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Naunyn-Schmiedeberg's Arch Pharmacol (1984) 327: 228- 233

Pharmacology 9 Springer-Verlag 1984

Effects of nerve stimulation on enzyme secretion from the in vitro rat pancreas and 3H-release after preincubation with catecholamines Jaipaul Singh and Geoffrey T. Pearson MRC Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, P.O. Box 147, Brownlow Hill, Liverpool, L69 3BX, UK

Summary. In the presence of the cholinergic antagonist atropine, electrical field stimulation (FS) ( 5 - 20 Hz) caused a marked, reversible increase in the amylase output from superfused rat pancreatic segments. Adrenaline and noradrenaline evoked dose-dependent increases in amylase output which were similar to those produced by FS. The FS- and catecholamine-evoked amylase secretions were abolished by the/~-adrenergic antagonist propranolol. The FS-evoked secretion could be abolished by either the removal of external Ca 2 § or the application of tetrodotoxin (TTX, 2 x 10 6 M). FS also resulted in a reversible increase in the fractional efflux of tritium (3H) from rat pancreatic tissues preincubated with either 3H-noradrenaline or 3Hadrenaline. The effects of FS ( 5 - 2 0 Hz) on 3H efflux were abolished by TTX (2 x 10 .6 M). TTX had no effect on the enhancement of 3H efflux caused by elevation of external potassium concentration (high K +, 75 mM). Removal of superfnsate Ca z+ completely abolished both the FS- and high K +-induced increases in 3H efflux. These observations suggest that intrinsic nerve stimulation (i.e. FS) results in the Ca 2 +-dependent release of sympathetic neurotransmitter, noradrenaline, which has a direct secretory action on the rat pancreas. Furthermore, the findings suggest that adrenaline can be taken up by nervous elements. This raises the possibility that uptake and re-release of circulating adrenaline might contribute to the control of rat pancreatic enzyme secretion by the adrenergic nervous system.

Key words: Nerve stimulation - Rat pancreas - Amylase secretion - 3 H - N o r a d r e n a l i n e - 3H-Adrenaline

Introduction It is well established that cholinergic nerves play an important role in the control of secretion from the exoerine pancreas (Thomas 1967; Lenninger 1974). A direct involvement of the adrenergie nervous system in the control of pancreatic secretion has not been clearly elucidated. It has been said that adrenergic stimulation only influences secretion indirectly via alterations of pancreatic bloodflow (Mann and McLachin 1917; Greengard et al. 1942; Barlow et al. 1974). Direct inhibitory effects have also been suggested (Harper and Vass 1941). Recently however, clear secretory effects of exogenously applied adrenergic agonists have been observed in the rat Offprint requests to J. Singh at the above address

pancreas (Furata et al. 1978; Lingard and Young 1983; Chariot et al. 1983). Interestingly, this is a species that was not used in the earlier investigations. In view of these findings, the aim of the present study was to question whether adrenergic nerves have a stimulatory effect on pancreatic secretion in the rat. Indeed, the existence of such a noncholinergic pathway would not be exceptional in the light of our previous demonstration that noncholinergic nonadrenergic nerves can evoke enzyme secretion in the guinea pig pancreas (Pearson et al. 1981 a, b). In the present study therefore, we have measured the enzyme secretion from in vitro preparations of rat pancreas and employed the technique of electrical field stimulation (FS) to excite intrinsic pancreatic nerves. Furthermore, and as an indication of the endogenous release of catecholamines we have studied the effiux of radioactivity from pancreatic tissue preloaded with tritium (3H)-labelled noradren~line and adrenaline. A preliminary report of our tritium efflux experiments has already appeared (Pearson and Singh 1983).

Methods Experiments were performed on isolated segments of rat pancreas. Sprague-Dawley adult rats (200-300 g) were killed by a blow to the head and the pancreas was quickly removed and placed in modified Krebs-Henseleit solution of the following composition (raM): NaC1 103, KC1 4.7, CaCI2 2.56, MgClz 1.13, NaHCO3 25, NaHzPO4 1.15, Dglucose, 2.8 N a pyruvate 4.9, Na fumarate 2.7, Na glutumate 4.9. The Krebs-Henseleit solution also contained atropine (10 .5 M) and was gassed with 95% 02 - 5% CO2 and maintained at 37 ~C. Amylase Secretion. The pancreas was cut into small segments ( 5 - 1 0 mg) and a total weight of about 2 5 0 300 mg was placed in a Perspex flow chamber and superfused with Krebs-Henseleit at a flow rate of 1 ml rain-~. The effluent from the flow chamber passed directly to an on-line automated fluorometric assay for the continuous measurement of amylase output (Matthews et al. 1974). This method, which is a modification of the original technique described in Rinderknecht and Marbach (1970) depends essentially upon the liberation of dialyzable fluorogenic products from amylopectin anthranilate used as a substrate. Amylase output is expressed in units (U) ml-1 100 mg of tissue- 1. One unit of amylase is defined as the amount of amylase which will liberate 1.0 mg of maltose from starch in 3 min at pH 6.9 and 20~ In the present

229 study, c~-amylase (Sigma type IIA) was used as a standard for calibration. Electrical field stimulation (FS) was achieved by connecting two silver electrodes, contained in the Perspex chamber to a Grass $44 stimulator (Pearson et al. 1981 b). This gave square-wave stimulation of the required frequency ( 5 20 Hz), pulse duration (1 ms) and voltage amplitude (50 V).

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3H-Noradrenaline and 3H-adrenaline efflux measurements. Pancreatic segments were incubated for 60 min in 3 ml of Krebs-Henseleit solution containing 30 laCi of either 3Hnoradrenaline or 3H-adrenaline. The specific activity of 3Hnoradrenaline was 10.7 Ci nmol - ~giving an incubation concentration of 0.93 nmol ml- z. The specific activity of 3Hadrenaline was 14.5 Ci nmol- ~ giving an incubation concentration of 0.69 nmol ml-~. The mixture was shaken (60 rain-1) and maintained at 37~ The procedure was similar to that described elsewhere for measurement of 45Ca2+ efflux (Matthews et al. 1973). After the loading period the segments ( 2 5 0 - 300 mg total) were transferred to a Perspex flow chamber and superfused (I ml rain-1) with label-free Krebs-Henseleit solution for 150 rain prior to the collection of effluent samples. This time was found adequate for the 3H efflux to attain a steady-state level (Matthews et al. 1973). Effluent fractions were collected at 2 rain intervals ~ into scintillation vials to which was added 10 ml of a mixture of Triton: Toluene (1:1) containing Scintol 2 (50 ml 1- ~) (Koch-Light Lab Ltd.). The 3H content of the samples was measured by liquid scintillation analysis. The residual tissue 3H content was measured after dissolving the pancreatic segments in 2 ml of Soluene-350 (Packard). The radioactivity readings were then processed by computer to yield values for 3H fractional effiux according to

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1A-C. Effects of electrical field stimulation (FS) on amylase output from rat pancreatic segments. The figure shows 3 separate experiments (A, B and C) in each of which 10- 5 M atropine was present in the superfusion solution. The horizontal lines labelled 0 represent zero amylase output in each experiment (also in Fig. 2). FS parameters: frequency as shown, pulse amplitude 50 V, pulse duration 1 ms, stimulation period (bar) 5 rain. A Effects of FS frequency variation. B Effects of FS in presence and absence of external Ca 2 +. The Ca z +-free Krebs-Henseleit solution contained 10-4 M EGTA. C FS in presence of 2 x 10-6 M tetrodotoxin (TTX) Fig,

A

Ax where Ax represents the d.p.m. At xt 3H released in the time interval At and x= the total tissue 3H

1.o unit ml-1 E (100 mg tissue) -1

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content at the midpoint of interval A t. Fractional efflux is expressed as a function of time and thus has units o f m i n - z . The tissue was electrically field stimulated in the manner described above. When high potassium-containing solution was required, the K + concentration was elevated from the normal level of 4.7 mM to 75 mM and the Na + concentration was adjusted to maintain iso-osmolarity. In experiments using Ca 2+-free Krebs-Henseleit solution, EGTA (ethylene gylcol-bis-/%amino ethyl ether N,N'-tetra acetic acid) was added to a final concentration of 10 =4 M.

Drugs used in this study. (-)-Noradrenaline bitartrate, (-)adrenaline bitartrate, atropine sulphate and tetrodotoxin were obtained from Sigma London Chemical Co., Poole, UK. Propranolol hydrochloride and pentagastrin (Peptavlon) were purchased from ICI Ltd., Macclesfield, UK. The isotopes DL-[7-3H]-noradrenaline and DL-[7-3H]-adrenaline were purchased from The Radioehemical Centre, Amersham, UK.

Results Amylase secretion In the presence of 10- 5 M atropine the mean basal amylase output from superfused segments of rat pancreas was 0.73 + 0.03 (SE) U m1-1 100 mg tissue 1 (n = 25). FS pro-

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