Samuel Chacko

Diabetic Bladder Dysfunction: Current Translational Knowledge

PURPOSE Diabetes mellitus, a metabolic disorder caused by an absolute or relative deficiency of insulin, is a debilitating and costly disease with multiple serious complications. Lower urinary tract complications are among the most common complications of diabetes mellitus. The most common, bothersome lower urinary tract complication of diabetes mellitus is diabetic cystopathy or diabetic bladder dysfunction. We reviewed the current translational knowledge of diabetic bladder dysfunction. MATERIALS AND METHODS We performed a search of the English literature through PubMed. The key words used were diabetes and bladder dysfunction or cystopathy. Our data and perspective are provided for consideration of the future direction of research. RESULTS Despite traditional recognition of diabetic bladder dysfunction as a voiding problem characterized by poor emptying and overflow incontinence, recent clinical and experimental evidence indicate storage problems such as urgency and urge incontinence in diabetes mellitus cases. Recent experimental evidence from studies of diabetic bladder dysfunction in small animal models of diabetes mellitus show a temporal effect on diabetic bladder dysfunction. Early phase diabetes mellitus causes compensated bladder function and the late phase causes decompensated bladder function. The temporal theory could plausibly provide the scientific road map to correlate clinical and experimental findings, and identify the role of mechanisms such as polyuria, hyperglycemia, oxidative stress, autonomic neuropathy and decompensation of the bladder contractile apparatus in the creation of clinical and experimental manifestations of diabetic bladder dysfunction. CONCLUSIONS Diabetic bladder dysfunction includes time dependent manifestations of storage and emptying problems. Identifying mechanistic pathways would lead to the identification of therapeutic intervention.

Enhanced Force Generation by Corpus Cavernosum Smooth Muscle in Rabbits With Partial Bladder Outlet Obstruction

PURPOSE Growing clinical evidence suggests that benign prostatic hyperplasia induced partial bladder outlet obstruction is associated with an increased incidence of erectile dysfunction. We determined whether corpus cavernosum smooth muscle from rabbits with partial bladder outlet obstruction show any molecular or functional differences versus controls. MATERIALS AND METHODS Force generation and relaxation of corpus cavernosum smooth muscle 2 weeks after partial bladder outlet obstruction by 125 mM. KCl, phenylephrine and field stimulation were determined. Expression of total smooth muscle myosin and alternatively spliced smooth muscle myosin isoforms were determined by reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative competitive RT-PCR, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Corpus cavernosum smooth muscle from sections of the penis were analyzed morphologically by immunofluorescence microscopy using antibodies to smooth muscle myosin and neurofilament protein. RESULTS Corpus cavernosum smooth muscle from rabbits with partial bladder outlet obstruction generated 40% to 50% more force than that of sham operated rabbits in response to KCl or phenylephrine and was more difficult to relax. Although quantitative competitive RT-PCR and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that corpus cavernosum smooth muscle from rabbits with partial bladder outlet obstruction expressed only slightly more total smooth muscle myosin at the messenger RNA and protein levels, expression of the high adenosine triphosphatase isoform SM-B increased 2-fold. Morphological examination of corpus cavernosum smooth muscle sections revealed decreased innervation and increased smooth muscle bundle size. CONCLUSIONS We present the novel finding of molecular and functional changes in the corpus cavernosum smooth muscle associated with partial bladder outlet obstruction. Although the inclusion of sham operation ruled out direct injury during surgery, a change in corpus cavernosum smooth muscle innervation induced by nerve compression by the ligature is likely to induce trophic changes in the corpus cavernosum smooth muscle leading to over expression of the SM-B smooth muscle myosin isoform, increased contractility and impaired relaxation.

DNA synthesis, mitosis, and differentiation in cardiac myogenesis

Abstract Cardiac muscle cells obtained by trypsinizing 5-day chick embryonic heart were cultured as single cells in separate culture dishes. Using this technique, problems of heterotypic cell interactions, “overgrowth” of one cell type, etc., are eliminated. Experiments performed on these single cell cultures show that the muscle cells in the embryonic chick hearts differ in morphology, including content of cross-striated myofibrils; in ability to synthesize DNA and undergo mitosis; and in frequency of contraction. Contracting cells containing cross-striated myofibrils undergo mitosis in vitro, giving rise to spontaneously beating daughter cells. These daughter cells contain cytoplasmic fibrils, which bind fluorescein-labeled antimyosin immediately after cytokinesis. Some cardiac muscle cells from 5-day heart do not divide in culture; the rest undergo 1–5 doublings. This preliminary investigation suggests that the new muscle cells formed during cardiac growth are derived from mitotically active “overtly” differentiated cardiac muscle cells.

The mechanism for the inhibition of actin-activated ATPase of smooth muscle heavy meromyosin by calponin.

Calponin, an actin-binding protein, inhibited the acto-heavy meromyosin (HMM) MgATPase and lowered the binding of HMM to actin. The amount of calponin bound to actin or tropomyosin-actin was the same when the ATPase was inhibited 80-90%. While the KATPase was diminished only less than 2-fold in the presence of calponin, the Vmax was decreased 6-fold and 2-fold with actin and tropomyosin-actin, respectively. A comparison of the kinetic constants for the ATP hydrolysis obtained in the presence of actin-calponin and tropomyosin-actin-calponin revealed that the tropomyosin augmented the Vmax 5-fold from the inhibited level, but there was no effect on the KATPase.

Smooth Muscle Hypertrophy Following Partial Bladder Outlet Obstruction Is Associated with Overexpression of Non-Muscle Caldesmon

Partial bladder outlet obstruction (PBOO) induces remodeling of urinary bladder smooth muscle (detrusor). We demonstrate an increase in bladder wall mass, muscle bundle size, and a threefold increase in the cross-sectional area of detrusor myocytes following PBOO in male New Zealand White rabbits compared to that of controls. Some bladders with detrusor hypertrophy function close to normal (compensated), whereas others were dysfunctional (decompensated), showing high intravesical pressure, large residual urine volume, and voiding difficulty. We analyzed the expression of smooth muscle-specific caldesmon (h-CaD) and non-muscle (l-CaD) by Western blotting, RT-PCR, and real-time PCR. The expression of l-CaD is increased significantly at the mRNA and protein levels in the decompensated bladders compared to that of normal and compensated bladders. The CaD was also co-localized with myosin containing cytoplasmic fibrils in cells dissociated from obstructed bladders and cultured overnight. Our data show that the inability of decompensated bladders to empty, despite detrusor hypertrophy, is associated with an overexpression of l-CaD. The level of l-CaD overexpression might be a useful marker to estimate the degree of detrusor remodeling and contractile dysfunction in PBOO.

Experimental colitis triggers the release of substance P and calcitonin gene-related peptide in the urinary bladder via TRPV1 signaling pathways.

Clinical data provide evidence of high level of co-morbidity among genitourinary and gastrointestinal disorders characterized by chronic pelvic pain. The objective of this study was to test the hypothesis that colonic inflammation can impact the function of the urinary bladder via activation of TRPV1 signaling pathways followed by alterations in gene and protein expression of substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory neurons and in the bladder. Inflammation was induced by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS, 12.5mg/kg), and desensitization of TRPV1 receptors was evoked by intracolonic resiniferatoxin (RTX, 10(-)(7)M). mRNA and protein concentrations of CGRP and SP were measured at 3, 5 and 30 days. RTX instillation in the colon caused 3-fold up-regulation of SP mRNA in the urinary bladder at day 5 (n=7, p ≤ 0.05) followed by 35-fold increase at day 30 (n=5, p ≤ 0.05). Likewise, TNBS colitis triggered 15.8-fold up-regulation of SP mRNA 1 month after TNBS (n=5, p ≤ 0.05). Desensitization of colonic TRPV1 receptors prior to TNBS abolished SP increase in the urinary bladder. RTX led to 4.3-fold increase of CGRP mRNA at day 5 (n=7, p ≤ 0.05 to control) in the bladder followed by 28-fold increase at day 30 post-RTX (n=4, p ≤ 0.05). Colitis did not alter CGRP concentration during acute phase; however, at day 30 mRNA level was increased by 17.8 ± 6.9-fold (n=5, p ≤ 0.05) in parallel with 4-fold increase in CGRP protein (n=5, p ≤ 0.01) in the detrusor. Protein concentration of CGRP in the spinal cord was diminished by 45-65% (p ≤ 0.05) during colitis. RTX pretreatment did not affect CGRP concentration in the urinary bladder; however, it caused a reduction in CGRP release from lumbosacral DRG neurons during acute phase (3 and 5 days post-TNBS). Our results clearly demonstrate that colonic inflammation triggers the release of pro-inflammatory neuropeptides SP and CGRP in the urinary bladder via activation of TRPV1 signaling mechanisms enunciating the neurogenic nature of pelvic organ cross-sensitization.

Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein

Large-conductance voltage- and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstruction-induced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 (MLC(20)) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel alpha- and beta-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BKbeta mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK beta-subunit was greater than that of the BK alpha-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK beta-subunit was employed to study the effect of BK depletion on MLC(20) phosphorylation. siRNA-mediated BK channel reduction was associated with an increased MLC(20) phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of MLC(20) phosphorylation.

Modulation of smooth muscle actomyosin ATPase by thin filament associated proteins

Caldesmon binds equally to both gizzard actin and actin containing stoichiometric amounts of bound tropomyosin. The binding of caldesmon to actin inhibits the actin-activation of the Mg-ATPase activity of phosphorylated myosin only when the actin contains bound tropomyosin. The reversal of this inhibition requires Ca2+-calmodulin; but it occurs without complete release of bound caldesmon. Although phosphorylation of the caldesmon occurs during the ATPase assay, a direct correlation between caldesmon phosphorylation and the release of the inhibited actomyosin ATPase is not consistently observed.

Expression of myosin isoforms in smooth muscle cells in the corpus cavernosum penis

Corpus cavernosum smooth muscle (CCSM) in the penis is unique in that it exhibits a high resting tone and, on stimulation, the muscle cells relax, allowing cavernous spaces to fill with blood, which results in an erection (tumescence). During detumescence, the muscle cells contract and return to the state of high resting tone. This study was undertaken to determine whether CCSM with these unique properties contains myosin isoforms typical of aorta or bladder smooth muscles, muscles that exhibit tonic and phasic characteristics, respectively. RT-PCR revealed that normal CCSM contains an SM2/SM1 mRNA ratio of 1.2:1 (similar to the rabbit aorta). Approximately 31% of the myosin heavy chain transcripts possess a 21-nt insert (predominant in bladder smooth muscle but not expressed in aorta) that encodes the seven-amino acid insert near the NH2-terminal ATP binding region in the head portion of the myosin molecule found in SMB, with the remaining mRNA being noninserted (SMA). Quantitative competitive RT-PCR revealed that the CCSM possesses approximately 4.5-fold less SMB than the bladder smooth muscle. Western blot analysis using an antibody specific for the seven-amino acid insert reveals that both SM1 and SM2 in the CCSM contain the seven-amino acid insert. Furthermore, SMB containing the seven-amino acid insert was localized in the CCSM by immunofluorescence microscopy using this highly specific antibody. The analysis of the expression of LC17 isoforms a and b in the CCSM revealed that it is similar to that of bladder smooth muscle. Thus the CCSM possesses an overall myosin isoform composition intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasiclike characteristics, respectively. Two-dimensional gel electrophoresis showed a relatively low level (approximately 10%) of Ca2+-dependent light-chain (LC20) phosphorylation at the basal tone, which reaches approximately 23% in response to maximal stimulation. The presence of noninserted and inserted myosin isoforms with low and high levels of actin-activated ATPase activities, respectively, in the CCSM may contribute to the ability of the CCSM to remain in a state of high resting tone and to relax rapidly for normal penile function.Corpus cavernosum smooth muscle (CCSM) in the penis is unique in that it exhibits a high resting tone and, on stimulation, the muscle cells relax, allowing cavernous spaces to fill with blood, which results in an erection (tumescence). During detumescence, the muscle cells contract and return to the state of high resting tone. This study was undertaken to determine whether CCSM with these unique properties contains myosin isoforms typical of aorta or bladder smooth muscles, muscles that exhibit tonic and phasic characteristics, respectively. RT-PCR revealed that normal CCSM contains an SM2/SM1 mRNA ratio of 1.2:1 (similar to the rabbit aorta). Approximately 31% of the myosin heavy chain transcripts possess a 21-nt insert (predominant in bladder smooth muscle but not expressed in aorta) that encodes the seven-amino acid insert near the NH2-terminal ATP binding region in the head portion of the myosin molecule found in SMB, with the remaining mRNA being noninserted (SMA). Quantitative competitive RT-PCR revealed that the CCSM possesses ∼4.5-fold less SMB than the bladder smooth muscle. Western blot analysis using an antibody specific for the seven-amino acid insert reveals that both SM1 and SM2 in the CCSM contain the seven-amino acid insert. Furthermore, SMB containing the seven-amino acid insert was localized in the CCSM by immunofluorescence microscopy using this highly specific antibody. The analysis of the expression of LC17isoforms a and b in the CCSM revealed that it is similar to that of bladder smooth muscle. Thus the CCSM possesses an overall myosin isoform composition intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasiclike characteristics, respectively. Two-dimensional gel electrophoresis showed a relatively low level (∼10%) of Ca2+-dependent light-chain (LC20) phosphorylation at the basal tone, which reaches ∼23% in response to maximal stimulation. The presence of noninserted and inserted myosin isoforms with low and high levels of actin-activated ATPase activities, respectively, in the CCSM may contribute to the ability of the CCSM to remain in a state of high resting tone and to relax rapidly for normal penile function.

Alteration of the PKC-mediated signaling pathway for smooth muscle contraction in obstruction-induced hypertrophy of the urinary bladder

Normal urinary bladder function requires contraction and relaxation of the detrusor smooth muscle (DSM). The DSM undergoes compensatory hypertrophy in response to partial bladder outlet obstruction (PBOO) in both men and animal models. Following bladder hypertrophy, the bladder either retains its normal function (compensated) or becomes dysfunctional (decompensated) with increased voiding frequency and decreased void volume. We analyzed the contractile characteristics of DSM in a rabbit model of PBOO. The protein kinase C (PKC) agonist phorbol 12, 13-dibutyrate (PDBu) elicited similar levels of contraction of DSM strips from normal and compensated bladders. However, PDBu-induced contraction decreased significantly in DSM strips from decompensated bladders. The expression and activity of PKC-α were also lowest in decompensated bladders. The PKC-specific inhibitor bisindolylmaleimide-1 (Bis) blocked PDBu-induced contraction and PKC activity in all three groups. Moreover, the phosphorylation of the phosphoprotein inhibitor CPI-17 (a 17-kDa PKC-potentiated inhibitory protein of protein phosphatase-1) was diminished in DSM from the decompensated bladder, which would result in less inhibitory potency of CPI-17 on myosin light chain phosphatase activity and contribute to less contractility. Immunostaining revealed the colocalization of PKC and phosphorylated CPI-17 in the DSM and confirmed the decreases of these signaling proteins in the decompensated bladder. Our results show a differential PKC-mediated DSM contraction with corresponding alterations of PKC expression, activity and the phosphorylation of CPI-17. Our finding suggests a significant correlation between bladder function and PKC pathway. An impaired PKC pathway appears to be correlated with severe bladder dysfunction observed in decompensated bladders.