GLP-1 inhibits proximal gastric motility but enhances perception

AGAAJl03

April 2000

1679

1681

CCKB·RECEPTORS (CCKBR) IN THE KID NE Y: IDENTIFICA· TION, LOCALIZATION, AND CC KBR· MEDIA TED CHANGES IN RENAL SODIUM AND POTASSIUM ABSORPTION. Tammo von Schrenck, Andreas de Weerth, Maike Ahre ns, Thomas Jock s, Gunter Wolf, Christoph Bobrowski, Christoph Neumaier, Mart ina Schulz, Heiner Greten, Rolf Ak Stahl, Univ Hosp Eppendorf, Hamburg , Germany.

CHARACTERIZATION OF SOMATOSTATIN RECEPTOR MEDI· ATED INHffiITION OF SMALL INTESTINAL PERISTALSIS IN THE RAT AND MOUSE. Faiza Abdu, Gareth A. Hicks, Jerem y P. Allen , David Grund y, Univ of Sheffield, Sheffield . United Kingdom ; Glaxo institute of applied pharm acology, Cambrid ge, United Kingd om.

CCKB receptors (CCKBR) have recently been detected in the kidney by PeR studies and by binding studies using '2SI_gastrin. However, the function of these CCKBR have not been established. Aim: I. To identify and localize CCKBR in the rat kidney by Northern blot and by imrnunhistochemi stry using a highly-specific polyclon al antibod y. II. To study the effects of gastrin on electrolyte transport in the isolated perfused rat kidney under in vitro conditions . Methods: I. RNA was isolated from rat kidney , stomach, brain, liver and muscle . Northern blot was performed using cDNA encod ing the rat CCKBR. II. Polyclonal subtype-s pecific antibodies against domains of the CCKBR (postion 39-56. extracellular N-termi nus, H 2N-CEP PRI RGA GTR ELE LAI CONH 2) were produced by imrnun zation of hens. III. Rat kidn eys were mounted in an orga n bath by attaching the renal artery to a perfusion syste m. The renal vein and the ureter were canulated to collect samples of the effluate which was analysed for the concentrations of electrol ytes. After a pre-perfu sion period of 20 min, gastrin-17-1 was given i.a. (10. 8 - 10.6 M), subsequently hemodynamic parameters (e. g. glomerular filtration rate) and changes in sodium and potassium absorption were determined . All data were analysed using an univariate ANOVA. Results: CCKBR-RNA was detected by Northern blot in kidney, stomach and brain. II. Immun ohistochemi stry localized CCKBR on proximal and distal tubuli and on collecting ducts. III. In comparison to controls. gastrin (10' 6 M) caused a marked decrease in the fracti onal sodium reabsorption: basal 80:t7.6%. 10 min after application of gastrin 71 :t 8.9%, after 20 min 62:t 13.5% (F<0.05). The most pronounce d changes in urinary potassium exc retion were caused by 10.8 M gastrin with a decrease from baseline values (100% ) to 6 1:t20% after 10 min and to 55:t 16% after 20 min (F < 0.0 I). The effec ts of gastrin were abolished by the CCKB receptor antagonist L-365.260. Gastrin reduced the perfusate flow at 10.6 M from 10:::I to 7.5:t3 mllm in. Conclusio n: Th is study provides evidence that the CCKB receptors on tubul ar structures and on collecting ducts in the kidney are of function al importance . In the rat kidney, gastrin interacts with CCKB receptors to increase renal potassium reabsorption and to decrease sodium absorption. It seems conc eivab le that gastrin interacts with the CCKBR to cause an inhibition of the NaIKATPase activity

Somatostatin is present within subpopulations of myenteri c neurone s and medi ates inhibition of intestinal peristalsis. In the present study , we investigated the mechani sm of this inhib ition, and the receptor subtype involved using receptor selective agonist and antagonist ligands , and sst2receptor knockout mice. Experim ent s were performed using a Trendelenburg type prepar ation in which 4-5cm segments of jejunum from Hooded Lister rats and C57 BL6 mice were mounted horizontall y in 20ml organ baths containing Kreb' s buffer maintained at 37°C. The oral and aboral end s were connected to a reservoir co ntaining kreb ' s buffer and a pressure transducer. respectively. Data are give n as mean :tSEM (n=4-5). After a 30min equilibration period (at OcmH 20), segments were distended (l 0- l lc mH 20 in rat, 2.5-5cmH 20 in mouse) to evoke contraction complexes (CC) which propogated from the oral to aboral end of the segment. CC had a duration of 22.66 :t2s, a maximum pressure of 1O.8:t1 cmH 20 and occurred at an interval of 113.36:t25 s in the rat. They were abolished by atropine (I/LM), TTX (0.6/LM ), w-conotoxin (O.l /LM) and nifedipine (l/LM). Somatostatin (SSTl 4) caused a dose-dependent increase in the CC interval, an effect abolished in the presence of L-NAME (J(lO/LM) and the selective sstyreceptor antagonist Cyanamid I54806 (IO/LM). The selective sst 2receptor agonists, Octreotide ( 100M) and BIM-23027(lOnM) increased the CC interval by 97.5 :t25% and 170:t57% respectively. In sst 2knockout mice, increases in CC interval produce d by SSTI4 ( IOnM) were significan tly less than in the wild-type litter mates (104.8:t42% versus 427.8:t 129%, n=4. P< 0.05). A similar differ ence was observed with BIM-23027 (30nM; 1.28:t7% versus 113.3:t38%, n=4, P
1680 GLP·l INHIBITS PROXIMAL GASTRIC MOTILITY BUT ENHANCES PERCEPTION. Uwe Wank, Joerg 1. Schirra, Rudolf Arno ld, Burkhard Goeke, Martin Katschinski, Philipp s Univ, Marburg. Germa ny; InselspitaJ. Univ of Bern, Berne , Switzerland. This study assessed the effect of physiological and supraphysiological loads of GLP-I on tonic and phasic motility of the fundus and perception of distention. Methods: In II healthy male volunteers, fundic tone was recorded with an electronic barostat against background infusions of saline or GLP-I at 0.3 or 0.9 pmol/rkgemin) on separate days (basal period: 1-60 min, peptide period: 61-120 min) . Subsequen tly, with ongoing infusions graded isobaric (18 D) and isovolumetric (IVD) distentions were performed . Fundic tone was derived from balloon volume excluding phasic volume waves. Endocrine parameters (blood glucose, insulin, PP) were regularl y determined. Results: GLP- l dose-dependently lowered fundic tone, markedly inhibited phasic contractile activity but increased compliance. It enhanced percept ion of isobaric but not of isovolumetric distentions. GLP-I slightly reduced blood glucose , but only transiently released insulin. The high load decreased IR-P P. Conclusions: These data support the role of GLP-I as a physio logical inhibitory regulator of tonic and phasic fundic motility. Diminution of fundic tone and volume waves most likely contributes to retarded gastric emptying. These inh ibitory motor effects cannot be accounted for by changes in blood glucose and plasma insulin. By contrast, reduction of PP release hints at impaired cholinergic tone as a mediatory mechanism. Fundic relaxation and stimulation of GLP-l receptors at circumventricular organs may both contribute to enhanced perception of distention.

Fundic Motility and PerceptionofDistention

Paramlller

Saline

GLP·l0.3

GLP·l0.9

121.2 ± 6.0 10.6±0.5 15606.2±1081.7 25.4 ± 4.1 9.9±1.3 28.0 ± 2.4

163.0± 15.0' 5.7± 0.6' 7363.4±1065 5' 40.6± 6.6' 13.6± 1.6' 28.7± 3.2

259.5 ± 17.2'# 3.1 ± 0.4'# 3566.3±556.6'# 59.8 ± 7.2'# 13.7 ± 1.5' 26.3 ± 3.6

1682 EPITHELIAL DISTRIBUTION OF NEURAL RECEPTORS IN THE GUINEA PIG SMALL INTESTINE. Carolyn J. Baglole , Joseph S. Davison, Jonathan B. Medd ings, Univ of Calgary, Calgary, AB, Canada. Introduction: It is well known that neural and paracrine agents such as dopamine. epinephrine and histam ine affect intestinal epitheli al functio n. It is unclear. however, if they are acting on receptors directl y on the enterocyte ; evidence linking the pharmac ological response is based on indirect functional, radioligand-bindin g and/or immun ocytochemical techn iques. Therefore , the precise localization of these receptors and their subtypes within the mucosa of the small intestine remains obscure and needs to be identified. Objectives: To characterize the cellular distribution of 0' 1- and J3-adrenergic, dopamine D, and D 2 and histamine receptors along the crypt-villus axis in the jejunum of the guinea pig. Methods: Single cell populations of villus or crypt epithelial cells were isolated from the jejunum of adult guinea pigs (n = 2-4) using a novel method developed by us (J Physiol 497 :161;1996 ). Enter ocytes (ENT) were separated from intraepithelial lymphocytes (JEL) by flow cytometry and specific binding to each cell population was determ ined using fluorescent-labe led probe s. Results: Alpha. -adrenergic receptors were located on villus and crypt IELs (56:t33% & 46.5:t180/0, respectively) and crypt ENTs (36.3:t2 1.9%) but not on villus ENTs « 10%). Beta-ad renergic receptors were found only on villus and crypt ENTs (43.9:t 15.1% & 43.2:t9.5%, respect ively). Dopa mine D , and D 2 recept ors were found on all cell types examined (Table 1) whereas histamin e recept ors were not detected «I0% for each cell population). Conclu sions: I . Recept ors for epinephrine and dopam ine have been demonstrated on epithelial cells of the guinea pig jejunum. 2. Betaadrenergic receptors are found on both villus and crypt ENT s whereas O' ,-adrenergic receptors are only located on crypt ENTs. 3. IELs have a I-adrenergic but no J3-adrenerg ic receptors. 4. The differential distribution of adrenergic recept ors may indicate that these agent s are acting direc tly, at least in part, at the level of the ENT or the IEL to modulate intestinal and immune function.

Table 1. Dopamine Receptors onIntestinal Epithelial Cells FundiC lone(ml) Volume waves (#110 min) Barostat Index (ml's) Compliance (mllmmHg) Perception score IBD Perception score IVD

'p<0.05 vs saline,• p<0.05 vs GLP1 0.3pmol/(kg'min); barostatindex: area under volume waves; perception score: cumulative value over alldistention steps

Crypt

Villus

01 02

ENT

IEL

ENT

IEL

67.7±9 84.9±5.3

25.17±18.6 50.1±14

80.8±12.5 80.1±8.5

50.9±15 55.8±8.6