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In Vitro and In Vivo Relaxation of Corpus Cavernosum Smooth Muscle by the Selective Myosin II Inhibitor, Blebbistatin
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In Vitro and In Vivo Relaxation of Corpus Cavernosum Smooth Muscle by the Selective Myosin II Inhibitor, Blebbistatin jsm_1424
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Xin-hua Zhang, MD, PhD,*† Memduh Aydin, MD,* Dwaraka Kuppam, BS,* Arnold Melman, MD,* and Michael E. DiSanto, PhD* *Department of Urology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY, USA; †Department of Urology, First Affiliated Hospital of Guangxi Medical University, Nanning, China DOI: 10.1111/j.1743-6109.2009.01424.x
ABSTRACT
Introduction. Blebbistatin (BLEB) is a small cell permeable molecule originally reported as a selective inhibitor of myosin II isoforms expressed by striated muscle and non-muscle cells (IC50 = 0.5–5 mM) with poor inhibition of turkey gizzard smooth muscle (SM) myosin II (IC50 ~80 mM). However, recently it was found that BLEB can potently inhibit mammalian arterial SM (IC50 ~5 mM). Aim. To investigate the effect of BLEB on corpus cavernosum SM (CCSM) tone and erectile function (EF). Methods. CC tissue obtained from penile implant patients along with CC, aorta and bladder from adult male rats were used for BLEB organ bath studies. Intracavernosal BLEB was administered to rats and EF was assessed via intracavernous pressure (ICP). Main Outcome Measures. Effects of BLEB on agonist-induced CCSM, aorta and bladder contraction in vitro and ICP in vivo. Results. BLEB completely relaxed human CCSM pre-contracted with phenylephrine (PE) in a dose-dependent manner decreasing tension by 76.5% at 10 mM. BLEB pre-incubation attenuated PE-induced contraction of human CC by ~85%. Human CC strips pre-contracted with endothelin-1 or KCl were almost completely relaxed by BLEB. Rat CCSM pre-contracted with PE showed BLEB relaxation comparable to human CCSM. BLEB inhibition was similar for rat aorta but slower for bladder. Both maximal ICP and ICP/mean arterial pressure were dosedependently increased by BLEB intracavernous injections with full erection at 1 micromole. Conclusion. Our novel data reveals that BLEB nearly completely relaxes rat and human CCSM pre-contracted with a variety of potent agonists and exhibits tissue selectivity. Coupled with our in vivo data in which nanomole doses of BLEB significantly increase ICP, our data substantiates an important role for the SM contractile apparatus in the molecular mechanism for EF and suggests the possibility of BLEB binding at myosin II as a therapeutic treatment for ED by targeting SM contractile pathways. Zhang X, Aydin M, Kuppam D, Melman A, and DiSanto ME. In vitro and in vivo relaxation of corpus cavernosum smooth muscle by the selective myosin II inhibitor, blebbistatin. J Sex Med 2009;6:2661–2671. Key Words. Erectile Function; Erectogenic Agent; Smooth Muscle Contraction; Myosin; Endothelin; Cross-Bridge Cycling
Introduction
T
he regulation of penile erection involves the relaxation of tonically contracted corpus cavernosum smooth muscle (CCSM) [1,2]. Although many studies have investigated CCSM relaxation pathways, few have focused on the role of the contractile apparatus in erectile function (EF). Similarly, the major therapeutic treatments available © 2009 International Society for Sexual Medicine
for erectile dysfunction (ED) have primarily targeted CCSM relaxation pathways with particular emphasis on nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling [3,4]. The goal of this study was to examine the effect of a recently discovered small molecule inhibitor of myosin II known as blebbistatin (BLEB) (See Figure 1) on CCSM contractility, compare its in vitro efficacy on CCSM to tonic-type aorta and phasic-type J Sex Med 2009;6:2661–2671
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Figure 1 Molecular structure of BLEB. Shown in this figure is the active (-) enantiomer form of blebbistain (1-Phenyl-1,2,3,4-tetrahydro-4-hydroxypyrrolo[2.3-b]-7methylquinolin-4-one) used in the current studies.
bladder smooth muscle (SM) and further to evaluate BLEB’s potential as an erectogenic agent in vivo. The molecular basis for SM contraction involves SM myosin (SMM) phosphorylation and dephosphorylation resulting from increases and decreases, respectively, in cytosolic (sarcoplasmic) free Ca2+ ([Ca2+]i) [5]. Norepinephrine released from nerve endings and endothelins and prostaglandins (e.g., PGF2a) released from the endothelium activate receptors on CCSM to increase inositol triphosphate (IP3) and diacylglycerol (DAG), increasing [Ca2+]I [6–8]. The Ca2+ binds to calmodulin which activates myosin light chain kinase (MLCK) which then phosphorylates the 20 kDa regulatory myosin light chains (MLC20) and induces the development of force [5,9]. In addition, phosphorylation of MLC20 activates myosin ATPase, which hydrolyzes ATP to provide energy for muscle contraction [5]. When the [Ca2+]i returns to basal levels, the calcium sensitivity increases, preventing myosin dephosphorylation which involves the RhoA/Rhokinase (ROK) mechanism, thus maintaining force [10]. On the other hand, decrease of free Ca2+ in the sarcoplasm mainly caused by the NO/cGMP/ phosphodiesterase-5 (PDE5) pathway, followed by calmodulin dissociation from MLCK leads to SMM dephosphorylation and subsequent SM relaxation [11]. SMM is composed of a pair of myosin heavy chains (MHCs) and two pairs of MLCs (MLC17 and MLC20) that are intimately intertwined [5]. It has been shown that the 5′ end of the SM MHC premRNA is alternatively spliced to generate isoforms known as SM-A and SM-B [12]. The SM-B isoform is predominantly found in SMs that demonstrate a more phasic contractile nature (e.g., urinary bladder), whereas the SM-A isoform is found in more tonic-type SM (e.g., aorta) and possesses lower ATPase activity [9,13–15]. We have demonstrated that the CCSM possesses a myosin isoform composition somewhat intermediate between J Sex Med 2009;6:2661–2671
Zhang et al. bladder and aortic SM, considered to exhibit tonic and phasic characteristics, respectively [9]. BLEB, discovered by means of a high throughput small molecule screen for inhibitors of nonmuscle myosin IIA [16], was recently reported to be a selective in vitro inhibitor of the myosin II isoforms expressed by striated muscle and nonmuscle cells (IC50 = 0.5–5 mM) but with reported poor inhibition of purified turkey gizzard SMM II (IC50 ~80 mM) [17]. Thus, BLEB, in the concentration range of 0.5–5 mM, was found to be an ATPase inhibiting agent specific for myosin II. Original studies showed BLEB exhibited an interesting specificity for several striated muscle and non-muscle myosins, including Dictyostelium myosin II, non-muscle myosins IIA and IIB, scallop striated muscle myosin II, porcine b cardiac muscle myosin II, and rabbit skeletal muscle myosin II, whereas SM and nonconventional myosins (I, V, and X) have been reported to be little influenced [17]. The fact that the four amino acid residues identified as the BLEB binding site on non-muscle myosin IIA and SMM II were found to be identical [18] suggested that there should be potent inhibition of SMM II by BLEB. As BLEB was found to be at least 16-fold less potent at inhibiting purified avian SMM II and since turkey gizzard expresses predominantly the SM-B SMM isoform [15] and the SM-A/SM-B alternative splice site occurs near the BLEB binding site, the question arose as to whether BLEB may be more efficacious at inhibiting SM tissues that express more of the SM-A isoform. Eddinger et al. later found that BLEB potently (IC50 ~3 mM) inhibited the actomyosin ATPase activities of recombinantly expressed SMM HC isoforms but that SM-A and SM-B type SMM were equally inhibited arguing against a SMM isoform selective effect [19]. BLEB also inhibited the KClinduced tonic contractions produced by rabbit femoral and renal arteries that express primarily SM-A and the weaker tonic contraction produced by the saphenous artery that expresses primarily SM-B, with an equivalent potency comparable with that identified for non-muscle myosin IIA (IC50 ~5 mM) [19]. Yet, KCl-induced contraction of chicken gizzard (almost completely SM-B) was less potently (IC50 ~20 mM) inhibited than the carotid artery (that expresses predominantly SM-A) [19]. To date, no studies have addressed the effect of BLEB on CCSM contractility or for that matter the effect on any urogenital SM. Accordingly, we examined the in vitro relaxation effect of BLEB on
In Vitro and In Vivo Relaxation of CCSM by Blebbistatin human and rat CCSM, as well as the in vivo proerectile potential of BLEB on rat CCSM. In addition, we also compared BLEB induced relaxation of CCSM (a SM that contains a mixture of SM-A and SM-B SMM HC isoforms) [9] to relaxation of urinary bladder (expressing predominantly SM-B) and aorta (expressing predominantly SM-A) SM [13]. Our findings reveal a potent CCSM relaxation effect and a novel pro-erectile role for BLEB and suggest the possibility of developing BLEB binding at myosin II as a novel therapeutic treatment for ED by targeting SM contractile pathways. Materials and Methods
Chemicals and Tissues Phenylephrine HCl (PE), carbamoylcholine chloride (carbachol) and endothelin 1 (ET-1) were purchased from Sigma (St. Louis, USA). (⫾) BLEB, (+) BLEB and (-) BLEB were from Tocris (Ellisville, MO). A stock solution of BLEB was made in dimethylsulphoxide (DMSO); the other substances were dissolved daily in double distilled water and further dilutions to the final concentrations were made in Krebs-Henseleit buffer (Krebs buffer). Control experiments showed that the final concentrations of DMSO used in these studies did not significantly modify the relaxation response induced by (⫾) BLEB. Human CC samples were obtained from patients undergoing penile prosthesis implantation (N = 18, age ranging 33 to 71 years, mean ⫾ SEM = 53.2 ⫾ 5.8) with informed consent and the approval of the Institutional Review Boards of Montefiore Medical Center and Albert Einstein College of Medicine of Yeshiva University. Male rat SM tissues including CC, aorta and urinary bladder were obtained from 275–300 g adult Sprague-Dawley (SD) rats (Charles River, Raleigh, NC, USA). All animal studies were approved by the Animal Institute Committee of the Albert Einstein College of Medicine of Yeshiva University. In Vitro Organ Bath Studies Rat CC, aorta and detrusor were dissected from 7 male rats. Briefly, the rats were sacrificed by CO2 overdose, the penis was dissected free at its base and the glans penis, urethra and connective tissue surrounding the shaft were removed. The bladder was also isolated and the detrusor dissected free at the level of the ureters. Rat thoracic aorta was also dissected free and cut into circular rings. The rat CC and bladder were mounted longitudinally while
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the aortic rings were mounted in a horizontal manner in a 5 mL organ bath—Multi-Myograph Model 610 M physiological force measuring apparatus (Danish Myo Technology, Aarhus, Denmark) by securing to the two pins. In addition, human CC strips, obtained as described above, were also mounted in the organ bath. One of the pins was attached to a force transducer which was calibrated to milligrams of force prior to the start of experimentation. The myograph was connected in line to a PowerLab 4/30 Data Acquisition System (ADInstruments; Colorado Springs, CO, USA), and in turn, to a dual-core processor Pentium computer for real-time monitoring of physiological force. The SM strips were equilibrated for at least 1 hour in Krebs buffer maintained at a mean temperature of 37 ⫾ 0.05°C with continuous bubbling of 95% O2 and 5% CO2 with buffer changes every 15 minutes. The buffer had the following mM composition: NaCl 110, KCl 4.8, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25 and dextrose 11. The strips were continuously adjusted to a resting tension (1,500 mg for human CC, 350 mg for rat CC, 500 mg for bladder and 700 mg for aorta) similar to that previously described [20–24], and the isometric tension was recorded. After equilibration, the tissues were contracted with 60 mM KCl. This degree of contractile response was taken as 100% and the force induced by different concentrations of the various agonists (PE, carbachol, and ET-1) was expressed as a percentage of this value. After washing several times to baseline with Krebs buffer, all strips precontracted with PE or carbachol (for bladder) at a concentration pre-determined to produce about 50% of maximum force (1 mM) were allowed to reach stable tension and then the relaxant effects of increasing doses (1, 10, 50 mM) of BLEB were evaluated. For human CC, after pre-incubation with BLEB (50 mM) for 15 minutes, its inhibitory effect on PE (1 mM) contractility was tested. Furthermore, for human CC pre-contracted with either KCl (60 mM) or ET-1 (10 nM), relaxation responses of BLEB (50 mM) were also determined. The racemic mixture (⫾) of BLEB was used in all studies as it was determined that the active (-) enantiomer form was equipotent to the (⫾) racemic mixture in the in vitro studies and that the inactive (+) form did not induce significant relaxation [16,17,19]. Because of the known light sensitivity of BLEB, the stock solution of BLEB was wrapped with aluminum foil and kept in darkness in the refrigerator until just prior to usage. During J Sex Med 2009;6:2661–2671
2664 the experiment, the organ bath chambers were covered.
In Vivo Studies A separate group of six male adult SD rats were anesthetized with pentobarbital (35 mg/kg) via an intraperitoneal injection. Mean arterial pressure (MAP) via carotid artery and intracavernous pressure (ICP) were continuously monitored, using methods described previously [25]. Briefly, an incision was made in the perineum, and a window was made in the ischiocavernosus muscle to expose both CC. The right crura was perforated with a 28-gauge needle connected to PE-50 tubing for ICP recording while the left crura was perforated with a 30-gauge needle connected to PE-10 tubing for drug delivery. MAP and ICP were recorded via pressure transducers connected in line to a PowerLab 4/30 data acquisition system (ADInstruments, Colorado Springs, CO) connected in turn to a Dual-Core processor Pentium computer for realtime monitoring of pressure changes. Pressure transducers were calibrated to cm H2O prior to each experiment. After a stable baseline ICP was obtained, intracavernous injection (ICI) of 50 mL aliquots of saline or DMSO were carried out to test volume and vehicle effects. Then, increasing doses (5, 50, 250, 500, 1,000 nmol) of (⫾) BLEB were ICI with 15 minutes intervals between with washout (saline flush). The ICP rise elicited with BLEB stimulation was quantified either by maximum ICP or by calculating the ratio of maximum ICP/MAP ¥ 100. The maximum ICP is the maximal ICP rise with MAP being the mean arterial pressure during the plateau phase. Statistical Analysis Results are expressed as mean ⫾ SEM for n experiments. Statistical analysis was performed using either the Student’s t-test (when two sample treatments were being compared) or using anova when multiple means were compared. P < 0.05 was considered significant. Results
Figure 2 shows typical force tracings of SM contraction induced by PE (1 mM) or carbachol (1 mM) on CCSM or aorta and bladder, respectively, followed by relaxation induced by BLEB. On rat bladder SM, carbachol produced a stronger phasic contraction (Figure 2D) while on rat aortic SM, PE produced more tonic-type contraction (Figure 2C). For rat (Figure 2A) and human J Sex Med 2009;6:2661–2671
Zhang et al. (Figure 2B) CC, PE administration resulted in a force contractile profile intermediate between tonic and phasic-type contraction. This is correlated to what we have found in rabbit CCSM, which possesses an overall myosin isoform composition intermediate between tonic-type aorta and phasic-type bladder characteristics [9]. However, (⫾) BLEB displayed an inhibition of all contractions including the tonic aorta, phasic bladder, and intermediate CC. In the present study, the racemic mixture (⫾) BLEB was used. (-) BLEB is the active enantiomer, while (+) BLEB, the inactive enantiomer, is a reliable control for (-) BLEB because it exhibits no myosin ATPase inhibitory activity even up to a dose of 100 mM [16,17]. In addition, we confirmed the inactivity of the (+) enantiomer of BLEB, as shown in Figure 2E, where we show that a 50 mM concentration of the (+) form of BLEB did not induce significant relaxation of human CCSM from the PE precontracted state. However, after washing and then recontracting the tissue with PE, a strong relaxation to a similar 50 mM dose of the (⫾) racemic mixture of BLEB is shown for the same CC strip. The averaged results for experiments like those shown in Figure 2A–D are summarized in Figure 2F and demonstrate a BLEB-induced dosedependent relaxation effect on all tissues studied. In fact, we started at 0.1 mM, but it did not show any significant relaxant responses (-0.5%, -4.8% and -1.8% for rat CCSM, aorta and bladder, respectively). At 1 mM, only a 7.6–14.1% relaxant effect was observed. However, BLEB at 10 mM potently relaxed all strips by around 76.5–94.6%. At a higher concentration (50 mM), for some SM tissues, it decreased the SM tension even lower than baseline tone (Figure 2F). From Figure 2F, it seems apparent that BLEB showed similar relaxant efficacy for all SM strips (human CC and rat CC, aorta, bladder). Interestingly, the time course of BLEB (10 mM) inhibition for all preparations is rather slow occurring over 30–60 minutes or more (Figure 2). Over a strictly controlled 30 minutes relaxation to BLEB it can be observed that bladder SM relaxation by BLEB was significantly slower compared to CCSM and aorta SM (Figure 3). For the latter tissues, 10 mM BLEB inhibited contraction to near zero levels within 30 minutes while 10 mM BLEB relaxed the bladder to only ~75%. Control pre-contractions treated with vehicle (0.1% DMSO) were not significantly inhibited (Figure 3). In general, the inhibition velocity of BLEB is much slower compared with sodium nitroprusside (SNP—data not shown). BLEB
In Vitro and In Vivo Relaxation of CCSM by Blebbistatin A
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Figure 2 BLEB relaxation effect on rat and human SM tissues pre-contracted with PE or carbachol. Typical tracings of BLEB in vitro relaxant response (Panels A–D). Tissues were pre-contracted with 1 mM PE (aorta and CCs) or 1 mM carbachol (bladder), then relaxed with increasing concentrations of BLEB. (A) rat CC; (B) human CC; (C) rat aorta; (D) rat bladder. Panel E is a typical tracing of the effect of (+) BLEB on human CC tissue where 1 mM PE pre-contracted CCSM was exposed to 50 mM (+) BLEB. The tissue was then washed and recontracted with 1 mM PE and then exposed to 50 mM racemic mixture (⫾) BLEB. The x-axis represents time (minutes) while the y-axis represents force (mg). Panel F is a summary graph for the data shown in Panels A–D (N = a minimum of 6 different animals per tissue). Maximal response to PE or carbachol was taken as 100%, while the relaxant effect of cumulative concentrations (0–50 mM) of BLEB was evaluated as a percentage of this response. Values are expressed as mean ⫾ SEM. Student’s t-test was used *P < 0.05 vs. vehicle, **P < 0.01 vs. vehicle.
J Sex Med 2009;6:2661–2671
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Figure 3 Time course of BLEB in vitro relaxant response. SMs were pre-contracted with 1 mM PE (aorta and CCs) or 1 mM carbachol (bladder) and then treated with 10 mM BLEB for 30 minutes. Maximal response to PE or carbachol was taken as 100%, while the relaxant effect of BLEB was evaluated as a percentage of this response. Each point represents the mean ⫾ SEM (N = at least 4 strips from 4 different rats or patients). ANOVA test was used for statistical analysis *P < 0.01 vs. all tissues, **P < 0.05 vs. all other tissues.
inhibitory effects on SM were further confirmed by its attenuation of PE-induced human CC contraction. As demonstrated in Figure 4, after 15 minutes pre-incubation with 50 mM BLEB, CC
Figure 4 BLEB in vitro inhibitory effect on PE-induced contraction of human CC. Human CC strips were contracted with 1 mM PE and maximum tension (normalized to KCl) determined. After several washes to return tension to the basal state, the CC was pre-incubated with 50 mM BLEB for 15 minutes and then the same dose of PE was again added. Responses to PE with or without BLEB were evaluated as a percentage of maximal contraction induced by 60 mM KCl. Student’s t-test was used **P < 0.01 vs. PE + BLEB (N = 5 strips obtained from 5 different patients).
J Sex Med 2009;6:2661–2671
Zhang et al.
Figure 5 BLEB in vitro relaxant effect on ET-1 and KCl pre-contracted human CC. Human CC strips were precontracted with 10 nM ET-1 or 60 mM KCl and then treated with 50 mM BLEB. The maximal response to ET-1 or KCl was taken as 100%, while the relaxant effect of 50 mM BLEB was evaluated as a percentage of this response. Values are expressed as mean ⫾ SEM (N = 6 or more different strips from 3 different patients for each agonist).
contraction elicited by PE was significantly abrogated (>85%). Moreover, Figure 5 shows that human CC pre-contracted with one of the strongest vasoconstrictors known (10 nM ET-1) or by the depolarization agent KCl (60 mM), was nearly completely relaxed by 50 mM BLEB. Finally, we extended our in vitro results to the in vivo setting by performing ICI of BLEB into the normal rat CC. Figure 6 is a typical tracing of ICP rise induced by 5–1,000 nmol of BLEB and concomitantly recorded blood pressure, from which we can see, there was a transient decrease in blood pressure after each ICI but it recovered within seconds (Panel A). However, overall MAP is only slightly decreased even at the highest dose of 1,000 nmol. We also observed that ICP was dosedependently increased and baseline ICP slightly elevated even after washout (Panel B). Data were further analyzed and summarized results from six different rats are shown graphically in Figure 7. Max ICP (Figure 7A) dose-dependently increased with a big rise (near 100 cm H2O) at the highest dose (1,000 nmol). At this highest concentration, we found that two of the rats developed long term erection. In these two rats ICP did not decrease for more than 1 hour even after flushing of the CC (data not shown). In control experiments we determined that the increase in ICP was not a volume effect as ICI of similar volumes of saline and
In Vitro and In Vivo Relaxation of CCSM by Blebbistatin
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Figure 6 Typical tracing of ICP and blood pressure changes induced by intracavernous injection (ICI) of BLEB. ICP rise was induced by ICI of increasing dose (5–1,000 nmol) of BLEB. Panel A: The related BP recording. Panel B: Intracavernous pressure recording. Each dose was observed at least 10 minutes with 2 minutes flush/ washout. In both graphs, the x-axis represents time (minutes) while the y-axis represents pressure (cm H2O).
solvent (DMSO) did not have any effect on ICP. When the maximal ICP was normalized to MAP, a similar dose-response curve was found (Figure 7B). Thus both the maximum ICP and the rise in ICP/MAP ratio are due to BLEB inhibitory activity, not because of the volume, vehicle, or MAP change. Discussion
Proper EF involves a complex, perfectly coordinated, regulation of neuronal and CCSM signal transduction pathways. Although proper neuronal input is clearly necessary to initiate erection, proper regulation of the CCSM contractile apparatus is also essential. To date, the majority of studies that have examined the molecular basis for CCSM contractility have focused on CCSM relaxation rather than contractile pathways. In part, this may be attributable to the availability of numerous NO/cGMP pathway associated agonists and antagonists. The recent finding of a small molecule, cell permeable inhibitor (BLEB) with selectivity for myosin type II isoforms and a low micromolar IC50 for vascular SM contraction provides a unique pharmacologic tool to examine the role of the CCSM contractile apparatus in EF as well as to evaluate the potential of BLEB as an erectogenic agent. Our study revealed that BLEB was able to relax normal rat aorta, bladder and CC by 76.5–94.6% at a dose of only 10 mM (Figure 2). The fact that
1 mM BLEB caused only a 7.6–14.1% relaxation of the same SM tissues corresponds well with the reported ~5 mM IC50 of BLEB against mammalian arterial SM [19]. At a higher dose of 50 mM BLEB, all three rat tissues were completely relaxed with some relaxation values below baseline, demonstrating a role for SM myosin in maintaining basal tone in these tissues (Figure 2). In order to demonstrate whether these findings in rat translated to the human condition, we obtained 18 CC specimens from men at the time of penile implant surgery. Consistent with our findings in rat, BLEB induced a similar dosedependent relaxation of human CC (Figure 2B). Of note, although 1 mM BLEB did not induce significant relaxation of human CC, it did consistently increase spontaneous contractile activity even at this low dose, which did not occur for rat CCSM. This may be due to the fact that the human CC was obtained by necessity from men with ED which has been associated with increased spontaneous contractile activity [26–28]. As can be seen in Figure 2A–D, the time course for BLEB-induced relaxation was rather slow, occurring over periods of approximately 30–60 minutes which is much slower than other vasorelaxing drugs such as the NO donor SNP that reaches maximum relaxation within 3–5 minutes [29]. In order to better quantify the time course for BLEB-induced relaxation, rat bladder, aorta and CC as well as human CC were exposed to 10 mM BLEB and percent relaxation determined over a J Sex Med 2009;6:2661–2671
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B
Figure 7 ICP induced by ICI of BLEB. Baseline ICP and ICP induced by 50 mL saline, 50 mL DMSO and increasing doses (5–1,000 nmol) of BLEB are plotted. Each dose was observed at least 10 minutes with 2 minutes washout. (A): Maximal ICP; (B): Maximal ICP normalized by MAP. (N = average of tracings from six different rats)
period of 30 minutes. As can be seen in Figure 3, it took approximately 30 minutes for all SM tissues to reach near complete relaxation with the exception of the rat bladder that only reached about 75% relaxation by 30 minutes. Moreover, both the rat and human CCSM relaxation curves fall between the rat bladder and rat aorta but closer to rat aorta, although not significantly different. One possible explanation for the above tissue difference may be the unique SM myosin isoform composition in the bladder (containing almost exclusively the high ATPase SM-B type SMM J Sex Med 2009;6:2661–2671
Zhang et al. heavy chain isoform) compared to the aorta and CCSM (which contain predominantly the slower SM-A SMM heavy chain isoform) [9,13–15]. This would support the findings of a higher IC50 in the turkey gizzard (mainly SM-B type SMM II) compared to arterial SMM II [17,19] as well as the observation that the KCl-induced contraction of chicken gizzard (almost completely SM-B) was less potently (IC50 ~20 mM) inhibited than the carotid artery (that expresses predominantly SM-A) [19]. Also, similar to our bladder finding, the chicken gizzard reached a maximum BLEB-induced inhibition of only 75% [19]. It is also interesting to note that the relaxation curve for CCSM (which has been reported to contain approximately 70% SM-A) falls closer to that for the aorta which contains almost exclusively SM-A [9]. As can also be seen in Figure 3, even at 30 minutes, there was no vehicle effect on SM relaxation. Our study also found that in addition to being able to almost completely relax human CCSM from the PE pre-contracted state, pre-incubation with 50 mM BLEB for 15 minutes attenuated 1 mM PE-induced CCSM contraction by >85% (Figure 4). Moreover, 50 mM BLEB relaxed KCl (near 100%) and ET-1 (>90%)—induced contraction (Figure 5). The strong inhibitory effect on endothelin-induced contraction is particularly noteworthy as ET-1 is the most powerful vasoconstricting agent known and it is also significantly up-regulated in currently hard-to-treat etiologies of ED including diabetes-induced ED and in hypertension [30–33]. Taken together, the above data demonstrates BLEB to be a potent and effective inhibitor of agonist-induced CCSM contraction in rat urogenital SM and human CC. Based upon our above in vitro observations, we hypothesized that BLEB may act as an erectogenic agent in vivo. As an initial step to elucidate a role for BLEB in modulating erection, we intracavernously injected BLEB into the left crura of anesthesized young male adult SD rats while simultaneously monitoring ICP in the right crura and MAP in the carotid artery as described in the Methods section. As can be seen in Figure 6B, ICI of BLEB dose dependently increased rat ICP. Even at our initial dose of 5 nmol a small increase in ICP above vehicle control was noted with a dose-dependent increase in ICP occurring through 1,000 nmol (Figure 6B). It can also be seen in the raw ICP tracing shown in Figure 6 that beginning at the 250 nmol dose the ICP did not return to baseline even after flushing the CC with saline which was a consistent observation in all rats.
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In Vitro and In Vivo Relaxation of CCSM by Blebbistatin Simultaneous monitoring of blood pressure during the dose response curve to ICI BLEB revealed only a slight ~5% decrease in MAP at the highest 1,000 nmol dose of BLEB (Figure 6A). This is an important finding since the aortic and CCSM tissue in vitro seemed to be equally sensitive to BLEB. However, we did note a rapid decrease in blood pressure occurring at the time of each ICI but the blood pressure rapidly recovered. Since this same rapid decrease in BP was also observed after DMSO vehicle injection in the current study, we conclude that this represents only a transient bolus effect which dissipates upon distribution throughout the blood stream. Thus when ICP is normalized to MAP there is still a dose-dependent increase in ICP/MAP ratio (Figure 7B) that reaches a value greater than 60% at 1,000 nmol. In general an ICP/MAP ratio greater than 0.6 is deemed sufficient for full erection in both rats [34] and humans [35], and we did visually observe a full erection in the majority of rats at the 1,000 nmol dose. Furthermore, we found that in two of the six rats that received ICI BLEB, the erection persisted for more than 1 hour even after flushing the CC with saline. In conclusion, we provide novel data that BLEB potently relaxes both rat and human CCSM precontracted with a variety of agonists and also can almost completely block KCl and ET-1 induced CCSM contraction at micromolar concentrations. We also demonstrate that nanomole doses of BLEB intracavernously injected into the CC significantly increases ICP as well as the ICP/MAP ratio and produces full erection at an ICI dose of 1,000 nmol. To our knowledge, this is the first contractile apparatus-targeted small molecule inhibitor that has been demonstrated as an erectogenic agent. Thus, our data clearly elucidates the important role of the SM contractile apparatus in the molecular mechanism for EF and suggests the possibility of BLEB binding at myosin II as a therapeutic treatment for ED by targeting SM contractile pathways. Future studies will include determining whether low doses of BLEB can potentiate current pharmacologic therapy (e.g., phosphodiesterase V inhibitors) for the treatment of ED. Acknowledgment
This work was supported by NIH grant R01 DK077116-01 to MD. Dr. Xin-hua Zhang is a Visiting Scientist from the Department of Urology of the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China.
Corresponding Author: Michael DiSanto, PhD, Urology, Albert Einstein College of Medicine, Forchheimer Building, Room 744, 1300 Morris Park Avenue, Bronx, New York, NY 10461, USA. Tel: +718-4303201; Fax: +718-828-2705; E-mail: mdisanto@ aecom.yu.edu Conflict of Interest: None declared.
Statement of Authorship
Category 1 (a) Conception and Design Xin-hua Zhang; Michael DiSanto (b) Acquisition of Data Xin-hua Zhang; Dwaraka Kuppam (c) Analysis an Interpretation of Data Xin-hua Zhang; Memduh Aydin; Dwaraka Kuppam; Arnold Melman; Michael DiSanto
Category 2 (a) Drafting the Manuscript Xin-hua Zhang; Michael DiSanto (b) Revising It for Intellectual Content Xin-hua Zhang; Memduh Aydin; Arnold Melman; Michael DiSanto
Category 3 (a) Final Approval of the Completed Manuscript Xin-hua Zhang; Memduh Aydin; Dwaraka Kuppam; Arnold Melman; Michael DiSanto
References
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2670 7 Morelli A, Filippi S, Vignozzi L, Mancina R, Maggi M. Physiology of erectile function: An update on intracellular molecular processes. EAU-EBU Update Ser 2006;4:96–108. 8 Wingard C, Fulton D, Husain S. Altered penile vascular reactivity and erection in the Zucker obesediabetic rat. J Sex Med 2007;4:348–62. 9 DiSanto ME, Wang Z, Menon C, Zheng Y, Chacko T, Hypolite J, Broderick G, Wein AJ, Chacko S. Expression of myosin isoforms in smooth muscle cells in the corpus cavernosum penis. Am J Physiol 1998;275:C976–87. 10 Sward K, Mita M, Wilson DP, Deng JT, Susnjar M, Walsh MP. The role of RhoA and Rho-associated kinase in vascular smooth muscle contraction. Curr Hypertens Rep 2003;5:66–72. 11 Ghalayini IF. Nitric oxide-cyclic GMP pathway with some emphasis on cavernosal contractility. Int J Impot Res 2004;16:459–69. 12 Babij P, Kelly C, Periasamy M. Characterization of a mammalian smooth muscle myosin heavy-chain gene: Complete nucleotide and protein coding sequence and analysis of the 5′ end of the gene. Proc Natl Acad Sci USA 1991;88:10676–80. 13 DiSanto ME, Cox RH, Wang Z, Chacko S. NH2terminal-inserted myosin II heavy chain is expressed in smooth muscle of small muscular arteries. Am J Physiol 1997;272:C1532–42. 14 Hypolite JA, DiSanto ME, Zheng YM, Chang SH, Wein AJ, Chacko S. Regional variation in myosin isoforms and phosphorylation at the resting tone in urinary bladder smooth muscle. Am J Physiol-Cell Physiol 2001;280:C254–64. 15 Kelley CA, Takahashi M, Yu JH, Adelstein RS. An insert of seven amino acids confers functional differences between smooth muscle myosins from the intestines and vasculature. J Biol Chem 1993;268: 12848–54. 16 Straight AF, Cheung A, Limouze J, Chen I, Westwood NJ, Sellers JR, Mitchison TJ. Dissecting temporal and spatial control of cytokinesis with a myosin II inhibitor. Science 2003;299:1743–7. 17 Limouze J, Straight AF, Mitchison T, Sellers JR. Specificity of blebbistatin, an inhibitor of myosin II. J Muscle Res Cell Motil 2004;25:337–41. 18 Allingham JS, Smith R, Rayment I. The structural basis of blebbistatin inhibition and specificity for myosin II. Nat Struct Mol Biol 2005;12:378–9. 19 Eddinger TJ, Meer DP, Miner AS, Meehl J, Rovner AS, Ratz PH. Potent inhibition of arterial smooth muscle tonic contractions by the selective myosin II inhibitor, blebbistatin. J Pharmacol Exp Ther 2007; 320:865–70. 20 Granchi S, Vannelli GB, Vignozzi L, Crescioli C, Ferruzzi P, Mancina R, Vinci MC, Forti G, Filippi S, Luconi M, Ledda F, Maggi M. Expression and regulation of endothelin-1 and its receptors in human penile smooth muscle cells. Mol Hum Reprod 2002;8:1053–64. J Sex Med 2009;6:2661–2671
Zhang et al. 21 Yamamoto M, Harm SC, Grasser WA, Thiede MA. Parathyroid hormone-related protein in the rat urinary bladder: A smooth muscle relaxant produced locally in response to mechanical stretch. Proc Natl Acad Sci USA 1992;89:5326–30. 22 Ghasemi M, Sadeghipour H, Shafaroodi H, Nezami BG, Gholipour T, Hajrasouliha AR, Tavakoli S, Nobakht M, Moore KP, Mani AR, Dehpour AR. Role of the nitric oxide pathway and the endocannabinoid system in neurogenic relaxation of corpus cavernosum from biliary cirrhotic rats. Br J Pharmacol 2007;151:591–601. 23 Ren J, Wang GJ, Petrovski P, Ren BH, Brown RA. Influence of hypertension on acetaldehyde-induced vasorelaxation in rat thoracic aorta. Pharmacol Res 2002;45:195–9. 24 Sandhu KS, Chua RG, Zhang X, Kanika ND, Collins SA, Mikhail M, Melman A, DiSanto ME. Regional heterogeneity in expression of the sphingosine-1phosphate pathway in the female rat lower urinary tract. Am J Obstet Gynecol 2009;200:576. 25 Melman A, Biggs G, Davies K, Zhao W, Tar MT, Christ GJ. Gene transfer with a vector expressing Maxi-K from a smooth muscle-specific promoter restores erectile function in the aging rat. Gene Ther 2008;15:364–70. 26 Yarnitsky D, Sprecher E, Barilan Y, Vardi Y. Corpus cavernosum electromyogram: Spontaneous and evoked electrical activities. J Urol 1995;153:653–4. 27 Holmquist F, Andersson KE, Hedlund H. Effects of pinacidil on isolated human corpus cavernosum penis. Acta Physiol Scand 1990;138:463–9. 28 Christ GJ, Maayani S, Valcic M, Melman A. Pharmacological studies of human erectile tissue: Characteristics of spontaneous contractions and alterations in alpha-adrenoceptor responsiveness with age and disease in isolated tissues. Br J Pharmacol 1990;101:375–81. 29 Karasu C. Time course of changes in endotheliumdependent and -independent relaxation of chronically diabetic aorta: Role of reactive oxygen species. Eur J Pharmacol 2000;392:163–73. 30 Francavilla S, Properzi G, Bellini C, Marino G, Ferri C, Santucci A. Endothelin-1 in diabetic and nondiabetic men with erectile dysfunction. J Urol 1997;158:1770–4. 31 Khan MA, Thompson CS, Sullivan ME, Dashwood MR, Jeremy JY, Morgan RJ, Mikhailidis DP. Endothelin and erectile dysfunction: A target for pharmacological intervention? Expert Opin Investig Drugs 1998;7:1759–67. 32 Chang S, Hypolite JA, Changolkar A, Wein AJ, Chacko S, DiSanto ME. Increased contractility of diabetic rabbit corpora smooth muscle in response to endothelin is mediated via Rho-kinase beta. Int J Impot Res 2003;15:53–62. 33 Carneiro FS, Nunes KP, Giachini FR, Lima VV, Carneiro ZN, Nogueira EF, Leite R, Ergul A, Rainey WE, Clinton WR, Tostes RC. Activation of
In Vitro and In Vivo Relaxation of CCSM by Blebbistatin the ET-1/ETA pathway contributes to erectile dysfunction associated with mineralocorticoid hypertension. J Sex Med 2008;5:2793–807. 34 Sato Y, Zhao W, Christ GJ. Central modulation of the NO/cGMP pathway affects the MPOA-induced intracavernous pressure response. Am J Physiol Regul Integr Comp Physiol 2001;281:R269–78.
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J Sex Med 2009;6:2661–2671
In Vitro and In Vivo Relaxation of Corpus Cavernosum Smooth Muscle by the Selective Myosin II Inhibitor, Blebbistatin jsm_1424
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Xin-hua Zhang, MD, PhD,*† Memduh Aydin, MD,* Dwaraka Kuppam, BS,* Arnold Melman, MD,* and Michael E. DiSanto, PhD* *Department of Urology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY, USA; †Department of Urology, First Affiliated Hospital of Guangxi Medical University, Nanning, China DOI: 10.1111/j.1743-6109.2009.01424.x
ABSTRACT
Introduction. Blebbistatin (BLEB) is a small cell permeable molecule originally reported as a selective inhibitor of myosin II isoforms expressed by striated muscle and non-muscle cells (IC50 = 0.5–5 mM) with poor inhibition of turkey gizzard smooth muscle (SM) myosin II (IC50 ~80 mM). However, recently it was found that BLEB can potently inhibit mammalian arterial SM (IC50 ~5 mM). Aim. To investigate the effect of BLEB on corpus cavernosum SM (CCSM) tone and erectile function (EF). Methods. CC tissue obtained from penile implant patients along with CC, aorta and bladder from adult male rats were used for BLEB organ bath studies. Intracavernosal BLEB was administered to rats and EF was assessed via intracavernous pressure (ICP). Main Outcome Measures. Effects of BLEB on agonist-induced CCSM, aorta and bladder contraction in vitro and ICP in vivo. Results. BLEB completely relaxed human CCSM pre-contracted with phenylephrine (PE) in a dose-dependent manner decreasing tension by 76.5% at 10 mM. BLEB pre-incubation attenuated PE-induced contraction of human CC by ~85%. Human CC strips pre-contracted with endothelin-1 or KCl were almost completely relaxed by BLEB. Rat CCSM pre-contracted with PE showed BLEB relaxation comparable to human CCSM. BLEB inhibition was similar for rat aorta but slower for bladder. Both maximal ICP and ICP/mean arterial pressure were dosedependently increased by BLEB intracavernous injections with full erection at 1 micromole. Conclusion. Our novel data reveals that BLEB nearly completely relaxes rat and human CCSM pre-contracted with a variety of potent agonists and exhibits tissue selectivity. Coupled with our in vivo data in which nanomole doses of BLEB significantly increase ICP, our data substantiates an important role for the SM contractile apparatus in the molecular mechanism for EF and suggests the possibility of BLEB binding at myosin II as a therapeutic treatment for ED by targeting SM contractile pathways. Zhang X, Aydin M, Kuppam D, Melman A, and DiSanto ME. In vitro and in vivo relaxation of corpus cavernosum smooth muscle by the selective myosin II inhibitor, blebbistatin. J Sex Med 2009;6:2661–2671. Key Words. Erectile Function; Erectogenic Agent; Smooth Muscle Contraction; Myosin; Endothelin; Cross-Bridge Cycling
Introduction
T
he regulation of penile erection involves the relaxation of tonically contracted corpus cavernosum smooth muscle (CCSM) [1,2]. Although many studies have investigated CCSM relaxation pathways, few have focused on the role of the contractile apparatus in erectile function (EF). Similarly, the major therapeutic treatments available © 2009 International Society for Sexual Medicine
for erectile dysfunction (ED) have primarily targeted CCSM relaxation pathways with particular emphasis on nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling [3,4]. The goal of this study was to examine the effect of a recently discovered small molecule inhibitor of myosin II known as blebbistatin (BLEB) (See Figure 1) on CCSM contractility, compare its in vitro efficacy on CCSM to tonic-type aorta and phasic-type J Sex Med 2009;6:2661–2671
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Figure 1 Molecular structure of BLEB. Shown in this figure is the active (-) enantiomer form of blebbistain (1-Phenyl-1,2,3,4-tetrahydro-4-hydroxypyrrolo[2.3-b]-7methylquinolin-4-one) used in the current studies.
bladder smooth muscle (SM) and further to evaluate BLEB’s potential as an erectogenic agent in vivo. The molecular basis for SM contraction involves SM myosin (SMM) phosphorylation and dephosphorylation resulting from increases and decreases, respectively, in cytosolic (sarcoplasmic) free Ca2+ ([Ca2+]i) [5]. Norepinephrine released from nerve endings and endothelins and prostaglandins (e.g., PGF2a) released from the endothelium activate receptors on CCSM to increase inositol triphosphate (IP3) and diacylglycerol (DAG), increasing [Ca2+]I [6–8]. The Ca2+ binds to calmodulin which activates myosin light chain kinase (MLCK) which then phosphorylates the 20 kDa regulatory myosin light chains (MLC20) and induces the development of force [5,9]. In addition, phosphorylation of MLC20 activates myosin ATPase, which hydrolyzes ATP to provide energy for muscle contraction [5]. When the [Ca2+]i returns to basal levels, the calcium sensitivity increases, preventing myosin dephosphorylation which involves the RhoA/Rhokinase (ROK) mechanism, thus maintaining force [10]. On the other hand, decrease of free Ca2+ in the sarcoplasm mainly caused by the NO/cGMP/ phosphodiesterase-5 (PDE5) pathway, followed by calmodulin dissociation from MLCK leads to SMM dephosphorylation and subsequent SM relaxation [11]. SMM is composed of a pair of myosin heavy chains (MHCs) and two pairs of MLCs (MLC17 and MLC20) that are intimately intertwined [5]. It has been shown that the 5′ end of the SM MHC premRNA is alternatively spliced to generate isoforms known as SM-A and SM-B [12]. The SM-B isoform is predominantly found in SMs that demonstrate a more phasic contractile nature (e.g., urinary bladder), whereas the SM-A isoform is found in more tonic-type SM (e.g., aorta) and possesses lower ATPase activity [9,13–15]. We have demonstrated that the CCSM possesses a myosin isoform composition somewhat intermediate between J Sex Med 2009;6:2661–2671
Zhang et al. bladder and aortic SM, considered to exhibit tonic and phasic characteristics, respectively [9]. BLEB, discovered by means of a high throughput small molecule screen for inhibitors of nonmuscle myosin IIA [16], was recently reported to be a selective in vitro inhibitor of the myosin II isoforms expressed by striated muscle and nonmuscle cells (IC50 = 0.5–5 mM) but with reported poor inhibition of purified turkey gizzard SMM II (IC50 ~80 mM) [17]. Thus, BLEB, in the concentration range of 0.5–5 mM, was found to be an ATPase inhibiting agent specific for myosin II. Original studies showed BLEB exhibited an interesting specificity for several striated muscle and non-muscle myosins, including Dictyostelium myosin II, non-muscle myosins IIA and IIB, scallop striated muscle myosin II, porcine b cardiac muscle myosin II, and rabbit skeletal muscle myosin II, whereas SM and nonconventional myosins (I, V, and X) have been reported to be little influenced [17]. The fact that the four amino acid residues identified as the BLEB binding site on non-muscle myosin IIA and SMM II were found to be identical [18] suggested that there should be potent inhibition of SMM II by BLEB. As BLEB was found to be at least 16-fold less potent at inhibiting purified avian SMM II and since turkey gizzard expresses predominantly the SM-B SMM isoform [15] and the SM-A/SM-B alternative splice site occurs near the BLEB binding site, the question arose as to whether BLEB may be more efficacious at inhibiting SM tissues that express more of the SM-A isoform. Eddinger et al. later found that BLEB potently (IC50 ~3 mM) inhibited the actomyosin ATPase activities of recombinantly expressed SMM HC isoforms but that SM-A and SM-B type SMM were equally inhibited arguing against a SMM isoform selective effect [19]. BLEB also inhibited the KClinduced tonic contractions produced by rabbit femoral and renal arteries that express primarily SM-A and the weaker tonic contraction produced by the saphenous artery that expresses primarily SM-B, with an equivalent potency comparable with that identified for non-muscle myosin IIA (IC50 ~5 mM) [19]. Yet, KCl-induced contraction of chicken gizzard (almost completely SM-B) was less potently (IC50 ~20 mM) inhibited than the carotid artery (that expresses predominantly SM-A) [19]. To date, no studies have addressed the effect of BLEB on CCSM contractility or for that matter the effect on any urogenital SM. Accordingly, we examined the in vitro relaxation effect of BLEB on
In Vitro and In Vivo Relaxation of CCSM by Blebbistatin human and rat CCSM, as well as the in vivo proerectile potential of BLEB on rat CCSM. In addition, we also compared BLEB induced relaxation of CCSM (a SM that contains a mixture of SM-A and SM-B SMM HC isoforms) [9] to relaxation of urinary bladder (expressing predominantly SM-B) and aorta (expressing predominantly SM-A) SM [13]. Our findings reveal a potent CCSM relaxation effect and a novel pro-erectile role for BLEB and suggest the possibility of developing BLEB binding at myosin II as a novel therapeutic treatment for ED by targeting SM contractile pathways. Materials and Methods
Chemicals and Tissues Phenylephrine HCl (PE), carbamoylcholine chloride (carbachol) and endothelin 1 (ET-1) were purchased from Sigma (St. Louis, USA). (⫾) BLEB, (+) BLEB and (-) BLEB were from Tocris (Ellisville, MO). A stock solution of BLEB was made in dimethylsulphoxide (DMSO); the other substances were dissolved daily in double distilled water and further dilutions to the final concentrations were made in Krebs-Henseleit buffer (Krebs buffer). Control experiments showed that the final concentrations of DMSO used in these studies did not significantly modify the relaxation response induced by (⫾) BLEB. Human CC samples were obtained from patients undergoing penile prosthesis implantation (N = 18, age ranging 33 to 71 years, mean ⫾ SEM = 53.2 ⫾ 5.8) with informed consent and the approval of the Institutional Review Boards of Montefiore Medical Center and Albert Einstein College of Medicine of Yeshiva University. Male rat SM tissues including CC, aorta and urinary bladder were obtained from 275–300 g adult Sprague-Dawley (SD) rats (Charles River, Raleigh, NC, USA). All animal studies were approved by the Animal Institute Committee of the Albert Einstein College of Medicine of Yeshiva University. In Vitro Organ Bath Studies Rat CC, aorta and detrusor were dissected from 7 male rats. Briefly, the rats were sacrificed by CO2 overdose, the penis was dissected free at its base and the glans penis, urethra and connective tissue surrounding the shaft were removed. The bladder was also isolated and the detrusor dissected free at the level of the ureters. Rat thoracic aorta was also dissected free and cut into circular rings. The rat CC and bladder were mounted longitudinally while
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the aortic rings were mounted in a horizontal manner in a 5 mL organ bath—Multi-Myograph Model 610 M physiological force measuring apparatus (Danish Myo Technology, Aarhus, Denmark) by securing to the two pins. In addition, human CC strips, obtained as described above, were also mounted in the organ bath. One of the pins was attached to a force transducer which was calibrated to milligrams of force prior to the start of experimentation. The myograph was connected in line to a PowerLab 4/30 Data Acquisition System (ADInstruments; Colorado Springs, CO, USA), and in turn, to a dual-core processor Pentium computer for real-time monitoring of physiological force. The SM strips were equilibrated for at least 1 hour in Krebs buffer maintained at a mean temperature of 37 ⫾ 0.05°C with continuous bubbling of 95% O2 and 5% CO2 with buffer changes every 15 minutes. The buffer had the following mM composition: NaCl 110, KCl 4.8, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25 and dextrose 11. The strips were continuously adjusted to a resting tension (1,500 mg for human CC, 350 mg for rat CC, 500 mg for bladder and 700 mg for aorta) similar to that previously described [20–24], and the isometric tension was recorded. After equilibration, the tissues were contracted with 60 mM KCl. This degree of contractile response was taken as 100% and the force induced by different concentrations of the various agonists (PE, carbachol, and ET-1) was expressed as a percentage of this value. After washing several times to baseline with Krebs buffer, all strips precontracted with PE or carbachol (for bladder) at a concentration pre-determined to produce about 50% of maximum force (1 mM) were allowed to reach stable tension and then the relaxant effects of increasing doses (1, 10, 50 mM) of BLEB were evaluated. For human CC, after pre-incubation with BLEB (50 mM) for 15 minutes, its inhibitory effect on PE (1 mM) contractility was tested. Furthermore, for human CC pre-contracted with either KCl (60 mM) or ET-1 (10 nM), relaxation responses of BLEB (50 mM) were also determined. The racemic mixture (⫾) of BLEB was used in all studies as it was determined that the active (-) enantiomer form was equipotent to the (⫾) racemic mixture in the in vitro studies and that the inactive (+) form did not induce significant relaxation [16,17,19]. Because of the known light sensitivity of BLEB, the stock solution of BLEB was wrapped with aluminum foil and kept in darkness in the refrigerator until just prior to usage. During J Sex Med 2009;6:2661–2671
2664 the experiment, the organ bath chambers were covered.
In Vivo Studies A separate group of six male adult SD rats were anesthetized with pentobarbital (35 mg/kg) via an intraperitoneal injection. Mean arterial pressure (MAP) via carotid artery and intracavernous pressure (ICP) were continuously monitored, using methods described previously [25]. Briefly, an incision was made in the perineum, and a window was made in the ischiocavernosus muscle to expose both CC. The right crura was perforated with a 28-gauge needle connected to PE-50 tubing for ICP recording while the left crura was perforated with a 30-gauge needle connected to PE-10 tubing for drug delivery. MAP and ICP were recorded via pressure transducers connected in line to a PowerLab 4/30 data acquisition system (ADInstruments, Colorado Springs, CO) connected in turn to a Dual-Core processor Pentium computer for realtime monitoring of pressure changes. Pressure transducers were calibrated to cm H2O prior to each experiment. After a stable baseline ICP was obtained, intracavernous injection (ICI) of 50 mL aliquots of saline or DMSO were carried out to test volume and vehicle effects. Then, increasing doses (5, 50, 250, 500, 1,000 nmol) of (⫾) BLEB were ICI with 15 minutes intervals between with washout (saline flush). The ICP rise elicited with BLEB stimulation was quantified either by maximum ICP or by calculating the ratio of maximum ICP/MAP ¥ 100. The maximum ICP is the maximal ICP rise with MAP being the mean arterial pressure during the plateau phase. Statistical Analysis Results are expressed as mean ⫾ SEM for n experiments. Statistical analysis was performed using either the Student’s t-test (when two sample treatments were being compared) or using anova when multiple means were compared. P < 0.05 was considered significant. Results
Figure 2 shows typical force tracings of SM contraction induced by PE (1 mM) or carbachol (1 mM) on CCSM or aorta and bladder, respectively, followed by relaxation induced by BLEB. On rat bladder SM, carbachol produced a stronger phasic contraction (Figure 2D) while on rat aortic SM, PE produced more tonic-type contraction (Figure 2C). For rat (Figure 2A) and human J Sex Med 2009;6:2661–2671
Zhang et al. (Figure 2B) CC, PE administration resulted in a force contractile profile intermediate between tonic and phasic-type contraction. This is correlated to what we have found in rabbit CCSM, which possesses an overall myosin isoform composition intermediate between tonic-type aorta and phasic-type bladder characteristics [9]. However, (⫾) BLEB displayed an inhibition of all contractions including the tonic aorta, phasic bladder, and intermediate CC. In the present study, the racemic mixture (⫾) BLEB was used. (-) BLEB is the active enantiomer, while (+) BLEB, the inactive enantiomer, is a reliable control for (-) BLEB because it exhibits no myosin ATPase inhibitory activity even up to a dose of 100 mM [16,17]. In addition, we confirmed the inactivity of the (+) enantiomer of BLEB, as shown in Figure 2E, where we show that a 50 mM concentration of the (+) form of BLEB did not induce significant relaxation of human CCSM from the PE precontracted state. However, after washing and then recontracting the tissue with PE, a strong relaxation to a similar 50 mM dose of the (⫾) racemic mixture of BLEB is shown for the same CC strip. The averaged results for experiments like those shown in Figure 2A–D are summarized in Figure 2F and demonstrate a BLEB-induced dosedependent relaxation effect on all tissues studied. In fact, we started at 0.1 mM, but it did not show any significant relaxant responses (-0.5%, -4.8% and -1.8% for rat CCSM, aorta and bladder, respectively). At 1 mM, only a 7.6–14.1% relaxant effect was observed. However, BLEB at 10 mM potently relaxed all strips by around 76.5–94.6%. At a higher concentration (50 mM), for some SM tissues, it decreased the SM tension even lower than baseline tone (Figure 2F). From Figure 2F, it seems apparent that BLEB showed similar relaxant efficacy for all SM strips (human CC and rat CC, aorta, bladder). Interestingly, the time course of BLEB (10 mM) inhibition for all preparations is rather slow occurring over 30–60 minutes or more (Figure 2). Over a strictly controlled 30 minutes relaxation to BLEB it can be observed that bladder SM relaxation by BLEB was significantly slower compared to CCSM and aorta SM (Figure 3). For the latter tissues, 10 mM BLEB inhibited contraction to near zero levels within 30 minutes while 10 mM BLEB relaxed the bladder to only ~75%. Control pre-contractions treated with vehicle (0.1% DMSO) were not significantly inhibited (Figure 3). In general, the inhibition velocity of BLEB is much slower compared with sodium nitroprusside (SNP—data not shown). BLEB
In Vitro and In Vivo Relaxation of CCSM by Blebbistatin A
B
C
D
E
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Figure 2 BLEB relaxation effect on rat and human SM tissues pre-contracted with PE or carbachol. Typical tracings of BLEB in vitro relaxant response (Panels A–D). Tissues were pre-contracted with 1 mM PE (aorta and CCs) or 1 mM carbachol (bladder), then relaxed with increasing concentrations of BLEB. (A) rat CC; (B) human CC; (C) rat aorta; (D) rat bladder. Panel E is a typical tracing of the effect of (+) BLEB on human CC tissue where 1 mM PE pre-contracted CCSM was exposed to 50 mM (+) BLEB. The tissue was then washed and recontracted with 1 mM PE and then exposed to 50 mM racemic mixture (⫾) BLEB. The x-axis represents time (minutes) while the y-axis represents force (mg). Panel F is a summary graph for the data shown in Panels A–D (N = a minimum of 6 different animals per tissue). Maximal response to PE or carbachol was taken as 100%, while the relaxant effect of cumulative concentrations (0–50 mM) of BLEB was evaluated as a percentage of this response. Values are expressed as mean ⫾ SEM. Student’s t-test was used *P < 0.05 vs. vehicle, **P < 0.01 vs. vehicle.
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Figure 3 Time course of BLEB in vitro relaxant response. SMs were pre-contracted with 1 mM PE (aorta and CCs) or 1 mM carbachol (bladder) and then treated with 10 mM BLEB for 30 minutes. Maximal response to PE or carbachol was taken as 100%, while the relaxant effect of BLEB was evaluated as a percentage of this response. Each point represents the mean ⫾ SEM (N = at least 4 strips from 4 different rats or patients). ANOVA test was used for statistical analysis *P < 0.01 vs. all tissues, **P < 0.05 vs. all other tissues.
inhibitory effects on SM were further confirmed by its attenuation of PE-induced human CC contraction. As demonstrated in Figure 4, after 15 minutes pre-incubation with 50 mM BLEB, CC
Figure 4 BLEB in vitro inhibitory effect on PE-induced contraction of human CC. Human CC strips were contracted with 1 mM PE and maximum tension (normalized to KCl) determined. After several washes to return tension to the basal state, the CC was pre-incubated with 50 mM BLEB for 15 minutes and then the same dose of PE was again added. Responses to PE with or without BLEB were evaluated as a percentage of maximal contraction induced by 60 mM KCl. Student’s t-test was used **P < 0.01 vs. PE + BLEB (N = 5 strips obtained from 5 different patients).
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Zhang et al.
Figure 5 BLEB in vitro relaxant effect on ET-1 and KCl pre-contracted human CC. Human CC strips were precontracted with 10 nM ET-1 or 60 mM KCl and then treated with 50 mM BLEB. The maximal response to ET-1 or KCl was taken as 100%, while the relaxant effect of 50 mM BLEB was evaluated as a percentage of this response. Values are expressed as mean ⫾ SEM (N = 6 or more different strips from 3 different patients for each agonist).
contraction elicited by PE was significantly abrogated (>85%). Moreover, Figure 5 shows that human CC pre-contracted with one of the strongest vasoconstrictors known (10 nM ET-1) or by the depolarization agent KCl (60 mM), was nearly completely relaxed by 50 mM BLEB. Finally, we extended our in vitro results to the in vivo setting by performing ICI of BLEB into the normal rat CC. Figure 6 is a typical tracing of ICP rise induced by 5–1,000 nmol of BLEB and concomitantly recorded blood pressure, from which we can see, there was a transient decrease in blood pressure after each ICI but it recovered within seconds (Panel A). However, overall MAP is only slightly decreased even at the highest dose of 1,000 nmol. We also observed that ICP was dosedependently increased and baseline ICP slightly elevated even after washout (Panel B). Data were further analyzed and summarized results from six different rats are shown graphically in Figure 7. Max ICP (Figure 7A) dose-dependently increased with a big rise (near 100 cm H2O) at the highest dose (1,000 nmol). At this highest concentration, we found that two of the rats developed long term erection. In these two rats ICP did not decrease for more than 1 hour even after flushing of the CC (data not shown). In control experiments we determined that the increase in ICP was not a volume effect as ICI of similar volumes of saline and
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Figure 6 Typical tracing of ICP and blood pressure changes induced by intracavernous injection (ICI) of BLEB. ICP rise was induced by ICI of increasing dose (5–1,000 nmol) of BLEB. Panel A: The related BP recording. Panel B: Intracavernous pressure recording. Each dose was observed at least 10 minutes with 2 minutes flush/ washout. In both graphs, the x-axis represents time (minutes) while the y-axis represents pressure (cm H2O).
solvent (DMSO) did not have any effect on ICP. When the maximal ICP was normalized to MAP, a similar dose-response curve was found (Figure 7B). Thus both the maximum ICP and the rise in ICP/MAP ratio are due to BLEB inhibitory activity, not because of the volume, vehicle, or MAP change. Discussion
Proper EF involves a complex, perfectly coordinated, regulation of neuronal and CCSM signal transduction pathways. Although proper neuronal input is clearly necessary to initiate erection, proper regulation of the CCSM contractile apparatus is also essential. To date, the majority of studies that have examined the molecular basis for CCSM contractility have focused on CCSM relaxation rather than contractile pathways. In part, this may be attributable to the availability of numerous NO/cGMP pathway associated agonists and antagonists. The recent finding of a small molecule, cell permeable inhibitor (BLEB) with selectivity for myosin type II isoforms and a low micromolar IC50 for vascular SM contraction provides a unique pharmacologic tool to examine the role of the CCSM contractile apparatus in EF as well as to evaluate the potential of BLEB as an erectogenic agent. Our study revealed that BLEB was able to relax normal rat aorta, bladder and CC by 76.5–94.6% at a dose of only 10 mM (Figure 2). The fact that
1 mM BLEB caused only a 7.6–14.1% relaxation of the same SM tissues corresponds well with the reported ~5 mM IC50 of BLEB against mammalian arterial SM [19]. At a higher dose of 50 mM BLEB, all three rat tissues were completely relaxed with some relaxation values below baseline, demonstrating a role for SM myosin in maintaining basal tone in these tissues (Figure 2). In order to demonstrate whether these findings in rat translated to the human condition, we obtained 18 CC specimens from men at the time of penile implant surgery. Consistent with our findings in rat, BLEB induced a similar dosedependent relaxation of human CC (Figure 2B). Of note, although 1 mM BLEB did not induce significant relaxation of human CC, it did consistently increase spontaneous contractile activity even at this low dose, which did not occur for rat CCSM. This may be due to the fact that the human CC was obtained by necessity from men with ED which has been associated with increased spontaneous contractile activity [26–28]. As can be seen in Figure 2A–D, the time course for BLEB-induced relaxation was rather slow, occurring over periods of approximately 30–60 minutes which is much slower than other vasorelaxing drugs such as the NO donor SNP that reaches maximum relaxation within 3–5 minutes [29]. In order to better quantify the time course for BLEB-induced relaxation, rat bladder, aorta and CC as well as human CC were exposed to 10 mM BLEB and percent relaxation determined over a J Sex Med 2009;6:2661–2671
2668 A
B
Figure 7 ICP induced by ICI of BLEB. Baseline ICP and ICP induced by 50 mL saline, 50 mL DMSO and increasing doses (5–1,000 nmol) of BLEB are plotted. Each dose was observed at least 10 minutes with 2 minutes washout. (A): Maximal ICP; (B): Maximal ICP normalized by MAP. (N = average of tracings from six different rats)
period of 30 minutes. As can be seen in Figure 3, it took approximately 30 minutes for all SM tissues to reach near complete relaxation with the exception of the rat bladder that only reached about 75% relaxation by 30 minutes. Moreover, both the rat and human CCSM relaxation curves fall between the rat bladder and rat aorta but closer to rat aorta, although not significantly different. One possible explanation for the above tissue difference may be the unique SM myosin isoform composition in the bladder (containing almost exclusively the high ATPase SM-B type SMM J Sex Med 2009;6:2661–2671
Zhang et al. heavy chain isoform) compared to the aorta and CCSM (which contain predominantly the slower SM-A SMM heavy chain isoform) [9,13–15]. This would support the findings of a higher IC50 in the turkey gizzard (mainly SM-B type SMM II) compared to arterial SMM II [17,19] as well as the observation that the KCl-induced contraction of chicken gizzard (almost completely SM-B) was less potently (IC50 ~20 mM) inhibited than the carotid artery (that expresses predominantly SM-A) [19]. Also, similar to our bladder finding, the chicken gizzard reached a maximum BLEB-induced inhibition of only 75% [19]. It is also interesting to note that the relaxation curve for CCSM (which has been reported to contain approximately 70% SM-A) falls closer to that for the aorta which contains almost exclusively SM-A [9]. As can also be seen in Figure 3, even at 30 minutes, there was no vehicle effect on SM relaxation. Our study also found that in addition to being able to almost completely relax human CCSM from the PE pre-contracted state, pre-incubation with 50 mM BLEB for 15 minutes attenuated 1 mM PE-induced CCSM contraction by >85% (Figure 4). Moreover, 50 mM BLEB relaxed KCl (near 100%) and ET-1 (>90%)—induced contraction (Figure 5). The strong inhibitory effect on endothelin-induced contraction is particularly noteworthy as ET-1 is the most powerful vasoconstricting agent known and it is also significantly up-regulated in currently hard-to-treat etiologies of ED including diabetes-induced ED and in hypertension [30–33]. Taken together, the above data demonstrates BLEB to be a potent and effective inhibitor of agonist-induced CCSM contraction in rat urogenital SM and human CC. Based upon our above in vitro observations, we hypothesized that BLEB may act as an erectogenic agent in vivo. As an initial step to elucidate a role for BLEB in modulating erection, we intracavernously injected BLEB into the left crura of anesthesized young male adult SD rats while simultaneously monitoring ICP in the right crura and MAP in the carotid artery as described in the Methods section. As can be seen in Figure 6B, ICI of BLEB dose dependently increased rat ICP. Even at our initial dose of 5 nmol a small increase in ICP above vehicle control was noted with a dose-dependent increase in ICP occurring through 1,000 nmol (Figure 6B). It can also be seen in the raw ICP tracing shown in Figure 6 that beginning at the 250 nmol dose the ICP did not return to baseline even after flushing the CC with saline which was a consistent observation in all rats.
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In Vitro and In Vivo Relaxation of CCSM by Blebbistatin Simultaneous monitoring of blood pressure during the dose response curve to ICI BLEB revealed only a slight ~5% decrease in MAP at the highest 1,000 nmol dose of BLEB (Figure 6A). This is an important finding since the aortic and CCSM tissue in vitro seemed to be equally sensitive to BLEB. However, we did note a rapid decrease in blood pressure occurring at the time of each ICI but the blood pressure rapidly recovered. Since this same rapid decrease in BP was also observed after DMSO vehicle injection in the current study, we conclude that this represents only a transient bolus effect which dissipates upon distribution throughout the blood stream. Thus when ICP is normalized to MAP there is still a dose-dependent increase in ICP/MAP ratio (Figure 7B) that reaches a value greater than 60% at 1,000 nmol. In general an ICP/MAP ratio greater than 0.6 is deemed sufficient for full erection in both rats [34] and humans [35], and we did visually observe a full erection in the majority of rats at the 1,000 nmol dose. Furthermore, we found that in two of the six rats that received ICI BLEB, the erection persisted for more than 1 hour even after flushing the CC with saline. In conclusion, we provide novel data that BLEB potently relaxes both rat and human CCSM precontracted with a variety of agonists and also can almost completely block KCl and ET-1 induced CCSM contraction at micromolar concentrations. We also demonstrate that nanomole doses of BLEB intracavernously injected into the CC significantly increases ICP as well as the ICP/MAP ratio and produces full erection at an ICI dose of 1,000 nmol. To our knowledge, this is the first contractile apparatus-targeted small molecule inhibitor that has been demonstrated as an erectogenic agent. Thus, our data clearly elucidates the important role of the SM contractile apparatus in the molecular mechanism for EF and suggests the possibility of BLEB binding at myosin II as a therapeutic treatment for ED by targeting SM contractile pathways. Future studies will include determining whether low doses of BLEB can potentiate current pharmacologic therapy (e.g., phosphodiesterase V inhibitors) for the treatment of ED. Acknowledgment
This work was supported by NIH grant R01 DK077116-01 to MD. Dr. Xin-hua Zhang is a Visiting Scientist from the Department of Urology of the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China.
Corresponding Author: Michael DiSanto, PhD, Urology, Albert Einstein College of Medicine, Forchheimer Building, Room 744, 1300 Morris Park Avenue, Bronx, New York, NY 10461, USA. Tel: +718-4303201; Fax: +718-828-2705; E-mail: mdisanto@ aecom.yu.edu Conflict of Interest: None declared.
Statement of Authorship
Category 1 (a) Conception and Design Xin-hua Zhang; Michael DiSanto (b) Acquisition of Data Xin-hua Zhang; Dwaraka Kuppam (c) Analysis an Interpretation of Data Xin-hua Zhang; Memduh Aydin; Dwaraka Kuppam; Arnold Melman; Michael DiSanto
Category 2 (a) Drafting the Manuscript Xin-hua Zhang; Michael DiSanto (b) Revising It for Intellectual Content Xin-hua Zhang; Memduh Aydin; Arnold Melman; Michael DiSanto
Category 3 (a) Final Approval of the Completed Manuscript Xin-hua Zhang; Memduh Aydin; Dwaraka Kuppam; Arnold Melman; Michael DiSanto
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