using a Hill fit protocol, allowing for the report of the estimated EC₅₀ values. Data were presented as means.e.m. Statistical differences were determined by ANOVA followed by Bonferroni's complementary analysis, where relevant, and Student's t-test using the SigmaStat Analysis Program (SPSS Science, Chicago, IL). A P-value <0.05 was considered to be significant.

Results

Identification and subcellular distribution of PKC isoforms

Using PKC isoform-specific antibodies, we have demonstrated the presence of PKC, , , , , , and in rat cavernosal smooth muscle (Figure 1). All the isoforms examined were also detected in rat brain extract, which was employed as a positive control (data not shown), suggesting that the antibodies had sufficient titer and binding affinity for visualization. PKC and isoforms showed strong immunoreactivity, whereas PKC, , , , and displayed minor immunostaining when equal amounts of protein were loaded onto the gels. The apparent molecular weights of PKC, , , , and were approximately 80 kDa. These values are consistent with reported molecular weights for these isozymes from vascular¹⁶ as well as nonvascular tissues.^{11, 17, 18} PKC showed a slightly higher molecular weight of approximately 90 kDa. This is consistent with that reported by Nishizuka⁷ and Singer.¹⁶ The molecular weight of PKC was approximately 76 kDa similar to that reported by Selbie et al¹⁹ in insulin-secreting cells. Comparison of compartmented expression levels of PKC isoforms was performed from unstimulated tissues. The quantitative analysis of different PKC isoforms in cytosolic and particulate fractions of rat cavernosal muscle is shown in Figure 1 (Bar graphs). PKC, , , and were predominantly present in the particulate fraction. PKC was almost equally distributed in soluble and particulate fractions, whereas PKC and PKC were mostly present in the soluble fraction.

Figure 1.

Western blot analysis of PKC isoforms in cytosolic (C) and particulate (P) fractions of rat corpora cavernosa. Equal amounts of protein (15 g) from cytosolic and particulate fractions were subjected to 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes and probed with antibodies specific to PKC, , , , , , and as described in Methods. Western blots are representative images from immunoblots. The graphs represent means.e.m. for densitometric quantitation of immunospecific bands with a sample size of 9–12.

Full figure and legend (68K)

Phenylephrine-induced force generation in isolated cavernosal muscle strips

As shown in Figures 2, 3 and 4, treatment of cavernosal smooth muscle strips with phenylephrine (0.1–10 M) led to a graded contraction of cavernosal tissues (active stress generation ranged from 2.60.7 to 9.91.3 mN/mm², n=16). A measure of 3 M phenylephrine resulted in a mean active stress generation of 9.61.5 mN/mm², which was 80% of the maximal response (Figure 4) and was the concentration utilized to examine the changes in isoform distribution under phenylephrine stimulation.

Figure 2.

Concentration-dependent contractile response of cavernosal muscle strips to phenylephrine in the presence of chelerythrine chloride. (a) Representative force traces from a cavernosal strip stimulated with phenylephrine (0.1–10 M) before (black line) and after incubation with 30 M chelerythrine chloride (gray line). (b) Active stress generation from cavernosal strips stimulated with phenylephrine (0.1–10 M) before and after 30 min incubation with 30 M chelerythrine chloride (Chel). Phenylephrine-induced muscle contraction (), phenylephrine+chelerythrine chloride (). The data represent the mean valuess.e.m. for a sample size of four.

Full figure and legend (61K)

Figure 3.

Concentration-dependent contractile response of cavernosal muscle strips to phenylephrine in the presence of PMA. (a) Representative force traces from a cavernosal strip stimulated with phenylephrine (0.1–10 M) before (black line) and after incubation with 0.1 M PMA (gray line). (b) Active stress generation from cavernosal strips stimulated with phenylephrine (0.1–10 M) before and after 30 min incubation with 0.1 M PMA. Phenylephrine-induced muscle contraction (), phenylephrine+PMA (). The data represent the mean valuess.e.m. for a sample size of four.

Full figure and legend (57K)

Figure 4.

Mean concentration-dependent contractile response of cavernosal muscle strips to phenylephrine in the presence of PMA and chelerythrine chloride (Chel). (a) Plot of the mean active stress generated from cavernosal strips stimulated with phenylephrine (0.1–10 M) before and after incubation with 30 M Chel and 0.1 M PMA. (b) The normalized active forces response of cavernosal strips stimulated with phenylephrine (0.1–10 M) before and after incubation with 30 M Chel and 0.1 M PMA. PMA (0.1 M) treatment was associated with a leftward shift of the concentration–response and Chel (30 M) treatment was associated with a rightward shift of the concentration–response. Lines on the plot represent Hill fit to mean response from which EC₅₀ values were reported. Phenylephrine-induced muscle contraction (), phenylephrine+PMA (), phenylephrine+chelerythrine chloride (). The data represent the mean valuess.e.m. for a sample size of 4–16.

Full figure and legend (52K)

Role of PKC in phenylephrine-induced muscle contraction in isolated cavernosal muscle strips

To examine a role of PKC in phenylephrine-induced muscle contraction, the nonselective PKC inhibitor (chelerythrine chloride, Chel) and activator (PMA) were used. The benzophenanthridine alkaloid chelerythrine is believed to inhibit PKC selectively, based on its lack of significant inhibitory action against cAMP-dependent protein kinase, and calcium/calmodulin-dependent protein kinase and others.²⁰ Chelerythrine appears to inhibit enzyme activity by a direct interaction with the PKC substrate and has nearly identical inhibitory potencies for several different PKC isozymes.²¹ PMA is a commonly used phorbol ester best known for its ability to activate the classical and novel isozymes of PKC. PMA acts by mimicking diacylglycerol, a natural ligand activator of PKCs. Because phorbol esters like PMA have 100-fold higher affinity for PKC than DAG and can activate PKC for longer periods of time, they have been used as probes for PKC activation both in vitro and in vivo.²²

As shown in Figures 2, 3 and 4 phenylephrine induced contractile responses in a concentration-dependent manner with an EC₅₀ value of 1.00.8 M (n=8, Figure 4b). The concentration-dependent phenylephrine force response in the presence of the chelerythrine chloride (30 M) resulted in a 50–90% reduction in the active stress generation for each concentration of phenylephrine examined (Figure 2b). The active stress generation ranged from -0.90.4 to 6.41.0 mN/mm² (n=4). The chelerythrine chloride treatment was associated with a significant reduction in the sensitivity to phenylephrine as the EC₅₀ value was increased to 5.72.4 M, ^*P=0.015 (n=4, Figure 4b).

In the presence of 0.1 M PMA, there was a 2- to 3-fold increase in the active stress generation for each concentration of phenylephrine examined (Figure 3b). Active stress generation ranged from 15.02.5 to 25.72.1 mN/mm² (n=4). Additionally, the EC₅₀ value for PE was shifted to 0.30.1 M (n=4, Figure 4b). These data suggest a potential role of PKC in phenylephrine-induced muscle contraction in rat cavernosal smooth muscle.

Effects of phenylephrine on PKC isoform immunoreactivity in the particulate fraction

To further investigate a role of PKC in phenylephrine-induced muscle contraction, we examined PKC isoform expression patterns in cytosolic and particulate fractions during agonist stimulation. It is well established in many systems that PKC isoforms are translocated to the plasma membrane and their immunoreactivity increased when they are activated.^{15, 18, 23} The level of PKC activation was estimated by Western blot analysis for particular isoform detected in the particulate fraction after phenylephrine treatment. As shown in Figures 5 and 6, immunoreactivity of PKC and PKC in the particulate fraction was significantly increased in the presence of phenylephrine (PKC: PSS (unstimulated) 1000 vs phenylephrine 1374 (n=12), P=0.024; and PKC: PSS (unstimulated) 1000 vs phenylephrine 1512.2 (n=9), P=0.039). We found that phenylephrine stimulation did not significantly alter the distribution of PKC, , , , and immunoreactivity in the particulate fraction (data not shown).

Figure 5.

Effect of phenylephrine and PMA stimulation on PKC immunoreactivity in the particulate fraction. Rat cavernosal muscle strips were exposed to the labeled stimulation condition for 10 min followed by isolation of the particulate (P) fraction. An equal amount of protein (15 g) from the particulate fraction was subjected to 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes and probed with specific anti-PKC antibodies as described in Methods. (a) Western blotting data are taken from a representative immunoblot. PSS indicates unstimulated with a physiological saline solution, PE identifies 3 M phenylephrine stimulation, PMA 0.1 M PMA stimulation, PC identifies positive control, rat brain extract. The quantitative data shown in panel b are means.e.m. of densitometric arbitrary units (n=12). ^*Statistical significance, P<0.05.

Full figure and legend (39K)

Figure 6.

Effect of phenylephrine and PMA stimulation on PKC immunoreactivity in the particulate fraction. Rat cavernosal muscle strips were exposed to the labeled stimulation condition for 10 min followed by isolation of the particulate (P) fraction. An equal amount of protein (15 g) from the particulate fraction was subjected to 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes and probed with specific anti-PKC antibodies as described in Methods. (a) Western blotting data are taken from a representative immunoblot. PSS indicates unstimulated with a physiological saline solution, PE identifies 3 M phenylephrine stimulation, PMA 0.1 M PMA stimulation, PC identifies positive control, rat brain extract. The quantitative data shown in panel b are means.e.m. of densitometric arbitrary units (n=9). ^*Statistical significance, P<0.05.

Full figure and legend (39K)

Effects of PMA on PKC isoform immunoreactivity in the particulate fraction

Rat cavernosal muscle expressed both PMA-sensitive PKC (, , , , , and ) and insensitive PKC () isoforms (Figure 1). Studies were undertaken in order to determine the effects of PMA on PKC isoform immunoreactivity in the particulate fraction. As shown in Figure 5, PMA increased immunoreactivity of PKC in the particulate fraction significantly (PSS 1000 vs PMA 14422 (n=9), P=0.024). In contrast, PMA did not result in a significant change of immunoreactivity of PKC in the particulate fraction (Figure 6).

Discussion

In the present study, we explored the role of PKC isoforms in 1-adrenergic receptor agonist phenylephrine-induced contractile response in isolated rat corpus cavernosum. The first approach to the question was to identify the PKC isoforms in rat cavernosal tissues. Using isoform-specific antibodies we demonstrated the presence of PKC, , , , , , and isoforms in rat corpus cavernosum. We also determined the subcellular distribution of each of these isoforms prior to phenylephrine or PMA treatment. We found that PKC, , , and isoforms were predominantly confined to the particulate fraction, whereas PKC and were confined mainly to the cytosolic fraction (Figure 1). The expression of PKC, , , , , and isoforms has been previously reported in various smooth muscles.^{11, 17, 24, 25, 26, 27, 28} Our results are similar to others who have reported a diverse expression of the various isozymes, which display at least one of each member of the three groups in the cell or tissues.^{13, 29} We did observe a moderate signal for PKC, an isozyme more often associated with neural tissue, which likely reflects the strong innervation of the cavernosal tissues.³⁰

Traditionally, PKCs were believed to reside in the cytosol in an inactive conformation and translocate to the membrane (or other subcellular sites) upon activation where they modify various cellular functions through phosphorylation of target substrates.^{16, 22} We observed that PKC, , , and were predominantly present in the particulate fraction of rat cavernosal smooth muscle in resting state. Based on this traditional view, our data suggested that these tissue preparations had a high level of PKC activity at rest. To rule out the possibility that translocation or activation of PKC isoforms occurred during manipulation of muscle for force measurements, PKC isoform distribution was examined in both hung strips (ie strips that were used to record contractile responses) and unhung strips (ie strips that were treated with agonists without setting tissue length). In general, we apply a set of stretches and length releases to establish the optimal tissue length and tissue passive force for recording force generation capacity.¹⁴ Stretch has been recognized to activate a variety of PKC-associated signaling pathways in other tissues.^{31, 32} We did not observe any significant difference in the PKC isoform distribution between hung and unhung tissues (data not shown). Our data indicate that at rest rat cavernosal smooth muscle contained high amounts of PKC, , , and in the particulate fraction. While we observed that these isozymes were preferentially found in the particulate fraction, we found no significant change in the unstimulated force in response to application of the PKC activator PMA or the inhibitor chelerythrine (data not shown). One possible interpretation for these results is based on the more recent understanding of signaling molecule complexes and scaffolds associated with PKC. As described in several recent reviews, there is strong evidence that these PKCs and other signaling molecules may be docked in a position to be rapidly activated following agonist binding and stimulation.^{33, 34} In particular, the role for A kinase anchoring proteins (ACAPs)³⁵ and receptors for inactive c proteins (RICs)³⁶ may allow for the localization of these PKC isoforms to the particulate fraction in an inactive state while in the presence of inhibitor molecules. Such a condition might be favorable for the cavernosal tissue where there needs to be rapid activation and inactivation of the vasoconstrictor signaling in order to allow for proper erectile responses. Additionally, the constant sympathetic stimulation this tissue receives to help maintain the penis in the flaccid state may favor the localization of elements of the constrictor pathway to membrane.²

The accumulated evidence reported in the literature suggests that in smooth muscle the bifurcating polyphosphoinositide signaling pathway, IP₃–Ca²⁺ and DAG–PKC, is a major mechanism for transducing signals by hormones, neurotransmitters, and other Ca²⁺-mobilizing agents. Additionally, there are other reports that demonstrated the important role PKC played in agonist-induced muscle contraction of various smooth muscles.^{27, 37, 38, 39, 40, 41} There is little information available about any role PKC may have in cavernosal smooth muscle function. We therefore examined the role of two most immunoreactive PKC isoforms, PKC and PKC, in phenylephrine-induced muscle contraction in cavernosal smooth muscle. Phenylephrine evoked contraction of cavernosal tissues from adult male rats. We also found that phenylephrine treatment increased the immunoreactivity of PKC and in the particulate fraction (Figures 5 and 6). Our results are similar to those reported for the ferret aorta smooth muscle, PKC was translocated to plasma membrane in response to stimulation by phenylephrine²⁵ and suggest a role for PKC in modulating cavernosal smooth muscle tone.²⁷

Tumor-promoting phorbol esters such as PMA can substitute for diacylglyerol as an activator of PKC. It has been demonstrated that atypical isoforms (, , ) are insensitive to PMA^{19, 42} while the classical PKC (, , ) and novel PKC (, , ) isoforms are sensitive to PMA.^{43, 44} We report here that PMA caused a potentiation of the stress response generated by phenylephrine in the corpus cavernosum (Figure 3). Additional evidence for a role of PKC in the agonist-induced contraction of rat corpus cavernosum was provided by the reduced stress response in the presence of the PKC inhibitor chelerythrine chloride (Figure 2). In rabbit femoral artery, phorbol ester-induced contraction could be inhibited by the PKC inhibitors GF109203x and Go6976.^{45, 46} Furthermore, PMA also increased PKC immunoreactivity significantly in the particulate fraction (Figure 5), with out any significant effect on PKC immunoreactivity (Figure 6).

The functional studies described above (Figures 2, 3 and 4) combined with increased immunoreactivity data for PKC (Figures 5 and 6) suggest that both Ca²⁺-dependent (PKC) and Ca²⁺-independent (PKC) PKC isoforms are involved in phenylephrine-induced contractile response in rat cavernosal smooth muscle. The role of individual PKC isoforms in muscle contraction is incompletely understood. However, many investigators have proposed distinct signaling mechanisms and a number of potential targets for PKC action during muscle contraction.¹⁶ A constitutively active PKC fragment could enhance contraction at constant [Ca²⁺]_i by directly inhibiting myosin light chain phosphatase (MLCP)⁴⁷ while others have suggested a role for the atypical PKCs.⁴⁸ PKC can inhibit MLCP, through convergent mechanisms, in both smooth muscle and nonmuscle cells. One of these convergent pathways could be the CPI-17/PKC pathway in which PKC may directly mediate contraction of vascular smooth muscle^{49, 50} or through a PKC-mediated effect on MLCP.^{51, 52} One interpretation of our data suggests that PKC activation may be involved in the increased calcium sensitization effect that augments force generation in the presence of PMA (Figure 3). There are several reports in other cell systems that RhoA- and PKC-mediated pathways interact.^{41, 53, 54} However, the mechanism of Rho–PKC interaction is still poorly defined.

The RhoA/Rho-kinase pathway in the penile circulation appears to play an important role in controlling erectile state of penis. Studies published earlier from our group have shown the presence of RhoA and Rho kinase in rat cavernosal smooth muscle.^{14, 55} Additionally, a selective Rho-kinase inhibitor, Y-27632, induced cavernosal smooth muscle relaxation via nitric oxide (NO)/cGMP-independent pathway.^{14, 50, 55, 56} It has also been suggested in the literature that RhoA can regulate Ca²⁺ sensitivity in smooth muscle via PKC-dependent pathway.^{51, 52}

Based on the data presented in this paper, we suggest that PKC and PKC play a role in phenylephrine-induced corpus cavernosum smooth muscle contraction. These effects may be heightened under the condition of erectile dysfunction, where there is an increased constrictor effect on the smooth muscle of the penis preventing adequate smooth muscle relaxation and erection. This is the first study to identify the expression pattern of PKC isoforms in cavernosal tissues and to demonstrate that pharmacological manipulation of PKC can alter the 1 adrenergic responsiveness of the rat corpus cavernosum. Our conclusions from these studies suggest PKC may play an important constrictive role in the corpus cavernosum and reveals another complex regulatory scheme for controlling penile circulation and erection.

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Acknowledgements

This work was supported by National Institute of Health (NIH DK59467) grant awarded to CJ Wingard. The authors are grateful to Dr John A Johnson for fruitful discussions during these studies.