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George J Christ

Publications and source records attributed to George J Christ.

At least 19 recordsLinked to original sources
PubMed2006

Diabetes-induced changes in the alternative splicing of the slo gene in corporal tissue.

OBJECTIVES: Erectile dysfunction is a common diabetic complication. Preclinical studies have documented that the Slo gene (encoding the BK or Maxi-K channel alpha-subunit) plays a critical role in erectile function. Therefore, we determined whether diabetes induces changes in the splicing of the Slo gene relevant to erectile function. METHODS: Reverse transcriptase-polymerase chain reaction was used to compare Slo splice variant expression in corporal tissue excised from control and streptozotocin (STZ)-induced diabetic Fischer F-344 rats. Splice variants were sequenced, characterized by patch clamping, and fused to green fluorescent protein to determine cellular localization. The impact of altered Slo expression on erectile function was further evaluated in vivo. RESULTS: A novel Slo splice variant (SVcyt, with a cytoplasmic location) was predominantly expressed in corporal tissue from control rats. STZ-diabetes caused upregulation of a channel-forming transcript SV0. Preliminary results suggest that SV0 was also more prevalent in the corporal tissue of human diabetic compared with nondiabetic patients. The change in isoform expression in STZ-treated rats was partially reversed by insulin treatment. Intracorporal injection of a plasmid expressing the SV0 transcript, but not SVcyt, restored erectile function in STZ-diabetic rats. CONCLUSIONS: Alternative splicing of the Slo transcript may represent an important compensatory mechanism to increase the ease with which relaxation of corporal tissue may be triggered as a result of a diabetes-related decline in erectile capacity.

Alternative Splicing↗
PubMed2006

Gap junction channel activity in short-term cultured human detrusor myocyte cell pairs: gating and unitary conductances.

Several independent lines of investigation indicate that intercellular communication through gap junctions modulates bladder physiology and, moreover, that altered junctional communication may contribute to detrusor overactivity. However, as far as we are aware, there are still no direct recordings of gap junction-mediated intercellular currents between human or rat detrusor myocytes. Northern and Western blots were used to identify connexin expression in frozen human bladder tissue and short-term cultured human detrusor myocytes. Double whole cell patch (DWCP) recording revealed that human detrusor myocyte cell pairs were well coupled with an average junctional conductance of 6.5 +/- 4.6 nS (ranging from 0.1 to 15 nS, n = 22 cell pairs). Macroscopic gap junction channel currents in human detrusor myocytes exhibited voltage dependence similar to homotypic connexin43. The normalized transjunctional conductance-voltage (G(j)-V(j)) relationship was symmetrical and well described by a two-state Boltzmann relation (G(min) approximately 0.33, V(0) = 63.6 mV, Z = 0.117 or equal to 2.95 gating charges), suggestive of a bilateral voltage-gated mechanism. In symmetric 165 mM CsCl, the measured single-channel slope conductance was approximately 120 pS for the fully open channel and approximately 26 pS for the major substate. Occasionally, other subconductance states were also observed. The single-channel mean open time declined with increasing V(j), accounting for the V(j)-dependent decline of macroscopic junctional current. Qualitatively similar electrophysiological characteristics were observed in DWCP of freshly isolated rat detrusor myocytes. These data confirm and extend previous observations and are consistent with reports in other smooth muscle cells types in which Cx43-mediated intercellular communication has been identified.

Animals↗
PubMed2006

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.

Animals↗
PubMed2006

Molecular mechanisms of detrusor and corporal myocyte contraction: identifying targets for pharmacotherapy of bladder and erectile dysfunction.

The Post-Genomic age presents many new challenges and opportunities for the improved understanding, diagnosis and treatment of human disease. The long-term goal is to identify molecular correlates of disease processes, and use this information to develop novel and more effective therapeutics. A major hurdle in this regard is ensuring that the molecular targets of interest are indeed relevant to the physiology and/or pathophysiology of the processes being studied, and, moreover, to determine if they are specific to the tissue/organ being investigated. As a first step in this direction, we have reviewed the literature pertaining to bladder and erectile physiology/pharmacology and dysfunction and attempted to summarize some of the critical molecular mechanisms regulating detrusor and corporal myocyte tone. Because of the vast amount of published data, we have limited the scope of this review to consideration of the calcium-mobilizing and calcium-sensitizing pathways in these cells. Despite obvious differences in phenotypic characteristics of the detrusor and corporal myocyte, there are some common molecular changes that may contribute to, for example, the increased myocyte contractility characteristic of bladder and erectile dysfunction (i.e. increased Rho kinase activity and decreased K(+) channel function). Of course, there are also some important distinctions in the pathways that modulate contractility in these two cell types (i.e. the contribution of ryanodine-sensitive calcium stores and the nitric oxide/cGMP pathways). This report highlights some of these similarities and distinctions in the hope that it will encourage scientific discourse and research activity in this area, eventually leading to an improved quality of life for those millions of individuals that are afflicted with bladder and erectile dysfunction.

Animals↗
PubMed2006

Effects of streptozotocin-induced diabetes on bladder and erectile (dys)function in the same rat in vivo.

OBJECTIVE: To establish the methods, feasibility and utility of evaluating the impact of diabetes on bladder and erectile function in the same rat, as more than half of diabetic patients have bladder dysfunction, and half of diabetic men have erectile dysfunction, but the severity of coincident disease has not been rigorously assessed. MATERIALS AND METHODS: In all, 16 F-344 rats had diabetes induced by streptozotocin (STZ), and were divided into insulin-treated (five) and untreated (11), and compared with age-matched controls (10), all assessed in parallel. All STZ rats were diabetic for 8-11 weeks. Cystometric studies were conducted on all rats, with cavernosometric studies conducted on a subset of rats. RESULTS: There were insulin-reversible increases in the following cystometric variables; bladder weight, bladder capacity, micturition volume, residual volume, micturition pressure and spontaneous activity (P < 0.05, in all, one-way analysis of variance, anova). Cavernosometry showed a diabetes-related, insulin-reversible decline in the cavernosal nerve-stimulated intracavernosal pressure (ICP) response at all levels of current stimulation (P < 0.05, in all one-way anova). Plotting erectile capacity (i.e. ICP) against bladder capacity showed no correlation between the extent of the decline in erectile capacity and the magnitude of the increase in bladder capacity. CONCLUSIONS: These studies extend previous work to indicate that the extent of diabetes-related bladder and erectile dysfunction can vary in the same rat. As such, these findings highlight the importance of evaluating the impact of diabetes on multiple organ systems in the lower urinary tract. Future studies using this model system should lead to a better understanding of the initiation, development, progression and coincidence of these common diabetic complications.

Animals↗
PubMed2006

Trypanosoma cruzi infection induces proliferation of vascular smooth muscle cells.

Trypanosoma cruzi infection causes cardiomyopathy and vasculopathy. Previous studies have demonstrated that infection of human umbilical vein endothelial and smooth muscle cells resulted in activation of extracellular signal-regulated kinase (ERK). In the present study, smooth muscle cells were infected with trypomastigotes, and immunoblot analysis revealed an increase in the expression of cyclin D1 and proliferating cell nuclear antigen (PCNA), important mediators of smooth muscle cell proliferation. Interestingly, after infection, the expression of caveolin-1 was reduced in both human umbilical vein endothelial cells and smooth muscle cells. Immunoblot and immunohistochemical analyses of lysates of carotid arteries obtained from infected mice revealed increased expression of PCNA, cyclin D1, its substrate, phospho-Rb (Ser780), and phospho-ERK1/2. The expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1), caveolin-1, and caveolin-3 was reduced in carotid arteries obtained from infected mice. There was an increase in the abundance of pre-pro-endothelin-1 mRNA in the carotid artery and aorta from infected mice. The ET(A) receptor was also elevated in infected arteries. ERK activates endothelin-1, which in turn exerts positive feedback activating ERK, and cyclin D1 is a downstream target of both endothelin-1 and ERK. There was significant incorporation of bromodeoxyuridine into smooth muscle cell DNA when treatment was with conditioned medium obtained from infected endothelial cells. Taken together, these data suggest that T. cruzi infection stimulates smooth muscle cell proliferation and is likely a result of the upregulation of the ERK-cyclin D1-endothelin-1 pathway.

Animals↗
PubMed2005

Proceedings of the Baltimore smooth muscle meeting: identifying research frontiers and priorities for the lower urinary tract.

PURPOSE: The myocyte is a major parenchymal cell of the lower urinary tract (LUT) in men and women. Significant phenotypic diversity ensures that myocytes subserve their important role in the physiologically distinct tissues and organs of the LUT, including the ureters, bladder, urethra, prostate, penis, vagina and myometrium. Coordinated contraction and relaxation of myocytes is required for normal organ function, while alterations in myocyte structure/function are implicated in the etiology of various LUT diseases/disorders. LUT diseases/disorders will continue to increase in an ever aging American population. The purpose of the Baltimore Smooth Muscle Meeting was to begin to identify some research frontiers and priorities. MATERIALS AND METHODS: A 1-day conference of some of the leading world experts in smooth muscle research was held at American Urological Association headquarters. These experts gave presentations in their areas of expertise and extensively discussed their work. This report details those interactions. RESULTS: There is astonishing diversity in the contribution of the myocyte to LUT physiology and dysfunction. Novel tools, technologies and ideas have produced increased understanding and identified new frontiers. CONCLUSIONS: An improved understanding of urogenital myocyte physiology, function and dysfunction is required better to elucidate disease mechanisms and develop novel therapeutics. The First Annual Baltimore Smooth Muscle Meeting provided the first step in this direction. More coordinated LUT myocyte funding initiatives, the further development of research resources, tools and technologies, and exploration of the urogenital system as a model system for studying systems biology and integrative physiology are among the highest research priorities.

Actin Cytoskeleton↗
PubMed2005

Trypanosoma cruzi infection induced changes in the innervation, structure and function of the murine bladder.

PURPOSE: The involvement of the lower urinary tract in chronic Chagas' disease has received little attention. Therefore, we investigated pathology and functional alterations in the bladder of Trypanosoma cruzi infected mice. MATERIALS AND METHODS: CD1 mice were infected with 5 x 10 T. cruzi trypomastigotes of the Brazil strain of T. cruzi. At day 100 after infection bladder structure and function were examined by pathological evaluation, magnetic resonance imaging and cystometric studies. RESULTS: The bladder in infected mice weighed more and were large, dilated, deformed, friable and thin walled compared with control mice. Magnetic resonance imaging confirmed these observations. Inflammation, fibrosis and ganglionitis was observed. Cystometric studies revealed that baseline, threshold and micturition pressures were increased in infected mice. Bladder overactivity and decreased bladder compliance were also noted in infected mice. There were no detectable differences in bladder capacity, micturition volume or residual volume between infected and uninfected mice. CONCLUSIONS: Bladder abnormalities may be a more common clinical sequelae of T. cruzi infection than previously appreciated.

Animals↗
PubMed2005

Role of endothelin 1 in the pathogenesis of chronic chagasic heart disease.

On the basis of previous observations, endothelin 1 (ET-1) has been suggested as contributing to the pathogenesis of Chagasic cardiomyopathy. Therefore, ET-1flox/flox;alpha-MHC-Cre(+) mice in which the ET-1 gene was deleted from cardiac myocytes and ET-1flox/flox;Tie 2 Cre(+) mice in which the ET-1 gene was deleted from endothelial cells were infected with Trypanosoma cruzi. Genetic controls for these cell-specific ET-1 knockout mice were used. Ninety percentage of all mice survived acute infection with the Brazil strain and were evaluated 130 days postinfection. Inflammation and fibrosis were observed in all infected mice; however, fibrosis was reduced in ET-1flox/flox;alpha-MHC-Cre(+) mice. Cardiac magnetic resonance imaging revealed that infection resulted in a significant increase in right ventricular internal diameter (RVID) in all mice except ET-1flox/flox;alpha-MHC-Cre(+) mice; i.e., RVID was not changed in infected ET-1flox/flox;alpha-MHC-Cre(+) mice. Echocardiography of the left ventricle demonstrated increased left ventricular end-diastolic diameter, reduced fractional shortening, and decreased relative wall thickness in infected mice. However, the magnitude of the changes was significantly less in ET-1flox/flox;alpha-MHC-Cre(+) mice compared to other groups. These data provide further evidence of a role for ET-1, particularly cardiac myocyte-derived ET-1, in the pathogenesis of chronic Chagasic cardiomyopathy.

Animals↗
PubMed2004

Gene therapy treatments for erectile and bladder dysfunction.

The postgenomic age presents many exciting challenges and opportunities for the application of molecular medicine to the treatment of lower urinary tract diseases. Chief among these are the therapeutic possibilities afforded to selectively modulate/alter gene expression in somatic cells to "normalize" aberrant cellular responses to the existing hormonal milieu. In this paper, this therapeutic strategy will be referred to as gene therapy or gene transfer. This article specifically reviews the potential use of gene therapy/transfer to the treatment of bladder and erectile dysfunction. Available treatments for both of these common urologic diseases/disorders have contraindications, untoward side effects, or limited efficacy in certain patient populations. Moreover, that genetic material can be locally administered in the bladder and penis removes a major therapeutic obstacle to the use of gene transfer. Thus far, "proof-of-concept" has been demonstrated in preclinical studies using cellular and molecular strategies on a variety of gene targets. The continuing evolution of gene transfer vectors and gene delivery technologies is expected to further enhance the selectivity, efficacy, and duration of gene therapy, making it a viable treatment option for the amelioration of lower urinary tract diseases/disorders.

Erectile Dysfunction↗
PubMed2004

Urogenital alterations in aged male caveolin-1 knockout mice.

PURPOSE: Caveolae are flask-shaped invaginations of the plasma membrane formed by the oligomerization of caveolins. Because only smooth muscle contains all caveolin (Cav) family members (Cav-1, 2 and 3), we examined the contribution of each caveolin to urogenital smooth muscle structure/function. MATERIALS AND METHODS: WT, Cav-1, 2, 3 and -1/3 knockout (KO) mouse bladders were characterized by Western blot, co-immunoprecipitation, immunofluorescence microscopy, electron microscopy, histochemistry and pharmacological techniques. Cystometric analysis was performed in conscious, freely moving mice. Other urogenital organs were investigated by histological analysis. RESULTS: The loss of bladder Cav-1 results in a marked decrease in Cav-2 but not Cav-3 expression. Ablation of Cav-3 fails to alter Cav-1 or Cav-2 expression. Deletion of Cav-1 results in the almost complete loss of caveolae, while Cav-2 KO and Cav-3 KO mouse smooth muscle showed a normal number of caveolae. The loss of Cav-1 generated caveolae led to significant urogenital changes in male mice (most marked by 12 months of age), namely 1) bladder weight-to-body weight ratios were increased, 2) the bladder smooth muscle layer was thickened, 3) the bladders had increased baseline, threshold and spontaneous pressures, 4) bladder strips showed a decreased contractile response to carbachol and KCl, and 5) these smooth muscle changes were accompanied by marked fluid accumulation in the prostate and seminal vesicles, with intracellular vacuolization in the kidneys. As such, male Cav-1 KO mice may be a useful animal model for studying LUTD (lower urinary tract dysfunction) that is so prevalent in aging male patients. CONCLUSIONS: The loss of Cav-1 and, thus, of most smooth muscle cell caveolae results in significant bladder dysfunction and urogenital organ changes in aged male mice.

Animals↗
PubMed2004

Trypanosoma cruzi infection activates extracellular signal-regulated kinase in cultured endothelial and smooth muscle cells.

Trypanosoma cruzi infection causes cardiomyopathy and vasculopathy. We examined the consequence of this infection for the mitogen-activated protein kinase (MAPK) pathways, which regulate cell proliferation in cultured human umbilical vein endothelial and vascular smooth muscle cells. Infection of these cells resulted in activation of extracellular signal-regulated kinases 1and 2 (ERK1/2) but not c-Jun N-terminal kinase or p38 MAPK. Treatment of these cells with the MAPK kinase inhibitor PD98059 prior to infection blocked the increase in phosphorylated ERK1/2 seen with infection. Heat-killed parasites did not activate ERK1/2, indicating that activation of ERK1/2 was dependent on infection of these cells by live parasites. Furthermore, transfection with dominant-negative Raf(301) or Ras(N17) constructs reduced the infection-associated levels of phospho-ERK1/2, indicating that the activation of ERK1/2 involved the Ras-Raf-ERK pathway. Infection also resulted in an increase in activator protein 1 (AP-1) activity, which was inhibited by transfection with a dominant-negative Raf(301) construct. T. cruzi-infected endothelial cells secreted endothelin-1 and interleukin-1beta, which activated ERK1/2 and induced cyclin D1 expression in uninfected smooth muscle cells. These data suggest a possible molecular paradigm for the pathogenesis of the vasculopathy and the cardiovascular remodeling associated with T. cruzi infection.

Animals↗
PubMed2004

Intracorporal injection of hSlo cDNA restores erectile capacity in STZ-diabetic F-344 rats in vivo.

The ability of gene transfer with the pore-forming subunit of the human maxi-K channel (hSlo) to ameliorate the decline in erectile capacity commensurate with 12-24 wk of streptozotocin (STZ)-diabetes was examined in 181 Fischer-344 rats. A 2-mo period of STZ-diabetes was induced before gene transfer, and erectile capacity was evaluated by measuring the intracavernous pressure response (ICP) to cavernous nerve (CN) stimulation (ranging from 0.5 to 10 mA). In the first series of experiments, ANOVA revealed increased CN-stimulated ICP responses at 1 and 2 mo postinjection of 100 microg pcDNA-hSlo compared with control values. A second series of experiments further examined the dose dependence and duration of gene transfer. The ICP response to submaximal (0.5 mA) and maximal (10 mA) nerve stimulation was evaluated 3 or 4 mo postinjection of a single dose of pcDNA-hSlo ranging from 10 to 1,000 microg. ANOVA again revealed that hSlo overexpression was associated with increased CN-stimulated ICP responses compared with responses in corresponding control animals. Histological studies revealed no immune response to the presence of hSlo. PCR analysis documented that expression of both plasmid and transcript were largely confined to the corporal tissue. In the third series of pharmacological experiments, hSlo gene transfer in vivo was associated with iberiotoxin-sensitive relaxation responses to sodium nitroprusside in corporal tissue strips in vitro. The latter data indicate that gene transfer produces functional maxi-K channels that participate in the modulation of corporal smooth muscle cell tone. Taken together, these observations suggest a fundamental diabetes-related change in corporal myocyte maxi-K channel regulation, expression, or function that may be corrected by expression of recombinant hSlo.

Animals↗
PubMed2003

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.

Animals↗