Neurotensin stimulates pancreatic exocrine secretion in rats

Regulatory Pepttdes, 15 (1986) 279-284 Elsewer

279

R P T 00510

Neurotensin stimulates pancreatic exocrine secretion in rats Talaat Khahl, Masakl Fujlmura*, George H Greeley, J r , C o u r t n e y M Townsend, Jr and James C T h o m p s o n Department of Surgery, The Umverslty of Texas Medwal Branch, Galveston, TX 77550, U S A (Received 16 April 1986, accepted for pubhcatmn 25 May 1986)

Summary Neurotensm (NT) stimulates pancreatic exocrlne secretion in dogs and humans The purpose of this study was to examine the effect of exogenous neurotensm on pancreatic exocnne secretion in rats. Five Sprague-Dawley male rats were prepared w~th pancreaUc, gastric and duodenal fistulas Bde was shunted into the duodenum m order to collect pure pancreatic juice 24 h later, neurotensm (0 05, 0 1, 0 2, 0 3, 1 0 nmol/kg) was infused intravenously in a random fasluon Pancreatic jmce was collected every 10 mm, and the volume was recorded and protein and bicarbonate were measured Neurotensm sUmulated, m a dose-related manner, the pancreatic secretmn of water, protein and b~carbonate Neurotensm may be involved in the physmlogqc control of pancreaUc secretion m rats b~carbonate, protein, fistula

Introduction Neurotensln (NT) is a tndecapeptlde found predominantly in the distal small intestine of many mammals including humans, dogs, and rats [1--4] Neurotensm-contalnmg neurons are found in rat pancreas [5]. The physiologm role of neurotensm has not yet been clearly defined, but neurotensm has a wide spectrum of biologic * Vlsltlng Scmntlst from the Second Department of Surgery, Shiga Unlverstty of Medical Scmnce, Otsu-Clty, Sbaga, Japan Address correspondence to James C Thompson, M D , Department of Surgery, The Umverslty of Texas Me&cal Branch, Galveston, TX 77550, U S A Tel (409) 761-1285 0167-0115/86/$03 50 © 1986 Elsewer ScmncePubhshers B V (Blome&cal I~vlslon)

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actions, including mhlbmon of gastric motfltty and secretion, intestinal vasodllat~on, and alteration of gastrointestinal motdlty [6,7] Several studies have demonstrated that neurotensm stimulates pancreatic exocrme secretion m dogs [8-12] and humans [13] To our knowledge, the stimulation of pancreaUc exocrme secretion by neurotensm has never been shown in rats m wvo In contrast to findings m dogs and humans, m fact, a dose-related mhlbmon of basal pancreatic secretion was observed when 1 5-10 nmol/kg neurotensm was rejected as a bolus m the conscious rat [14,15] On the other hand, Feurle and Remecke [5] reported that neurotensln stimulated enzyme secretion m a preparatmn of isolated rat pancreatic lobules The purpose of this study was to determine the effect of neurotensm on pancreatic exocrlne secretion in conscious rats

Materials and Methods Male Sprague-Dawley rats (Zwlc-Mdler, Alhson Park, PA) (40(0450 g) were maretamed m an air conditioned, temperature-controlled room with hghtmg schedule of 14 h of dark (hghts were on from 0500 to 1900 h) Rats had free access to laboratory rat chow and tap water Five rats were prepared with pancreaUc fistulas according to the method described by Colwell [16] For this procedure, the rats were anesthetazed with ether after an 18 h fast A polyethylene catheter (PE 50, I D 0 58 mm, OD 0 9 mm, length 15 cm) was introduced into the duodenum and then into the pancreaUc duct through a duodenostomy 5 cm proximal to the opening of the duct, and secured with a hgature The mare bile-pancreatic duct was hgated m the mesentery just proxtmal to the point at whlch it becomes surrounded by pancreatic tissue The bile was then shunted directly into the duodenum by cannulatlon of the bile duct 2-3 mm above the ligature with a sdastlc tube (ID 0 61 mm) with a teflon Up. This tube was then inserted into the duodenum near the openmg of the pancreatic duct through another duodenostomy, which was closed with a purse stnng The rats were also prepared with gastric and duodenal fistulas The pancreatic and duodenal tubes were extenonzed through the lower end of the wound Rats were placed m Bollman cages [17] and a tall veto was cannulated using a 23-gauge catheter Rats were kept under a heat lamp until they recovered and then were maintained at room temperature (23 + 2"C) The pancreatic and duodenal fistulas were connected to allow the pancreatic juice to enter the duodenum All rats were gwen a continuous infusion of 5% dextrose m lactated Ringer's solution vm the taft veto (1-2 ml/kg/h) until compleUon of neurotensm challenge studies All rats were studied 24 h after surgery We have prewously shown that 24 h after operatmn Is an acceptable t~me to perform such studies [18] D u n n g the experiment, the pancreatnc and duodenal fistulas were d~sconneeted so as to allow collectmn of pancreatic fluid Pancreatic jmce was collected m nonheparlmzed capillary tubes (ID 0 1-1 2 mm, OD 1 4-1 6 mm, length 74 mm) The pancreatic flow rate was measured every 10 mm and was recorded m #1 every 10 mm Three 10-mm specimens of pancreatic jmce were combined every 30 nun and 120 #1 was saved and placed into sealed plasuc tubes for later measurement o f bicarbonate

28l

and protein The excess juice was re-infused into the duodenum through the duodenostomy tube dunng the next 30 mln Basal pancreatic exocnne secretions were collected every 10 mln for 60 mln Graded doses o f syntheuc neurotensin (NT 1-13) (Bachem, Torrance, CA) (0 05, 0 l, 0 2, 0 5, 1 0 nmol/kg) were given intravenously as bolus mjectlons m the tall vem in a random fashion PancreaUc secretmns were collected every l0 mm for 60 mln after neurotensm challenges 30 mm were allowed after each dose of neurotensin as a recovery penod Pancreatic protein concentrations were measured by the Lowry method [19] Pancreatlc juice bicarbonate concentrations were measured by adding 1 ml of 0 1 N HC1 to 0 1 ml samples of pancreatic juice, swirling the mixture, and back-titratmg to pH 7 with 0 1 M N a O H using an automatic titrator with a pH meter (Radmmeter, Copenhagen, Denmark) [20] Total protein and btcarbonate outputs were calculated by multiplying their concentrations by the volume o f pancreatic juice collected m 30 rain

Statistical analysts Data are expressed as the mean + S E M Slgmficant differences were identified by Student's t-test Differences with a P value of less than 0 05 were considered significant Results The results are summarized in Table I and m Figs 1 and 2 The adnunlstratlon of A e-

E 0

m

NT DOSES • O A • []

120

110.


100

I-ill tj

"' (J m

Or)

90

U.I

~ Z

05 n m o l / k g 1 nmol/kg 2 nmol/kg 5 nmol/kg 0 nmol/kg

N = 5 RATS

.-I

o> I Z 0m

0 0 0 0 1

80

O-

0

10 ) 2 ~0 30 ~ 4 ~0 5~06 ~0 7 0~ 80 ~ 9 ~0 MINUTES

F~g 1 Effect of neurotensm on volume of pancreatic secreuon m rats *P < 0 05 versus basal or lower dose

282

N = 5 RATS

20

2

15

8~ 10

<

o_

B Z

0 05

O1

02

O

10

A ~

,T, E

N = 5 RATS S-

432-

=<8

1B

005

O1

02

DOSE OF NEUROTENSlN

05

10

(nmol/kg)

Fig 2 Effect o f neurotensm on pancreatic bicarbonate output (upper panel) and protein output (lower panel) m rats *P < 005 versus basal or lower dose

graded doses of neurotensm (0 05, 0 1, 0 2, 0 5 and 1.0 nmol/kg) to rats resulted in a dose-related elevation of pancreatic seeretaon volume (Fig. 1), btcarbonate and protein outputs (Fig 2) m comparison to basal levels Maximal sttmulataon was achieved with a dose of 0 5 nmol/kg In order to make sure that the pancreatic response was not due to contammaUon with cholecystoklmn (CCK)-hke matenal, we tested our neurotensan substance on rabbit gallbladder stnps in vitro, according to the method described by Amer and Becvar [21] It was found to have no effect on the rabbit gallbladder strip

TABLE I Effects of neurotensm on pancreatic secretion in rats

NT dose (nmol/kg)

Pancreatac volume (pl/kg, 10 nun)

B~carbonate output ~Eq/kg, 30 nun)

Protein output (mg/kg, 30 ram)

Basal 005 01 02 05 10

823 1000 1077 1173 1239 1096

64 85 104 131 160 107

13 20 28 32 37 23

+ 44444-

11 1 161" 171" 181" 182" 222*

Values are mean 4- S E M of maxtmum response * P < 0 05 versus basal or lower dose

44+ 444-

12 13* 15" 17" 19* 17*

44+ 444-

04 05* 08 09* 09* 06*

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Discussion The results of this study demonstrate clearly that neurotensln stimulates pancreatic exocrme secretion of water, protein and bicarbonate in conscious rats in a doserelated manner The data suggest that neurotensln may be involved in the physiologic control of pancreatic exocrlne secretion In earlier studies, Demol and colleagues [15] reported that neurotensln inhibited basal and stimulated pancreatic secretion m rats They claimed that the Inhibitory effect of neurotensln reached a maximum one hour after bolus injection and was sustained for 2 5 h In order to test the effect on endogenously or exogenously stlmulated pancreatic secretion with duodenal infusion of either HCI m olelc acid or i v injection of secretm or CCK, they gave neurotensln 1 h prior to stimulation They used much higher doses ofneurotensm (125 pg/kg) in their experiment The inhibitory effect they reported may have resulted from the hypotenslve effect o f neurotensln Moreover, they failed to return the pancreatic juice to the duodenum in their experiments, which was reflected in the weak response of their model to secretln and CCK stimulation The stlmulatory effect of neurotensln on pancreatic exocrlne secretion Jn dogs and humans is well established [8-13] by several studies The results of our expenment concur with these studies and show that there is no species difference between rats and dogs or human In the dog, neurotensln can act in an additive manner with endogenously released CCK on pancreatic protein secretion, and neurotensm can potentiate the stimulatory action of secretln on pancreatic bicarbonate secretion [12] The stlmulatory actions of neurotensln in pancreatic exocnne secretion are blocked by somatostatm (unpublished data from our laboratory) The mechanism of action of neurotensln on the pancreas, as well as the question of whether it acts directly or indirectly on pancreatic aclnar cells, is not understood Feurle and Relnecke [5] demonstrated that isolated pancreatic lobules respond to neurotensln On the other hand, Weaver and colleagues [22] from our laboratory, as well as Demol and colleagues [14], found that neurotensm alone had no effect on amylase release from either dispersed pancreatic acml or pancreatic lobules In fact, Weaver and colleagues [22] found that neurotensln inhibited CCK-stlmulated amylase release from dispersed pancreatic aclnl Feurle and Relnecke [5], however, found slgmflcant elevation in amylase only after 90 mln of neurotensln stimulation, which is a prolonged penod compared to the 30 mln incubation as described by Pelkm and colleagues [23] and used by our group [22] In summary, neurotensln in rats stimulates the intact pancreas in VlVO, but has no effect on isolated pancreatic aclnl This suggests that neurotensln may act indirectly or may require the presence of neural or humoral contnbutlons In order to effect pancreatic secretion in conscious rats

Acknowledgements This work was supported by grants from the National Institutes of Health (RO1 AM 15241, PO1 AM 35608, and R C D A 008557), and a grant from the Amencan Cancer Society (PDT-220)

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