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β-NICOTINE AMIDE ADENINE DIPHOSPHATE ACTIVATES AN ATP-SENSITIVE POTASSIUM CHANNEL IN THE SINGLE CHANNEL STUDY OF HUMAN URINARY BLADDER SMOOTH MUSCLE CELLS
THE JOURNAL OF UROLOGY®
148
ALPHA-1A-AR EXPRESSION HIGH ALPHA-1A LOW OR NO ALPHA-1A SMOOTH MUSCLE CELLS IN: SMOOTH MUSCLE CELLS IN: Intra-renal Arteries Intra-renal Veins Distal Ureter Renal Pelvis Proximal Urethra Proximal Ureter Seminal Vesicle Bladder Body Prostate Bladder Base Penis Prepuce Membranous Urethra Dorsal Penile Artery Cavernous Penile Artery Deep Dorsal Penile Vein Corpus Cavernosum Corpus Spongiosum Vas Deferens NON-MUSCLE CELLS IN: Epididymis Collecting Tubules Seminal Ves. Epithelium NON-MUSCLE CELLS IN: Prostate Epithelium Glomerulus Vas Deferens Epithelium Bowman’s Capsule Cavernous Trabeculae Prox. Convoluted Tubules Tunica Albuginea Spermatogenic Cells Testis Interstitial Cells
Source of Funding: NIH, VA, and AHA
412 DSYREGULATION OF ADRENERGIC SIGNALING BY SMOOTH MUSCLE CAVEOLAE IN SPONTANEOUSLY HYPERTENSIVE RATS WITH DETRUSOR OVERACTIVITY Vivian Cristofaro*, Subbarao V Yalla, Maryrose P Sullivan, West Roxbury, MA INTRODUCTION AND OBJECTIVE: Abnormalities in caveolae, specialized membrane signaling microdomains, have been implicated in the pathophysiology of various diseases. In spontaneously hypertensive rats (SHR), decreased caveolae and altered expression of caveolin (proteins forming caveolae) are described in several tissues. Along with systemically increased sympathetic activity, the SHR exhibits marked detrusor overactivity (DO). Since altered adrenergic mechanisms have been implicated in the pathogenesis of DO and adrenergic signaling in the bladder is modulated by caveolae, we investigated whether bladder smooth muscle (bsm) caveolae are altered in SHR and determined its functional consequence on adrenergically-mediated contraction. METHODS: Caveolar density in bsm cells from SHR and control Wistar Kyoto Rat (WKY) was compared by electron microscopy. Caveolin (cav) expression, distribution and interaction with adrenoceptors (AR) were investigated by immunofluorescence, western blotting and immunoprecipitation. Responses to A- and B-AR agonists were measured in bladder tissue from SHR and WKY before and after caveolae were disrupted, using methyl-ß-cyclodextrin (mßcd). RESULTS: SHR bsm cells had significantly less caveolae than WKY. Cav-1 and cav-3 protein expression decreased in SHR relative to WKY. A-and B-ARs precipitated with caveolin proteins in bsm tissue from both strains, however caveolin-AR interactions were altered in SHR. Relaxation response to isoproterenol (ISO) was shifted leftward in SHR compared with WKY. However mßcd resulted in a greater decrease in the relaxation response to ISO in WKY compared with SHR. Contractile response to phenylephrine (PE) at baseline in SHR was higher than WKY. Moreover, response to PE in WKY was significantly increased by disruption of caveolae compared with SHR, which was unaffected by the same concentration of mßcd. Higher mßcd concentrations were needed in SHR to observe a difference in PE response. CONCLUSIONS: Adrenergic receptor-mediated responses were regulated by caveolae in WKY with normal bladders. In overactive bladders of SHR, contractile and relaxation responses to ARs agonists were altered. The loss of caveolar elements in SHR, together with the lack of effect of mBcd on adrenergically induced responses and altered AR-caveolin interactions in SHR, suggest that caveolae-mediated regulation of adrenergic signaling is impaired in overactive bladders. Thus pathologic aberrations of caveolar elements may lead to DO. Source of Funding: Supported by Research Service, Department of Veterans Affairs, Washington, DC
Vol. 181, No. 4, Supplement, Sunday, April 26, 2009
413 B-NICOTINE AMIDE ADENINE DIPHOSPHATE ACTIVATES
AN ATP-SENSITIVE POTASSIUM CHANNEL IN THE SINGLE CHANNEL STUDY OF HUMAN URINARY BLADDER SMOOTH MUSCLE CELLS. Shunichi Kajioka*, Nouval Shahab, Ryosuke Takahashi, Fukuoka, Japan; Alison F Brading, Oxford, United Kingdom; Narihito Seki, Seiji Naito, Fukuoka, Japan INTRODUCTION AND OBJECTIVE: Pharmacological and molecular studies have suggested the existence of ATP-sensitive potassium channel (KATP) in human urinary bladder smooth muscle cells however there is no report in single channel studies regardless of its therapeutic possiblity. On the other hand, the KATP channels are known to be regulated by various intracellular factors. Above all, intracellular nucleotide diphosphates (NDPis) activate or recover the KATP channels. Nicotinic acid (pyridine nucleotide) such as B-nicotine amide adenine diphosphate (BNAD) obviously has a diphosphate-structure and plays an important role in electron transfer reactions leading the cell metabolism. However its effect on the KATP channel has not been investigated. The aim of this study is not only to present the single channel recordings of KATP channels in human detrusor but also to investigate the effect of BNAD. METHODS: Patch clamp procedures (whole-cell configuration, cell-attached and inside-out patch) have been performed to dispersed single smooth muscle cells of human urinary bladder. RESULTS: Whole-cell voltage-clamp experiments were performed in human detrusor cells. 10μM levcromakalim, a K channel opener induced a long-lasting outward current of 58.7±20.2pA at the holding potential of -40mV (n=4). This outward current was completely inhibited by glibenclamide. In cell-attached configuration, levcromakalim activated K+ channels with a unitary conductance of ~12 pS. When the patch configuration was changed to inside-out mode, the K+ channel activity ran down. Subsequent application of guanosine diphosphate (GDP) re-activated the channels. The openings of the 12 pS K+ channels in the presence of GDP was suppressed by ATP and glibenclamide. BNAD also reactivate this 12 pS K+ channel. The open probability was 0.047±0.015 and 0.053± 0.009 in the presence of 1mM BNAD and 500μM GDP, respectively (n=4). CONCLUSIONS: This is the first report of single channel study of KATP channels in human detrusor. The unitary conductance of this channel was 12 pS. The properties of this channel, i.e. ATP-sensitivity and NDPi-activation were also consistent with those of other smooth muscle organs. Another type of diphosphate, BNAD has a potency to activate KATP channels in human detrusor. It is very likely that this channel plays some role during not only ischemic condition but also physiological muscle motion leading the activation of cell metabolism. Source of Funding: grants-in-aid for scientific research from the Japan Society for the promotion of Science
414 AUGMENTED POLYAMINE SIGNALING BLOCKS THE LARGE CONDUCTANCE CALCIUM ACTIVATED POTASSIUM (BK) CHANNEL IN BLADDER UROTHELIAL CELLS FROM PATIENTS WITH OVERACTIVE BLADDER SYNDROME Mingkai Li*, Yan Sun, J Marc Simard, Jian Ying Wang, Toby C Chai, Baltimore, MD INTRODUCTION AND OBJECTIVE: To determine how polyamine signaling affects BK channel activity in bladder urothelial cells (BUC) and whether this is altered in OAB using cell culture and tissue from human controls and OAB subjects. METHODS: BUC were cultured from biopsies of OAB and control subjects. Electrophysiologic techniques were utilized to study outward currents mediated by BK in cells. Exogenous spermine was used to augment and DFMO was used to block polyamine signaling. Immunohistofluorescence was utilized to measure expression of ODC, the rate limiting enzyme in polyamine synthesis, in bladder urothelial biopsies.
148
ALPHA-1A-AR EXPRESSION HIGH ALPHA-1A LOW OR NO ALPHA-1A SMOOTH MUSCLE CELLS IN: SMOOTH MUSCLE CELLS IN: Intra-renal Arteries Intra-renal Veins Distal Ureter Renal Pelvis Proximal Urethra Proximal Ureter Seminal Vesicle Bladder Body Prostate Bladder Base Penis Prepuce Membranous Urethra Dorsal Penile Artery Cavernous Penile Artery Deep Dorsal Penile Vein Corpus Cavernosum Corpus Spongiosum Vas Deferens NON-MUSCLE CELLS IN: Epididymis Collecting Tubules Seminal Ves. Epithelium NON-MUSCLE CELLS IN: Prostate Epithelium Glomerulus Vas Deferens Epithelium Bowman’s Capsule Cavernous Trabeculae Prox. Convoluted Tubules Tunica Albuginea Spermatogenic Cells Testis Interstitial Cells
Source of Funding: NIH, VA, and AHA
412 DSYREGULATION OF ADRENERGIC SIGNALING BY SMOOTH MUSCLE CAVEOLAE IN SPONTANEOUSLY HYPERTENSIVE RATS WITH DETRUSOR OVERACTIVITY Vivian Cristofaro*, Subbarao V Yalla, Maryrose P Sullivan, West Roxbury, MA INTRODUCTION AND OBJECTIVE: Abnormalities in caveolae, specialized membrane signaling microdomains, have been implicated in the pathophysiology of various diseases. In spontaneously hypertensive rats (SHR), decreased caveolae and altered expression of caveolin (proteins forming caveolae) are described in several tissues. Along with systemically increased sympathetic activity, the SHR exhibits marked detrusor overactivity (DO). Since altered adrenergic mechanisms have been implicated in the pathogenesis of DO and adrenergic signaling in the bladder is modulated by caveolae, we investigated whether bladder smooth muscle (bsm) caveolae are altered in SHR and determined its functional consequence on adrenergically-mediated contraction. METHODS: Caveolar density in bsm cells from SHR and control Wistar Kyoto Rat (WKY) was compared by electron microscopy. Caveolin (cav) expression, distribution and interaction with adrenoceptors (AR) were investigated by immunofluorescence, western blotting and immunoprecipitation. Responses to A- and B-AR agonists were measured in bladder tissue from SHR and WKY before and after caveolae were disrupted, using methyl-ß-cyclodextrin (mßcd). RESULTS: SHR bsm cells had significantly less caveolae than WKY. Cav-1 and cav-3 protein expression decreased in SHR relative to WKY. A-and B-ARs precipitated with caveolin proteins in bsm tissue from both strains, however caveolin-AR interactions were altered in SHR. Relaxation response to isoproterenol (ISO) was shifted leftward in SHR compared with WKY. However mßcd resulted in a greater decrease in the relaxation response to ISO in WKY compared with SHR. Contractile response to phenylephrine (PE) at baseline in SHR was higher than WKY. Moreover, response to PE in WKY was significantly increased by disruption of caveolae compared with SHR, which was unaffected by the same concentration of mßcd. Higher mßcd concentrations were needed in SHR to observe a difference in PE response. CONCLUSIONS: Adrenergic receptor-mediated responses were regulated by caveolae in WKY with normal bladders. In overactive bladders of SHR, contractile and relaxation responses to ARs agonists were altered. The loss of caveolar elements in SHR, together with the lack of effect of mBcd on adrenergically induced responses and altered AR-caveolin interactions in SHR, suggest that caveolae-mediated regulation of adrenergic signaling is impaired in overactive bladders. Thus pathologic aberrations of caveolar elements may lead to DO. Source of Funding: Supported by Research Service, Department of Veterans Affairs, Washington, DC
Vol. 181, No. 4, Supplement, Sunday, April 26, 2009
413 B-NICOTINE AMIDE ADENINE DIPHOSPHATE ACTIVATES
AN ATP-SENSITIVE POTASSIUM CHANNEL IN THE SINGLE CHANNEL STUDY OF HUMAN URINARY BLADDER SMOOTH MUSCLE CELLS. Shunichi Kajioka*, Nouval Shahab, Ryosuke Takahashi, Fukuoka, Japan; Alison F Brading, Oxford, United Kingdom; Narihito Seki, Seiji Naito, Fukuoka, Japan INTRODUCTION AND OBJECTIVE: Pharmacological and molecular studies have suggested the existence of ATP-sensitive potassium channel (KATP) in human urinary bladder smooth muscle cells however there is no report in single channel studies regardless of its therapeutic possiblity. On the other hand, the KATP channels are known to be regulated by various intracellular factors. Above all, intracellular nucleotide diphosphates (NDPis) activate or recover the KATP channels. Nicotinic acid (pyridine nucleotide) such as B-nicotine amide adenine diphosphate (BNAD) obviously has a diphosphate-structure and plays an important role in electron transfer reactions leading the cell metabolism. However its effect on the KATP channel has not been investigated. The aim of this study is not only to present the single channel recordings of KATP channels in human detrusor but also to investigate the effect of BNAD. METHODS: Patch clamp procedures (whole-cell configuration, cell-attached and inside-out patch) have been performed to dispersed single smooth muscle cells of human urinary bladder. RESULTS: Whole-cell voltage-clamp experiments were performed in human detrusor cells. 10μM levcromakalim, a K channel opener induced a long-lasting outward current of 58.7±20.2pA at the holding potential of -40mV (n=4). This outward current was completely inhibited by glibenclamide. In cell-attached configuration, levcromakalim activated K+ channels with a unitary conductance of ~12 pS. When the patch configuration was changed to inside-out mode, the K+ channel activity ran down. Subsequent application of guanosine diphosphate (GDP) re-activated the channels. The openings of the 12 pS K+ channels in the presence of GDP was suppressed by ATP and glibenclamide. BNAD also reactivate this 12 pS K+ channel. The open probability was 0.047±0.015 and 0.053± 0.009 in the presence of 1mM BNAD and 500μM GDP, respectively (n=4). CONCLUSIONS: This is the first report of single channel study of KATP channels in human detrusor. The unitary conductance of this channel was 12 pS. The properties of this channel, i.e. ATP-sensitivity and NDPi-activation were also consistent with those of other smooth muscle organs. Another type of diphosphate, BNAD has a potency to activate KATP channels in human detrusor. It is very likely that this channel plays some role during not only ischemic condition but also physiological muscle motion leading the activation of cell metabolism. Source of Funding: grants-in-aid for scientific research from the Japan Society for the promotion of Science
414 AUGMENTED POLYAMINE SIGNALING BLOCKS THE LARGE CONDUCTANCE CALCIUM ACTIVATED POTASSIUM (BK) CHANNEL IN BLADDER UROTHELIAL CELLS FROM PATIENTS WITH OVERACTIVE BLADDER SYNDROME Mingkai Li*, Yan Sun, J Marc Simard, Jian Ying Wang, Toby C Chai, Baltimore, MD INTRODUCTION AND OBJECTIVE: To determine how polyamine signaling affects BK channel activity in bladder urothelial cells (BUC) and whether this is altered in OAB using cell culture and tissue from human controls and OAB subjects. METHODS: BUC were cultured from biopsies of OAB and control subjects. Electrophysiologic techniques were utilized to study outward currents mediated by BK in cells. Exogenous spermine was used to augment and DFMO was used to block polyamine signaling. Immunohistofluorescence was utilized to measure expression of ODC, the rate limiting enzyme in polyamine synthesis, in bladder urothelial biopsies.