Bioprinting on sheet-based scaffolds applied to the creation of implantable tissue-engineered constructs with potentially diverse clinical applications: Tissue-Engineered Muscle Repair (TEMR) as a representative testbed.

Purpose: This report explores the overlooked potential of bioprinting to automate biomanufacturing of simple tissue structures, such as the uniform deposition of (mono)layers of progenitor cells on sheetlike decellularized extracellular matrices (dECM). In this scenario, dECM serves as a biodegradable celldelivery matrix to provide enhanced regenerative microenvironments for tissue repair. The Tissue-Engineered Muscle Repair (TEMR) technology-where muscle progenitor cells are seeded onto a porcine bladder acellular matrix (BAM), serves as a representative testbed for bioprinting applications. Previous work demonstrated that TEMR implantation improved functional outcomes following VML injury in biologically relevant rodent models.Materials and Methods: In the described bioprinting system, a cell-laden hydrogel bioink is used to deposit high cell densities (1.4 × 105-3.5 × 105 cells/cm2), onto both sides of the bladder acellular matrix as proof-of-concept.Results: These bioprinting methods achieve a reproducible and homogeneous distribution of cells, on both sides of the BAM scaffold, after just 24hrs, with cell viability as high as 98%. These preliminary results suggest bioprinting allows for improved dual-sided cell coverage compared to manual-seeding.Conclusions: Bioprinting can enable automated fabrication of TEMR constructs with high fidelity and scalability, while reducing biomanufacturing costs and timelines. Such bioprinting applications are underappreciated, yet critical, to expand the overall biomanufacturing paradigm for tissue engineered medical products. In addition, biofabrication of sheet-like implantable constructs, with cells deposited on both sides, is a process that is both scaffold and cell-type agnostic, and furthermore, is amenable to many geometries, and thus, additional tissue engineering applications beyond skeletal muscle.

Gene transfer with a vector expressing Maxi-K from a smooth muscle-specific promoter restores erectile function in the aging rat.

Previous reports have demonstrated that gene transfer with the alpha, or pore-forming, subunit of the human Maxi-K channel (hSlo) restores the decline in erectile capacity observed in established rat models of diabetes and aging. Preliminary data from a human clinical trial also showed safety and potential efficacy in 11 men treated with the same plasmid construct expressing the Maxi-K channel. In all instances, the original plasmid was driven by the heterologous cytomegalovirus promoter which is broadly active in a wide variety of cell and tissue types. To more precisely determine the contribution of the corporal myocyte to the observed physiological effects in vivo, we report here our initial work using a distinct vector (pSMAA-hSlo) in which hSlo gene expression was driven off the mouse smooth muscle alpha-actin (SMAA) promoter. Specifically, older rats, with diminished erectile capacity, were given a single intracorporal injection with either 100 mug pVAX-hSlo or 10, 100 or 1000 mug pSMAA-hSlo, or vector or vehicle alone. Significantly increased intracavernous pressure (ICP) responses to cavernous nerve stimulation were observed for all doses of both plasmids encoding hSlo, relative to control injections. These data confirm and extend previous observations to document that smooth muscle cell-specific expression of hSlo in corporal tissue is both necessary and sufficient to restore erectile function in aging rats.

Physiological roles for K+ channels and gap junctions in urogenital smooth muscle: implications for improved understanding of urogenital function, disease and therapy.

Smooth muscle cells constitute a heterogeneous collection of effector cells that, by virtue of both their constituency in blood vessels and presence as primary parenchymal cells in diverse tissues, affect the function of all organs. Thus, perhaps it is not surprising that alterations in, and/or dysfunction of, smooth muscle cells are quite common, and responsible, at least in part, for the morbidity and mortality associated with a very wide range of human diseases. These facts point to the necessity for improved understanding of the mechanism(s) governing the control of myocyte contractility (i.e., tone). Such understanding has been rapidly forthcoming in recent years, and has indicated that in many smooth muscle cell types intercellular communication through gap junctions acts in concert with nonjunctional (K+) ion channels to make important contributions to the control of myocyte tone and tissue homeostasis in physiologically diverse organs. Intercellular communication through connexin43-derived gap junction channels and K+ flux through the KCa and KATP channel subtypes, in particular, appear to play prominent roles in this process. The goal of this report, therefore, is to review the data concerning junctional and nonjunctional ion channels on the detrusor myocytes of the urinary bladder, as well as on the specialized vascular myocytes of the corpus cavernosum. The choice of an excitable (i.e., bladder detrusor myocytes) and nonexcitable (i.e., corporal smooth muscle) smooth muscle cell type ensures that the discussion will at least encompass consideration of a large portion of the spectrum of physiological possibilities for the participation of junctional and nonjunctional ion channels in the initiation, maintenance and modulation of smooth muscle tone. A central thesis of this communication is that detailed knowledge of the myocyte- and tissue-specific properties of K+ channels and gap junctions will likely lead to the improved understanding and treatment of human smooth muscle diseases/disorders.

The physiology, pathophysiology and therapeutic potential of gap junctions in smooth muscle.

Phenotypic variability in smooth muscle cells accounts, in large part, for the incredible functional diversity required of the involuntary hollow organs of the body (i.e., respiratory passages, blood vessels, gastrointestinal tract, urogenital tract, etc.). In all instances coordination of smooth muscle cell responses, that is, contraction and relaxation, is critical to normal organ function. While numerous biological mechanisms exist for coordinating smooth muscle cell responses, intercellular communication through gap junctions represents a common denominator present in all organ systems. In this report, we review the evidence documenting the presence and functional significance of myocyte gap junctions to physiologically distinct organ systems, and furthermore, provide some examples of their putative roles in organ pathology. Finally, we advance the thesis that despite their ubiquity and heterogeneous expression, gap junctions are nonetheless potentially attractive therapeutic targets for the treatment of certain smooth muscle disorders. Their therapeutic efficacy will necessarily hinge on the existence of connexin isoform-selective junctional effects. The overall rationale for targeting the intercellular pathway is therefore analogous to strategies that target other ubiquitously expressed ion channels, such as calcium or potassium channels. Such strategies have proved efficacious for the treatment of a wide range of human smooth muscle disorders including hypertension, urinary incontinence and sexual function.

Cholinergic modulation of immunoglobulin secretion from avian plasma cells: the role of calcium.

The existence of a functional connection between the nervous and immune systems has long been argued. To determine if such a link exists in the secretory immune system, we have examined the avian lacrimal gland (Harderian gland) which contains large numbers of plasma cells. We have shown that these plasma cells bind an antibody to muscarinic acetylcholine receptor and that carbachol, an acetylcholine agonist, increases the secretion rate of IgG by these cells above a constitutive baseline level. This neurotransmitter-dependent increase of immunoglobulin secretion requires an influx of Ca2+, whereas the constitutive baseline secretion is apparently less dependent on such a flux. Furthermore, the Ca2+ flux appears to be mediated by voltage-dependent calcium channels. These data support the hypothesis that plasma cells can respond to neurotransmitters and, in the case of acetylcholine, increase immunoglobulin secretion.

Pharmacological studies of human erectile tissue: characteristics of spontaneous contractions and alterations in alpha-adrenoceptor responsiveness with age and disease in isolated tissues.

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 degrees C. These spontaneous oscillations were markedly attenuated by indomethacin, BW755C, nifedipine, removal of extracellular Ca2+, or lower temperatures (less than or equal to 32 degrees C), 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 (Emax) 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 much greater than clonidine. These data indicate the presence of a population of membrane receptors that are predominantly of the alpha 1-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, a Winer 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 alpha x-adrenoceptor population. Lastly, the responsiveness of isolated HET to phenylephrine was shown to be altered by both age and disease.

Genetic influences on agonist binding to cardiac beta-receptors.

Regulation of beta-adrenoceptor-agonist function in the Maudsley Reactive (MR/Har) and the Maudsley Non-Reactive (MNRA/Har) rat strains was assessed by comparison of isoproterenol competition for [125I]iodocyanopindolol (ICYP) binding sites in crude left ventricular homogenate preparations. Non-linear, least-squares analysis of isoproterenol competition for ICYP binding in the absence of guanine nucleotide revealed different proportions of high- and low-affinity receptors in the two strains; MR/Har rats (59 +/- 3.3%) had a significantly greater proportion of receptors in the high-affinity state than the MNRA/Har rats (41 +/- 4.5%). Addition of the non-hydrolyzable guanine nucleotide analog guanylylimidodiphosphate (Gpp(NH)p) converted receptors to the low-affinity state. Analysis of Gpp(NH)p concentration-response curves in left ventricular homogenates of the two strains revealed that the MR/Har strain had a significantly (P less than 0.02) lower EC50 for guanyl nucleotide inhibition of isoproterenol competition for ICYP binding than the MNRA/Har. Confirming previous experimental results, a significantly (P less than 0.04) greater density of ventricular beta-receptors was found in MR/Har rats (13.16 +/- 0.92 fmol/mg protein) than in MNRA/Har rats (10.81 +/- 0.63 fmol/mg protein). Left ventricular catecholamine levels were found to be correlated inversely with beta-adrenoceptor density in the two strains; norepinephrine (NE) and epinephrine (EPI) concentrations (ng/mg protein) in left ventricle were 12.19 +/- 0.94 for NE and 0.165 +/- 0.038 for EPI in MNRA/Har, and 8.73 +/- 0.95 and 0.018 +/- 0.018, respectively, in MR/Har. All other parameters of agonist interactions with the cardiac beta-adrenoceptor for the MR/Har and MNRA/Har rat strains were similar [the IC50 for displacement of ICYP binding by isoproterenol, the accompanying Hill coefficients in the Gpp(NH)p present and absent condition, the Kd of the high- and low-affinity states in the absence of Gpp(NH)p, and the Kd of the uniform low-affinity state in the presence of Gpp(NH)p]. We hypothesize that the strain-dependent differences in high-affinity state formation reported here may account for some of the in vivo differences in cardiovascular function previously demonstrated in the Maudsley rats.

The putative mechanistic basis for the modulatory role of endothelin-1 in the altered vascular tone induced by Trypanosoma cruzi.

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.

Myocardial expression of endothelin-1 in murine Trypanosoma cruzi infection.

Chagas' disease, caused by Trypanosoma cruzi, is an important cause of myocarditis and chronic cardiomyopathy and is accompanied by microvascular spasm and myocardial ischemia. We reported previously that infection of cultured endothelial cells with T. cruzi increased the synthesis of biologically active endothlein-1 (ET-1). In the present study, we examined the role of ET-1 in the cardiovascular system of CD1 mice infected with the Brazil strain of T. cruzi and C57BL/6 mice infected with the Tulahuen strain during acute infection. In the myocardium of infected mice myonecrosis and multiple pseudocysts were observed. There was also an intense vasculitis of the aorta, coronary artery, smaller myocardial vessels and the endocardial endothelium. Immunohistochemistry studies employing anti-ET-1 antibody revealed increased expression of ET-1 that was most intense in the endocardial and vascular endothelium. Elevated levels of mRNA for preproET-1, endothelin converting enzyme and ET-1 were observed in the same myocardial samples. Plasma ET-1 levels were significantly elevated in infected CD1 mice 10-15 days post infection. These observations suggest that increased levels of ET-1 are a consequence of the initial invasion of the cardiovascular system and provide a mechanism for infection-associated myocardial dysfunction.

K+ channels and gap junctions in the modulation of corporal smooth muscle tone.

Changes in the contractile status (i.e., contraction and relaxation) of corporal and arterial smooth muscle cells (myocytes) govern the flow of blood to and from the penis and, thus, ultimately have a major impact on erectile capacity. As with many other smooth muscle cell types, corporal myocyte contractility is inextricably linked to ion channel activity. Corporal smooth muscle cells possess a rich repertoire of ion channels, including calcium, chloride and potassium channels, as well as gap junction (intercellular) channels. Among these, the K(ATP) (i.e., the metabolically regulated K(+) channel) and the K(Ca) (i.e., maxi-K or large conductance, calcium-sensitive K(+) channel) nonjunctional channel subtypes, as well as connexin43-derived gap junction (intercellular) channels, are thought to be particularly relevant to the control of corporal myocyte contractility. In fact, whereas K(+) channels are an important convergence point for modulating cellular function, gap junctions are a major conduit for ensuring coordinated cellular, and thus tissue, function. The evidence documenting the presence and physiological relevance of K(+) channels and gap junctions to human erectile physiology and function is reviewed. Finally, one potentially revolutionary therapeutic strategy that takes advantage of the important contribution of K(+) channels and gap junctions to erectile physiology is described: maxi-K ion channel (gene) therapy.

Relationship between contraction and relaxation in human and rabbit corpus cavernosum.

Although alterations in corporeal smooth muscle tone undoubtedly play an important role in the etiology of erectile dysfunction, the relationship between the degree of corporeal smooth muscle contraction and the magnitude of the observed relaxation response has never been quantitated. Thus, in vitro studies were conducted to examine the relationship between alpha 1-adrenergic contractility, and relaxations elicited by the clinically and physiologically relevant vasorelaxants, nitroglycerin, nitroprusside, and prostaglandin E1. Corporeal tissues strips were isolated from impotent and potent men, as well as sexually mature rabbits, and precontracted over a wide range of phenylephrine doses, prior to exposure of each tissue to the same dose of vasorelaxant. Plots of percent contraction versus percent relaxation revealed that the relationship between contraction and relaxation was accurately described by a first order linear equation, and characterized by an inverse relationship in all tissues studied, for all vasorelaxants examined. Statistical analysis indicated that the slope of the regression line was significantly greater than unity in all corporeal tissues obtained from patients with organic impotence; however, corporeal tissues obtained from patients with documented erections and from sexually mature rabbits had significantly lower slopes that were indistinguishable from unity. The existence of an inverse relationship between contraction and relaxation, even in the absence of organic disease, emphasizes the importance of the level of basal corporeal smooth muscle tone per se. These studies provide further evidence implicating heightened adrenergic tone as a significant etiologic factor in erectile dysfunction.

Characterization of opioid receptor binding in adult and fetal sheep brain regions.

Opioid receptor binding was studied in 3 brain regions from maternal and fetal sheep at various gestational ages. [3H]dihydromorphine [( 3H]DHM) and [3H]D-Ala2,D-Leu5-enkephalin ([3H]DADLE) were employed as radioligands to characterize mu- and delta-opioid receptor binding sites, respectively. [3H]DHM binding was found to be highest in maternal cerebellum, intermediate in frontal cortex, and lowest in hippocampus. [3H]DADLE binding was highest in frontal cortex, intermediate in hippocampus and lowest in cerebellum. Cerebellum was the only tissue studied which contained more [3H]DHM than [3H]DADLE binding sites. Dissociation constants for [3H]DHM binding were similar in all 3 brain regions from both maternal and fetal sheep, while the dissociation constant for [3H]DADLE binding was significantly higher in cerebellum than in frontal cortex or hippocampus. Binding of both mu- and delta-receptor-selective ligands was 70% of maternal values in fetal cerebellum at 97-101 days of gestation and gradually increased over the remainder of the gestational period studied. Levels of [3H]DHM binding in frontal cortex and hippocampus were also similar to maternal levels at all timepoints studied. In contrast, [3H]DADLE binding was only 40-45% of maternal levels in fetal frontal cortex and hippocampus prior to 110 days of gestation, followed by a rapid increase in binding in both brain regions.

Frontiers in gene therapy for erectile dysfunction.

Complete sequencing of the human genome has made possible a new age of molecular medicine. The utilization of sophisticated genomic technologies has important implications to the understanding, diagnosis and treatment of erectile dysfunction. This report will review one aspect of the impact of the genomic revolution on urology, to wit, the preclinical evidence emerging from several laboratories indicating that gene therapy for erectile dysfunction may well provide the first safe and effective application of gene therapy to the treatment of human smooth muscle disease. The molecular targets explored thus far have concentrated largely on manipulating various aspects of the nitric oxide/guanylate cyclase/cGMP system, although genetic modulation of growth factors, calcium sensitization mechanisms and potassium channel expression have also been explored. Cell-based gene therapy techniques are also being explored. The apparent preclinical success of virtually all of these gene-based strategies reflects the multifactorial nature of erectile disease as well as the numerous regulatory mechanisms available for restoring erectile capacity. While technical hurdles remain with respect to the choice of delivery vectors, molecular target validation and duration of efficacy, 'proof-of-concept' has clearly been documented. The ultimate goal of gene therapy is to provide a safe, effective and specific means for altering intracavernous pressure 'on demand', while simultaneously eliminating the necessity for other forms of therapy, and moreover, without altering resting penile function, or the physiology of other organ systems. It is in these arenas that the groundbreaking potential of gene transfer technology to the treatment of erectile dysfunction will be fully tested. In fact, the potential benefits of the application of gene transfer techniques to this important medical problem is just now beginning to be appreciated/recognized.

Gap junctions and ion channels: relevance to erectile dysfunction.

The corporal myocyte is a critical determinant of erectile capacity whose functional integrity, in the vast majority of impotent patients, is sufficient to guarantee its relevance as a therapeutic target. As with numerous other smooth muscle cell types, ion channels are important modulators of corporal smooth muscle tone/contractility. As such, the transmembrane flow of ions (ie Ca(2+), K(+) and Cl(-)) plays an important role in modulating membrane potential and contractile status in individual human corporal smooth muscle cells, while intercellular ion flow ensures the functionality of myocyte cellular networks. The integral membrane proteins that selectively regulate many aspects of these critical transmembrane (eg K(+) and Ca(2+) channels) and intercellular (eg gap junctions) ionic movements have been identified. To date, the large conductance calcium-sensitive K(+) channel (ie K(Ca)), the metabolically regulated K+ channel (ie K(ATP)), and the L-type voltage-dependent Ca(2+) channel appear to be the most physiologically relevant nonjunctional ion channels. With respect to intercellular ionic/solute/second messenger movement, connexin43-derived gap junction channels are widely recognized as an obligatory component to normal integrative erectile biology. The presence of an intercellular pathway ensures that individual cellular alterations are carefully orchestrated in the rapid and syncytial fashion required for normal erectile function. This report reviews the known details concerning junctional and nonjunctional ion channels in human corporal tissue, and illustrates how one particular application of this knowledge, that is, preclinical studies utilizing low efficacy gene therapy (ie low transfection efficiency) with the K(Ca) channel has further confirmed the physiological relevance and therapeutic potential of gap junctions and ion channels to erectile physiology/dysfunction. International Journal of Impotence Research (2000) 12, Suppl 4, S15-S25.

Integrative erectile biology: the role of signal transduction and cell-to-cell communication in coordinating corporal smooth muscle tone and penile erection.

The contractility of corporal smooth muscle plays a critical role in the entire erectile process in man. Moreover, in the absence of severe vascular disease, or congenital or other structural abnormalities/malformations, relaxation of the corporal smooth muscle is both necessary and sufficient to elicit a sustained erection. As such, understanding the initiation, maintenance and modulation of corporal smooth muscle tone is an absolute prerequisite to the improved understanding, diagnosis and treatment of erectile dysfunction. Despite this fact, identification of both the precise mechanistic basis by which endogenous and exogenous vasomodulators exert their effects on individual corporal smooth muscle cells, and moreover, the process by which these signals are spread among the diverse array of parenchymal cells in the paired corpora, remains somewhat of a physiological enigma. Therefore, the goal of this report is two-fold: first, to review current knowledge of the regulation of corporal smooth muscle tone at the cellular and molecular level; and second, to outline a cogent explanation for the rapid and syncytial integration of the effects of diverse stimuli among corporal smooth muscle cells in the human penis.

Identification of a down-regulated mRNA transcript in corpus cavernosum from diabetic patients with erectile dysfunction.

Fifty per cent of men with diabetes have erectile dysfunction. Previous studies demonstrated that cultured smooth muscle cells from corpus cavernosum display significantly altered K+ channel function, PGE-induced cAMP accumulation, and endothelin-1 induced Ca2+ mobilization that are consistent with the pathophysiology of erectile dysfunction. Since defects in signal transduction frequently lead to altered gene expression, we examined differences in gene expression in corporal tissue excised from three diabetic patients with erectile dysfunction function and one patient with neurogenic erectile dysfunction. Using differential display, we identify a transcript expressed in tissue derived from the patient with impotence secondary to a radical prostectomy, but which was greatly reduced or absent in corporal tissue from all three diabetic patients examined. DNA sequence analysis indicates that this transcript has no significant homology to sequences presently deposited in the GenBank database. This suggests that altered gene expression may play a significant part in the etiology of erectile dysfunction.

Comparative studies of the maxi-K (K(Ca)) channel in freshly isolated myocytes of human and rat corpora.

Patch clamp techniques in freshly isolated myocytes from human corpora have documented that the large conductance calcium-sensitive K channel (K(Ca)) subtype represents an important convergence point for the modulation of corporal smooth muscle tone, and therefore, erectile capacity. Other recent studies indicate a similar role for the K(Ca) channel in the modulation of smooth muscle tone in the rat penis. Therefore, the explicit aim of this investigation was to evaluate and compare the characteristics of the K(Ca) channel subtype present in freshly isolated myocytes from rat and human corpora. In short, myocytes isolated from rat and human corpora retain their characteristic morphology and contractility in vitro, as evidenced by light microscopic studies of their respective responses to activation of the alpha1-adrenergic receptor subtype by phenylephrine (PE). Large conductance K+ currents commensurate with the presence of the K(Ca) channel were readily apparent in myocytes from both preparations. I-V curves constructed from cell-attached patches utilizing symmetric KCl solutions revealed the presence of a single channel slope conductance of approximately 200 pS for both rat and human myocytes. 1 mM TEA applied in the bath solution reversibly diminished whole cell outward K+ currents by approximately 50%, and also blocked the unitary K(Ca) channel activity observed in the outside-out patch mode. Addition of 2 mM 8-bromo-cAMP elicited a TEA-sensitive (1 mM) approximately 2-3 fold increase in the magnitude of the whole cell outward K+ currents in rat myocytes. Taken together, these data confirm and extend previous observations and provide strong evidence that the rat corporal smooth muscle K(Ca) channel has many similarities to its counterpart in the human penis.

The role of gap junctions and ion channels in the modulation of electrical and chemical signals in human corpus cavernosum smooth muscle.

Intercellular communication through aqueous intercellular channels, known as gap junctions, has been postulated to provide an important mechanism for coordinating the rapid and synchronous responses of corporal smooth muscle during human penile erection and detumescence. Mathematical modeling analyses of drug diffusion were utilized to examine the potential physiological importance of the intercellular pathway to the regulation of smooth muscle tone in the human corpus cavernosum. In addition, patch clamp analyses and optical imaging studies were conducted to assess the ionic basis for cellular excitability and homeostasis in cultured corporal smooth muscle cells. In short, the computer modeling studies demonstrated that intercellular communication through gap junctions is likely to be the 'preferred' pathway for coordination of cellular activation and syncytial smooth muscle responses in this tissue. Moreover, the observed ion channel diversity reveals even further complexities to the modulation of corporal smooth muscle tone.

The genetics and immunology of Peyronie's disease.

Peyronie's disease (PD) is a condition characterized by localized and often progressive fibrosis and scarring of the penis. This condition has an unknown etiology although several hypotheses have been proposed. These include traumatic, immunologic and genetic causes. We studied the genetics and immunology of PD using both molecular biologic and molecular genetic techniques. Men (n=283) with PD were identified by retrospective chart review of one physician's office practice. These men were contacted by telephone and asked to submit to an interview and blood test for genetic studies. Simultaneously, tissue and cells collected in the laboratory were examined by Western and Northern blot analysis for examination of protein and RNA for expression of HLA. Of the first 107 men contacted, 24 were available and consented to interview and blood testing. The mean age was 60.3 y with an average duration of PD of 4.9 y. One patient had a family history of PD while no patients had Dupuytren's contracture. Twenty patients were considered to have primary disease while four were secondary. Eleven patients had tissue prepared for Northern blot analysis and nine patients were the subject of Western blot analysis. All tissue, both Peyronie's and control expressed class I MHC while no tissue expressed class II MHC. The expression of mRNA of class I MHC was equal for Peyronie's and control patients while the expression at the protein level was less in the PD patients. We conclude that PD may have multiple etiologic agents. One cannot exclude a class II MHC association but in our population, HLA DQ is not expressed. Class I MHC may be involved as the expression of class I MHC protein is different in Peyronie's patients than in controls. Genetic studies are ongoing. International Journal of Impotence Research (2000) 12, Suppl 4, S127-S132.

Characterization of ATP-sensitive potassium channels in human corporal smooth muscle cells.

Potassium (K) channels play a significant role in modulating human corporal smooth muscle tone, and thus, erectile capacity. Recent pharmacological studies indicate that the metabolically-regulated K channel (KATP) may be an important modulator of human penile erection with significant therapeutic potential. The goal of these initial studies, therefore, was to utilize patch clamp techniques to characterize the putative KATP subtype(s) present in cultured and freshly isolated human corporal smooth muscle cells. In the cell-attached patch mode, two distinct unitary K+ currents were identified whose respective conductance values were similar in cultured and freshly isolated smooth muscle cells. In cultured myocytes, the measured conductance values in symmetric KCl (140 mM) solutions were 59.1 +/- 2.7 pS and 18.4 +/- 2.1 pS (n = 5 cells). Under identical experimental conditions in freshly isolated myocytes, corresponding conductance values were 59.2 +/- 3.7 pS and 18.5 +/- 2.4 pS, respectively (n = 4 cells). I-V curves constructed during step depolarization (-60 to +80 mV), revealed a linear I-V relationship for both unitary conductances. Single channel records documented that both conductances were reversibly inhibited by the application of ATP (1-3 mM) to the bath solution in the inside-out attached patch configuration. The unitary activity of both K channel subtypes was significantly increased by the application of pinacidil (10 microM) and levcromakalim (10 microM). Whole cell patch recordings documented a glibenclamide-sensitive, pinacidil- and levcromakalim-induced increase in the whole cell outward K+ current during step depolarization (-70 mV to +130 mV) of 105 +/- 37%, 139 +/- 42%, respectively. These data confirm and extend our previous observations, and provide the first evidence for the presence of KATP channel subtypes in human corporal smooth muscle cells.