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Membrane guanylate cyclase in exocrine pancrease
April 1 9 9 5
Hormones and Receptors
• PLASMA CCK AND SECRETIN LEVELS ARE ELEVATED IN TYPE II DIABETES. D. Guan. W. T. Phillips, J. G. Schwartz, E. Paul, J. Rehfeld, G. M. Green. Depts. of Physiology, Pathology and Radiology, Univ. Texas Hlth. Sci. Ctr., San Antonio, TX, and Dept. of Clin. Biochemistry, Rigshospitalet, Copenhagen, Denmark. Last year (Gastr0. 106:A811, 1994) we reported that basal and postprandial plasma CCK levels (bioassay) were elevated in a rat model of type II diabetes, in contrast to Rushakoff et al (J Clin Endo Metab 76:489-93,1993), who reported markedly decreased postprandial CCK levels in type II diabetes patients. The present study further examined this question to help explain accelerated gastric emptying in patients with early type II diabetes (New Eng J Med 324:130-1,1991). AIMS: To determine the plasma CCK andsecretin responses to a liquid mixednutrient test meal in Zucker type II diabetic and lean rats and the plasma CCK response to a solid mixed meal in diabetic patients and controls, and to relate these values to gastric emptying rates. METHODS: Rat studies: Twelve Zucker type II diabetic fatty rats and 12 Zuoker lean controls were ted (by garage) a liquid mixed nutrient test meal (~2 kcal/ml, 1 ml/100 g BW). Plasma CCK and secretin levels were determined by RIA and gastric emptying rates measured by gamma scintigraphy at intervals up to 60 min~ Human studies: Six recently diagnosed type II diabetes patients and age- and weight-matched controls were fed test meals consisting of pancakes (344 kcal) labeled with 99mTc-sulfur colloid. Stomach emptying rates and plasma CCK (bioassay) were measured at 15 min intervals for 2 hr. RESULTS: Rats: Plasma CCK and secretin levels were significantly elevated in type II diabetic rats (Figs. 1&2), yet in the same rats gastric emptying rate was significantly increased at each interval. Humans: Plasma CCK levels were significantly elevated in diabetic patients at 15 and 105 min after feeding (Fig. 3), yet gastric emptying was significantly accelerated (tl/2:42.2+14.8 vs. 62.7+15.5, diabetes vs. control). CONCLUSIONS: Accelerated gastric emptying in early type II diabetes is associated with increased, rather than decreased, levels of gastrointestinal hormones that inhibit gastric emptying. This suggests resistence of the target organ (stomach) to inhibitory effects of secretin and CCK in type II diabetes. $
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• MEMBRANE GUANYLATE CYCLASE IN EXOCRINE PANCREAS.A.S.Gukovskaya and S.J.Pandol. Dept. of Medicine, University of California & VA Medical Center, San Diego, CA Cyclic G M P r i s e observed in different signaltransduction pathways is mediated by either soluble or particulate guanylate cyclase (GC). Recently, we have shown that in p a n c r e a t i c a c i n a r cells activation of nitric oxide synthase (NOS) a n d soluble GC mediates cyclic G M P r i s e induced by carbachol and that the cyclic GMP rise mediates Ca 2+ influx. This study addresses the role of particulate GC in the regulation of cyclic GMP in p a n c r e a t i c acini from guinea pig. Subcellular distribution shows that GC activity exists in membrane fractions (i0 pmol/mg protein/min) that possess m o r e t h a n 30% of the total GC activity. In m e m b r a n e s isolated from pancreas. GC activity was stimulated 1.8±0.3 times b y I~M heat stable enterotoxin and 1.3±0.04 times by I~M atrial natriu r e t i e peptide (ANP).ImM ATP-y-S did not affect b a s a l GC activity but stimulated the response to Ip24 ANP by 3~0.I times. This suggests that isoforms of A and C membrane GC are present in guinea pig pancreas. NO donor nitroprusside (100~M) stimulated the activity of the soluble GC up 5o i0 fold but did not affect the activity of membrane GC. Changes in [Ca 2+] from 20 to 1000nM decreased the activity of soluble GC by 80%; the particulate GC was not affected. Depletion of intracellular Ca 2+ p o o l s w i t h 2~M thapsigargin stimulated the activity of m e m b r a n e GC by 2 fold. Thus, receptor-induced t r a n s f e r of stored Ca 2+ to the cytoplasm appears to activate both soluble GC via NOS and particulate GC. Together with negative regulation of soluble GC by Ca 2+ these findings demonstrate several pathways involved in the regulation of cyclic GMP formation and Ca 2+ influx in the pancreatic acinar cell.
A973
CORTICOTROPIN RELEASING FACTOR (CRF) AND VASOPRESSINE (AVP) ARE PROTECTIVE IN STRESS-INDUCED ENHANCEMENT OF COLITIS IN RATS. M. G u t * . C. Bonbonne, C. Del Rio, J.L. Junien*, J. Fioramonti and L. B u t n o . Department of Pharmacology, INRA, Toulouse, France and *Institut de Recherches JOUVEINAL, Fresnes, France. Clinical and experimental observations suggest that emotional stress is associated with exacerbation of inflammatory bowel disease but the involvement of hypothalamo-pituitary-adrenal (HPA) axis a n d of the key stress factors, such as CRF a n d AVP in the stress-induced modulation of colitis are poorly documented. This work was performed to study the role of CRF and AVP in stressinduced enhancement of colitis induced by trinitrobenzene sulfonic acid (TNB) in rats chronically equipped with intracerebroventricular (ICV) catheter. The chronic partial restraint stress (PRS) used consists to restrain forelimbs together, during 2 hours per day; PRS was applied for 4 consecutive days and was assimilated to a chronic stress. The severity of colitis was assessed by blind macroscopic scoring and measurement of myeloperoxidase (MPO) activity. Five groups of 6 adult male Wistar rats (275-300 g) were used and were distributed as follow: Group A: rats were rectally instilled with saline (control group); group B: corresponded to non-stressed rats ; group C: rats were submitted to chronic PRS; group D and E: rats received ICV injection of CRF-antagonist (c~-helical CRF9-41, 5 Ilg) or AVP antagonist [(deaminoPen3, Val4, D-ArgS)Vasopressin, 5 ~tg], respectively, before each session of PRS; thereafter, except for group A, rats were rectally instilled with TNB (40 mg/kg) for colitis induction and were killed 4 days later for colitis evaluation. In non stressed rats, TNB (40 rng/kg) induced colonic damage (ulcer index: 3.5 + 0.2) and an increase in MPO activity (346 + 57 v s 58 -+ 11 U MPO/g of proteins, in controls). When the colitis was induced after chronic PRS, we observed an increase in ulceration score (4.8 + 0.3 v s 3.5 + 0.2) as well as in MPO activity compared to non-stressed group (687 -+ 36 v s 346 _+ 57 U MPO/g of proteins). Central injection of either c~-helical CRF9-41 or AVP. antagonist before PRS session, dramatically increased the MPO activity, which reached 1429 +- 347 and 1328 + 175 U MPO/g of proteins, respectively; in addition, ulceration index was increased in both group (5.0 + 0.1 and 4.9 -+ 0.2), compared to group B. We concluded that chronic PRS applied betbre TNB instillation enhances colitis and that CRF a n d AVP are not involved in the stress-induced exacerbation of colitis but may have a protective effect in such situation.
IDENTIFICATION BY [3H]-SR 27897B OF HIGH AFFINITY CCK A BINDING SITES ON RAT PANCREATIC MEMBRANES. D. Gully, C. Marcy, D. Frthel, J.P. Maffrand and G. Le Fur*. Sanofi Recherche, 195 route d'Espagne, 31036 Toulouse cddex and ~;32-34 rue Marbeuf, 75008 Paris, France. We previously reported pharmacological data concerning SR 27897B, a new highly selective, orally active non peptide CCK A receptor antagonist. In the present study we used its radiolabeled form [3I-I]SR 27897B (35 Ci/mmole) to characterize CCK A receptors on rat pancreatic membranes. [3H]-SR 27897B binding was time-dependent (steady state at 10 min at 25°C) and fully reversible (dissociation rate constant (k-I) 0.049 ± 0.013 mind). Specific binding measured with 2 nM of [3H]-SR 27897B, was linear up to 100 rag of protein/assay and represented 80 % of total binding. CCKSS completely displaced [3H]-SR 27897B binding with a lower affinity than measured with [125I]-CCK8S. As previously described in studies performed with [3H]-L-364,718, CCK8S reveals two binding sites of low and very low affinities (IC50 values 187 ± 68 nM and 20 ± 1 laM), that represents respectively 46 % and 56 % of the whole population of binding sites (n=3). On the opposite; antagonist compounds such as L-364,718 and SR 27897B maintained affinities in the nanomolar range. However, in the case of SR 27897B, displacement curves (n=5) better fitted with a two sites- than a one site-binding model with respective 1C50 values of 0.17 ± 0.15 nM (21%) and 5.8 4- 1.7 nM (77%). Scatchard analysis of the saturation carve performed with increasing concentrations of [3H]-SR 27897B (0.04 to 6 nM) confirmed the existence of two populations of binding sites with respective Kd values of 10 ± 9 pM (6%) and 0.63 4- 0.26 nM (94%). In the same conditions and on the same menthrane preparation, [3H]-L-364,718 displacement and saturation curves revealed only one population of binding sites (Kd = 0.29 ± 0.01 nM). The total number of binding sites (Bmax value) was higher in the antagonist model than in the agonist one. [3H]-SR 27897B is the first selective ligand of CCK A receptors able to demonstrate the existence of a population of very high affinity binding sites on rat pancreatic membranes. These data obtained by the use of radiolabeled agonist and antagonists ligands strengthened the possibility of different binding sites not only for agonist and antagonist ligands but also for chemically unrelated antagonist molecules such as L-364,718 and SR 27897B.
Hormones and Receptors
• PLASMA CCK AND SECRETIN LEVELS ARE ELEVATED IN TYPE II DIABETES. D. Guan. W. T. Phillips, J. G. Schwartz, E. Paul, J. Rehfeld, G. M. Green. Depts. of Physiology, Pathology and Radiology, Univ. Texas Hlth. Sci. Ctr., San Antonio, TX, and Dept. of Clin. Biochemistry, Rigshospitalet, Copenhagen, Denmark. Last year (Gastr0. 106:A811, 1994) we reported that basal and postprandial plasma CCK levels (bioassay) were elevated in a rat model of type II diabetes, in contrast to Rushakoff et al (J Clin Endo Metab 76:489-93,1993), who reported markedly decreased postprandial CCK levels in type II diabetes patients. The present study further examined this question to help explain accelerated gastric emptying in patients with early type II diabetes (New Eng J Med 324:130-1,1991). AIMS: To determine the plasma CCK andsecretin responses to a liquid mixednutrient test meal in Zucker type II diabetic and lean rats and the plasma CCK response to a solid mixed meal in diabetic patients and controls, and to relate these values to gastric emptying rates. METHODS: Rat studies: Twelve Zucker type II diabetic fatty rats and 12 Zuoker lean controls were ted (by garage) a liquid mixed nutrient test meal (~2 kcal/ml, 1 ml/100 g BW). Plasma CCK and secretin levels were determined by RIA and gastric emptying rates measured by gamma scintigraphy at intervals up to 60 min~ Human studies: Six recently diagnosed type II diabetes patients and age- and weight-matched controls were fed test meals consisting of pancakes (344 kcal) labeled with 99mTc-sulfur colloid. Stomach emptying rates and plasma CCK (bioassay) were measured at 15 min intervals for 2 hr. RESULTS: Rats: Plasma CCK and secretin levels were significantly elevated in type II diabetic rats (Figs. 1&2), yet in the same rats gastric emptying rate was significantly increased at each interval. Humans: Plasma CCK levels were significantly elevated in diabetic patients at 15 and 105 min after feeding (Fig. 3), yet gastric emptying was significantly accelerated (tl/2:42.2+14.8 vs. 62.7+15.5, diabetes vs. control). CONCLUSIONS: Accelerated gastric emptying in early type II diabetes is associated with increased, rather than decreased, levels of gastrointestinal hormones that inhibit gastric emptying. This suggests resistence of the target organ (stomach) to inhibitory effects of secretin and CCK in type II diabetes. $
%) 2 d 0/4
1
~151
...Diabetic 2b 4"0 Minutes
b
2
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I ' - - " .--II
~
2'0 4'0 6'0 Minutes
0 6
~
51
%)
3
- . . Diabetes Pts.
" 04
3"0 6'0 9"0 l~O Minutes
• MEMBRANE GUANYLATE CYCLASE IN EXOCRINE PANCREAS.A.S.Gukovskaya and S.J.Pandol. Dept. of Medicine, University of California & VA Medical Center, San Diego, CA Cyclic G M P r i s e observed in different signaltransduction pathways is mediated by either soluble or particulate guanylate cyclase (GC). Recently, we have shown that in p a n c r e a t i c a c i n a r cells activation of nitric oxide synthase (NOS) a n d soluble GC mediates cyclic G M P r i s e induced by carbachol and that the cyclic GMP rise mediates Ca 2+ influx. This study addresses the role of particulate GC in the regulation of cyclic GMP in p a n c r e a t i c acini from guinea pig. Subcellular distribution shows that GC activity exists in membrane fractions (i0 pmol/mg protein/min) that possess m o r e t h a n 30% of the total GC activity. In m e m b r a n e s isolated from pancreas. GC activity was stimulated 1.8±0.3 times b y I~M heat stable enterotoxin and 1.3±0.04 times by I~M atrial natriu r e t i e peptide (ANP).ImM ATP-y-S did not affect b a s a l GC activity but stimulated the response to Ip24 ANP by 3~0.I times. This suggests that isoforms of A and C membrane GC are present in guinea pig pancreas. NO donor nitroprusside (100~M) stimulated the activity of the soluble GC up 5o i0 fold but did not affect the activity of membrane GC. Changes in [Ca 2+] from 20 to 1000nM decreased the activity of soluble GC by 80%; the particulate GC was not affected. Depletion of intracellular Ca 2+ p o o l s w i t h 2~M thapsigargin stimulated the activity of m e m b r a n e GC by 2 fold. Thus, receptor-induced t r a n s f e r of stored Ca 2+ to the cytoplasm appears to activate both soluble GC via NOS and particulate GC. Together with negative regulation of soluble GC by Ca 2+ these findings demonstrate several pathways involved in the regulation of cyclic GMP formation and Ca 2+ influx in the pancreatic acinar cell.
A973
CORTICOTROPIN RELEASING FACTOR (CRF) AND VASOPRESSINE (AVP) ARE PROTECTIVE IN STRESS-INDUCED ENHANCEMENT OF COLITIS IN RATS. M. G u t * . C. Bonbonne, C. Del Rio, J.L. Junien*, J. Fioramonti and L. B u t n o . Department of Pharmacology, INRA, Toulouse, France and *Institut de Recherches JOUVEINAL, Fresnes, France. Clinical and experimental observations suggest that emotional stress is associated with exacerbation of inflammatory bowel disease but the involvement of hypothalamo-pituitary-adrenal (HPA) axis a n d of the key stress factors, such as CRF a n d AVP in the stress-induced modulation of colitis are poorly documented. This work was performed to study the role of CRF and AVP in stressinduced enhancement of colitis induced by trinitrobenzene sulfonic acid (TNB) in rats chronically equipped with intracerebroventricular (ICV) catheter. The chronic partial restraint stress (PRS) used consists to restrain forelimbs together, during 2 hours per day; PRS was applied for 4 consecutive days and was assimilated to a chronic stress. The severity of colitis was assessed by blind macroscopic scoring and measurement of myeloperoxidase (MPO) activity. Five groups of 6 adult male Wistar rats (275-300 g) were used and were distributed as follow: Group A: rats were rectally instilled with saline (control group); group B: corresponded to non-stressed rats ; group C: rats were submitted to chronic PRS; group D and E: rats received ICV injection of CRF-antagonist (c~-helical CRF9-41, 5 Ilg) or AVP antagonist [(deaminoPen3, Val4, D-ArgS)Vasopressin, 5 ~tg], respectively, before each session of PRS; thereafter, except for group A, rats were rectally instilled with TNB (40 mg/kg) for colitis induction and were killed 4 days later for colitis evaluation. In non stressed rats, TNB (40 rng/kg) induced colonic damage (ulcer index: 3.5 + 0.2) and an increase in MPO activity (346 + 57 v s 58 -+ 11 U MPO/g of proteins, in controls). When the colitis was induced after chronic PRS, we observed an increase in ulceration score (4.8 + 0.3 v s 3.5 + 0.2) as well as in MPO activity compared to non-stressed group (687 -+ 36 v s 346 _+ 57 U MPO/g of proteins). Central injection of either c~-helical CRF9-41 or AVP. antagonist before PRS session, dramatically increased the MPO activity, which reached 1429 +- 347 and 1328 + 175 U MPO/g of proteins, respectively; in addition, ulceration index was increased in both group (5.0 + 0.1 and 4.9 -+ 0.2), compared to group B. We concluded that chronic PRS applied betbre TNB instillation enhances colitis and that CRF a n d AVP are not involved in the stress-induced exacerbation of colitis but may have a protective effect in such situation.
IDENTIFICATION BY [3H]-SR 27897B OF HIGH AFFINITY CCK A BINDING SITES ON RAT PANCREATIC MEMBRANES. D. Gully, C. Marcy, D. Frthel, J.P. Maffrand and G. Le Fur*. Sanofi Recherche, 195 route d'Espagne, 31036 Toulouse cddex and ~;32-34 rue Marbeuf, 75008 Paris, France. We previously reported pharmacological data concerning SR 27897B, a new highly selective, orally active non peptide CCK A receptor antagonist. In the present study we used its radiolabeled form [3I-I]SR 27897B (35 Ci/mmole) to characterize CCK A receptors on rat pancreatic membranes. [3H]-SR 27897B binding was time-dependent (steady state at 10 min at 25°C) and fully reversible (dissociation rate constant (k-I) 0.049 ± 0.013 mind). Specific binding measured with 2 nM of [3H]-SR 27897B, was linear up to 100 rag of protein/assay and represented 80 % of total binding. CCKSS completely displaced [3H]-SR 27897B binding with a lower affinity than measured with [125I]-CCK8S. As previously described in studies performed with [3H]-L-364,718, CCK8S reveals two binding sites of low and very low affinities (IC50 values 187 ± 68 nM and 20 ± 1 laM), that represents respectively 46 % and 56 % of the whole population of binding sites (n=3). On the opposite; antagonist compounds such as L-364,718 and SR 27897B maintained affinities in the nanomolar range. However, in the case of SR 27897B, displacement curves (n=5) better fitted with a two sites- than a one site-binding model with respective 1C50 values of 0.17 ± 0.15 nM (21%) and 5.8 4- 1.7 nM (77%). Scatchard analysis of the saturation carve performed with increasing concentrations of [3H]-SR 27897B (0.04 to 6 nM) confirmed the existence of two populations of binding sites with respective Kd values of 10 ± 9 pM (6%) and 0.63 4- 0.26 nM (94%). In the same conditions and on the same menthrane preparation, [3H]-L-364,718 displacement and saturation curves revealed only one population of binding sites (Kd = 0.29 ± 0.01 nM). The total number of binding sites (Bmax value) was higher in the antagonist model than in the agonist one. [3H]-SR 27897B is the first selective ligand of CCK A receptors able to demonstrate the existence of a population of very high affinity binding sites on rat pancreatic membranes. These data obtained by the use of radiolabeled agonist and antagonists ligands strengthened the possibility of different binding sites not only for agonist and antagonist ligands but also for chemically unrelated antagonist molecules such as L-364,718 and SR 27897B.