Gintautas Grabauskas

Synergistic interaction between leptin and cholecystokinin in the rat nodose ganglia is mediated by PI3K and STAT3 signaling pathways: implications for leptin as a regulator of short term satiety.

Research has shown that the synergistic interaction between vagal cholecystokinin-A receptors (CCKARs) and leptin receptors (LRbs) mediates short term satiety. We hypothesize that this synergistic interaction is mediated by cross-talk between signaling cascades used by CCKARs and LRbs, which, in turn, activates closure of K+ channels, leading to membrane depolarization and neuronal firing. Whole cell patch clamp recordings were performed on isolated rat nodose ganglia neurons. Western immunoblots elucidated the intracellular signaling pathways that modulate leptin/CCK synergism. In addition, STAT3, PI3K, Src, and MAPK genes were silenced by lentiviral infection and transient Lipofectamine transfection of cultured rat nodose ganglia to determine the effect of these molecules on leptin/CCK synergism. Patch clamp studies showed that a combination of leptin and CCK-8 caused a significant increase in membrane input resistance compared with leptin or CCK-8 alone. Silencing the STAT3 gene abolished the synergistic action of leptin/CCK-8 on neuronal firing. Leptin/CCK-8 synergistically stimulated a 7.7-fold increase in phosphorylated STAT3 (pSTAT3), which was inhibited by AG490, C3 transferase, PP2, LY294002, and wortmannin, but not PD98059. Silencing the Src and PI3K genes resulted in a loss of leptin/CCK-stimulated pSTAT3. We conclude that the synergistic interaction between vagal CCKARs and LRbs is mediated by the phosphorylation of STAT3, which, in turn, activates closure of K+ channels, leading to membrane depolarization and neuronal firing. This involves the interaction between CCK/Src/PI3K cascades and leptin/JAK2/PI3K/STAT3 signaling pathways. Malfunctioning of these signaling molecules may result in eating disorders.

Protein kinase signalling requirements for metabotropic action of kainate receptors in rat CA1 pyramidal neurones

Hippocampal pyramidal neurones display a Ca2+‐dependent K+ current responsible for the slow afterhyperpolarization (IsAHP), a prominent regulator of excitability. There is considerable transmitter convergence onto IsAHP but little information about the interplay between the kinase‐based transduction mechanisms underlying transmitter action. We have added to existing information about the role of protein kinase C (PKC) in kainate receptor actions by demonstrating that direct postsynaptic activation of PKC with either 1‐oleoyl‐2‐acethylsn‐glycerol (OAG) or indolactam is sufficient to inhibit IsAHP. The physiological correlate of this action – activation of PKC by kainate receptors – requires Gαi/o proteins. The cAMP/PKA system is well documented to subserve the actions of monoamine transmitters. We have found an additional role for the cAMP/PKA system as a requirement for kainate receptor‐mediated inhibition of IsAHP. Inhibition of adenylyl cyclase with dideoxyadenosine or PKA with either H89 or RpcAMPs blocked kainate receptor‐mediated actions but did not prevent the actions of direct PKC activation with either OAG or indolactam. We therefore propose that the PKA requirement is upstream from the actions of PKC. We additionally report a downstream link in the form of increased mitogen‐activated protein (MAP) kinase activity, which may explain the long duration of metabotropic actions of kainate receptors on IsAHP.

Prolactin‐releasing peptide affects gastric motor function in rat by modulating synaptic transmission in the dorsal vagal complex

Prolactin‐releasing peptide (PrRP) is a recently discovered neuropeptide implicated in the central control of feeding behaviour and autonomic homeostasis. PrRP‐containing neurones and PrRP receptor mRNA are found in abundance in the caudal portion of the nucleus tractus solitarius (NTS), an area which together with the dorsal motor nucleus of the vagus (DMV) comprises an integrated structure, the dorsal vagal complex (DVC) that processes visceral afferent signals from and provides parasympathetic motor innervation to the gastrointestinal tract. In this study, microinjection experiments were conducted in vivo in combination with whole‐cell recording from neurones in rat medullary slices to test the hypothesis that PrRP plays a role in the central control of gastric motor function, acting within the DVC to modulate the activity of preganglionic vagal motor neurones that supply the stomach. Microinjection of PrRP (0.2 pmol (20 nl)−1) into the DMV at the level of the area postrema (+0.2 to +0.6 mm from the calamus scriptorius, CS) markedly stimulated gastric contractions and increased intragastric pressure (IGP). Conversely, administration of peptide into the DMV at sites caudal to the obex (0.0 to –0.3 mm from the CS) decreased IGP and reduced phasic contractions. These effects occurred without change in mean arterial pressure and were abolished by ipsilateral vagotomy, indicating mediation via a vagal‐dependent mechanism(s). The pattern of gastric motor responses evoked by PrRP mimicked that produced by administration of l‐glutamate at the same sites, and both the effects of l‐glutamate and PrRP were abolished following local administration of NMDA and non‐NMDA‐type glutamate receptor antagonists. On the other hand, microinjection of PrRP into the medial or comissural nucleus of the solitary tract (mNTS and comNTS, respectively) resulted in less robust changes in IGP in a smaller percentage of animals, accompanied by marked alterations in arterial pressure. Superfusion of brain slices with PrRP (100–300 nm) produced a small depolarization and increased spontaneous firing in 10 of 30 retrogradely labelled gastric‐projecting DMV neurones. The excitatory effects were blocked by administration of TTX (2 μm) or specific glutamate receptor antagonists, indicating that they resulted from interactions of PrRP at a presynaptic site. Congruent with this, PrRP increased the amplitude of excitatory postsynaptic currents (EPSCs, 154 ± 33%, 12 of 25 neurones) evoked by electrical stimulation in mNTS or comNTS. In addition, administration of PrRP decreased the paired‐pulse ratio of EPSCs evoked by two identical stimuli delivered 100 ms apart (from 0.95 ± 0.08 to 0.71 ± 0.11, P < 0.05), whereas it did not affect the amplitude of inward currents evoked by exogenous application of l‐glutamate to the slice. The frequency, but not amplitude of spontaneous EPSCs and action potential‐independent miniature EPSCs was also increased by administration of PrRP, suggesting that the peptide was acting at least in part at receptors on presynaptic nerve terminals to enhance glutamatergic transmission. In recordings obtained from a separate group of slices, we did not observe any direct effects of PrRP on spontaneous discharge or postsynaptic excitability in either mNTS or comNTS neurones (n= 31). These data indicate that PrRP may act within the DVC to regulate gastric motor function by modulating the efficacy of conventional excitatory synaptic inputs from the NTS onto gastric‐projecting vagal motor neurones.

Diabetic Visceral Hypersensitivity Is Associated With Activation of Mitogen-Activated Kinase in Rat Dorsal Root Ganglia

OBJECTIVE Diabetic patients often experience visceral hypersensitivity and anorectal dysfunction. We hypothesize that the enhanced excitability of colon projecting dorsal root ganglia (DRG) neurons observed in diabetes is caused by a decrease in the amplitude of the transient A-type K+ (IA) currents resulting from increased phosphorylation of mitogen-activated protein kinases (MAPK) and reduced opening of Kv4.2 channels. RESEARCH DESIGN AND METHODS We performed patch-clamp recordings of colon projecting DRG neurons from control and streptozotocin-induced diabetic (STZ-D) rats. Western blot analyses and immunocytochemistry studies were used to elucidate the intracellular signaling pathways that modulate the IA current. In vivo studies were performed to demonstrate that abnormal MAPK signaling is responsible for the enhanced visceromotor response to colorectal distention in STZ-D rats. RESULTS Patch-clamp studies demonstrated that IA current was diminished in the colon projecting DRG neurons of STZ-D rats. Western blot analysis of STZ-D DRG neurons revealed increases in phosphorylated MAPK and KV4.2. In diabetic DRG neurons, increased intracellular Ca2+ ([Ca2+]i), protein kinase C (PKC), and MAPK were involved in the regulation of IA current through modulation of Kv4.2. Hypersensitive visceromotor responses to colorectal distention in STZ-D rats were normalized by administration of MAPK inhibitor U0126. CONCLUSIONS We demonstrated that reduction of the IA current in STZ-D DRG neurons is triggered by impaired [Ca2+]i ion homeostasis, and this in turn activates the PKC-MAPK pathways, resulting in decreased opening of the Kv4.2 channels. Hence, the PKC-MAPK–Kv4.2 pathways represent a potential therapeutic target for treating visceral hypersensitivity in diabetes.

Mo2033 Marked Elevation in Mucosal Proinflammatory PGE2 Is Responsible for Pain in Diarrhea Predominant IBS (IBS-D) Patients

Background: Patients with irritable bowel syndrome (IBS) suffer from abdominal pain and associated constipation, diarrhea, and bloating. Some IBS patients (~35%) have visceral hypersensitivity to noxious experimental stimuli (e.g., colonic distension). We previously reported that colonic NMDA receptor signaling in IBS patients may be an underlying mechanism leading to visceral hypersensitivity that is reversed with Dextromethorphan (J Pain 2012;13:901-9). The current study was performed to determine if down regulation of in vivo NMDA receptor signaling via upregulation of miR-23a using miRNA mimics reverses visceral hypersensitivity in an IBS animal model. Methods: Male rats (n=12) received onehour water avoidance (WA) stress per day, for 10 consecutive days to induce visceral hypersensitivity as measured by colonic distension using a barostat. Intrathecal injection of miR-23a mimics (n=6 rats) or miRNA control (n=6 rats) was then performed and visceral hypersensitivity was measured 7 days later following injections. Following colonic barostat testing, all rats were sacrificed to measure colonic NMDA receptors and miR-23a expression using fluorescence in situ hybridization (FISH) using tyramide signal amplification (TSA) technology. Results: Rats treated with intrathecal injection of miR-23a mimics had decreased visceral hypersensitivity compared to rats that received intrathecal control miRNA (p<0.01). There was a significant increase in colonic miR-23a expression and a decrease in NMDA receptor signaling in rats that received miR-23a mimcs that correlated with decreased visceral hypersensitivity.Conclusion:Down regulation of NMDA receptor signaling through augmentation of miR-23a reverses visceral hypersensitivity in WA rats. Aberrant expression of key miRNAs dysregulate downstream targets such as NMDA receptors which contribute to visceral hypersensitivity in IBS patients. This data suggests that miR-23a could be a new therapeutic target for attenuating visceral hypersensitivity in IBS patients. Work supported by NIH grant (R01DK099052).

887 Analgesic Action of Resolvin D1 to Combat Visceral Pain in a Rat Model of Visceral Hypersensitivity

G A A b st ra ct s root ganglia (DRG) neurons. The aim of this study was to examine the downstream signaling pathways mediating these effects, with the focus on Smad 3 and TRPV1. Methods: In vitro patch clamp studies were performed on dissociated DRG neurons. For in vivo studies, CP was induced by intraductal injection of trinitrobenzene sulfonic acid (TNBS) in adult male rats, with controls receiving the same volume of vehicle, followed by intrapancreatic injection of DiI, a retrograde neuronal tracer, to label the DRG neurons that project to pancreas. Three weeks later, SIS3 (2.5mg/kg i.p. daily) was administered for 1 week. Subsequently, thoracic DRGs were harvested and stained for pSmad3 using immunohistochemistry. Phosphorylated Smad3 (pSmad3), Smad3 and TRPV1 protein levels in the thoracic DRGs (T8T12) were measured using immunoblot assay. Results: DRG cells were cultured with vehicle (1μg/ml DMSO-solvent for SIS3), TGF β1(10ng/ml), SIS3-TGF β1(10ng/ml-0.3μM), respectively for 48 hours and then TRPV1 currents were determined by the response to 1μM of capsaicin. TGFβ1 significantly enhanced TRPV1 currents in DRG neurons in culture (99.06±24 pA/pF; n=6) compared to controls (44.77±14 pA/pF; n=8), an effect that was blocked by SIS3 (26.23±4.4 pA/pF; n=6) (p<0.01 by ANOVA). However, sis3 was unable to block TRPA1 currents induced by mustard oil, attesting to the relative specificity of this effect. In vivo, as shown previously, SIS3 significantly attenuates both somatic referred hypersensitivity (measured by VFF) and pancreatic hyperalgesia (measured by behavioral response to pancreatic electrical stimulation) in rats with CP. pSmad3 positive neurons in the DiI-labeled thoracic neurons were significantly increased in CP rats (79.06±4.72% compare with control 67.15±1.53%, p<0.05), which can be reduced by treatment with SIS3 (63.64±2.21%, P<0.05). Immunoblot confirmed that pSmad3 levels, but not total Smad3, were significantly reduced in the DRGs (T8-T12) of rats treated with SIS3 relative to vehicle treated rats (P<0.05). Finally, compared with vehicle treatment we found that SIS3 significantly reduced TRPV1 protein levels in rats with CP (106.11±9.44 vs 70.32±6.17, P<0.05). Conclusions: The present results demonstrate a novel link between TGFβ1 signaling and TRPV1 function and expression, an effect that appears to be mediated by Smad3. These results provide the fundamental basis for developing novel therapeutic approaches for CPinduced pain.

Butyrate-Induced Colonic Hypersensitivity is Maintained by ERK1/2-CREB Pathway-Dependent Upregulation of CGRP Expression in Rat Dorsal Root Ganglia Neurons

mesolimbic nodes). Lean subjects demonstrated greater connectivity relative to obese between caudate and executive control network (associated with control processes) in both conditions. Lean subjects relative to obese showed more positive connectivity between posterior cingulate cortex and frontoparitetal network (FPN) during LOW, but not HIGH. Obese subjects showed: 1) more positive connectivity between INS and FPN during HIGH; 2) more positive connectivity between putamen and DMN during LOW and 3) more positive connectivity between the parahippocampal gyrus (PHG) and FTPN during LOW. Conclusions: Ingestion of a high caloric drink is associated with reduced connectivity in both lean and obese subjects within multiple networks. The networks showing nutrient-induced reduction of connectivity include key regions of an interoceptive network (INS and MCC). In addition, obese subjects show greater connectivity of regions involved in reward-motivated and emotional learning (PHG, putamen, INS). fcMRI is a potential powerful tool to study brain mechanisms involved in obesity. Supported by NIH grants P30 DK041301, T32 DK007180, R24 AT002681, R01 DK048351, P50DK64531

658 Chronic Hyperglycemia in Diabetes Alters Electrophysiological Properties of a Subset of DRG Neurons in Rats. Implication in Disturbed Anorectal Functions in Diabetes

muscle, in the basal state, via computerized Power Lab chart recording system. In addition, we investigated the transcriptional (mRNA levels) and translational expressions (protein levels) of COX-1 and COX-2 in the IAS vs. the rectal smooth muscle (RSM), in the basal state. COX-1 and COX-2 specific mRNA and protein levels were determined using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses, respectively. We also determined and compared the effects of selective deletion of COX-1 and COX-2 genes on the murine IAS basal tone. Results: SC-560 caused significantly more efficacious and potent decrease in the IAS tone as compared with rofecoxib. The concentrations causing 50% of the maximal response (pIC50in the case of SC-560 and rofecoxib were 6.7 ± 0.1 and 5.0 ± 0.1, respectively. Additional detailed data show significantly higher expressions of COX-1 mRNA and proteins as compared with those of COX-2, in the rat IAS vs. the RSM. The IAS basal tone in the wild type mice 0.17 ± 0.02 mN//mg, was significantly less (0.08 ± 0.01 mN/mg) in the COX-1-/mice (*; p < 0.05; n =5). However, the basal tone in the COX-2-/mice was not significantly different from their counterpart wild type. In the wild type mice SC-560 (1x10-5 M) caused 41.4 ± 3.4% (mean ± SE) significant decrease (p < 0.05; n = 5) in the IAS tone vs. 5.4 ± 2.2% decrease caused by rofecoxib. In COX-1-/mice, SC-560 caused no further significant decrease while it produced a significant decrease in the IAS tone of COX-2-/mice. These latter data further validate the selective genetic deletions of COX-1 and COX-2, and of the selectivity of COX-1 and COX-2 inhibitors, in the IAS tone. Finally, both thromboxane A2 (TXA2) and prostaglandin F2α (PGF2α) antagonists SQ-29598 and AL-8810 respectively, produce significant decrease in the IAS tone. Conclusions: Collectively, we conclude that COX-1-related products (especially TXA2 and PGF2α) provide a significant contribution to the IAS tone.