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Central thyrotropin-releasing hormone (TRH) enhances the hepatic blood flow in rats
A1010
AGA ABSTRACTS
• G PROTEINS REGULATE CHOLECYSTOKININ (CCK) RECEPTOR AFFINITY AND ACTIVATE SIGNAL TRANSOUCTION BY DIFFERENT MECHANISMS. V.D. Talkad, K.P. Fortune, J.D, Gardner. Saint Louis University, St.Louis, MO. 63104. Occupation of G protein-coupled receptors by an agonist causes GTP to occupy and activate the G protein. The G protein then activates a'signal transduction mechanism and also reduces the affinity of the receptor for the agonist. Receptor-mediated activation of signal transduction is a relatively well characterized process resulting from an interaction between the third intracellular loop of the receptor and the G protein. In contrast, knowledge of G protein regulation of receptor affinity is limited and it is not known if G protein activation of signal transduction is congruent with its effect on receptor affinity. In the presen t , study we used rat pancreatic acini permeabilized with streptolysin 0 to compare different agents that activate G proteins (guanine nucleotides, mastoparan, AIF4") in terms of their abilities to influence the effinfty =of the low affinity state of the pancreatic CCK receptor for agonists and tO activate phospholipase C(PLC). Receptor affinity for agonists was measured as binding of 12SI-CCK-8 to the low affinity state of the CCK receptor. Activation of PLC was measured as increased [3H]inositol phosphate in acini that were prelabeled with [3H]myoin0sitol. We have shown in other studies that CCK-8 activates PLC and causes a 5-fold increase in [ZH]inositol phosphate by occupying the low affinity state of the CCK receptor in pancreatic acini. AIF4" at 3 mM caused maximal activation of PLC but continued to decrease binding of 1=51-CCK-8 at concentrations up to 30 raM. In contrast, GTPFS continued to activate PLC at concentrations up to lOOpM but caused maximal inhibition of binding of'~sI-CCK-8 at 10pM. GDP/YSand mastoparan did not activate PLC but were as effective as GTPyS in decreasing binding of ~2~1-CCK-8. GDPBS inhibited activation of PLC by CCK-8 and GTPys but did not alter activation by AIF4-. Mastoparan inhibited activation of PLC by CCK-8 and GTPFS but enhanced activation by AIF4".The present results demonstrate that G protein activation of signal transduction is not congruent with G protein regulation of CCK receptor affinity. Thus, regulation of receptor affinity involves a different mechanism from that that activates signalling and these differences probably involve different domains of the CCK receptor molecule.
• G-PROTEINS COUPLE EXCITATORY AND INHIBITORY RECEPTORS TO ADENYLATE CYCLASE IN AH/TYPE 2 MYENTERIC NEURONS OF GUINEA-PIG SMALL INTESTINE. K. Tamura, H. Itoh and J.D. Wood. Depts. Physiol., The Ohio State Univ. Col. Med., Columbus, OH and Tokai Univ. Med. Sch., Isehara, Japan. Several lines of evidence suggest that receptor activation of adenylate cyclase and 2nd messenger function of cAMP are involved in signal transduction for slow synaptic excitation (slow EPSP) in AH/type 2 neurons, Adenosine A 1 receptors suppress stimulation of adenylate cyclase by excitatory messengers. Stimulation of adenylate cyclase by forskoli~ is suppressed by adenosine A 1 receptors. Our study tested the hypothesis that G-proteins couple both excitatory and inhibitory receptors to adenylate cyclase. Intracellular methods of electrical recording were used to investigate the effects of GTP-T-S, GDP-I~-S and pertussis toxin (PTX) on slow EPSPs, as well as excitatory responses to substance P (SP) and forskolin. Effects on the inhibitory action of the adenosine Atagonist CCPA were also studied. GTP-r-S and GDP-P=-S (20 mM) were injected from the microelectrodes. Impalements were maintained for 5.5 hours to evaluate the effects of 1/Jg/ml PTX. Stimulation of slow EPSPs was alternated with "puffs" of SP at 10 min intervals. Slow EPSPs and SP responses declined progressively and disappeared after 3.75 hrs. Forskolln (1 pM) at this point excited the neurons. CCPA (10 /JM) did not suppress the action of forskolin after prolonged exposure to PTX. Hyperpolarizing after-potentials were unaffected by PTX. GTP-r-S injection resulted in a progressive increase in slow EPSP-like behavior, characterized by slowly activating depolarization and increased input resistance. CCPA (10/IM) did not suppress the excitatory responses to forskoli~, nor was basal excitability affected by CCPA after GTP-r-S. SP-induced depolarization was infinitely prolonged resulting in inability to evoke responses after an initial "puff". Iniection of GDP-13-S also suppressed slow EPSPs coincident with progressive increase tn input resistance. Forskolin activation Of the neurons after injection of GDP-13-S was not suppressed by CCPA. The overall results are consistent with the involvement of G-proteins in coupling both excitatory and inhibitory adenosine receptors to adenylate cyclase in AH/type 2 neurons. (Supported by Grant 02044134 from M.E.S.C., Japan and NIH R01 DK37238)
GASTROENTEROLOGY, VOI. I O 8 , NO. 4
• CENTRAL
T H Y R O T R O P I N - R E L E A S I N G H O R M O N E (TRH) ENHANCES THE HEPATIC BLOOD FLOW IN RATS. K. Tamo.fi., M. Yoneda, S. Yokohama, Y. Sato, K. Nakamura, A. Kimura, K. Akiyama and I. Makino Dept. of Medicine It, Asahikawa Medical College, Japan Central neuropeptides plays a role in many physiological and pathophysiological regulations through autonomic nervous systems. TRH is distributed in the central nervous system and act as neurotransmitter to regulate gastric functions through vagaI and muscarinic pathways. Liver is also richly innervated and a role of central neuropeptide in hepatic functions is expected. However, stilt very little is known about central regulation of hepatic functions by neuropeptides. Purpose: To investigate the effect of central TRH on hepatic blood flow (HBF). Methods: HBF was measured by the hydrogen gas-clearance technique. Male Wistar fasted rats (240-350 g) were anesthetized with urethane (1.5 g/kg, ip), and a midline abdominal incision was performed. A platinum wire type electrode was inserted into the hepatic left lateral lobe and an Ag-AgCI reference electrode was placed inside the peritoneal cavity. The experimental protocol involved alternating 15-min periods of saturation and desaturation of the tissue with hydrogen gas. After 60 rain stabilization, two measurement of basal HBF Were performed. Either the stable TRH analog, RX 77368 (5, 10, 100 or 500 rig) or saline vehicle was injected intracistemally or intravenously. HBF response was observed for 90 rain after the peptide. Atropine methyl nitrate (0.15 mg/kg, ip), indomethacin (5 mg/kg, ip) or NG-nitro-L-arginine methyl ester (L-NAME, l0 mg/kg, iv) pretreatment was performed 15 rain, 30 rain or 15 min before the peptide, respectively. Vagotomy or sham operation was also performed 120 rain before the peptide. The change of HBF was expresse d as percent compared to basal HBF. Results: Intracistemal injection of TRH (10 ng) significantly enhanced HBF with peak response at 30 min after the peptide, and the enhanced HBF was returned to basal at 60 rain (Mean + SEM; %: 15 min 136 + 7; 30 min 142 _+6; 45rain 117 + l l; 60 min 106 _+ 18i 75 rain 115 + 14; 90 min 101 + 15). This stimulatory effect of central TRH was dose-related (Mean + SEM; peak response: saline 104 -+ 12; 5 ng 117 -+7; 10 ng 147 --+2; 100 ng 174 -+ 24; 500 ng 155 --+12). The stimulation of HBF by central TRH was abolished by atropine, indomethacin, L-NAME and vagotomy. Intravenous injection of TRH did not have any effect on HBF. Conclusion: TRH acts in the brain to enhance HBF through vagal-muscarinic, prostaglandin and nitric oxide pathways. These results suggest the central regulation of hepatic hemodynamics by neuropeptides.
DIFFERENT EXPRESSION OF PEPTIDE-RECEPTORS IN HUMAN PANCREATIC ENDOCRINE TUMORS. C. Tanq, I. Siemond, C.B.H.W. Lamers. Department of 'Gastroenterology, University Hospital, Leiden, The Netherlands Pancreatic endocrine tumors secrete several peptides which cause the corresponding clinical syndrome. Up to now, the peptide-receptor status of the tumors is largely unknown except for the somatostatin receptor. In this study, 11 pancreatic endocrine tumors from 7 patients have been analyzed for their receptors for cholecystokinin (CCK), somatostatin (SST), bombesth (BBS) and secretin using storage phosphor autoradiography. C~ostat sections of the tumors were incubated with radioactive iodine-labeled CCK-33, SST-14, BBS and secretin (100 pM) in the absence o r presence' of excess unlabeled corresponding peptides(1 p.M). The incidence and measurement of peptide-receptors in tumors Tumor
n
CCK n*
SST n*
Gastdnoma
8 0
8
VlPoma
3 3
5.3+4.0 3
BBS n*
Secretin n*
4.5+1.2
8 7.7+7.4 7
1.~.5
1 0.4
1.5+1.2
1 1.3
n': The positive numbers; Mean+SD (fmol/mg protein); Gastrin caused an inhibition of labeled CCK-33 binding to VIPoma sections at a little higher half-maximal inhibition concentration (IC50) than that of CCK (8+1 nM vs 1+0.8 nM). The IC50 of L365,260, a CCKB(gastrin) receptor antagonist, of 40+1 nM was much lower than that of Iorglumide, a CCK-A receptor antagonist, [9_+0.4 (xl03) nM] in the VIPomas. Thus, in the VIPomas tested in this study, the labeled CCK appeared to be bound to CCK-B (gastdn)receptors; Conclusions: 1)Gastrinomas present' receptors for SST, BBS and Secretin. 2)VlPomas present receptors for CCK-B(gastdn) and SST. The lack of CCK-B (gastrin) receptors in gastdnomas suggests an absence of the autecdne loop in this tumor-type, The different expression of peptidereceptors in pancreatic endocrine tumors may provide opportunities for receptor agonists or antagonists in the diagnosis and treatment of these tumors in man.
AGA ABSTRACTS
• G PROTEINS REGULATE CHOLECYSTOKININ (CCK) RECEPTOR AFFINITY AND ACTIVATE SIGNAL TRANSOUCTION BY DIFFERENT MECHANISMS. V.D. Talkad, K.P. Fortune, J.D, Gardner. Saint Louis University, St.Louis, MO. 63104. Occupation of G protein-coupled receptors by an agonist causes GTP to occupy and activate the G protein. The G protein then activates a'signal transduction mechanism and also reduces the affinity of the receptor for the agonist. Receptor-mediated activation of signal transduction is a relatively well characterized process resulting from an interaction between the third intracellular loop of the receptor and the G protein. In contrast, knowledge of G protein regulation of receptor affinity is limited and it is not known if G protein activation of signal transduction is congruent with its effect on receptor affinity. In the presen t , study we used rat pancreatic acini permeabilized with streptolysin 0 to compare different agents that activate G proteins (guanine nucleotides, mastoparan, AIF4") in terms of their abilities to influence the effinfty =of the low affinity state of the pancreatic CCK receptor for agonists and tO activate phospholipase C(PLC). Receptor affinity for agonists was measured as binding of 12SI-CCK-8 to the low affinity state of the CCK receptor. Activation of PLC was measured as increased [3H]inositol phosphate in acini that were prelabeled with [3H]myoin0sitol. We have shown in other studies that CCK-8 activates PLC and causes a 5-fold increase in [ZH]inositol phosphate by occupying the low affinity state of the CCK receptor in pancreatic acini. AIF4" at 3 mM caused maximal activation of PLC but continued to decrease binding of 1=51-CCK-8 at concentrations up to 30 raM. In contrast, GTPFS continued to activate PLC at concentrations up to lOOpM but caused maximal inhibition of binding of'~sI-CCK-8 at 10pM. GDP/YSand mastoparan did not activate PLC but were as effective as GTPyS in decreasing binding of ~2~1-CCK-8. GDPBS inhibited activation of PLC by CCK-8 and GTPys but did not alter activation by AIF4-. Mastoparan inhibited activation of PLC by CCK-8 and GTPFS but enhanced activation by AIF4".The present results demonstrate that G protein activation of signal transduction is not congruent with G protein regulation of CCK receptor affinity. Thus, regulation of receptor affinity involves a different mechanism from that that activates signalling and these differences probably involve different domains of the CCK receptor molecule.
• G-PROTEINS COUPLE EXCITATORY AND INHIBITORY RECEPTORS TO ADENYLATE CYCLASE IN AH/TYPE 2 MYENTERIC NEURONS OF GUINEA-PIG SMALL INTESTINE. K. Tamura, H. Itoh and J.D. Wood. Depts. Physiol., The Ohio State Univ. Col. Med., Columbus, OH and Tokai Univ. Med. Sch., Isehara, Japan. Several lines of evidence suggest that receptor activation of adenylate cyclase and 2nd messenger function of cAMP are involved in signal transduction for slow synaptic excitation (slow EPSP) in AH/type 2 neurons, Adenosine A 1 receptors suppress stimulation of adenylate cyclase by excitatory messengers. Stimulation of adenylate cyclase by forskoli~ is suppressed by adenosine A 1 receptors. Our study tested the hypothesis that G-proteins couple both excitatory and inhibitory receptors to adenylate cyclase. Intracellular methods of electrical recording were used to investigate the effects of GTP-T-S, GDP-I~-S and pertussis toxin (PTX) on slow EPSPs, as well as excitatory responses to substance P (SP) and forskolin. Effects on the inhibitory action of the adenosine Atagonist CCPA were also studied. GTP-r-S and GDP-P=-S (20 mM) were injected from the microelectrodes. Impalements were maintained for 5.5 hours to evaluate the effects of 1/Jg/ml PTX. Stimulation of slow EPSPs was alternated with "puffs" of SP at 10 min intervals. Slow EPSPs and SP responses declined progressively and disappeared after 3.75 hrs. Forskolln (1 pM) at this point excited the neurons. CCPA (10 /JM) did not suppress the action of forskolin after prolonged exposure to PTX. Hyperpolarizing after-potentials were unaffected by PTX. GTP-r-S injection resulted in a progressive increase in slow EPSP-like behavior, characterized by slowly activating depolarization and increased input resistance. CCPA (10/IM) did not suppress the excitatory responses to forskoli~, nor was basal excitability affected by CCPA after GTP-r-S. SP-induced depolarization was infinitely prolonged resulting in inability to evoke responses after an initial "puff". Iniection of GDP-13-S also suppressed slow EPSPs coincident with progressive increase tn input resistance. Forskolin activation Of the neurons after injection of GDP-13-S was not suppressed by CCPA. The overall results are consistent with the involvement of G-proteins in coupling both excitatory and inhibitory adenosine receptors to adenylate cyclase in AH/type 2 neurons. (Supported by Grant 02044134 from M.E.S.C., Japan and NIH R01 DK37238)
GASTROENTEROLOGY, VOI. I O 8 , NO. 4
• CENTRAL
T H Y R O T R O P I N - R E L E A S I N G H O R M O N E (TRH) ENHANCES THE HEPATIC BLOOD FLOW IN RATS. K. Tamo.fi., M. Yoneda, S. Yokohama, Y. Sato, K. Nakamura, A. Kimura, K. Akiyama and I. Makino Dept. of Medicine It, Asahikawa Medical College, Japan Central neuropeptides plays a role in many physiological and pathophysiological regulations through autonomic nervous systems. TRH is distributed in the central nervous system and act as neurotransmitter to regulate gastric functions through vagaI and muscarinic pathways. Liver is also richly innervated and a role of central neuropeptide in hepatic functions is expected. However, stilt very little is known about central regulation of hepatic functions by neuropeptides. Purpose: To investigate the effect of central TRH on hepatic blood flow (HBF). Methods: HBF was measured by the hydrogen gas-clearance technique. Male Wistar fasted rats (240-350 g) were anesthetized with urethane (1.5 g/kg, ip), and a midline abdominal incision was performed. A platinum wire type electrode was inserted into the hepatic left lateral lobe and an Ag-AgCI reference electrode was placed inside the peritoneal cavity. The experimental protocol involved alternating 15-min periods of saturation and desaturation of the tissue with hydrogen gas. After 60 rain stabilization, two measurement of basal HBF Were performed. Either the stable TRH analog, RX 77368 (5, 10, 100 or 500 rig) or saline vehicle was injected intracistemally or intravenously. HBF response was observed for 90 rain after the peptide. Atropine methyl nitrate (0.15 mg/kg, ip), indomethacin (5 mg/kg, ip) or NG-nitro-L-arginine methyl ester (L-NAME, l0 mg/kg, iv) pretreatment was performed 15 rain, 30 rain or 15 min before the peptide, respectively. Vagotomy or sham operation was also performed 120 rain before the peptide. The change of HBF was expresse d as percent compared to basal HBF. Results: Intracistemal injection of TRH (10 ng) significantly enhanced HBF with peak response at 30 min after the peptide, and the enhanced HBF was returned to basal at 60 rain (Mean + SEM; %: 15 min 136 + 7; 30 min 142 _+6; 45rain 117 + l l; 60 min 106 _+ 18i 75 rain 115 + 14; 90 min 101 + 15). This stimulatory effect of central TRH was dose-related (Mean + SEM; peak response: saline 104 -+ 12; 5 ng 117 -+7; 10 ng 147 --+2; 100 ng 174 -+ 24; 500 ng 155 --+12). The stimulation of HBF by central TRH was abolished by atropine, indomethacin, L-NAME and vagotomy. Intravenous injection of TRH did not have any effect on HBF. Conclusion: TRH acts in the brain to enhance HBF through vagal-muscarinic, prostaglandin and nitric oxide pathways. These results suggest the central regulation of hepatic hemodynamics by neuropeptides.
DIFFERENT EXPRESSION OF PEPTIDE-RECEPTORS IN HUMAN PANCREATIC ENDOCRINE TUMORS. C. Tanq, I. Siemond, C.B.H.W. Lamers. Department of 'Gastroenterology, University Hospital, Leiden, The Netherlands Pancreatic endocrine tumors secrete several peptides which cause the corresponding clinical syndrome. Up to now, the peptide-receptor status of the tumors is largely unknown except for the somatostatin receptor. In this study, 11 pancreatic endocrine tumors from 7 patients have been analyzed for their receptors for cholecystokinin (CCK), somatostatin (SST), bombesth (BBS) and secretin using storage phosphor autoradiography. C~ostat sections of the tumors were incubated with radioactive iodine-labeled CCK-33, SST-14, BBS and secretin (100 pM) in the absence o r presence' of excess unlabeled corresponding peptides(1 p.M). The incidence and measurement of peptide-receptors in tumors Tumor
n
CCK n*
SST n*
Gastdnoma
8 0
8
VlPoma
3 3
5.3+4.0 3
BBS n*
Secretin n*
4.5+1.2
8 7.7+7.4 7
1.~.5
1 0.4
1.5+1.2
1 1.3
n': The positive numbers; Mean+SD (fmol/mg protein); Gastrin caused an inhibition of labeled CCK-33 binding to VIPoma sections at a little higher half-maximal inhibition concentration (IC50) than that of CCK (8+1 nM vs 1+0.8 nM). The IC50 of L365,260, a CCKB(gastrin) receptor antagonist, of 40+1 nM was much lower than that of Iorglumide, a CCK-A receptor antagonist, [9_+0.4 (xl03) nM] in the VIPomas. Thus, in the VIPomas tested in this study, the labeled CCK appeared to be bound to CCK-B (gastdn)receptors; Conclusions: 1)Gastrinomas present' receptors for SST, BBS and Secretin. 2)VlPomas present receptors for CCK-B(gastdn) and SST. The lack of CCK-B (gastrin) receptors in gastdnomas suggests an absence of the autecdne loop in this tumor-type, The different expression of peptidereceptors in pancreatic endocrine tumors may provide opportunities for receptor agonists or antagonists in the diagnosis and treatment of these tumors in man.