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OBJECTIVE To investigate the in vitro and in vivo effects of blebbistatin (a small cell-permeable molecule with high affinity and selectivity toward the myosin II contractile molecule) on bladder smooth muscle (SM) contractility, as antimuscarinic therapy is only 65-75% effective in treating overactive bladder (OAB) and is associated with considerable side-effects, with a < 25% continuation rate at 1 year. MATERIALS AND METHODS Bladder and aortic strips from adult male rats, and human bladder strips obtained from open prostatectomy, were used for organ-bath studies of blebbistatin. Awake cystometry was also used in rats in both the presence and absence of intravesically delivered blebbistatin. RESULTS Blebbistatin dose-dependently and completely relaxed both KCl- and carbachol-induced rat detrusor and endothelin-1-induced human bladder contraction. Pre-incubation with 10 µm blebbistatin attenuated carbachol responsiveness by ≈ 65% while blocking electrical field stimulation-induced bladder contraction reaching 50% inhibition at 32 Hz. The basal tone and amplitude of spontaneous contraction were also significantly diminished. Urodynamic variables were obviously altered by intravesical infusion with blebbistatin. CONCLUSION Our novel data show that blebbistatin strongly relaxes both rat and human bladder contraction induced by various physiological stimuli. Coupled with our in vivo data showing that nanomole doses of blebbistatin significantly alter urodynamic variables to produce a less active bladder, our results suggest the possibility of intravesically administered blebbistatin binding at myosin II being developed as a therapeutic treatment for OAB via a novel targeting of the SM contractile apparatus.

Intracorporal injection of plasmids encoding opiorphins into retired breeder rats can result in animals developing a priapic-like condition. Microarray analysis demonstrated that following intracorporal gene transfer of plasmids expressing opiorphins the most significantly upregulated gene in corporal tissue was the ornithine decarboxylase gene (ODC). Quantitative RT-PCR confirmed the upregulation of ODC, as well as other genes involved in polyamine synthesis, such as arginase-I and -II, polyamine oxidase, spermidine synthase, spermidine acetyltransferase (SAT), and S-adenosylmethionine decarboxylase. Western blot analysis demonstrated upregulation of arginase-I and -II, ODC, and SAT at the protein level. Levels of the polyamine putrescine were upregulated in animals treated with opiorphin-expressing plasmids compared with controls. A direct role for the upregulation of polyamine synthesis in the development of the priapic-like condition was supported by the observation that the ODC inhibitor 1,3-diaminopropane, when added to the drinking water of animals treated with plasmids expressing opiorphins, prevented experimental priapism. We also demonstrate that in sickle cell mice, another model of priapism, there is increased expression of the mouse opiorphin homologue in corporal tissue compared with the background strain at a life stage prior to evidence of priapism. At a life stage when there is onset of priapism, there is increased expression of the enzymes involved in polyamine synthesis (ODC and arginase-I and -II). Our results suggest that the upregulation of enzymes involved in the polyamine synthetic pathway may play a role in the development of experimental priapism and represent a target for the prevention of priapism.

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 (IC 50 = 0.5–5 µM) with poor inhibition of turkey gizzard smooth muscle (SM) myosin II (IC 50 ∼80 µM). However, recently it was found that BLEB can potently inhibit mammalian arterial SM (IC 50 ∼5 µM). 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 µM. 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 dose-dependently 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.