The Ca2+-sensitive K+ channel (maxi-K+) is an important modulator of corporal smooth muscle tone. The goal of these studies was twofold: 1) to determine the feasibility of transfecting corporal smooth muscle cells in vivo with the hSlo cDNA, which encodes for the human smooth muscle maxi-K+channel, and 2) to determine whether transfection of the maxi-K+channel would affect the physiological response to cavernous nerve stimulation in a rat model in vivo. Intracorporal microinjection of pCMVβ/Lac Z DNA in 10-wk-old rats resulted in significant incorporation and expression of β-galactosidase activity in 10 of 12 injected animals for up to 75 days postinjection. Moreover, electrical stimulation of the cavernous nerve revealed that, relative to the responses obtained in age-matched control animals ( N = 12), intracavernous injection of naked pcDNA/ hSlo DNA was associated with a statistically significant elevation in the mean amplitude of the intracavernous pressure response at all levels of current stimulation (range 0.5–10 mA) at both 1 mo ( N= 5) and 2 mo ( N = 8) postinjection. Furthermore, qualitatively similar observations were made at 3 mo ( N = 2) and 4 mo ( N = 2) postinjection. These data indicate that naked hSlo DNA is quite easily incorporated into corporal smooth muscle and, furthermore, that expression is sustained for at least 2 mo in corporal smooth muscle cells in vivo. Finally, after expression, hSlo is capable of measurably altering nerve-stimulated penile erection. Taken together, these data provide compelling evidence for the potential utility of gene therapy in the treatment of erectile dysfunction.

1 The pathophysiology of impotence related to vascular smooth muscle dysfunction in the male corpus cavernosum was studied on human isolated erectile tissue (HET). Studies were conducted on 140 sections of HET obtained from 38 male patients undergoing surgery for implantation of penile prostheses to correct underlying erectile dysfunction. 2 Spontaneous myotonic oscillations were characteristic of greater than 90% of all HET preparations at 37°C. These spontaneous oscillations were markedly attenuated by indomethacin, BW755C, nifedipine, removal of extracellular Ca2+, or lower temperatures (< 320C), but were not sensitive to inhibition by atropine, phentolamine or tetrodotoxin. Our data suggest that the oscillations may, at least in part, result from the generation and/or release of a stable cyclo-oxygenase product and a consequent increase in transmembrane Ca2 + influx. 3 The phenylephrine-induced contractions in HET may be reliably assayed up to 24 h after surgical removal, without significant alterations in the EC50, maximum response (E,,^x) or slope index of the steady-state concentration-response curve to phenylephrine. 4 The competitive and surmountable nature of the antagonism of phenylephrine-induced contractions by prazosin and yohimbine allowed calculation of antagonist dissociation constants. The calculated pKb values for prazosin and yohimbine, respectively, were 9.47 + 0.49 and 5.54 + 0.22. The rank order of agonist potency in HET was: noradrenaline = phenylephrine > clonidine. These data indicate the presence of a population of membrane receptors that are predominantly of the a,-adrenoceptor subtype.5 The entire patient population was stratified on a decennial basis into five age groups, and each age group was subsequently subdivided into diabetic and nondiabetic diagnostic categories. With respect to the steady-state phenylephrine concentration-response curves, a Winer two-factor analysis of variance revealed a significant effect of age on the calculated pEC50 value, as well as a significant age-diagnosis interaction. A post hoc statistical analysis for unpaired samples yielded significant differences between pEC50 values for diabetic and nondiabetic patients in age groups 41-50 and 61-70 years. In addition, aWiner two-factor analysis of variance also detected a significant effect of age on the calculated E.., value. 6 In conclusion, our studies demonstrate that spontaneous contractions in HET are likely to be mediated by the generation and release of a stable cyclo-oxygenase product. Furthermore, the results of both agonist and antagonist studies are consistent with the presence of a homogeneous ax-adrenoceptor population. Lastly, the responsiveness of isolated HET to phenylephrine was shown to be altered by both age and disease.

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The goal of these studies was to examine the potential utility of bladder instilled K+ channel gene therapy with hSlo cDNA (i.e., the maxi-K channel) to ameliorate bladder overactivity in a rat model of partial urinary outlet obstruction. Twenty-two female Sprague-Dawley rats were subjected to partial urethral (i.e., outlet) obstruction, with 17 sham-operated control rats run in parallel. After 6 wk of obstruction, suprapubic catheters were surgically placed in the dome of the bladder in all rats. Twelve obstructed rats received bladder instillation of 100 microg of hSlo/pcDNA in 1 ml PBS during catheterization, and another 10 obstructed rats received 1 ml PBS (7 rats) or 1 ml PBS containing pcDNA only (3 rats). Two days after surgery cystometry was performed on all animals to examine the characteristics of the micturition reflex in conscious and unrestrained rats. Obstruction was associated with a three- to fourfold increase in bladder weight and alterations in virtually every micturition parameter estimate. PBS-injected obstructed rats routinely displayed spontaneous bladder contractions between micturitions. In contrast, hSlo injection eliminated the obstruction-associated bladder hyperactivity, without detectably affecting any other cystometric parameter. Presumably, expression of hSlo in rat bladder functionally antagonizes the increased contractility normally observed in obstructed animals and thereby ameliorates bladder overactivity. These initial observations indicate a potential utility of gene therapy for urinary incontinence.

Intracavernous pharmacotherapy relies heavily on the use of vasoactive agents which act by increasing intracellular cAMP levels in human corpus cavernosum smooth muscle. Yet little is known about the cAMP generating system in this tissue, and how it may affect observed patient variability. Thus, the goal of these studies was to better characterize the biochemistry of cAMP formation in human corpus cavernosum smooth muscle, and thus provide more insight into the mechanisms of corporal smooth muscle relaxation in vivo. We studied both receptor and nonreceptor mediated increases in cAMP formation in short-term cultures of human corpus cavernosum smooth muscle cells. Both isoproterenol (ISO) and prostaglandin E1 (PGE1) produced concentration-dependent increases in cAMP, but histamine, serotonin and vasoactive intestinal polypeptide did not. Forskolin, a relatively specific activator of adenylate cyclase, was also a potent stimulant of cAMP formation in these cells. Moreover, there was a direct correlation between the degree of forskolin-induced cAMP accumulation in cultured corporal smooth muscle cells and the magnitude of the forskolin-induced relaxation response of precontracted isolated corporal smooth muscle strips. Prostaglandin E1 and ISO concentration response curves (CRCs) were then assayed in the absence and presence of subthreshold forskolin (0.1 microM.). In the presence of forskolin, the calculated maximal PGE1-induced cAMP accumulation (Emax) was significantly greater than that elicited by PGE1 alone, ISO alone, or ISO + forskolin (p < or = 0.02). In addition, a fixed molar ratio (FMR) (PGE1:ISO) protocol was used to demonstrate that both 80:20 and 70:30 FMRs (but not 95:5 or 90:10), were associated with significantly greater cAMP Emax values than that observed for PGE1 alone (p < or = 0.01). These data provide direct evidence that the degree of cAMP formation in cultured corporal smooth muscle cells is strongly correlated with the magnitude of relaxation of isolated corporal smooth muscle strips. In addition, since simultaneous activation of distinct components of the cAMP generating system produces significant increases in maximal intracellular cAMP accumulation, this suggests that such drug combinations may also augment corporal smooth muscle relaxation in vitro and in vivo.

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OBJECTIVE To identify early diabetes‐related alterations in gene expression in bladder and erectile tissue that would provide novel diagnostic and therapeutic treatment targets to prevent, delay or ameliorate the ensuing bladder and erectile dysfunction. MATERIALS AND METHODS The RG‐U34A rat GeneChip® (Affymetrix Inc., Sunnyvale, CA, USA) oligonucleotide microarray (containing ≈8799 genes) was used to evaluate gene expression in corporal and male bladder tissue excised from rats 1 week after confirmation of a diabetic state, but before demonstrable changes in organ function in vivo. A conservative analytical approach was used to detect alterations in gene expression, and gene ontology (GO) classifications were used to identify biological themes/pathways involved in the aetiology of the organ dysfunction. RESULTS In all, 320 and 313 genes were differentially expressed in bladder and corporal tissue, respectively. GO analysis in bladder tissue showed prominent increases in biological pathways involved in cell proliferation, metabolism, actin cytoskeleton and myosin, as well as decreases in cell motility, and regulation of muscle contraction. GO analysis in corpora showed increases in pathways related to ion channel transport and ion channel activity, while there were decreases in collagen I and actin genes. CONCLUSIONS The changes in gene expression in these initial experiments are consistent with the pathophysiological characteristics of the bladder and erectile dysfunction seen later in the diabetic disease process. Thus, the observed changes in gene expression might be harbingers or biomarkers of impending organ dysfunction, and could provide useful diagnostic and therapeutic targets for a variety of progressive urological diseases/conditions (i.e. lower urinary tract symptoms related to benign prostatic hyperplasia, erectile dysfunction, etc.).

Chagas' disease, caused by Trypanosoma cruzi, is an important cause of heart disease in Latin America. T. cruzi-induced microvascular compromise, in turn, is thought to play a major role in chagasic heart disease. Previous in vitro studies have implicated endothelin-1 (ET-1) as a potentially important vasomodulator present in increased levels in the supernatant of T. cruzi infected cultured human umbilical vein endothelial cells (HUVEC). Thus, the goal of the present investigation was to further evaluate the potentially important contribution of ET-1 to T. cruzi-induced alterations in vascular tone in vitro. Bioassay studies once again documented that exposure of isolated rat aortic rings to infected HUVEC supernatants elicited contractile responses whose steady-state magnitude was significantly greater than contractile responses elicited by exposure of aortic rings to uninfected HUVEC supernatants. Furthermore, the increased aortic contractility was significantly attenuated by the presence of the ET(A) subtype selective antagonists BMS-182,874 or BQ-123. Additionally, incubation of HUVEC with either verapamil or phosphoramidon prior to infection was also associated with reduced aortic contractility, upon exposure to the supernatant. Phosphoramidon, but not verapamil, produced a significant decrease in the measured ET-1 levels in the HUVEC supernatant. Consistent with the bioassay results, preincubation of Fura-2-loaded cultured rat aortic vascular smooth muscle cells with verapamil resulted in a near complete ablation of ET-1-induced transmembrane Ca2+ flux. Taken together, these data are consistent with the hypothesis that ET-1-induced vasoconstriction may play an important modulatory role in the vascular compromise characteristic of T. cruzi infection.