Weixin Zhao

Endothelin-1 as Putative Modulator of Erectile Dysfunction. II. Calcium Mobilization in Cultured Human Corporal Smooth Muscle Cells

PURPOSE To gain mechanistic insight into the actions of endothelin-1 (ET-1) in human erectile tissue. MATERIALS AND METHODS Fura-2 based digital imaging microscopy was used to measure intracellular calcium levels in discrete cellular regions (cytosol and nucleus) in single cultured human corporal smooth muscle cells derived from corporal tissue excised from 3 patients with organic erectile dysfunction, as well as 2 patients with documented erections. RESULTS Endothelin-1 elicited a transient and concentration-dependent increase in cytosolic and nuclear calcium levels in cells from both patient populations. However, while the peak amplitudes of the ET-1-induced cytosolic calcium increases were similar in cells from the 2 patient populations, the observed increases in nuclear calcium levels were much more pronounced in cells from patients with organic erectile dysfunction than in patients with documented erections. CONCLUSIONS Endothelin-1, by virtue of its ability to increase both cytosolic and nuclear calcium levels, may serve not only as a critical determinant of corporal tissue tone, but also as a modulator of cellular proliferation and phenotypic expression.

POTASSIUM CHANNELS AND HUMAN CORPOREAL SMOOTH MUSCLE CELL TONE: DIABETES AND RELAXATION OF HUMAN CORPUS CAVERNOSUM SMOOTH MUSCLE BY ADENOSINE TRIPHOSPHATE SENSITIVE POTASSIUM CHANNEL OPENERS

PURPOSE Sustained contraction of human corporeal smooth muscle depends on continuous transmembrane calcium flux through voltage gated calcium channels. K channels modulate corporeal smooth muscle membrane potential and, thus, ultimately affect transmembrane calcium flux. Therefore, we characterized relaxation responses elicited by the K channel modulators pinacidil and levcromakalim on isolated human corporeal tissue strips. We also evaluated the possibility that there may be alterations in adenosine triphosphate sensitive K channel pharmacology/function related to the presence of diabetes mellitus. MATERIALS AND METHODS A total of 215 isolated human corporeal tissue strips obtained from 57 male patients with organic erectile dysfunction were investigated. Cumulative concentration-response curves were constructed at half log increments for steady state relaxation responses elicited by pinacidil and levcromakalim on equivalently phenylephrine pre-contracted (to approximately 75% of maximum) isolated corporeal tissue strips. Potassium currents were measured using the cell attached whole cell patch clamp technique on freshly isolated corporeal smooth muscle cells. RESULTS A concentration dependent, glibenclamide sensitive relaxation response of phenylephrine pre-contracted corporeal tissue strips was observed for pinacidil and levcromakalim. Consistent with such observations, electrophysiological recordings on freshly isolated myocytes revealed that pinacidil (10 microM.) and levcromakalim (10 microM.) induced whole cell potassium currents that were blocked by glibenclamide (10 microM.). In addition, statistical analysis revealed that phenylephrine pre-contracted corporeal tissue strips from patients without diabetes were more sensitive to relaxation by both compounds than corporeal tissue strips excised from those with diabetes. Furthermore, relaxation responses elicited by pinacidil and levcromakalim were not affected by charybdotoxin or 4-aminopyridine but were completely reversed by KCl or tetraethylammonium chloride. CONCLUSIONS These data indicate that the adenosine triphosphate sensitive K channel subtype is likely to have an important role in the relaxation of isolated corporeal tissue strips and, moreover, they are the molecular target for the K channel modulators/openers levcromakalim and pinacidil. Such observations are consistent with the supposition that alterations in the structure/function/activity of these potassium channels may underlie at least some aspects of observed diabetes related differences in tissue sensitivity to K channel modulators.

Experimental Diabetes Alters Connexin43 Derived Gap Junction Permeability in Short-Term Cultures of Rat Corporeal Vascular Smooth Muscle Cells

PURPOSE Intercellular communication through gap junctions was assessed in 8 to 10-week STZ diabetic rats to evaluate diabetes related effects on gap junctional conductance and permeability in short-term cultures of corporeal myocytes. MATERIALS AND METHODS Rats were made diabetic by a single intraperitoneal injection of STZ. Eight to 10 weeks later erectile function was evaluated in vivo and corporeal tissue was harvested to isolate corporeal myocytes. Dual whole cell patch clamp studies of intercellular communication through connexin43 (Cx43) derived gap junction channels were done in short-term, ie passages 0 to 2, cultured corporeal myocytes excised from STZ diabetic rats with documented erectile impairment as well as in myocytes from age matched control rats. RESULTS No differences in macroscopic junctional conductance, single channel conductance or open probability were detected between myocytes from age matched control and STZ diabetic rats, confirming the lack of diabetes related alterations in Cx43 gating or conductance. However, fluorescence dye transfer experiments revealed a marked 3-fold increase in Cx43 mediated junctional permeability in the absence of any detectable change in Cx43 protein expression. CONCLUSIONS These data suggest that an alteration in the selectivity filter of Cx43 in diabetic animals affects the permeability of specifically sized and charged solutes. To our knowledge these studies provide the first evidence of a diabetes related increase in intercellular permselectivity in corporeal myocytes and, thus, they may have important implications for diabetes related erectile dysfunction.

Bladder instillation of "naked" hSlo/pcDNA3 ameliorates detrusor hyperactivity in obstructed rats in vivo.

Christ, G. J., N. S. Day, M. Day, C. Santizo, W. Zhao, T. Sclafani, J. Zinman, K. Hsieh, K. Venkateswarlu, M. Valcic, and A. Melman. Bladder injection of “naked” hSlo/ pcDNA3 ameliorates detrusor hyperactivity in obstructed rats in vivo. Am J Physiol Regulatory Integrative Comp Physiol 281: R1699–R1709, 2001.—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 mg 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 threeto 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.

Intercellular communication and bladder function.

There is now considerable experimental and clinical evidence supporting the supposition that overactivity of the bladder is associated with detectable alterations in the electrical properties of the detrusor smooth muscle cells. The preliminary data described in this report indicates that intercellular communication through gap junctions might play an important role in this process. Moreover, alterations in Cx43 mRNA expression may represent a tissue response to a physiologic insult (i.e., increased after load) in an attempt to further increase the syncytial nature and force of detrusor contractility to compensate for an increased pressure load. Finally, this report elucidates the rationale for suspecting that intercellular communication through gap junctions may play a role in normal bladder physiology and the pathophysiology of urinary incontinence caused by partial outlet obstruction.