- Email: [email protected]
Histamine H3 receptors mediate both gastro-protective and antiinflammatory effects
Al136 AGA ABSTRACTS • G4648 SOMATOSTATIN RECEPTOR SUBTYPE 5 MEDIATES INHIBITION OF PEPTIDE YY SECRETION FROM INTESTINAL CULTURES. C. Chisolm and G.R.Greenberg, Dept. of Medicine and Physiology University of Toronto, Toronto, Canada. Background/Aims: Five somatostatin receptors subtypes (SSTR) have been characterized in the gastrointestinal tract. Both bioactive forms of somatostatin, S-14 and S-28, bind to all SSTR's, but S-28 localized predominantly in ileum/colon has preferential affinity for SSTR5. This study examined whether SSTR-5 mediates somatostatin inhibition of PYY, also found in ileum/colon. Methods: Rat intestinal cultures were treated with somatostatin agonists with relatively high specificity for SSTR1-5. Results: Administration of GRP (10-TM) stimulated PYY secretion to 161 _+ 12% of paired controls; this response was not influenced by pertussis toxin (200 ng/ml) or nitredipine (50 uM). PYY secretion was more potently inhibited by S-28 (ICso 0.1nM) then by S-14 (IC5o 100nM). Phosphoramidon and amastatin did not alter the potencies of S-28 or S-14. The SSTR5 agonist, BIM 23052 inhibited PYY secretion (ICs0 0.1nM) with similar potency to S-28. Whereas the SSTR5 agonist L372, 588 also inhibited PYY (IC5o 0.1nM), the structurally-related peptides L-372, 587 (tyrosine conversion: 7 from 2) and L362, 855 (phenylalanines: both 2 and 7) caused no inhibition at I00 nM. The SSTR agonist NC8-12 was marginally more potent (ICso 50nM) than S-14 while an SSTR3 agonist was without effect at 100nM. Pertussis toxin blocked PYY inhibition by S-28 and the SSTR5 agonists; nitredipine was without effect. PYY stimulated by PMA was abolished by S-28 and the SSTR-5 agonists at lnM, but after forskolin-stimulated PYY secretion, S-28 and BIM 23052 caused two-fold less inhibition and L372,588 was without effect. Conclusions: S-28, acting through a Gi protein mediates inhibition of GRP and protein-kinase C stimulated PYY secretion through SSTR5 activation. Hydroxyl group conversions of SSTR5 peptides may facilitate the development of more potent agonists or antagonists. Supported in part by a grant from MRC Canada. • G4649 INTERNALIZATION IS NOT IMPORTANT FOR DESENSITIZATION OR RESENSITIZATION OF THE CHOLECYSTOKININ TYPE B RECEPTOR (CCKBR). J.-K. Choi. C. Linehan, N. Perin, M. Pohl and S.A. Wank. DDB, NIDDK, NIH Bethesda, MD.
For some GPCRs, such as the 13 adrenergic receptor, immediate uncoupling from G proteins is the predominant mechanism for desensitization while for others, such as the CCKAR, intermediate term compartmentalization predominates. CCKBRs expressed in neurons undergo rapid desensitization and we have shown in a number of transformed cell lines that CCKBRs undergo rapid internalization that is in large part dependent on serine and threonine residues in the cytoplasmic tail. At present, there is no convenient source of either pure primary cells or transformed cell lines expressing high levels of only type B CCKRs. Therefore, to characterize the process of internalization and desensitization of CCKBRs, we studied the rat CCKBR stably expressed in CHO-K1 cells. To determine the role of internalization in the desensitization process, an internalization impaired mutant CCKBR with all 10 potential Ser/Thr phosphorylation sites mutated to Ala, CCKBR(AS/T), was also expressed in CHO-K1 cells. Clones expressing similar numbers of CCKBR and CCKBR(AS/T) displayed similar affinity for CCK-8 with IC50s of 0.5 and 1.0 nM, respectively, in 125I-CCK-8 binding displacement studies. CCKBR(AS/T) internalization was markedly reduced to = 5% at 5 minutes compared to = 60% for CCKBR as measured by stripping of surface bound 125I-CCK-8 with 0.5 M KSCN. Both the CCKBR and CCKBR(AS/T) stimulated total inositol phosphates with similar potencies (EC50 = 2.2 and 0.8 nM), respectively. To study short term desensitization and resensitization in the same cells in real time, we measured CCK-8 stimulated changes in acidification rate (AR) using a microphysiometer (Molecular Devices). Similar to total inositol phosphate stimulation, CCK-8 exhibited a dosedependent increase in the AR with EC50s of 8 and 4 nM, respectively. For both the CCKBR and CCKBR(AS/T), a maximal desensitizing dose of 100 nM CCK-8 for one rain. caused an = 85% and = 65% reduction of the expected response when rechallenged with a half-maximal and maximal dose of CCK-8 at 5 min. with full resensitization by 15 and 30 rain., respectively. The degree of desensitization and rate of resensitization were independent of the desensitizing dose of CCK-8 when rechallenged with an equal dose. Desensitization by 1 nM CCK-8 was nearly completely overcome by rechallenging with a maximal dose of 100 nM CCK-8 or stimulation of another endogenous GPCR receptor. Increasing the duration of the desensitizing dose from 1 to 10 min had a minimal effect upon either the degree of desensitization or rate of resensitization. These data indicate that the CCKBR undergoes rapid internalization and desensitization and suggest that, unlike the CCKAR, receptor internalization is not an important component of short term desensitization or resensitization.
GASTROENTEROLOGY Vol. 114, No. 4 G4650
SIMULATED MICROGRAVITY SUPPRESSES INDUCIBLE CYTOCHROME OXIDASE ACTIVITY IN A HEPATOMA CELL LINE. S.W. Cohen, Y.F. Shiau, M. Yeh, B. Hakim, LSUMC Shreveport LA. Inhibition of cellular activity has previously been demonstrated in microgravity environments. Scant data is available conceming the effect of gravity on hepatic function, particularly regarding the P450 mixed function oxidases that play a crucial role in drug biotransformation. The hepatoma cell line serves as a useful model for examining these inducible hepatic enzymes. Briefly, hepatoma (Hep G2) cells were grown on glass coverslips and incubated with 3-methylcholanthrene (MCA), a polycyclic aromatic hydrocarbon inducer of the P450 enzyme aryl hydrocarbon hydroxylase (AHH). Cell Cultures were exposed to lg or 0g environments throughout the 30 hour incubation period. A rapidly rotating clinostat was used to simulate 0g conditions. Fluorescence spectrophotometry was used to measure AHH activity, as determined by production of the 3-hydroxylated metabolite of benzo[a]pyrene. Activity was expressed as units of AHH activity per milligram of protein. As compared to lg cultures, cells undergoing induction in 0g demonstrated a 4-fold reduction in AHH activity (p<0.01). No activity was detected in solvent controls. Inhibition of hepatic P450 cytochrome oxidase activity may have important implications in long duration manned spaceflight through disruption of normal hepatic homeostatic mechanismsand its pharmacologicconsequences. G4651
ROLE OF NITRIC OXIDE IN THE ANTIPROLIFERATIVE SIGNAL MEDIATED BY THE SOMATOSTATIN RECEPTOR sst5. P. Cordelier J.-P. Est~ve, P. Rochaix, Jj. Voigt, A. V. Schally*, N. Vaysse, C. Susini, L. Buscail. INSERM U151, IFR 31, CHU Rangueil; Department of Pathology, Institut Claudius Regaud, 31403 Toulouse, France and *Tulane University, New Orleans, LA. Somatostatin exerts inhibitory effects on the exocrine and endocrine secretory processes of GI tract as well as on cell proliferation. We recently demonstrated that the human sst5 somatostatin receptor, stably expressed in CHO cells, mediated the antiproliferative effect of the somatostatin analogue RC-160. This effect depends on the inhibition of successively: a soluble guanylate cyclase, protein kinase G and p42 MAP kinase activities (PNAS, 1997;94:9343). Aim of the present study was thus to investigate, the implication of nitric oxide (NO), a known activator of soluble guanylate cyclase, in the antiproliferative effect mediated by sst5 receptor. The cell proliferation of CHO cells stably expressing the human sst5 receptor (CHO/sst5) was stimulated by agents known to increase the intracellular levels of NO and/or cGMP such as L-arginine (10 nM; + 63%), sodium nitropmssiate (SNP) (0.1 ~,i; + 47%) and CCK-8 (0.1 p/vl; + 43 %). CCK-induced cell proliferation was inhibited by the NO synthase inhibitors such as L-NAME (100 laM) and GGA (0.1 laM) suggesting a role for NO in the proliferation of CHO/sst5 ceils. The analogue RC-160 inhibited cell proliferation stimulated by CCK-8, L-arginine and SNP, this effect being abolished by a pre-treatment of ceils by pertussis toxin. NO synthase activity of CHO/sst5 cells was stimulated by a CCK-8 treatment (0.1 laM; + 177%). RC-160 inhibited CCK-induced stimulation of NO synthase activity (maximal: - 83%) in a dose dependent manner, the ED50 (0.33 nM) being related to those observed for inhibition of CCK-induced cell proliferation (1.1 nM) and CCK-induced increase of cGMP levels (0.32 nM), as well as to the affinity of the analogue for recombinant sst5 receptor. In parallel, expression studies at the mRNA (RT-PCR) and/or protein level (Immunocytochemistry) were performed in human normal and tumor tissues: we observed that sst5 receptor was expressed not only in pituitary but also in stomach, endocrine and exocrine pancreas, gallbladder, and colon. In conclusion, our results suggest that inhibition of cell proliferation mediated by sst5 somatostatin receptor results from the inhibition of NO synthase activity. This negative coupling may have important implications, not only in the negative regulation of cell proliferation but also in the regulation of digestive tract secretory, vasoactive and motor functions regulated by somatostatin and its analogues in human. G4652
HISTAMINE H 3 RECEPTORS MEDIATE BOTH GASTROPROTECTIVE AND ANTHNFLAMMATORY EFFECTS. G. Coruzzi, G. Bertaccini. Institute of Pharmacology, Parma, Italy. Histamine H3 receptors have been involved in the negative regulation of gastric acid secretion and intestinal motility (1). A reduction of neurotransmitter release from different types of neurons, as well as an inhibitory effect on histamine synthesis and release were demonstrated in a variety of tissues. In the present study the effects of the histamine H3 receptor selective agonists (R)cc-methylhistamine (MHA) and immepip (IMM) were investigated in the rat on the gastric lesions induced by different noxious stimuli and on the hindpaw oedema induced by 1% carrageenan. MHA (1-100 mg/kg i.g.) significantly reduced the gastric damage induced by absolute ethanol (100% reduction at 100 mg/kg i.g) and, to a lesser extent, that induced by acetylsalicylic acid and indomethacin. Histological
April 1998 examination of MHA-treated stomachs by light and electron microscopy revealed that MHA promotes a rapid process of re-epithelization, increases volume and number of surface and neck mucous cells, and increases adherent mucus layer thickness. IMM (3-100 mg/kg i.g.) was virtually ineffective. The protective effect of MHA 10 and 30 mg/kg was prevented by the H 3 antagonist clobenpropit (3 mg/kg i.g.). Both MHA and IMM (3-10 mg/kg i.g. or s.c.) significantly reduced paw swelling produced by subplantar injection (0.1 ml) of carrageenan (maximum inhibition was 48.6 +- 10.3%, after MHA 10 mg/kg s.c.). The antiinfiammatory effects of H a agonists were more pronounced when these compounds were administered before carrageenan injection, rather than on established inflammation. The antiinflammatory effects of the H 3 agonists were counteracted by clobenpropit (3 mg/kg s.c.), which per se did not modify carrageenan-induced paw oedema. Thermal hyperalgesia induced by carrageenan was not significantly modified by Ha ligands. In conclusion, histamine H 3 receptors may mediate antiinflammatory effects in the rat; it is of interest that this action is not accompanied by gastric damage, but actually, at least in the case of MHA, by a definite gastroprotective effect. [1] Bertaccini, G., Coruzzi, G~ Dig. Dis. Sci. 40:2052-2063 (1995) • G4653
PROTEINASE-ACTIVATED RECEPTORS (PARs) ON MYENTERIC NEURONS: ACTIVATION BY TItROMBIN AND MAST CELL TRYPTASE. C,U. Corvera, K. McConalogue, P. Gamp, G. H. Caughey, N.W. Bunnett. Depts. of Surg., Physiol. & Med. University of California, San Francisco, CA. PARs are a new family of G-protein coupled receptors that are activated by proteolysis. Thrombin cleaves PAR-1 in neurons and astrocytes of the CNS to regulate morphology, growth and survival. Trypsin and tryptase, a major secretory granule protease of mast cells, cleave and trigger PAR-2. Cleavage exposes tethered ligand domains that bind and activate the receptors. Myenteric neurons may be exposed to thrombin during injury and to tryptase when mast cells degranulate. We examined the hypothesis that thrombin and tryptase excite myenteric neurons by cleaving and activating PAR-1 and PAR-2. Neurons were isolated from the myenteric plexus of the guinea pig small intestine and examined after 7-14 days in culture. PARs were localized in cultured neurons and whole mounts of the myenteric plexus by immunofluorescence and confocal microscopy, using region specific antibodies to PAR-1 and PAR-2. [Ca2+]i was measured in individual cultured neurons using Fura-2/AM. Immunoreactive PAR-1 and PAR-2 were detected in many neurons. PAR-1 was mostly detected in processes, whereas there were large intracellular stores of PAR-2 in the soma. Specificity of staining was confirmed by preabsorption with the peptides that were used for immunization. Thrombin and a peptide corresponding to the PAR-1 tethered ligand (SFLLRN) caused a prompt and sustained increase in [Ca~+]j in many cells (ECs0 ~1 nM and ~100 nM, respectively). Of the cells that responded to 10 nM thrombin, 70% (n=104 cells) also responded to 55 mM KC1, indicating that they are neurons, Other PAR-1 responsive cells may be glial cells. Similarly, trypsin, tryptase and a peptide corresponding to the PAR-2 tethered ligand (SLIGKV-NH2) caused a rapid and transient increase in [Ca2+]i in many cells (ECs0 ~0.1 nM, ~1 nM and ~ 1 ~tM, respectively). Of the cells that responded to PAR-2 agonists, 72% (n=86 cells) also responded to 55 mM KCI, indicating that they are neurons, and 14% responded to 100 laM ACh, indicating that they express cholinergic receptors. Proteases that were inactivated by selective inhibitors and scrambled sequences of the agonist peptides were inactive. Our results indicate that a large proportion of myenteric neurons express PAR-1 and PAR-2. During trauma and inflammation, when prothrombin is activated and mast cells degranulate, thrombin and tryptase may excite myenteric neurons by cleaving and triggering PAR-1 mad PAR-2, respectively. The consequences of this novel mechanism of regulation remain to be determined, but may be related to neuronal responses to injury and inflammation. Supported by DK39957, DK43207 and the Crohns and Colitis Foundation.
Hormones, Transmitters, Growth Factors, and their Receptors Al137
SP or [Sar9MetO2H]-SP (NK1-R agonist) labeled with cyanine 3. G~q/H, GRK-2/-3 and 13-arrestin-1/-2 were localized by immunofluorescence. SP caused a prompt increase in [Ca2+]i in 40.6 ___4.3 % (54 neurons) of all neurons. This was due to activation of the NK1-R, verified with specific agonists and antagonists. Exposure to 100 nM SP for 5 min strongly desensitized the response to 100 nM SP applied 10 min later. Desensitization was specific to the NKI-R, since these neurons responded normally to 100 [aM ACh. Neurons expressing the NK1-R also expressed G~,CH, GRK-2/-3 and 13-arrestin-l/-2, although these proteins were also localized m many other neurons, where they may interact with other receptors. When cells were incubated with 100 nM cy3-SP or -[Sar9MetO2H]-SP at 4°C, peptides were detected at the cell surface, where they colocalized with NK1-R and G,~,/H, whereas GRK-2/-3 and 13-arrestin-l/-2 were principally cytosolic. ,~ier 30 s - 30 min at 37°C, cy3-SP and NK1-R internalized into the same early endosomes, whereas Gaq/l l remained at the cell surface. After 30 s, GRK-2/-3 were localized in superficial vesicles containing the NK1-R, but by 5 min GRK-2#3 were cytosolic. Although 13-arrestin-1/-2 similarly translocated from the cytosol to endosomes containing the NK1-R, this striking redistribution was prolonged. Thus, SP activates neuronal NK1-R causing receptor desensitization and endocytosis, and redistribution of GRK-2/-3 and 13-arrestin-l/-2 in a manner that suggests they regulate receptor function. This regulation will determine if NK1-R expressing neurons can participate in important reflexes including peristalsis and nociception. Supported by NIH grants DK 43207 and DK 39957. G4655
ENDOGENOUS CHOLECYSTOKININ (CCK) DOES NOT STIMULATE PANCREATIC ENZYME SECRETION VIA CAPSAICINSENSITIVE AFFERENT PATHWAYS IN ANESTHETIZED RATS. T. Coskun*, P. Gong, Y. Zong, T.E. Solomon. *Marmara University School of Medicine, Department of Physiology, Haydarpasa 81326, Istanbul, Turkey; Res. Serv., West Los Angeles VAMC and CURE, Los Angeles, CA 90073. BACKGROUND: It has been reported by others (Gastroenterology, 107:525,1994) that endogenously released CCK stimulates pancreatic enzyme secretion by activation of a capsaicin-sensitive afferent pathway in anesthetized rats. This mechanism could not be demonstrated in conscious rats, however (Am.J.Physiol.,261:G735,1991; Am.J.Physiol., 270:G881, 1996). AIM: To test the hypothesis that endogenously released CCK stimulates pancreatic secretion by activating afferent neurons in anesthetized rats. METHODS: Rats were anesthetized with Inactin (100 mg/kg, IP) and prepared with catheters in the bile-pancreatic duct, duodenum, and jugular vein. Bile-pancreatic juice was collected in 15 min portions for measurement of volume and amylase and continuously returned to the duodenum. After a 1 h basal period, pancreatic amylase responses to diversion of bile-pancreatic juice (BPJD) or intraduodenal (ID) perfusion (3ml/h) of 10% casein or 20% Intralipid for 2 h were determined. These experiments were performed in rats treated with vehicle or atropine (50 btg/kg-h IV from -30 min; ATR), sub-diaphragmatic vagotomy (-3 h; VX), systemic capsaicin (25, 50, and 50 mg/kg SC on successive days beginning at -14 d; CAP) or the CCK-A receptor antagonist L364,718 (1 mg/kg IV at -30 min; L364). RESULTS: BPJD, ID casein, and ID Intralipid significantly increased amylase output over basal levels (2460+-221 U/120 min, n=26). L364 at a dose that completely blocked amylase response to an ED50 dose of CCK-8 (90 pmol/kg-h) abolished responses to BPJD, casein, and Intralipid. ATR had no statistically significant effect on these responses. VX reduced responses to all stimulants, but statistically significant effects were seen only with ID casein. CAP had no inhibitory effects on these responses; in fact, the response to BPJD was significantly (p<0.05) increased.
o
I I
12O0O ~
EIpJ DIv gre IOn 2(J% L i p i d
soeo
i ...... i , • G4654 SUBSTANCE P (SP)-INDUCED DESENSITIZATION OF THE NEUROKININ-1 RECEPTOR (NK1-R) AND T R A F F I C K I N G OF G-PROTEIN RECEPTOR KINASES (GRK) AND 13-ARRESTIN IN NEURONS. C.U. Corvera, K. McConalogue, P.D. Gamp, E.F. Grady, N.W. Bunnett. Depts. Surg. & Physiol., University of California, San Francisco, CA. To prevent uncontrolled stimulation of neurons, signaling by G-protein coupled receptors for neurotransmitters is attenuated by receptor desensitization and endocytosis. Studies of reconstituted and transfected systems suggest that GRKs and 13-arrestins translocate from the cytosol to mediate desensitization and endocytosis of surface receptors. It is not known if they are expressed in neurons with the receptors they are thought to regulate, and receptor desensitization and trafficking of these proteins have not been studied in neurons. We examined SP-induced desensitization and trafficking of the NK1-R, Gaq/ll, GRK-2/-3, and 13-arrestin-l/-2 in neurons from the myenteric plexus of the guinea pig small intestine after 7-14 d in culture. [Ca2+]i was measured in individual neurons using Fura-2/AM. The NK1-R was localized by confocal microscopy using immunofluorescence and
.
i ~o4 • p(i.Jl,
oil
p|rl!
II llltln
m14 IrQIp. ~ pql~l,
ms4
I b m p l ~ l t t o I P ~ P I r n v p . " p q J . I t , l e e p l r o v ~ (¥1d i t . u p
CONCLUSIONS: The stimulatory actions of endogenously released CCK on pancreatic secretion in Inactin-anesthetized rats are not mediated by capsaicinsensitive afferent pathways. A non-cholinergic vagal efferent mechan-ism may contribute to stimulation of pancreatic secretion by intestinal protein.
For some GPCRs, such as the 13 adrenergic receptor, immediate uncoupling from G proteins is the predominant mechanism for desensitization while for others, such as the CCKAR, intermediate term compartmentalization predominates. CCKBRs expressed in neurons undergo rapid desensitization and we have shown in a number of transformed cell lines that CCKBRs undergo rapid internalization that is in large part dependent on serine and threonine residues in the cytoplasmic tail. At present, there is no convenient source of either pure primary cells or transformed cell lines expressing high levels of only type B CCKRs. Therefore, to characterize the process of internalization and desensitization of CCKBRs, we studied the rat CCKBR stably expressed in CHO-K1 cells. To determine the role of internalization in the desensitization process, an internalization impaired mutant CCKBR with all 10 potential Ser/Thr phosphorylation sites mutated to Ala, CCKBR(AS/T), was also expressed in CHO-K1 cells. Clones expressing similar numbers of CCKBR and CCKBR(AS/T) displayed similar affinity for CCK-8 with IC50s of 0.5 and 1.0 nM, respectively, in 125I-CCK-8 binding displacement studies. CCKBR(AS/T) internalization was markedly reduced to = 5% at 5 minutes compared to = 60% for CCKBR as measured by stripping of surface bound 125I-CCK-8 with 0.5 M KSCN. Both the CCKBR and CCKBR(AS/T) stimulated total inositol phosphates with similar potencies (EC50 = 2.2 and 0.8 nM), respectively. To study short term desensitization and resensitization in the same cells in real time, we measured CCK-8 stimulated changes in acidification rate (AR) using a microphysiometer (Molecular Devices). Similar to total inositol phosphate stimulation, CCK-8 exhibited a dosedependent increase in the AR with EC50s of 8 and 4 nM, respectively. For both the CCKBR and CCKBR(AS/T), a maximal desensitizing dose of 100 nM CCK-8 for one rain. caused an = 85% and = 65% reduction of the expected response when rechallenged with a half-maximal and maximal dose of CCK-8 at 5 min. with full resensitization by 15 and 30 rain., respectively. The degree of desensitization and rate of resensitization were independent of the desensitizing dose of CCK-8 when rechallenged with an equal dose. Desensitization by 1 nM CCK-8 was nearly completely overcome by rechallenging with a maximal dose of 100 nM CCK-8 or stimulation of another endogenous GPCR receptor. Increasing the duration of the desensitizing dose from 1 to 10 min had a minimal effect upon either the degree of desensitization or rate of resensitization. These data indicate that the CCKBR undergoes rapid internalization and desensitization and suggest that, unlike the CCKAR, receptor internalization is not an important component of short term desensitization or resensitization.
GASTROENTEROLOGY Vol. 114, No. 4 G4650
SIMULATED MICROGRAVITY SUPPRESSES INDUCIBLE CYTOCHROME OXIDASE ACTIVITY IN A HEPATOMA CELL LINE. S.W. Cohen, Y.F. Shiau, M. Yeh, B. Hakim, LSUMC Shreveport LA. Inhibition of cellular activity has previously been demonstrated in microgravity environments. Scant data is available conceming the effect of gravity on hepatic function, particularly regarding the P450 mixed function oxidases that play a crucial role in drug biotransformation. The hepatoma cell line serves as a useful model for examining these inducible hepatic enzymes. Briefly, hepatoma (Hep G2) cells were grown on glass coverslips and incubated with 3-methylcholanthrene (MCA), a polycyclic aromatic hydrocarbon inducer of the P450 enzyme aryl hydrocarbon hydroxylase (AHH). Cell Cultures were exposed to lg or 0g environments throughout the 30 hour incubation period. A rapidly rotating clinostat was used to simulate 0g conditions. Fluorescence spectrophotometry was used to measure AHH activity, as determined by production of the 3-hydroxylated metabolite of benzo[a]pyrene. Activity was expressed as units of AHH activity per milligram of protein. As compared to lg cultures, cells undergoing induction in 0g demonstrated a 4-fold reduction in AHH activity (p<0.01). No activity was detected in solvent controls. Inhibition of hepatic P450 cytochrome oxidase activity may have important implications in long duration manned spaceflight through disruption of normal hepatic homeostatic mechanismsand its pharmacologicconsequences. G4651
ROLE OF NITRIC OXIDE IN THE ANTIPROLIFERATIVE SIGNAL MEDIATED BY THE SOMATOSTATIN RECEPTOR sst5. P. Cordelier J.-P. Est~ve, P. Rochaix, Jj. Voigt, A. V. Schally*, N. Vaysse, C. Susini, L. Buscail. INSERM U151, IFR 31, CHU Rangueil; Department of Pathology, Institut Claudius Regaud, 31403 Toulouse, France and *Tulane University, New Orleans, LA. Somatostatin exerts inhibitory effects on the exocrine and endocrine secretory processes of GI tract as well as on cell proliferation. We recently demonstrated that the human sst5 somatostatin receptor, stably expressed in CHO cells, mediated the antiproliferative effect of the somatostatin analogue RC-160. This effect depends on the inhibition of successively: a soluble guanylate cyclase, protein kinase G and p42 MAP kinase activities (PNAS, 1997;94:9343). Aim of the present study was thus to investigate, the implication of nitric oxide (NO), a known activator of soluble guanylate cyclase, in the antiproliferative effect mediated by sst5 receptor. The cell proliferation of CHO cells stably expressing the human sst5 receptor (CHO/sst5) was stimulated by agents known to increase the intracellular levels of NO and/or cGMP such as L-arginine (10 nM; + 63%), sodium nitropmssiate (SNP) (0.1 ~,i; + 47%) and CCK-8 (0.1 p/vl; + 43 %). CCK-induced cell proliferation was inhibited by the NO synthase inhibitors such as L-NAME (100 laM) and GGA (0.1 laM) suggesting a role for NO in the proliferation of CHO/sst5 ceils. The analogue RC-160 inhibited cell proliferation stimulated by CCK-8, L-arginine and SNP, this effect being abolished by a pre-treatment of ceils by pertussis toxin. NO synthase activity of CHO/sst5 cells was stimulated by a CCK-8 treatment (0.1 laM; + 177%). RC-160 inhibited CCK-induced stimulation of NO synthase activity (maximal: - 83%) in a dose dependent manner, the ED50 (0.33 nM) being related to those observed for inhibition of CCK-induced cell proliferation (1.1 nM) and CCK-induced increase of cGMP levels (0.32 nM), as well as to the affinity of the analogue for recombinant sst5 receptor. In parallel, expression studies at the mRNA (RT-PCR) and/or protein level (Immunocytochemistry) were performed in human normal and tumor tissues: we observed that sst5 receptor was expressed not only in pituitary but also in stomach, endocrine and exocrine pancreas, gallbladder, and colon. In conclusion, our results suggest that inhibition of cell proliferation mediated by sst5 somatostatin receptor results from the inhibition of NO synthase activity. This negative coupling may have important implications, not only in the negative regulation of cell proliferation but also in the regulation of digestive tract secretory, vasoactive and motor functions regulated by somatostatin and its analogues in human. G4652
HISTAMINE H 3 RECEPTORS MEDIATE BOTH GASTROPROTECTIVE AND ANTHNFLAMMATORY EFFECTS. G. Coruzzi, G. Bertaccini. Institute of Pharmacology, Parma, Italy. Histamine H3 receptors have been involved in the negative regulation of gastric acid secretion and intestinal motility (1). A reduction of neurotransmitter release from different types of neurons, as well as an inhibitory effect on histamine synthesis and release were demonstrated in a variety of tissues. In the present study the effects of the histamine H3 receptor selective agonists (R)cc-methylhistamine (MHA) and immepip (IMM) were investigated in the rat on the gastric lesions induced by different noxious stimuli and on the hindpaw oedema induced by 1% carrageenan. MHA (1-100 mg/kg i.g.) significantly reduced the gastric damage induced by absolute ethanol (100% reduction at 100 mg/kg i.g) and, to a lesser extent, that induced by acetylsalicylic acid and indomethacin. Histological
April 1998 examination of MHA-treated stomachs by light and electron microscopy revealed that MHA promotes a rapid process of re-epithelization, increases volume and number of surface and neck mucous cells, and increases adherent mucus layer thickness. IMM (3-100 mg/kg i.g.) was virtually ineffective. The protective effect of MHA 10 and 30 mg/kg was prevented by the H 3 antagonist clobenpropit (3 mg/kg i.g.). Both MHA and IMM (3-10 mg/kg i.g. or s.c.) significantly reduced paw swelling produced by subplantar injection (0.1 ml) of carrageenan (maximum inhibition was 48.6 +- 10.3%, after MHA 10 mg/kg s.c.). The antiinfiammatory effects of H a agonists were more pronounced when these compounds were administered before carrageenan injection, rather than on established inflammation. The antiinflammatory effects of the H 3 agonists were counteracted by clobenpropit (3 mg/kg s.c.), which per se did not modify carrageenan-induced paw oedema. Thermal hyperalgesia induced by carrageenan was not significantly modified by Ha ligands. In conclusion, histamine H 3 receptors may mediate antiinflammatory effects in the rat; it is of interest that this action is not accompanied by gastric damage, but actually, at least in the case of MHA, by a definite gastroprotective effect. [1] Bertaccini, G., Coruzzi, G~ Dig. Dis. Sci. 40:2052-2063 (1995) • G4653
PROTEINASE-ACTIVATED RECEPTORS (PARs) ON MYENTERIC NEURONS: ACTIVATION BY TItROMBIN AND MAST CELL TRYPTASE. C,U. Corvera, K. McConalogue, P. Gamp, G. H. Caughey, N.W. Bunnett. Depts. of Surg., Physiol. & Med. University of California, San Francisco, CA. PARs are a new family of G-protein coupled receptors that are activated by proteolysis. Thrombin cleaves PAR-1 in neurons and astrocytes of the CNS to regulate morphology, growth and survival. Trypsin and tryptase, a major secretory granule protease of mast cells, cleave and trigger PAR-2. Cleavage exposes tethered ligand domains that bind and activate the receptors. Myenteric neurons may be exposed to thrombin during injury and to tryptase when mast cells degranulate. We examined the hypothesis that thrombin and tryptase excite myenteric neurons by cleaving and activating PAR-1 and PAR-2. Neurons were isolated from the myenteric plexus of the guinea pig small intestine and examined after 7-14 days in culture. PARs were localized in cultured neurons and whole mounts of the myenteric plexus by immunofluorescence and confocal microscopy, using region specific antibodies to PAR-1 and PAR-2. [Ca2+]i was measured in individual cultured neurons using Fura-2/AM. Immunoreactive PAR-1 and PAR-2 were detected in many neurons. PAR-1 was mostly detected in processes, whereas there were large intracellular stores of PAR-2 in the soma. Specificity of staining was confirmed by preabsorption with the peptides that were used for immunization. Thrombin and a peptide corresponding to the PAR-1 tethered ligand (SFLLRN) caused a prompt and sustained increase in [Ca~+]j in many cells (ECs0 ~1 nM and ~100 nM, respectively). Of the cells that responded to 10 nM thrombin, 70% (n=104 cells) also responded to 55 mM KC1, indicating that they are neurons, Other PAR-1 responsive cells may be glial cells. Similarly, trypsin, tryptase and a peptide corresponding to the PAR-2 tethered ligand (SLIGKV-NH2) caused a rapid and transient increase in [Ca2+]i in many cells (ECs0 ~0.1 nM, ~1 nM and ~ 1 ~tM, respectively). Of the cells that responded to PAR-2 agonists, 72% (n=86 cells) also responded to 55 mM KCI, indicating that they are neurons, and 14% responded to 100 laM ACh, indicating that they express cholinergic receptors. Proteases that were inactivated by selective inhibitors and scrambled sequences of the agonist peptides were inactive. Our results indicate that a large proportion of myenteric neurons express PAR-1 and PAR-2. During trauma and inflammation, when prothrombin is activated and mast cells degranulate, thrombin and tryptase may excite myenteric neurons by cleaving and triggering PAR-1 mad PAR-2, respectively. The consequences of this novel mechanism of regulation remain to be determined, but may be related to neuronal responses to injury and inflammation. Supported by DK39957, DK43207 and the Crohns and Colitis Foundation.
Hormones, Transmitters, Growth Factors, and their Receptors Al137
SP or [Sar9MetO2H]-SP (NK1-R agonist) labeled with cyanine 3. G~q/H, GRK-2/-3 and 13-arrestin-1/-2 were localized by immunofluorescence. SP caused a prompt increase in [Ca2+]i in 40.6 ___4.3 % (54 neurons) of all neurons. This was due to activation of the NK1-R, verified with specific agonists and antagonists. Exposure to 100 nM SP for 5 min strongly desensitized the response to 100 nM SP applied 10 min later. Desensitization was specific to the NKI-R, since these neurons responded normally to 100 [aM ACh. Neurons expressing the NK1-R also expressed G~,CH, GRK-2/-3 and 13-arrestin-l/-2, although these proteins were also localized m many other neurons, where they may interact with other receptors. When cells were incubated with 100 nM cy3-SP or -[Sar9MetO2H]-SP at 4°C, peptides were detected at the cell surface, where they colocalized with NK1-R and G,~,/H, whereas GRK-2/-3 and 13-arrestin-l/-2 were principally cytosolic. ,~ier 30 s - 30 min at 37°C, cy3-SP and NK1-R internalized into the same early endosomes, whereas Gaq/l l remained at the cell surface. After 30 s, GRK-2/-3 were localized in superficial vesicles containing the NK1-R, but by 5 min GRK-2#3 were cytosolic. Although 13-arrestin-1/-2 similarly translocated from the cytosol to endosomes containing the NK1-R, this striking redistribution was prolonged. Thus, SP activates neuronal NK1-R causing receptor desensitization and endocytosis, and redistribution of GRK-2/-3 and 13-arrestin-l/-2 in a manner that suggests they regulate receptor function. This regulation will determine if NK1-R expressing neurons can participate in important reflexes including peristalsis and nociception. Supported by NIH grants DK 43207 and DK 39957. G4655
ENDOGENOUS CHOLECYSTOKININ (CCK) DOES NOT STIMULATE PANCREATIC ENZYME SECRETION VIA CAPSAICINSENSITIVE AFFERENT PATHWAYS IN ANESTHETIZED RATS. T. Coskun*, P. Gong, Y. Zong, T.E. Solomon. *Marmara University School of Medicine, Department of Physiology, Haydarpasa 81326, Istanbul, Turkey; Res. Serv., West Los Angeles VAMC and CURE, Los Angeles, CA 90073. BACKGROUND: It has been reported by others (Gastroenterology, 107:525,1994) that endogenously released CCK stimulates pancreatic enzyme secretion by activation of a capsaicin-sensitive afferent pathway in anesthetized rats. This mechanism could not be demonstrated in conscious rats, however (Am.J.Physiol.,261:G735,1991; Am.J.Physiol., 270:G881, 1996). AIM: To test the hypothesis that endogenously released CCK stimulates pancreatic secretion by activating afferent neurons in anesthetized rats. METHODS: Rats were anesthetized with Inactin (100 mg/kg, IP) and prepared with catheters in the bile-pancreatic duct, duodenum, and jugular vein. Bile-pancreatic juice was collected in 15 min portions for measurement of volume and amylase and continuously returned to the duodenum. After a 1 h basal period, pancreatic amylase responses to diversion of bile-pancreatic juice (BPJD) or intraduodenal (ID) perfusion (3ml/h) of 10% casein or 20% Intralipid for 2 h were determined. These experiments were performed in rats treated with vehicle or atropine (50 btg/kg-h IV from -30 min; ATR), sub-diaphragmatic vagotomy (-3 h; VX), systemic capsaicin (25, 50, and 50 mg/kg SC on successive days beginning at -14 d; CAP) or the CCK-A receptor antagonist L364,718 (1 mg/kg IV at -30 min; L364). RESULTS: BPJD, ID casein, and ID Intralipid significantly increased amylase output over basal levels (2460+-221 U/120 min, n=26). L364 at a dose that completely blocked amylase response to an ED50 dose of CCK-8 (90 pmol/kg-h) abolished responses to BPJD, casein, and Intralipid. ATR had no statistically significant effect on these responses. VX reduced responses to all stimulants, but statistically significant effects were seen only with ID casein. CAP had no inhibitory effects on these responses; in fact, the response to BPJD was significantly (p<0.05) increased.
o
I I
12O0O ~
EIpJ DIv gre IOn 2(J% L i p i d
soeo
i ...... i , • G4654 SUBSTANCE P (SP)-INDUCED DESENSITIZATION OF THE NEUROKININ-1 RECEPTOR (NK1-R) AND T R A F F I C K I N G OF G-PROTEIN RECEPTOR KINASES (GRK) AND 13-ARRESTIN IN NEURONS. C.U. Corvera, K. McConalogue, P.D. Gamp, E.F. Grady, N.W. Bunnett. Depts. Surg. & Physiol., University of California, San Francisco, CA. To prevent uncontrolled stimulation of neurons, signaling by G-protein coupled receptors for neurotransmitters is attenuated by receptor desensitization and endocytosis. Studies of reconstituted and transfected systems suggest that GRKs and 13-arrestins translocate from the cytosol to mediate desensitization and endocytosis of surface receptors. It is not known if they are expressed in neurons with the receptors they are thought to regulate, and receptor desensitization and trafficking of these proteins have not been studied in neurons. We examined SP-induced desensitization and trafficking of the NK1-R, Gaq/ll, GRK-2/-3, and 13-arrestin-l/-2 in neurons from the myenteric plexus of the guinea pig small intestine after 7-14 d in culture. [Ca2+]i was measured in individual neurons using Fura-2/AM. The NK1-R was localized by confocal microscopy using immunofluorescence and
.
i ~o4 • p(i.Jl,
oil
p|rl!
II llltln
m14 IrQIp. ~ pql~l,
ms4
I b m p l ~ l t t o I P ~ P I r n v p . " p q J . I t , l e e p l r o v ~ (¥1d i t . u p
CONCLUSIONS: The stimulatory actions of endogenously released CCK on pancreatic secretion in Inactin-anesthetized rats are not mediated by capsaicinsensitive afferent pathways. A non-cholinergic vagal efferent mechan-ism may contribute to stimulation of pancreatic secretion by intestinal protein.