Muscarinic and purinergic receptor expression in the urothelium of rats with detrusor overactivity induced by bladder outlet obstruction

Kim, Joon Chul,Yoo, Jae Suk,Park, Eun Young,Hong, Sung Hoo,Seo, Seong Il,Hwang, Tae-Kon Blackwell Publishing Ltd 2008 BJU international Vol.101 No.3

<P>OBJECTIVE</P><P>To investigate the expression of muscarinic and purinergic receptors in rat urothelium, and changes in their distribution and expression following detrusor overactivity induced by bladder outlet obstruction (BOO).</P><P>MATERIALS AND METHODS</P><P>Thirty Sprague-Dawley rats were divided into control (10) and BOO groups (20). Partial BOO was induced for 3 weeks and the rats assessed by cystometrography. A portion of the bladder was stained using immunofluorescence for M<SUB>2</SUB> and M<SUB>3</SUB> muscarinic receptors, and P2X<SUB>3</SUB> purinergic receptors. The remainder was dissected into bladder urothelium and the smooth muscle layer, and the expression of the receptor proteins analysed by Western blotting.</P><P>RESULTS</P><P>Cystometrography showed a significant decrease in contraction interval and increase in contraction pressure in the BOO group. On immunofluorescence staining, muscarinic and purinergic receptors were localized in both the urothelium and the muscle layer. Immunoreactivity of M<SUB>2</SUB> and M<SUB>3</SUB> muscarinic receptors was greater in the urothelium of the BOO group than in the control group; there was a smaller increase in P2X<SUB>3</SUB> immunoreactivity. On Western blotting, the expression of M<SUB>2</SUB>, M<SUB>3</SUB> and P2X<SUB>3</SUB> receptors was increased in the urothelium of the BOO group, and there was increased M<SUB>3</SUB> receptor expression in the muscle layer of the BOO group.</P><P>CONCLUSIONS</P><P>There were detectable changes in muscarinic and purinergic receptors with bladder overactivity induced by BOO. Our results suggest that changes in urothelium receptor expression could have a role in mediating the afferent sensory responses in the urinary bladder.</P>

Modulators of Ion Transport in Nasal Polyps: An in situ Measurement of Short-Circuit Current

이준호,이철희,이재서,김대우 대한이비인후과학회 2008 Clinical and Experimental Otorhinolaryngology Vol.1 No.2

Objectives. To examine possible modulators of the ion transport through the apical membrane of the nasal polyps. Methods. The study was conducted using the freshly-excised nasal polyps from the patients with chronic sinusitis. A voltage- sensitive vibrating probe technique was introduced to monitor the short-circuit current across the apical membrane of the polyp at 37℃. Results. In the presence of amiloride, Adenosine 5’-triphosphate induced 4,4’-Diisothiocyanatostilbene-2,2’-disulfonic acidsensitive chloride current. Uridine 5’-diphosphate was less potent than Uridine 5’-triphosphate, and adenosine increased chloride secretion, which was blocked by the antagonist, 8-(p-sulfophenyl) theophylline on adenosine receptor. Based on the pharmacologic profiles, multiple purinergic receptors, including P2Y2, P2Y6, and P1 receptors, were functionally expressed. However, P2X receptor agonists ( , -methyleneadenosine 5’-triphosphate and 2’- & 3’-O-[4-benzoyl-benzoyl] adenosine 5’-triphosphate), Cystic fibrosis conductance regulator (CFTR) activator (genistein), nitric oxide substrate (L-arginine), and nitric oxide donor (sodium nitroprusside) had no significant effect on the short circuit current. Conclusion. Among tested drugs, P2Y receptor agonists were major modulators of ion transport in nasal polyps in situ. Objectives. To examine possible modulators of the ion transport through the apical membrane of the nasal polyps. Methods. The study was conducted using the freshly-excised nasal polyps from the patients with chronic sinusitis. A voltage- sensitive vibrating probe technique was introduced to monitor the short-circuit current across the apical membrane of the polyp at 37℃. Results. In the presence of amiloride, Adenosine 5’-triphosphate induced 4,4’-Diisothiocyanatostilbene-2,2’-disulfonic acidsensitive chloride current. Uridine 5’-diphosphate was less potent than Uridine 5’-triphosphate, and adenosine increased chloride secretion, which was blocked by the antagonist, 8-(p-sulfophenyl) theophylline on adenosine receptor. Based on the pharmacologic profiles, multiple purinergic receptors, including P2Y2, P2Y6, and P1 receptors, were functionally expressed. However, P2X receptor agonists ( , -methyleneadenosine 5’-triphosphate and 2’- & 3’-O-[4-benzoyl-benzoyl] adenosine 5’-triphosphate), Cystic fibrosis conductance regulator (CFTR) activator (genistein), nitric oxide substrate (L-arginine), and nitric oxide donor (sodium nitroprusside) had no significant effect on the short circuit current. Conclusion. Among tested drugs, P2Y receptor agonists were major modulators of ion transport in nasal polyps in situ.

Functional Expression of P2Y Receptors in WERI-Rb1 Retinoblastoma Cells

Na-Hyun Kim,Kyu-Sang Park,Joon Hyung Sohn,Byung-Il Yeh,Chang Mann Ko,In Deok Kong 대한생리학회-대한약리학회 2011 The Korean Journal of Physiology & Pharmacology Vol.15 No.1

P2Y receptors are metabotropic G-protein-coupled receptors, which are involved in many important biologic functions in the central nervous system including retina. Subtypes of P2Y receptors in retinal tissue vary according to the species and the cell types. We examined the molecular and pharmacologic profiles of P2Y purinoceptors in retinoblastoma cell, which has not been identified yet. To achieve this goal, we used Ca²⁺ imaging technique and western blot analysis in WERI-Rb-1 cell, a human retino</SUP>blastoma cell line. ATP (10ՌM) elicited strong but transient [Ca²⁺]<i>_i</i> increase in a concentration- dependent manner from more than 80% of the WERI-Rb-1 cells (n=46). Orders of potency of P2Y agonists in evoking [Ca²⁺]<i>_i</i> transients were 2MeS-ATP>ATP>>UTP=ՁՂ-MeATP, which was compatible with the subclass of P2Y₁ receptor. The [Ca²⁺]<i>_i</i> transients evoked by applications of 2MeS-ATP and/or ATP were also profoundly suppressed in the presence of P2Y₁ selective blocker (MRS 2179; 30ՌM). P2Y₁ receptor expression in WERI-Rb-1 cells was also identified by using western blot. Taken together, P2Y₁ receptor is mainly expressed in a retinoblastoma cell, which elicits Ca²⁺ release from internal Ca²⁺ storage sites via the phospholipase C-mediated pathway. P2Y₁ receptor activation in retinoblastoma cell could be a useful model to investigate the role of purinergic [Ca²⁺]<i>_i</i> signaling in neural tissue as well as to find a novel therapeutic target to this lethal cancer.

Functional Expression of P2Y Receptors in WERI-Rb1 Retinoblastoma Cells

Kim, Na-Hyun,Park, Kyu-Sang,Sohn, Joon-Hyung,Yeh, Byung-Il,Ko, Chang-Mann,Kong, In-Deok The Korean Society of Pharmacology 2011 The Korean Journal of Physiology & Pharmacology Vol.15 No.1

P2Y receptors are metabotropic G-protein-coupled receptors, which are involved in many important biologic functions in the central nervous system including retina. Subtypes of P2Y receptors in retinal tissue vary according to the species and the cell types. We examined the molecular and pharmacologic profiles of P2Y purinoceptors in retinoblastoma cell, which has not been identified yet. To achieve this goal, we used $Ca^{2+}$ imaging technique and western blot analysis in WERI-Rb-1 cell, a human retinoblastoma cell line. ATP ($10\;{\mu}M$) elicited strong but transient $[Ca^{2+}]_i$ increase in a concentration dependent manner from more than 80% of the WERI-Rb-1 cells (n=46). Orders of potency of P2Y agonists in evoking $[Ca^{2+}]_i$ transients were 2MeS-ATP>ATP>>UTP=${\alpha}{\beta}$-MeATP, which was compatible with the subclass of $P2Y_1$ receptor. The $[Ca^{2+}]_i$ transients evoked by applications of 2MeS-ATP and/or ATP were also profoundly suppressed in the presence of $P2Y_1$ selective blocker (MRS 2179; $30\;{\mu}M$). $P2Y_1$ receptor expression in WERI-Rb-1 cells was also identified by using western blot. Taken together, $P2Y_1$ receptor is mainly expressed in a retinoblastoma cell, which elicits $Ca^{2+}$ release from internal $Ca^{2+}$ storage sites via the phospholipase C-mediated pathway. $P2Y_1$ receptor activation in retinoblastoma cell could be a useful model to investigate the role of purinergic $[Ca^{2+}]_i$ signaling in neural tissue as well as to find a novel therapeutic target to this lethal cancer.

Molecular Vibration-Activity Relationship in the Agonism of Adenosine Receptors

지현근,오석준 한국유전체학회 2013 Genomics & informatics Vol.11 No.4

The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated bystructure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. Theresulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram resultproduced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study ofadenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibrationpatterns showed its potential for the functional classification of adenosine receptor ligands.

Molecular Vibration-Activity Relationship in the Agonism of Adenosine Receptors

Chee, Hyun Keun,Oh, S. June Korea Genome Organization 2013 Genomics & informatics Vol.11 No.4

The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands.

Extracellular ATP Stimulates Na<SUP>⁢</SUP> and Cl<SUP>⁣</SUP> Transport through the Activation of Multiple Purinergic Receptors on the Apical and Basolateral Membranes in M-1 Mouse Cortical Collecting Duct Cells

Jin Sup Jung,Sook Mi Hwang,Ryang Hwa Lee,Soo Kyung Kang,Jae Suk Woo,Yong Keun Kim 대한생리학회-대한약리학회 2001 The Korean Journal of Physiology & Pharmacology Vol.5 No.3

<P> The mammalian cortical collecting duct (CCD) plays a major role in regulating renal NaCl reabsorption, which is important in Na<SUP>⁢</SUP> and Cl<SUP>⁣</SUP> homeostasis. The M-1 cell line, derived from the mouse cortical collecting duct, has been used as a mammalian model of the study on the electrolytes transport in CCD. M-1 cells were grown on collagen-coated permeable support and short circuit current (I<SUB>sc</SUB>) was measured. M-1 cells developed amiloride-sensitive current 5∼7 days after seeding. Apical and basolateral addition of ATP induced increase in I<SUB>sc</SUB> in M-1 cells, which was partly retained in Na<SUP>⁢</SUP>-free or Cl<SUP>⁣</SUP>-free solution, indicating that ATP increased Na<SUP>⁢</SUP> absorption and Cl<SUP>⁣</SUP> secretion in M-1 cells. Cl<SUP>⁣</SUP> secretion was mediated by the activation of apical cystic fibrosis transmembrane regulator (CFTR) chloride channels and Ca<SUP>2⁢</SUP>- activated chloride channels, but Na<SUP>⁢</SUP> absorption was not mediated by activation of epithelal sodium channel (ENaC). ATP increased cAMP content in M-1 cells. The RT-PCR analysis demonstrated that M-1 cells express P2Y<SUB>2</SUB>, P2X<SUB>3</SUB> and P2Y<SUB>4</SUB> receptors. These results showed that ATP regulates Na<SUP>⁢</SUP> and Cl<SUP>⁣</SUP> transports via multiple P2 purinoceptors on the apical and basolateral membranes in M-1 cells.

Octyl Gallate Inhibits ATP-induced Intracellular Calcium Increase in PC12 Cells by Inhibiting Multiple Pathways

구유지,홍이재,장현종,김명준,이덕주,조양혁,한상준,윤신희 대한약리학회 2010 The Korean Journal of Physiology & Pharmacology Vol.14 No.1

Phenolic compounds affect intracellular free Ca2+ concentration ([Ca2+]i) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca2+ signaling in PC12 cells using fura-2-based digital Ca2+ imaging and whole-cell patch clamping. Treatment with ATP (100μM) for 90 s induced increases in [Ca2+]i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20μM) for 10 min inhibited the ATP-induced [Ca2+]i response in a concentration-dependent manner (IC50=2.84μM). Treatment with octyl gallate (3μM) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca2+ with nominally Ca2+-free HEPES HBSS or depletion of intracellular Ca2+ stores with thapsigargin (1μM). Treatment for 10 min with the L-type Ca2+ channel antagonist nimodipine (1μM) significantly inhibited the ATP-induced [Ca2+]i increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca2+]i increase induced by 50 mM KCl. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein (50μM) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced [Ca2+]i increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca2+ from extracellular space and P2Y receptor-induced release of Ca2+ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca2+ responses by inhibiting the secondary activation of voltage-gated Ca2+ channels.

Octyl Gallate Inhibits ATP-induced Intracellular Calcium Increase in PC12 Cells by Inhibiting Multiple Pathways

Yujie Guo,Yi Jae Hong,Hyun-Jong Jang,Myung-Jun Kim,Duck-Joo Rhie,Yang-Hyeok Jo,Sang June Hahn,Shin Hee Yoon 대한생리학회-대한약리학회 2010 The Korean Journal of Physiology & Pharmacology Vol.14 No.1

Phenolic compounds affect intracellular free Ca²⁺ concentration ([Ca²⁺]_i) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca²⁺ signaling in PC12 cells using fura-2-based digital Ca²⁺ imaging and whole-cell patch clamping. Treatment with ATP (100</SUP>ՌM) for 90 s induced increases in [Ca²⁺]_i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20ՌM) for 10 min inhibited the ATP-induced [Ca²⁺]_i response in a concentration-dependent manner (IC₅₀=2.84ՌM). Treatment with octyl gallate (3ՌM) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca²⁺ with nominally Ca²⁺-free HEPES HBSS or depletion of intracellular Ca²⁺ stores with thapsigargin (1ՌM). Treatment for 10 min with the L-type Ca²⁺ channel antagonist nimodipine (1ՌM) significantly inhibited the ATP-induced [Ca²⁺]_i increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca²⁺]_i increase induced by 50 mM KCl. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein (50ՌM) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced [Ca²⁺]_i increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca²⁺ from extracellular space and P2Y receptor-induced release of Ca²⁺ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca²⁺ responses by inhibiting the secondary activation of voltage-gated Ca²⁺ channels.

Octyl Gallate Inhibits ATP-induced Intracellular Calcium Increase in PC12 Cells by Inhibiting Multiple Pathways

Guo, Yujie,Hong, Yi-Jae,Jang, Hyun-Jong,Kim, Myung-Jun,Rhie, Duck-Joo,Jo, Yang-Hyeok,Hahn, Sang-June,Yoon, Shin-Hee The Korean Society of Pharmacology 2010 The Korean Journal of Physiology & Pharmacology Vol.14 No.1

Phenolic compounds affect intracellular free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced $Ca^{2+}$ signaling in PC12 cells using fura-2-based digital $Ca^{2+}$ imaging and whole-cell patch clamping. Treatment with ATP ($100\;{\mu}M$) for 90 s induced increases in $[Ca^{2+}]_i$ in PC12 cells. Pretreatment with octyl gallate (100 nM to $20\;{\mu}M$) for 10 min inhibited the ATP-induced $[Ca^{2+}]_i$ response in a concentration-dependent manner ($IC_{50}=2.84\;{\mu}M$). Treatment with octyl gallate ($3\;{\mu}M$) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular $Ca^{2+}$ with nominally $Ca^{2+}$-free HEPES HBSS or depletion of intracellular $Ca^{2+}$ stores with thapsigargin ($1\;{\mu}M$). Treatment for 10 min with the L-type $Ca^{2+}$ channel antagonist nimodipine ($1\;{\mu}M$) significantly inhibited the ATP-induced $[Ca^{2+}]_i$ increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the $[Ca^{2+}]_i$ increase induced by 50 mM KCI. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein ($50\;{\mu}M$) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced $[Ca^{2+}]_i$ increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of $Ca^{2+}$ from extracellular space and P2Y receptor-induced release of $Ca^{2+}$ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced $Ca^{2+}$ responses by inhibiting the secondary activation of voltage-gated $Ca^{2+}$ channels.