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.

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.

Molecular studies of human connexin 43 (Cx43) expression in isolated corporal tissue strips and cultured corporal smooth muscle cells.

Intercellular communication plays an important role in erectile function. The goal of this study, therefore, was two-fold. Firstly, to determine if cultured corporal smooth muscle cells provide a valid model system for evaluating the role of junctional communication to erectile physiology, and secondly, to explore the possibility that there may be age-related alterations in Cx43 mRNA expression. Human corpus cavernosum tissue was obtained from 31 patients with a mean age of 58 (range 27-89), while cell cultures were developed from 21 distinct patients with a mean age of 57 (range 26-59). Northern blots revealed that mRNA for Cx43 was expressed at detectable levels in all samples examined. It migrated as a transcript with an apparent size of 3.1 Kb. Western blots revealed the presence of multiple bands of Cx43 protein in both tissues and cells. However, Cx43 protein in tissue predominantly migrated as a 45 kDa band, while the Cx43 from cultured cells predominantly migrated as 41 kDa band. Cx43 mRNA expression was similarly heterogeneous in both frozen tissues and cultured cells. An approximately 3-5-fold increase in Cx43 mRNA levels was observed in cultured cells relative to frozen tissue, but the expression of Cx43 mRNA was not further altered upon passaging (p1-5). When Cx43 mRNA levels were normalized, and expressed as a ratio of the Cx43/beta-tubulin mRNA, there was a significant negative correlation between patient age and Cx43 levels on frozen tissues, but not on cultured cells. We conclude that: (1) There is similar heterogeneity/variability in Cx43 mRNA levels in frozen tissues and cultured cells derived from human corpus cavernosum. (2) That the expression of Cx43 mRNA in cultured cells is sufficiently stable, and similar to, expression levels in tissue as to provide a valid and physiologically relevant model system for further studying the role(s) of Cx43 in the regulation of penile erection. (3) There is a statistically significant, albeit modest, negative correlation between the Cx43/beta-tubulin ratio and patient age in frozen corporal tissue strips, but not on cultured corporal smooth muscle cells. Such observations provide further evidence for the plasticity of intercellular communication in the erectile process. Moreover, the similarities in the apparent regulation of Cx43 mRNA levels and that of the putative 'housekeeping' gene beta-tubulin, may suggest that Cx43 is constitutively synthesized in this tissue.

Membrane-associated inositol hexakisphosphate binding in bovine retina.

We investigated the InsP6 binding proteins in bovine retinal membranes and rod outer segments (ROS) by radioligand binding assay and western blotting. The relative affinity of InsP6 for the binding protein was determined by competitive binding of [3H]-InsP6 with increasing concentrations of the unlabeled InsP6 or other isomers. InsP6 specifically binds to both bovine retinal membranes and ROS; maximum binding was achieved after one-hour incubation at 4 degrees C and was unchanged up to 2 h. Tris-HCl or acetate buffer was equally suitable for the binding assay over a broad range of pH, although specific binding was slightly increased at acidic pH. The order of potencies of displacement was InsP6 > Ins(1,3,4,5,6)P5 > Ins(1,3,4,6)P4 = Ins(1,3,4,5)P4, whereas Ins(1,4,5)P3, Ins(1,4)P2, Ins(4,5)P2, and Ins(1)P were not effective displacers. Scatchard analyses of the binding data were consistent with an equilibrium dissociation constant (Kd) of 2.5 +/- 0.2 microM and maximal binding capacity (Bmax) of 123.7 +/- 25.0 pmol/mg at pH 7.4. Western blotting was used to detect whether AP-2 (an InsP6 binding protein) is present in the retina. Immunoreactivity to AP-2 alpha and beta subunits was found in retinal membranes and ROS. Thus, bovine retinal membranes and ROS contain membrane-associated InsP6 binding protein(s) which is distinct from proteins that bind InsP5, InsP4, or InsP3.

Gi proteins and the response to 5-hydroxytryptamine in porcine cultured endothelial cells with impaired release of EDRF.

1. The receptor-mediated release of endothelium-derived relaxing factor(s) (EDRF) requires the presence of different functional G proteins in endothelial cells. Release of EDRF in response to 5-hydroxytryptamine (5-HT), which involves activation of pertussis toxin-sensitive Gi proteins, is impaired in both regenerated endothelium of the coronary artery following balloon catheterization and in porcine cultured endothelial cells. This study used porcine cultured endothelial cells as a model of regenerated endothelium to determine if the abnormal release of EDRF in response to 5-HT may be associated with the loss of functional pertussis toxin-sensitive Gi proteins. 2. Binding studies on porcine cultured endothelial cells demonstrated specific binding sites for [3H]-5-HT. Scatchard analyses revealed a single binding site for [3H]-5-HT with Kd of 7.2 +/- 3.5 nM and maximal binding (Bmax) of 121.4 +/- 51.3 fmol mg-1 protein. Binding of [3H]-5-HT was displaced by methiothepin (5-HT1 and 5-HT2 antagonist; Ki = 6.2 +/- 1.2 nM), but not by ketanserin (preferential 5-HT2 antagonist). 3. Gi alpha 1 protein was expressed in cultured but not in native endothelial cells. Gi alpha 2 and Gi alpha 3 proteins were expressed to significant levels in porcine native and cultured endothelial cells, as detected by Northern and Western blot analysis. 4. In membranes from cultured endothelial cells, two bands of 40 and 41 kDa, which corresponded to the Gi alpha 2 and the combination of Gi alpha 3-Gi alpha 1 proteins, respectively, were ADP-ribosylated by pertussis toxin. The labelling intensity was Gi alpha 2>Gi alpha 3-Gi alpha l and the amount of ADP-ribosylation was not different between porcine native and cultured endothelial cells. Stimulation of the cultured cells with 5-HT (3 x 10-6 M; 4 min) decreased significantly further ADP-ribosylation of Gi alpha 2 by pertussis toxin, but not that of Gi alpha 3 and/or Gi alpha l.5. The present results suggest that porcine endothelial cell culture may lead to the abnormal expression of Gi alpha l protein and that the dysfunctional release of EDRF from cultured porcine endothelial cells in response to 5-HT is not associated with the loss of Gi alpha proteins or the absence of 5-HT binding sites.

Inositol-1,4,5-trisphosphate receptors in the vertebrate retina.

Evidence has shown an activation of phosphatidylinositol 4,5-bisphosphate (PIP2) specific phospholipase C (PtdIns-PLC) by light in the vertebrate retina and rod outer segments (ROS), suggesting important roles for its two metabolites, 1,2-diacylglycerol (DG) and inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]. DG activates protein kinase C (PKC) and Ins(1,4,5)P3 releases bound intracellular calcium. Since Ca2+ plays an important role in light adaptation, the presence of Ins(1,4,5)P3 receptors in ROS may indicate a regulatory role of Ins(1,4,5)P3 to the free Ca2+ content. In the present study, we investigated the Ins(1,4,5)P3 receptors in whole retinal membranes and several subcellular fractions prepared from bovine retinas. Scatchard analyses of binding data for retinal membrane preparations showed a single, high-affinity binding site with equilibrium dissociation constant (Kd) of 24 +/- 2 nM and maximal binding capacity (Bmax) of 353 +/- 15 fmol/mg protein at pH 7.4. Specific binding was found in both small and large synaptosomal preparations representing inner and outer plexiform layers, respectively. A detectable, but low abundance of Ins(1,4,5)P3-specific binding in ROS was observed at both pH 7.4 and 8.3, but no specific binding of Ins(1,4,5)P3 was found in isolated outer segment discs. The binding of Ins(1,4,5)P3 in ROS was reduced by addition of ATP, suggesting a regulatory role for this nucleotide. Addition of calcium, sodium, and potassium ions also reduced specific binding of Ins(1,4,5)P3. Immunocytochemical studies indicate intense staining in the inner segment and extending to the ROS. Inner and outer plexiform layers were also stained. These findings show that the Ins(1,4,5)P3 receptor is present in photoreceptor cells and inner and outer plexiform layers in the vertebrate retina.

Muscarinic cholinergic receptor-mediated phosphoinositide metabolism in peripheral nerve.

Few receptor-mediated phenomena have been detected in peripheral nerve. In this study, the ability of the muscarinic cholinergic receptor agonist carbamylcholine to enhance phosphoinositide (PPI) breakdown in sciatic nerve was investigated by measuring the accumulation of inositol phosphates. Rat sciatic nerve segments were prelabeled with myo-[3H]inositol and then incubated either with or without carbamylcholine in the presence of Li+. [3H]Inositol monophosphate ([3H]IP) accumulation contained most of the radioactivity in inositol phosphates, with [3H]inositol bisphosphate ([3H]IP2) and [3H]inositol trisphosphate ([3H]IP3) accounting for 7-8% and 1-2% of the total, respectively. In the presence of 100 microM carbamylcholine, [3H]IP accumulation increased by up to 150% after 60 min. The 50% effective concentration for the response was determined to be 20 microM carbamylcholine and stimulated IP generation was abolished by 1 microM atropine. Enhanced accumulation of IP2 and IP3 was also observed. Determination of the pA2 values for the muscarinic receptor antagonists atropine (8.9), pirenzepine (6.5), AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (5.7), and 4-diphenylacetoxy-N-methylpiperidinemethiodide (4-DAMP) (8.6) strongly suggested that the M3 muscarinic receptor subtype was predominantly involved in mediating enhanced PPI degradation. Following treatment of nerve homogenates and myelin-rich fractions with pertussis toxin and [32P]NAD+, the presence of an ADP-ribosylated approximately 40-kDa protein could be demonstrated. The results indicate that peripheral nerve contains key elements of the molecular machinery needed for muscarinic receptor-mediated signal transduction via the phosphoinositide cycle.

Characterization of the train-of-four response in fast and slow muscles: effect of d-tubocurarine, pancuronium, and vecuronium.

The in vivo cat soleus and gastrocnemius muscles were used to compare isometric contraction strength and the train-of-four (T4) response (2 Hz for 2 s) of two muscle types (fast and slow) during onset of competitive neuromuscular blockade in order to determine the extent of the correlation between twitch depression and T4 fade. Prior to drug administration the muscles that were studied differed significantly in that the T4 ratio was 1.0 in the gastrocnemius and only 0.87 in the soleus. Three competitive neuromuscular-blocking agents were compared: d-tubocurarine, pancuronium, and vecuronium. d-Tubocurarine was found to produce a close correlation between the degrees of twitch strength depression and T4 for both muscles. However, these muscles demonstrated significantly different ED50 values (105 micrograms/kg for gastrocnemius, 150 micrograms/kg for soleus). Pancuronium also produced a similar relationship between twitch strength depression and T4 decrement for each muscle. In this case, however, there was little difference in their ED50 values for twitch depression (11.5 micrograms/kg for gastrocnemius, 13 micrograms/kg for soleus). The effects of vecuronium were quite different from the other two muscle relaxants. Although vecuronium produced a comparable correlation between twitch tension and T4 fade in fast muscle, no such relationship was found to exist in slow muscle. Even when the twitch strength was blocked to 18% of control, the soleus T4 response was depressed to only 75% of control. These results highlight major differences among competitive neuromuscular-blocking agents and suggest multiple sites of action.