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Cross-regulation of cAMP and cGMP levels by cAMP-specific phosphodiesterase-3 (PDE3) and cGMP-specific PDE5
1054 Sodium Current in Human Small Intestinal Interstitial Cells of Cajal Peter R. Strege, Adam J. Rich, Yiiun Ou, Simon J. Gibbons, Michael G. Sarr, Joseph H. Szurszewski, Gianrico Farrugia, Enteric NeumScience Program, Mayo Clin, Rochester, MN Background/Objectives: Interstitial cells of Cajal generate the electrical slow wave and are required for normal gastrointestinal motility. Human small intestinal interstitial cells of Cajal express calcium channels but the other ionic conductances expressed in human interstitial cells of Cajalare unknown. Recentdata suggest that together with an inward calcium current an inward sodium current is present in human jejunal circular smooth muscle cells. The aim of this study was to determine if a sodium current is present in human interstitial cells of CajaL Methods: Human jejunal interstitial cells of Cajalwere dissociatedfrom circular muscle strips obtained from normal human jejuna. Interstitial cells of Cajal were identified by the number of processes (greater than 3), shape and size, and presence of cKit mRNA. Whole cell currents were recorded using standard patch clamp techniqueswith cesium in the pipette to block potassium currents and normal Ringer's solution in the bath. Cells were held at 100 mV between pulse protocols. Results: At least two components of inward current were identified. One component activated at about -35 mV, peaked at about +5 mV and was blocked by nifedipine (1/~M), suggesting it was due to calcium current from L-type calcium channels. The other major component activated at about -55 mV, peaked at -30 mV and inactivated more rapidly compared to the calcium current. Replacementof sodium by NmethyI-D-glucamineabolished the more negativelyactivating component (-164+-63 to -9+_2 pA, n =9, P 0.05) but blocked the calcium current (-118 +-37 to - 13+-7 pA, n = 5, P< 0.05). The effects of the selectivesodium channel blocker GX 314 were tested on the sodium current in the presence of nifedipine (1 p.M). External 0X314 (500/~M) blockedthe sodium current in a time dependentfashion (151 +_50 to 58+_11 pA, at 25 min, n = 3, P
pretreetment(1/~M for 60 min) causeda concentration-dependentincrease in GTPaseactivity of 70+-8% (maximal effect) over basal levels. Similar pretreatment with PDBU resulted in a 43_+5% reduction in the maximal activation of GTPaseand a significant rightward shift in the dose-responsecurve (p
1057 Inhibition of RheA-Dependent Sustained Smooth Muscle Contraction by cAMP- and oGMP-Dependent Protein Kinase (PKA and PKG). Karnam S. Murthy, Huiping Zhou, John R. Grider, Gabriel M. Makhlouf, Medical Coil of Virginia, VCU, Richmond, VA BACKGROUND.Both PKAand PKG inhibit agonist-stimulatedphospholipaseC (PLC-/3)activity and IPrdependent Ca2+ releasewhich mediate the initial contraction of gastric and intestinal smooth muscle. Sustainedcontraction, however, is mediatedby a different signaling pathway involving sequentialactivation of G13and RhoA, leadingto sequentialactivation of Rho-kinase, phospholipaseD (PLD) and various isozymesof protein kinaseC (PKC). A Ca2+-independent PKC isozyme (PKC-~) inhibits myosin light chain (MLC)phosphatase, resulting in MLC2o phosphorylation and sustained smooth muscle contraction. AIM. The aim of this study was to determine whether PKA and PKG induce relaxation of sustained muscle contraction in dispersed gastric smooth muscle cells by regulating the upstream steps in this pathway. RESULTS.Eorskolin(10/.&l), which activatesPKAand cross-activatesPKGcausedphosphorylotion of RhoA and abolished RhoA activity induced by acetylcholine (ACh). ACh-stimulated Rho-kinase activity was inhibited by cBIMPS (64+_4%), which selectivelyactivates PKA, and by pS-pcCPT-cGMP(76+_2%) and Na nitroprusside (SNP;92+_4%),which selectivelyactivates PKG. VIP, which activates both PKA and PKG, and forskolin inhibited Rho-kinaseactivity by 76+_5% and 64-+3%, respectively,rIP and SNP inhibited Rho-kinase-dependentPLD activity and sustained contraction induced by ACh in a concentration-dependentfashion (ECso5 nM and 10 nM, respectively).NeitherVIP nor SNP causeddirect phosphoryletionof PLD, implying that inhibition of PLD activity was dependenton inhibition of RhoAand Rho-kinase. Inhibition of PLD activity and sustainedcontraction by SNPwas completelyblockedby the PKGinhibitor, KT5823, whereas inhibition of PLD activity and sustained contraction by VIP was partly blocked by KT5823, and the PKA inhibitor, myristoylated PKI, and completely blocked by a combination of both inhibitors. CONCLUSION.Both PKA and PKG inhibit agonist-dependent activation of RhoA, leading to inhibition of Rho-kinase and PLD activities, and resulting in inhibition of sustained contraction (i.e., relaxation).
1055 Association of Translocated RhoA-PKCo~in the Particulate Fraction in Smooth Muscle Expressing Gain of Function and Loss of Function of HSP27 Phospbofflation Mutants. Mercy Pawar, PeedikayilE. Thoma, Khalil N. Bitar, Univ of Michigan, Ann Arbor, MI BACKGROUND:We haveinvestigated:t) The signaling pathwaysinvolving a possibleassociation of translocated PKC a and of RhoA on the membrane,and 2) The role of phosphoryleted Hsp27 in maintainingand modulating this functional association. METHODS:Smooth muscle cells were isolated from the circular muscle of the rabbit colon and from the colons of transgenic mice overexprsssingthe phosphorylatedform of Hsp27. In other experimentswe studied the gain-of-function and loss-of-function of Hsp27 phosphorylationmutants in smooth muscle cells. We havetransfectedsmooth musclecells from the rabbit colon with phosphorylation mutants of human Hsp27. In the 3G Hsp27 mutant, all three-serine phosphorylationsites (Ser-15, Ser-78, and Set-82) of the human Hsp27 cDNAwere replacedwith glycine, to mimic non-phosphorylatableserine residues. In the 3D mutant, Ser-15, Ser-78, and Ser-82 were mutated into aspartate; to mimic constitutively phosphorylated residues. RESULTS:A) In normal cells: Both PKC a and RhoA translocated to the membrane upon stimulation with acetylcholine (10-TM) as seen on western blotting. Immunoprecipitation of the particulate fraction with anti-RhoAantibodyfollowed by immunoblotting with anti-PKCaantihodyindicated that acetylcholine induced an immunocomplexing of PKC~-RhoA in the particulate fraction. Similar results were obtainedwhenthe particulatefraction was immunoprecipitatedwith PKCc~ antibody followed by immunoblotting with RhoA antibody. B) In 3G transfected cells, PKCa failed to translocate in response to stimulation with asetylcholine, and RhoA translocation was inhibited. The inhibition of translocation was associatedwith a significant decrease(48.4 _+ 4% P
1058 Cross-RegutaUon of cAMP and cGMP Levels by cAMP-Specific Phosphodiecterase-3 (PDE3) and cGMP-Speoifio PDE5. Kamam S. Murthy, Huiping Zhou, Medical Coil of Virginia, VCU, Richmond, VA BACKGROUND.cGMP activates cGMP-dependentprotein kinase (PKG) and cAMP activates cAMP-dependentprotein kinase (PKA) and can cross-activate PKG. The levels of cAMP and cGMP are determined by degradation via phosphodiestrases(PDEs), which are susceptible to regulation by both kinases and by cGMP. We have previously shown that cAMP-specific PDE3 is activated by PKA, and that cGMP-specific PDE5 is activated by PKG but only in the presence of cGMP. AIM. To determine whether cGMP can regulate cAMP levels by inhibiting PDE3, and whether cAMP can regulate cGMP levels by activating PDE5. METHODS.PDE3 and PDE5expression, PDE activity, and cAMP and cGMP levels were measured in dispersed gastric muscle cells. RESULTS.RT-PCRshowed that both PDE3and PDE5were expressed in gastric musclecells. ForskolinstimulatedcAMP and activatedboth PKAand PDE3;activation of PDE3 was mediated by PKA. ForskoUn-stimulated PDE3 activity was inhibited by Na nitroprusside (SNP) which selectively stimulates cGMP; the inhibition of PDE3 was reversed by blockade of soluble guanylyl cyclase with LY-83583. Consistent with inhibition of PDE3, forskolin-stimulated cAMP levels were augmentedby SNP;the augmentationcould be further potentiatedwhen cGMP levelswere increasedby blockadeof PDE5with zaprinast.The pattern implied that a simultaneous increase in cGMP can augment cAMP levels by inhibiting PDE3 activity. SNP stimulated cGMP and activated both PKG and PDE5; activation of PDE5 was mediated by PKG and could be blocked by the PKG inhibitor, KT-5823. Forskolin decreased SNP-stimulated cGMP levels and increased PDE5activity; the increase in PDE5 activity was reversed by the PKA inhibitor, myristoylated PKI. The pattern implied that a simultaneous increase in cAMP acting via PKA increased PDE5activity and thus, decreasedcGMP levels. CONCLUSION.As depicted in the schema, concurrent stimulation of cAMP and cGMP leads to further increase in cAMP levels and decreasein cGMP levels.The increasein cAMP reflects the ability of cGMP to inhibit cAMP-specific PDE3.The decreasein cGMP reflects the ability of PKA to stimulate cGMP-specific PDES.
1056 Protein Kinase C (PKC)-dependent Phosphorylation of Go Is Associated with inhibition of Function. Yao Liu, Chunfang Guo, Yu Shangguan, Karen E. Hall, John W. Wiley, Univ of Michigan, Ann Arbor, MI Background: We observed recently that diabetic neuropathy is associated with enhanced calcium influx coupledto activationof apoptosis,impairedopioid-medietedinhibiton of calcium influx in diabetic DRG neurons, inhibition of opioid-medietedactivation of Goin diabetic DRGs and increased PKG-dependentphosphorylation of the alpha subunit of Goin diabetic DRG neurons. Opioid receptors are tightly coupled to inhibition of calcium channels via Go in neurons. We hypothesizedthat increased PKC-dependentphosphorylation of Go would be associated with inhibition of Gofunction. Methods: We examined opioid (DAMGO)-mediated activation of Go in cultured S¥5Y neurons using a GTPaseassay employing GTP~/~Sin the presence and absence of the PKC activator phorbol ester PDBU, the inactive analogue 4~ phorbol and the PKC inhibitor, NPC-15437. Parallel opioid receptor binding studies were performed using 3H-naloxone.DAMGO-mediatedinhibition of calcium influx was examined in acutely dissociated rat dorsal root ganglion (DRG) neurons in the presenceand absence of PDBU,4a-phorbol and NPC. Results: DAMGO(1 nM-lO/~M) in the presenceof 4~phorbol
PDE5 +
PKG
PDE3
/
+,/t,
c~MP ~
A-201
" PKA
cAMP
pretreetment(1/~M for 60 min) causeda concentration-dependentincrease in GTPaseactivity of 70+-8% (maximal effect) over basal levels. Similar pretreatment with PDBU resulted in a 43_+5% reduction in the maximal activation of GTPaseand a significant rightward shift in the dose-responsecurve (p
1057 Inhibition of RheA-Dependent Sustained Smooth Muscle Contraction by cAMP- and oGMP-Dependent Protein Kinase (PKA and PKG). Karnam S. Murthy, Huiping Zhou, John R. Grider, Gabriel M. Makhlouf, Medical Coil of Virginia, VCU, Richmond, VA BACKGROUND.Both PKAand PKG inhibit agonist-stimulatedphospholipaseC (PLC-/3)activity and IPrdependent Ca2+ releasewhich mediate the initial contraction of gastric and intestinal smooth muscle. Sustainedcontraction, however, is mediatedby a different signaling pathway involving sequentialactivation of G13and RhoA, leadingto sequentialactivation of Rho-kinase, phospholipaseD (PLD) and various isozymesof protein kinaseC (PKC). A Ca2+-independent PKC isozyme (PKC-~) inhibits myosin light chain (MLC)phosphatase, resulting in MLC2o phosphorylation and sustained smooth muscle contraction. AIM. The aim of this study was to determine whether PKA and PKG induce relaxation of sustained muscle contraction in dispersed gastric smooth muscle cells by regulating the upstream steps in this pathway. RESULTS.Eorskolin(10/.&l), which activatesPKAand cross-activatesPKGcausedphosphorylotion of RhoA and abolished RhoA activity induced by acetylcholine (ACh). ACh-stimulated Rho-kinase activity was inhibited by cBIMPS (64+_4%), which selectivelyactivates PKA, and by pS-pcCPT-cGMP(76+_2%) and Na nitroprusside (SNP;92+_4%),which selectivelyactivates PKG. VIP, which activates both PKA and PKG, and forskolin inhibited Rho-kinaseactivity by 76+_5% and 64-+3%, respectively,rIP and SNP inhibited Rho-kinase-dependentPLD activity and sustained contraction induced by ACh in a concentration-dependentfashion (ECso5 nM and 10 nM, respectively).NeitherVIP nor SNP causeddirect phosphoryletionof PLD, implying that inhibition of PLD activity was dependenton inhibition of RhoAand Rho-kinase. Inhibition of PLD activity and sustainedcontraction by SNPwas completelyblockedby the PKGinhibitor, KT5823, whereas inhibition of PLD activity and sustained contraction by VIP was partly blocked by KT5823, and the PKA inhibitor, myristoylated PKI, and completely blocked by a combination of both inhibitors. CONCLUSION.Both PKA and PKG inhibit agonist-dependent activation of RhoA, leading to inhibition of Rho-kinase and PLD activities, and resulting in inhibition of sustained contraction (i.e., relaxation).
1055 Association of Translocated RhoA-PKCo~in the Particulate Fraction in Smooth Muscle Expressing Gain of Function and Loss of Function of HSP27 Phospbofflation Mutants. Mercy Pawar, PeedikayilE. Thoma, Khalil N. Bitar, Univ of Michigan, Ann Arbor, MI BACKGROUND:We haveinvestigated:t) The signaling pathwaysinvolving a possibleassociation of translocated PKC a and of RhoA on the membrane,and 2) The role of phosphoryleted Hsp27 in maintainingand modulating this functional association. METHODS:Smooth muscle cells were isolated from the circular muscle of the rabbit colon and from the colons of transgenic mice overexprsssingthe phosphorylatedform of Hsp27. In other experimentswe studied the gain-of-function and loss-of-function of Hsp27 phosphorylationmutants in smooth muscle cells. We havetransfectedsmooth musclecells from the rabbit colon with phosphorylation mutants of human Hsp27. In the 3G Hsp27 mutant, all three-serine phosphorylationsites (Ser-15, Ser-78, and Set-82) of the human Hsp27 cDNAwere replacedwith glycine, to mimic non-phosphorylatableserine residues. In the 3D mutant, Ser-15, Ser-78, and Ser-82 were mutated into aspartate; to mimic constitutively phosphorylated residues. RESULTS:A) In normal cells: Both PKC a and RhoA translocated to the membrane upon stimulation with acetylcholine (10-TM) as seen on western blotting. Immunoprecipitation of the particulate fraction with anti-RhoAantibodyfollowed by immunoblotting with anti-PKCaantihodyindicated that acetylcholine induced an immunocomplexing of PKC~-RhoA in the particulate fraction. Similar results were obtainedwhenthe particulatefraction was immunoprecipitatedwith PKCc~ antibody followed by immunoblotting with RhoA antibody. B) In 3G transfected cells, PKCa failed to translocate in response to stimulation with asetylcholine, and RhoA translocation was inhibited. The inhibition of translocation was associatedwith a significant decrease(48.4 _+ 4% P
1058 Cross-RegutaUon of cAMP and cGMP Levels by cAMP-Specific Phosphodiecterase-3 (PDE3) and cGMP-Speoifio PDE5. Kamam S. Murthy, Huiping Zhou, Medical Coil of Virginia, VCU, Richmond, VA BACKGROUND.cGMP activates cGMP-dependentprotein kinase (PKG) and cAMP activates cAMP-dependentprotein kinase (PKA) and can cross-activate PKG. The levels of cAMP and cGMP are determined by degradation via phosphodiestrases(PDEs), which are susceptible to regulation by both kinases and by cGMP. We have previously shown that cAMP-specific PDE3 is activated by PKA, and that cGMP-specific PDE5 is activated by PKG but only in the presence of cGMP. AIM. To determine whether cGMP can regulate cAMP levels by inhibiting PDE3, and whether cAMP can regulate cGMP levels by activating PDE5. METHODS.PDE3 and PDE5expression, PDE activity, and cAMP and cGMP levels were measured in dispersed gastric muscle cells. RESULTS.RT-PCRshowed that both PDE3and PDE5were expressed in gastric musclecells. ForskolinstimulatedcAMP and activatedboth PKAand PDE3;activation of PDE3 was mediated by PKA. ForskoUn-stimulated PDE3 activity was inhibited by Na nitroprusside (SNP) which selectively stimulates cGMP; the inhibition of PDE3 was reversed by blockade of soluble guanylyl cyclase with LY-83583. Consistent with inhibition of PDE3, forskolin-stimulated cAMP levels were augmentedby SNP;the augmentationcould be further potentiatedwhen cGMP levelswere increasedby blockadeof PDE5with zaprinast.The pattern implied that a simultaneous increase in cGMP can augment cAMP levels by inhibiting PDE3 activity. SNP stimulated cGMP and activated both PKG and PDE5; activation of PDE5 was mediated by PKG and could be blocked by the PKG inhibitor, KT-5823. Forskolin decreased SNP-stimulated cGMP levels and increased PDE5activity; the increase in PDE5 activity was reversed by the PKA inhibitor, myristoylated PKI. The pattern implied that a simultaneous increase in cAMP acting via PKA increased PDE5activity and thus, decreasedcGMP levels. CONCLUSION.As depicted in the schema, concurrent stimulation of cAMP and cGMP leads to further increase in cAMP levels and decreasein cGMP levels.The increasein cAMP reflects the ability of cGMP to inhibit cAMP-specific PDE3.The decreasein cGMP reflects the ability of PKA to stimulate cGMP-specific PDES.
1056 Protein Kinase C (PKC)-dependent Phosphorylation of Go Is Associated with inhibition of Function. Yao Liu, Chunfang Guo, Yu Shangguan, Karen E. Hall, John W. Wiley, Univ of Michigan, Ann Arbor, MI Background: We observed recently that diabetic neuropathy is associated with enhanced calcium influx coupledto activationof apoptosis,impairedopioid-medietedinhibiton of calcium influx in diabetic DRG neurons, inhibition of opioid-medietedactivation of Goin diabetic DRGs and increased PKG-dependentphosphorylation of the alpha subunit of Goin diabetic DRG neurons. Opioid receptors are tightly coupled to inhibition of calcium channels via Go in neurons. We hypothesizedthat increased PKC-dependentphosphorylation of Go would be associated with inhibition of Gofunction. Methods: We examined opioid (DAMGO)-mediated activation of Go in cultured S¥5Y neurons using a GTPaseassay employing GTP~/~Sin the presence and absence of the PKC activator phorbol ester PDBU, the inactive analogue 4~ phorbol and the PKC inhibitor, NPC-15437. Parallel opioid receptor binding studies were performed using 3H-naloxone.DAMGO-mediatedinhibition of calcium influx was examined in acutely dissociated rat dorsal root ganglion (DRG) neurons in the presenceand absence of PDBU,4a-phorbol and NPC. Results: DAMGO(1 nM-lO/~M) in the presenceof 4~phorbol
PDE5 +
PKG
PDE3
/
+,/t,
c~MP ~
A-201
" PKA
cAMP