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Biomedical subjects

G J Christ

Publications and source records attributed to G J Christ.

At least 19 recordsLinked to original sources
PubMed2003

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.

Erectile Dysfunction↗
PubMed2003

The successful long-term treatment of age related erectile dysfunction with hSlo cDNA in rats in vivo.

PURPOSE: We have previously reported that 1 intracorporeal injection of 100 microg hSlo/pcDNA reversed the effect of aging on erectile function in a rat model in vivo for at least 2 months. We report our further investigations of the amplitude, duration and physiological relevance of this novel gene transfer approach. MATERIALS AND METHODS: A total of 191 retired breeder Sprague-Dawley rats were given a single intracavernous injection of phosphate buffered saline, 1,000 microg pcDNA, or 10, 100 or 1,000 microg pcDNA/hSlo. The animals were studied 1 to 6 months after injection. The intracorporeal pressure (ICP) response to cavernous nerve stimulation and immunostaining as well as hematoxylin and eosin staining were done to evaluate effector nerve integrity and tissue histology, respectively. RESULTS: Gene transfer prevented an age related decrease in resting ICP and a physiologically relevant, significant effect on normalizing erection in vivo, as determined by submaximal (0.5 mA) and maximal (4.0 mA) cavernous nerve stimulation. The effects were observed 1 month after transfection and sustained for 6 months at the 100 and 1,000 microg doses of pcDNA/hSlo (p <0.026). CONCLUSIONS: The physiological manifestations of gene transfer were detected as an amelioration of the age related decrease in resting ICP, and parallel increase in the magnitude of the cavernous nerve stimulated an ICP response to a level at which visible erections were again observed in this rat model of aging in vivo.

Animals↗
PubMed2002

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.

Animals↗
PubMed2002

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.

Adenosine Triphosphate↗
PubMed2001

Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.

Caveolin-1 is the principal structural protein of caveolae membranes in fibroblasts and endothelia. Recently, we have shown that the human CAV-1 gene is localized to a suspected tumor suppressor locus, and mutations in Cav-1 have been implicated in human cancer. Here, we created a caveolin-1 null (CAV-1 -/-) mouse model, using standard homologous recombination techniques, to assess the role of caveolin-1 in caveolae biogenesis, endocytosis, cell proliferation, and endothelial nitric-oxide synthase (eNOS) signaling. Surprisingly, Cav-1 null mice are viable. We show that these mice lack caveolin-1 protein expression and plasmalemmal caveolae. In addition, analysis of cultured fibroblasts from Cav-1 null embryos reveals the following: (i) a loss of caveolin-2 protein expression; (ii) defects in the endocytosis of a known caveolar ligand, i.e. fluorescein isothiocyanate-albumin; and (iii) a hyperproliferative phenotype. Importantly, these phenotypic changes are reversed by recombinant expression of the caveolin-1 cDNA. Furthermore, examination of the lung parenchyma (an endothelial-rich tissue) shows hypercellularity with thickened alveolar septa and an increase in the number of vascular endothelial growth factor receptor (Flk-1)-positive endothelial cells. As predicted, endothelial cells from Cav-1 null mice lack caveolae membranes. Finally, we examined eNOS signaling by measuring the physiological response of aortic rings to various stimuli. Our results indicate that eNOS activity is up-regulated in Cav-1 null animals, and this activity can be blunted by using a specific NOS inhibitor, nitro-l-arginine methyl ester. These findings are in accordance with previous in vitro studies showing that caveolin-1 is an endogenous inhibitor of eNOS. Thus, caveolin-1 expression is required to stabilize the caveolin-2 protein product, to mediate the caveolar endocytosis of specific ligands, to negatively regulate the proliferation of certain cell types, and to provide tonic inhibition of eNOS activity in endothelial cells.

Albumins↗
PubMed2001

Schistosoma hematobium soluble egg antigens induce proliferation of urothelial and endothelial cells.

Bladder carcinoma accounts for 26% of reported human malignancies in Egypt, and has been strongly associated with urinary schistosomiasis. Nevertheless, the immediate role of schistosomal egg proteins in bladder carcinogenesis is unexplored. We investigated the effects of crude soluble egg antigens (SEA) of Schistosoma hematobium on urothelial cell proliferation. The proliferation of bovine endothelial Endo, human urothelial J82 and smooth muscle SMC cell lines was assessed by low-density growth assays. SEA induced proliferation of both J82 and Endo cells in a dose-dependent fashion, but not SMC. Preboiling or proteinase K treatment of SEA abolished its effect. In addition, SEA enhanced urothelial expression of B-cell translocation protein (BTG1) and human proliferating cell nuclear antigen (PCNA) mRNAs. Given the strong correlation between cell proliferation and carcinogenesis, the findings suggest that crude SEA may play some role in schistosomal bladder carcinogenesis.

Animals↗
PubMed2001

Integrative erectile biology. The effects of age and disease on gap junctions and ion channels and their potential value to the treatment of erectile dysfunction.

Initiation, maintenance, and modulation of corporal smooth muscle tone are critically dependent upon agonist-induced changes in intracellular calcium levels and mobilization as well as transmembrane calcium flux. The transient control of myocyte excitability and contractility at the cellular level is inextricably linked to membrane potential, which, in turn, is modulated by potassium ion efflux through one of the four known corporeal smooth muscle potassium ion channels. Corporal tissue responses are subsequently coordinated by means of the movement of intracellular second messenger molecules (i.e., IP3, cAMP, cGMP) and ions (i.e., K+ and Ca2+) among the corporal myocytes by means of intercellular communication through gap junction channels. Knowledge of the critical contribution of these interlinking cellular (nonjunctional ion channels [e.g., maxi-K]) and tissue (gap junction channels [e.g., connexin 43]) systems to the modulation of erectile capacity has provided the scientific rationale for the promulgation of the successful preclinical testing of hSlo ion channel gene therapy for the normalization of erectile status in both aged and diabetic rats.

Aging↗
PubMed2001

Significant physiological roles of ancillary penile nerves on increase in intracavernous pressure in rats: experiments using electrical stimulation of the medial preoptic area.

The objectives of this work were to evaluate the contributions of the ancillary penile nerves to penile erection in male rats in vivo. We investigated the effects of unilateral and bilateral transection of the cavernous nerve (main penile nerve) on the increase in intracavernous pressure (ICP) following electrical stimulation of the medial preoptic area (MPOA) in male rats in vivo. After unilateral or bilateral transection of the cavernous nerve (main penile nerve), the ICP responses showed decreases of 28% and 55%, respectively compared to those ICP responses before transection. In other words, even after bilateral transection of the cavernous nerve, significant increases in the ICP response following central stimulation were observed. In contrast to these findings, the ICP response was completely eliminated following bilateral pelvic nerve transection. These data suggested that the ancillary penile nerves, which originate from the major pelvic ganglia, have a complementary role to the cavernous nerves in the autonomic motor innervation of the penis.

Animals↗
PubMed2001

Intercellular communication in cultured human vascular smooth muscle cells.

Intercellular communication through gap junction channels plays a fundamental role in regulating vascular myocyte tone. We investigated gap junction channel expression and activity in myocytes from the physiologically distinct vasculature of the human internal mammary artery (IMA, conduit vessel) and saphenous vein (SV, capacitance vessel). Northern and Western blots documented the presence of connexin43 (Cx43) in frozen tissues and cultured cells from both vessels. Northern blots also confirmed the presence of Cx40 mRNA in cultured IMA and SV myocytes. Dual whole cell patch-clamp experiments revealed that macroscopic junctional conductance was voltage dependent and characteristic of that observed for Cx43. In the majority of records, in both vessels, single-channel activity was dominated by a main-state conductance of 120 pS, with subconducting events comprising less than 10% of the amplitude histograms. However, some records showed "atypical" unitary events that had a conductance similar to Cx40 (approximately 140-160 pS), but gating behavior like that of Cx43. As such, it is conceivable that the presence and coexpression of Cx40 and Cx43 in IMA and SV myocytes may result in heteromeric channel formation. Nonetheless, in terms of gating, Cx43-like behavior clearly dominates.

Aged↗
PubMed2001

Central modulation of the NO/cGMP pathway affects the MPOA-induced intracavernous pressure response.

Alterations in the nitric oxide (NO)/cGMP levels in hypothalamic nuclei, including the medial preoptic area (MPOA), regulate critical aspects of sexual behavior and penile reflexes. However, the effects of altered central nervous system (CNS) NO/cGMP levels at the end organ level, that is, on the magnitude/quality of the erection so achieved [intracavernous pressure (ICP) response], has yet to be evaluated. The goal of this report was to evaluate the effects of intrathecal administration of modulators of NO and cGMP levels on ICP responses to stimulation of the MPOA and cavernous nerve in rats in vivo. In all cases, intrathecal administration of compounds that increase and decrease cGMP and NO levels, respectively, was associated with corresponding increases and decreases in the MPOA-stimulated ICP response. Specifically, sodium nitroprusside (SNP), 8-bromo-cGMP, and sildenafil increased the MPOA-stimulated ICP response, whereas N(omega)-nitro-L-arginine methyl ester reduced it. None of the intrathecal treatments had detectable effects on blood pressure or the cavernous nerve-stimulated ICP response, although intravenous sildenafil increased the latter. These data clearly indicate that intrathecal drug administration affects central and not peripheral neural mechanisms and, moreover, documents that CNS NO/cGMP levels can affect erectile capacity per se (i.e., ICP) in the rat model.

Animals↗
PubMed2001

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

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.

Administration, Intravesical↗
PubMed2001

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.

Animals↗
PubMed2000

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.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

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.

Aging↗