Thyrotropin-releasing hormone (TRH) in the left dorsal vagal complex (DVC) increases the hepatic blood flow in rats

Al194 AGA ABSTRACTS G4878 EFFECT OF CENTRAL UROCORT1N ON CCL4-INDUCED ACUTE LIVER INJURY IN RATS. S . Yokohama. M. Yoneda, K. Tamori, Y. Nakade, S. Takamoto, K. Aso, Y. Sato, M. Aoshima, K. Nakamura and I. Makino. Second Department of Medicine, Asahikawa Medical College, Japan. Central corticotropin-releasing factor (CRF) is known as a key chemical messenger in response to stress, and plays important roles in physiological regulation mediated through the autonomic nervous system. We have demonstrated that intracistemal injection of CRF exacerbates CC14-induced acute liver injury through the sympathetic nervous system. Although CRF receptors, CRF1 and CRF2, have recently identified, a role of these receptors in the brain remains unclear. Urocortin, recently discovered peptide, has been revealed to dominantly binds CRF2 receptor. However, nothing is known about the specific receptor of action for CRF to elicit aggravation of CC14induced acute liver injury, Purpose: To investigate the effect of central urocortin on CC14-induced acute liver injury in rats. Method: Male Wistar rats (280-320 g) were injected with CC14 (2 ml/kg) subcutaneously. Either urocortin (0.5-10 pg) or saline vehicle was injected intracisternally or intravenously just before and 6 h after CC14 injection. The liver tissues were removed 24 h after CC14 injection and the specimens were stained with H&E. Degeneration and necrosis areas were observed under a light microscope, and measured by a computerized image analyzer. The blood samples were obtained before and 24 h after CCI4 injection and serum AST and ALT levels were determined. Hepatic sympathectomy (-3 days), hepatic branch vagotomy (-3 days), or respective sham operation was performed. Results: Administration of CC14 induced regeneration and necrosis in the hepatic tissue 24 h later. Intracisternal urocortin dose-dependently enlarged the regeneration and necrosis areas induced by CC14 (Mean ± SE, %: saline 5.5 ± 1.2; 0.5 pg 6.3 ± 3.0; l lag 10.5 ± 2.2; 3 lag 17.5 ± 1.9; 5 lag 24.3 ± 5.0; 10 jag 23.5 ± 2.6, n=5-6). Elevations of serum AST and ALT levels were also dose-dependently enhanced by intracisternal urocortin. Intravenous urocortin did not influence CCl4-induced acute liver injury. The aggravating effect of central urocortin on CC14-induced acute liver injury was abolished by sympathectomy, but not by vagotomy. Conclusion: Urocortin acts in the brain to exacerbate acute liver injury through sympathetic nervous system and these results suggest the involvement of CRF2 receptor in the exacerbating effect of central CRF on CC14-induced acute liver injury. • G4879 THYROTROPIN-RELEASING HORMONE (TRH) IN THE LEFT DORSAL VAGAL COMPLEX (DVC) INCREASES THE HEPATIC BLOOD FLOW IN RATS. M. Yoneda, K. Tamori, Y. Nakade, S. Takamoto, S. Yokohama, K. Aso, Y. Sato, M. Aoshima, K. Nakamura and I. Makino Dept. of Med. II, Asahikawa Med. Coll., Japan. Intracisterual injection of TRH increases hepatic blood flow (HBF) through the vagus. TRH nerve fibers and receptors are localized in the DVC, which is an important site for vagal regulation, and retrograde tracing technique has revealed that the hepatic vagal efferent nerves mainly originate from the left DVC. However, nothing is known about the site of action for TRH to elicit the increase in HBF. Purpose: To investigate the effect of microinjection of TRH into the DVC on HBF. Methods: HBF was determined by hydrogen gas-clearance technique and laser doppler flowmeter. Male Wistar rats (250-280 g) were anesthetized with urethane (1.5 g/kg, ip), and positioned on stereotaxic apparatus. A abdominal incision was performed, and platinum wire type electrode was inserted into the hepatic left lateral lobe. A probe of laser doppler flowmeter was placed on the surface of the liver. After 60 min stabilization, two measurement of basal HBF by hydrogen gas clearance technique was performed. The stable TRH analog, RX 77368 (0.1, 0.3, 0.5, 1, 5, 10 ng) or vehicle (saline) was microinjected into either the left or tight DVC. HBF was observed for 120 min post peptide injection. Either left or right cervical vagotomy, or hepatic branch vagotomy was performed 120 min before the peptide. Results: Microinjection of TRH (5 ng) into the left DVC significantly increased HBF with peak response at 30 rain, and the enhanced HBF was returned to basal at 90 rain (Mean ± SE; %: base 100 ± 0; 15 min 146_+7; 30 min 178±12; 45 min 168±11; 60 rnin 145±10; 75 min 125±14; 90 min 109+5; 105 rain 101±7; 120 min 98+-5). This stimulatory effect by microinjection of TRH into the left DVC was doserelated ranging from 0.3 to 5 ng (at 30 rain; saline 104 ± 12; 0.1 ng 102 ± 6; 0.3 ng 112±8; 0.5 ng 144±14; 1 ng 155±12; 5 ng 178±12; 10 ng 181 ± 15). However, TRH (5 ng) microinjection into the right DVC induce only a slight increase in HBF, but it is not significant change. Stimulation of HBF by TRH microinjection into the left DVC was abolished by left cervical and hepatic branch vagotomy but not by right cervical vagotomy. Conclusion: TRH acts in the left DVC to stimulate HBF through the left trancal and hepatic vagus, and these findings suggest that neuropeptides act in the specific brain nuclei to regulate hepatic microcirculations.

GASTROENTEROLOGYVol. 114, No. 4 • G4880 CALMODULIN-DEPENDENT PROTEIN KINASE AND DIACYLGLYCEROL-ACTIVATED PROTEIN KINASE C ARE INVOLVED IN CHOLECYSTOKININ SECRETION BY PHENYLALANINE IN STC-1 CELLS. H. Yoshida, M Iida, J. Niikawa, S. Tanaka, K. Mitamura. Second Int. Med., Showa Univ., Tokyo, Japan. BACKGROUND: Secretion of cholecystokinin (CCK), a key gastrointestinal hormone, is stimulated by amino acids, diazepam binding inhibitor (DBI), luminal CCK-releasing factor (LCRF) or fatty acids from small intestine to regulate pancreatic secretion, growth, gastrointestinal motility and satiety. We had proposed, DBI33.5o, a CCK-releasing peptide (CCK-RP), directly acts on STC-1, an intestinal endocrine tumor cell line, developed in mice, to evoke Ca2+ via L-type voltage-dependent Ca2÷ channels (VDCCs) of plasma membrane which result in secretion of CCK (Gastroenterol 110: AI135, 1995). AIM: To investigate responsible cellular mechanism involved in the simulation of CCK secretion by an aromatic amino acid, L-phenylalanine (L-Phe) which penetrates through plasma membrane, we compared signal tranduction pathways activated by L-Phe to those by bombesin (BBS) utilizing STC-1 ceils. METHODS: STC-1 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) with horse serum and fetal calf serum, then incubated with 1 ml of release medium containing BSA and aprotinin. CCK was measured by enzyme immunoassay using anti-CCK-8specific antiserum (OAL-656) and alkaline phosphatase (ALP)-CCK-8 or the ALP-CCK-33 conjugate solution. The calmodulin-dependent protein kinase (CaMK) II activity was measured by 32P-incorporation transferred from [~,_32p] ATP using autocamtide II as the substrate. RESULTS: A dosedependent stimulation in CCK secretion was observed for 60 rain-incubation of STC-1 cells with L-Phe. No significant increase in CCK secretion was observed with D-Phe. BAPTA/AM (1 and 10 pM), an intracellular chelator, significantly inhibited CCK secretion stimulated by 50 raM L-Phe. W-7 (50 taM), a calmodulin antagonist, significantly inhibited CCK secretion. Pretreatment with various dosages, 0.5, 1 and 5 pM of KN-62, a selective inhibitor of CaMK II, reduced L-Phe-stimulated CCK secretion to 69.9, 38.9 and 19.3 % of those levels without KN-62, respectively. This secretion was inhibited in a dose-dependent manner. R59022 (5 raM), the diacylglycerol (DAG) kinase inhibitor I, induced a significant augmentation of the maximal CCK secretion stimulated by 50 mM L-Phe. H-7, calphostin C and staurosporine, protein kinase C (PKC) inhibitors, significantly inhibited CCK secretion stimulated by L-Phe. CaMK II activity was increased by 50 and 75 mM L-Phe. A maximal stimulation was observed at 20 minincubation with L-Phe. BBS, 1 pM upto 100 nM, which dose-dependently evoked CCK secretion did not affect CaMK II activity. CONCLUSIONS: We conclude that L-Phe dose-dependently stimulated CCK secretion from STC-1 ceils utilizing intracellular Ca2+. This study also indicates that DAG-activated PKC and CaMK II mainly mediated L-Phe-stimulated CCK secretion but CaMK II failed to do BBS-stimulated CCK secretion. G4881 EVIDENCE FOR EFFERENT FUNCTION OF DORSAL ROOT GANGLION (DR(;) NEURONS ONTO COLONIC SMOOTH MUSCLE CELLS IN PRIMARY CULTURE. S.H. Young, H.S. Ennes, and E.A. Mayer. UCLA/CURE Neuroenteric Disease Program, Depts. of Medicine and Physiology, and Brain Research Institute, UCLA School of Medicine, and West LAVA Medical Center, Los Angeles, CA 90024. Background: Retrograde (efferent) signaling from peripheral terminals of primary afferent neurons plays a role in communication between DRG neurons and various target ceils. Such signaling can occur in form of axon reflexes resulting in action potential-mediated peripheral neurosecretion, or may involve exchange of intracellular messenger molecules (Ca2÷, Ins(l,4,5)P3) via heterologous gap junctions (Neuroreport 8:733; 1997). Aims: To determine if mechanical or electrical stimulation of DRG somas cocultured with colonic myocytes results in changes in [Ca2÷]i in neuritecontacted myocytes. Methods: Primary rat colonic smooth muscle ceils were grown in culture to sub-confluent densities. Freshly dissociated DRG neurons were then added to these cell cultures and grown for an additional 48-72 hours in the presence of 5ng/ml NGF. [Ca2+]i changes in response to mechanical stimulation were monitored using fura-2 fluorescence videomicroscopy. Somas of selected DRG neurons were placed under patch clamp; mechanical stimulation of the soma was accomplished by suction applied to the patch electrode in cell attached mode; electrical stimulation of the soma was accomplished by a train of depolarizing current pulses with the patch clamp configuration of whole cell mode (duration 50 msecs, frequency 2 Hz). Results: Distance between soma and myocyte was always >50pm. Average neuritic [Ca2+]i wave velocity was 17 larn/sec. Both trains of depolarizing pulses and suction resulted in somatic action potentials in -20% of the neurons. Suction on cell-attached membrane patches of DRG somas resulted in 2 different responses: a) small amplitude, slowly rising [Ca2÷]j transients in 37% of the contacted myocytes (n=16). Time to peak ranged from 1 to 55 sees (median 6.5 see), and soma to myocyte delay ranged from 4 to 6 sees. b) fast transient increases in [Ca2*]i in 25% of the nerve muscle contacts. Time to peak was < 1.5 sees, and soma to myocyte delay was < 0.2 sees. Conclusions: Since the soma-myocyte distance was never less