mo1648 protein kinase d1 mediates mitogenic signaling in intestinal epithelial cells

Mo1646

Mechanisms of Relaxant Activity by cGMP Stimuator in Guinea-PigGallbladder Smooth MuscleWoo Jin Jeong, Gab Jin Cheon, Koonhee Han, Young Don Kim, Sang Jin Lee, Jong KyuPark

The soluble guanylyl cyclase is expressed in guinea-pig gallbladder smooth muscle andagents that stimulate this enzyme activity cause gallbladder relaxation. The compound 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine(BAY 41-2272) is a potent nitric oxide (NO)-independent soluble guanylyl cyclase stimulator,but little is known about its effects in gallbladder smooth muscle. This study investigatedthe mechanisms underlying the relaxations of guinea-pig gallbladder smooth muscle inducedby BAY 41-2272. Gallbladder strips were mounted in 20-ml organ baths for isometricforce recording. BAY 41-2272 concentration-dependently relaxed histamine-precontractedgallbladder strips. Prior incubation with the NO synthesis inhibitor L-NAME (0.1 mM) orthe soluble guanylyl cyclase inhibitor ODQ (10 μM) caused significant rightward shifts inthe concentration-response curves to BAY 41-2272. NO-donor, sodium nitroprusside causedconcentration-dependent relaxations, which were greatly potentiated by BAY 41-2272 andalmost completely inhibited by ODQ. To evaluate the effect of K+ channels, the relaxantresponses of gallbladder muscle to Bay 41-2272 remained unchanged selective K+ channelinhibitor such as glibenclamide (10 μM), 4-aminopyridine (1 mM) or the co-incubation ofcharybdotoxin (0.1 μM) with apamin (1 μM). To evaluate the participation of prostanoidsand cAMP pathway, the cyclooxygenase inhibitor indomethacin (10 μM) and selectivephosphodiesterase type 4 inhibitor rolipram (1 μM) failed to significantly affect the BAY41-2272-induced gallbladder relaxations. BAY 41-2272 shifted to the right the gallbladdercontractile responses to either histamine (0.0001-10 μM). BAY 41-2272 (10 μM) also causeda marked rightward shift and decreased the maximal contractile responses to extracellularCaCl2. The effects of an inhibitor of the PKA, Rp-8CPT-cAMPS (10 μM), or the PKG, Rp-8-pCPT-cGMPS (10 μM) on relaxation induced by the cumulative adminstration of BAY41-2272 were studied in precontracted with 1 μM histamine. The relaxing effect of BAY41-2272 were significantly attenuated by Rp-8-pCPT-cGMPS, but not by Rp-8CPT-cAMPS.These results indicate that BAY 41-2272 causes cGMP-dependent guinea-pig gallbladdersmooth muscle relaxations in a synergistic fashion with NO. BAY 41-2272 has also anadditional mechanism independently of soluble guanylyl cyclase activation possibly involvingCa2+ entry blockade.

Mo1647

Neurotensin Signaling Activates the HIF-1/VEGF Pathway in Human ColonicEpithelial CellsKyriaki Bakirtzi, Ivy Law, Charalabos Pothoulakis

Background and Aims: Neurotensin (NT) is a brain-gut neuropeptide that plays an importantrole in the pathophysiology of colonic inflammation and colon cancer via activation of its highaffinity receptor, NT-receptor-1 (NTR1). Hypoxia, and its link to angiogenesis, represents andimportant component of intestinal inflammation and colon cancer. To date, however, thereis no evidence to suggest involvement of NTR1 signaling in hypoxia-related pathways. Herewe investigated the possibility that NT is capable of inducing the HIF-1a/VEGF signalingpathway in human colonocytes Methods: NCM460-overexpressing NTR1 (NCM460-NTR1)cells were treated with NT (10-7 M) with or without NTR1 inhibitor, SR48692. HIF-1amRNA expression was detected by RT-PCR. HIF-1a protein stabilization/activation wasdetected following NT (10-7M) exposure of NCM460-NTR1 cells usingWestern Blot analysisof cytoplasmic and nuclear cell fractions. HIF-1 transcriptional activity was evaluated byluciferase assay using reporter plasmid containing hypoxia response elements (HREs)upstream of luciferase gene. Expression of VEGFA was determined by RT-PCR. Results:Levels of total HIF-1a protein were increased in NCM460-NTR1 cells 6 hours following NTtreatment, but there was no increased expression of HIF-1a at the mRNA level. Nuclearlocalization of HIF-1a was apparent 30min and 6 hours following NT treatment. Pretreatmentof NCM460-NTR1 cells with SR48692 showed this effect to be dependent on NTR1 activation.Evaluation of HIF-1a transcriptional activity showed that NT treatment of NCM460-NTR1cells transfected with HIF-1a luciferase reporter vector resulted in increased luciferase activity30 min (2.5 fold) and 6 hours (2.3 fold) after treatment. Since the promoter region of theangiogenic factor VEGFA contains HIF-1a binding sites, we next examined whether NTstimulates VEGFA expression. We found that mRNA levels of VEGFA in NCM460-NTR1cells were significantly increased 30 min (p=0.033) and 6 hours (p=0.004) following NTexposure. Conclusions: This is the first report that NT/NTR1 coupling causes protein stabiliza-tion and nuclear translocation of HIF-1a in human colonocytes. Moreover, VEGFA levelsof expression are increased in response to NT suggesting a role for NT in angiogenesis-related signaling pathways initiated by hypoxia.

Mo1648

Protein Kinase D1 Mediates Mitogenic Signaling in Intestinal Epithelial CellsJames Sinnett-Smith, Robert K. Kui, Enrique Rozengurt

Background: Protein kinase D1 (PKD1), the founding member of a new protein kinasefamily within the CAMK group, can be activated rapidly within intact cells by multiplestimuli, including G protein-coupled receptor (GPCR) agonists and growth factors via proteinkinase C (PKC) phosphorylation of Ser744 and Ser748 in the PKD1 activation loop (residuenumber corresponding to the mouse PKD1). Rapid PKC-dependent PKD1 activation isfollowed by a late, PKC-independent phase of activation induced by Gq-coupled receptoragonists. Here, we exploited novel chemical inhibitors of the PKD family to examine theregulation and function of PKD in intestinal epithelial cells. Results: To determine the roleand regulation of endogenous PKD family members in intestinal epithelial cells, IEC-6 andIEC-18 cells were treated with increasing concentrations (0.3-5 μM) of the potent andrecently identified PKD family inhibitors kb NB 142-70 and CRT0066101 prior to stimulationwith the Gq-coupled receptor agonist angiotensin II (ANG II) at 50 nM, a maximal concentra-tion for inducing DNA synthesis. Treatment with kb NB 142-70 or CRT0066101 prevented

S-649 AGA Abstracts

PKD1 phosphorylation at residues Ser748 and Ser916 in a dose-dependent manner, in linewith our model predicting that these sites are autophosphorylated by PKD1. In contrast,phosphorylation of Ser744, the primary site targeted by novel PKCs (δ, ε, θ, η), was notaffected by either CRT0066101 or kb NB 142-70, corroborating that the activation loopof PKD1 integrates PKC-mediated transphosphorylation and PKD1 autophosphorylationmechanisms. Accordingly, treatment with the preferential PKC inhibitors GFI (also knownas bisindolylmaleimide I or GF109203X) or Go6983 profoundly inhibited early but not latePKD activation, as shown by Ser748 and Ser916 phosphorylation and by In Vitro kinase assays.Critically, treatment of IEC-18 or IEC-6 cells with either CRT0066101 or kb NB 142-70,at concentrations that inhibited PKD family activity within these cells, abrogated GPCR-induced ERK pathway activation, c-Fos gene product accumulation and DNA synthesis inthese cells. Accordingly, knockdown of PKD1 with siRNAs prevented mitogenic signaling(ERK, c-Fos, DNA synthesis) in response to GPCR agonists in intestinal epithelial cells.Conclusion: These studies, using recently identified PKD family inhibitors, probe furtherthe mechanism of PKD1 activation by multi-site phosphorylation within intact intestinalepithelial cells and provide novel evidence for a major role of the PKD family in mediatingmitogenic signaling, including ERK in these cells.

Mo1649

Dopaminergic Control of Motility in Human and Guinea Pig IntestineGuo-Du Wang, Xiyu Wang, Yun Xia, Dean J. Mikami, Bradley Needleman, W. S. Melvin,Jackie D. Wood

Background and Aims: Dopamine is a major neurotransmitter in the integrative functionsof the brain and, based on our preliminary work, has equivalent importance in the neurophysi-ological control of a complex of intestinal secretory and motility functions by the entericnervous system. Parkinson's disease reflects malfunction of the dopaminergic system in thebrain, which is likely to be the case also in the enteric nervous system and be an underlyingfactor in the pathophysiology of gut dysfunction associated with Parkinson's disease. Wehave reported that dopamine DR1 and DR2 receptors are expressed by enteric neurons inguinea pig and human intestine and to have differential actions on neuronal electrical andsynaptic behavior when stimulated (J. Neurogasterol Mot 2007; 19:41). Understanding theseactions becomes important in terms of the therapeutic efficacy of dopaminergic drugs, suchas domperidone in treatment of gastroparesis and the irritable bowel syndrome. Methods:Classical pharmacological analysis In Vitro was done for circular and longitudinal musclestrips from guinea pig distal ileum and colon and human jejunal segments discarded duringRoux-En-Y gastric bypass surgeries. The numbers of circular and longitudinal strips testedin each of the series presented below ranged from 4 to 20. Results: Application of theselective DR2 agonists, quinpirole (20μM) or sumanirole (10μM), evoked, in concentration-dependent manner, significant increases in baseline contractile tension and in the amplitudeof spontaneously-occurring contractions, without changes in the frequency of contractionsfor all muscle strips. The stimulatory action of the DR2 agonists, in all of the preparationswas suppressed significantly, but not abolished, by the presence of 1μM tetrodotoxin in thebathing medium. Preincubation with the DR2 antagonist, L741626 (20μM), but not theputative DR2 antagonist, domperidone (20μm), suppressed the stimulatory action of 20 μMquinpirole and 10μM sumanirole in all muscle strips. Electrical field stimulation (0.1Hz,0.5 ms, 10 mA) evoked contractile responses in each kind of muscle strip. The responsesto electrical stimulation were enhanced in the presence quinpirole or sumanirole and weresuppressed in the presence of tetrodotoxin. Neither of the DR2 agonists altered contractileresponses to carbachol. Concentrations of domperidone >20μM (i.e., 20-100 μM) suppressedthe action of the DR2 agonists on the electrically-evoked contractile responses. Conclusion:Stimulation of enteric musculomotor neurons by dopaminergic DR2 receptor agonists stimu-lates longitudinal and circular muscle contractile activity in guinea pig and human intestine.(Supported by NIH RO1 DK037238, RO1 DK-068258 and KO8 DK060468)

Mo1650

Purinergic Inhibitory Neuromuscular Transmission in Human Jejunum andGuinea Pig ColonGuo-Du Wang, Yun Xia, Xiyu Wang, Bradley Needleman, Dean J. Mikami, W. S. Melvin,Jackie D. Wood

Background and Aims: Evidence supports ATP and beta-nicotinamide adenine dinucleotide(β-NAD) as neurotransmitters, which act at P2Y1 purinergic receptors to evoke inhibitoryjunction potentials in the intestinal circular muscle coat (Am J Physiol Gastrointest LiverPhysiol. 2007; 292:G1483; Proc Natl Acad Sci U S A. 2007 Oct 9;104:16359). We aimedto compare the actions of the two putative inhibitory musculomotor neurotransmitters inhuman jejunum and guinea pig colon. Methods: Muscle strips cut from the longitudinaland circular axes of guinea pig colon and human jejunal segments, discarded during Roux-En-Y gastric bypass surgeries, were studied in organ baths In Vitro. Intracellular “sharp”microelectrodes were used to record neurally evoked junction potentials in the circularmuscle coat. Results: Bath application of ATP, β-NAD andMRS2365, a selective P2Y1 receptoragonist, each evoked hyperpolarizing responses in a concentration-dependent manner incircular muscle from 22 human and 27 guinea pig preparations. The potency order forguinea pig colon was MRS2365 (IC50=0.7μM) > ATP (IC50=24 μM) > β-NAD (IC50=37μM). The potency order for human jejunum was MRS2365 (IC50=1.3μM) > ATP (IC50=28μM) > β-NAD (IC50=44μM). Preincubation with the selective P2Y1 receptor antagonists,MRS 2179 (20 μM), or MRS 2500 (2μM) suppressed the hyperpolarizing responses to β-NAD or ATP, without any significance difference between 12 human and 16 guinea pigpreparations. Application of 1μM MRS2365, 30μM ATP or 50μM β-NAD each suppressedspontaneously-occurring contractile activity and relaxed baseline contractile tension in circu-lar and longitudinal muscle strips (n=16 longitudinal strips and 16 circular strips removedfrom 4 human jejunal segments; n=24 longitudinal strips and 24 circular strips removedfrom the colon of 6 guinea pigs). Pretreatment with MRS2179 (20μM) or MRS2500 (2μM)reversed the inhibitory actions of MRS2365, ATP or β-NAD. Exposure to apamin, whichblocks small conductance Ca2+-activated K+ channels in smooth muscle, significantly sup-pressed or abolished the actions of β-NAD and ATP to inhibit ongoing contractile activityand relax baseline tension. Conclusions: The results are consistent with a hypothesis that

AG

AA

bst

ract

s