SUMMARYInterlobular ducts were isolated from the pancreas of copper-deficient rats and maintained in culture on polycarbonate filter rafts. Within 8 h the ends of the ducts had sealed. This was followed by a marked dilatation of the lumen, a flattening of the epithelium against the surrounding connective tissue layer and an over-all swelling of the duct. Apart from a reduction in their height, a fall in the number of intracellular fat droplets and a widening of intercellular spaces, epithelial cells within the cultured ducts retained all the ultrastructural characteristics of those in freshly isolated preparations. The basal concentration of adenosine 3',5'-phosphate (cyclic AMP) in the cultured ducts was 43 3 +6 8 ,umol .1-1 duct epithelium (n = 5) and was increased to 1889 + 55-2 ,umol . 1-1 duct epithelium (n = 5) in the presence of 10-8 M secretin. The basal rate of fluid secretion, measured using micropuncture techniques, was 0-16 + 0 03 nl. h-. nl-l duct epithelium (n = 12). This was increased 14-fold by 10-8 M secretin while the dose of the hormone required for half-maximal secretion was about 2 x 10-11 mol . 1-1. The concentration of chloride ions in secreted fluid and perifusion buffer were similar. Variation in culture time up to 52 h had no effect on fluid secretion, and the response to secretin was dependent on the presence of bicarbonate ions in the perifusion fluid. N6,02-dibutyryl adenosine 3',5'-phosphate (dibutyryl cyclic AMP) also increased fluid secretion but caerulein had no effect. We suggest that isolated ducts secrete fluid at comparable rates to ducts in situ within the pancreas of copper-replete rats.

SUMMARY1. We have studied the effects of acetylcholine (ACh) on fluid secretion and intracellular messengers in interlobular ducts isolated from the rat pancreas and maintained in short-term tissue culture.2. Ductal fluid secretion was measured using micropuncture techniques.Intracellular free calcium ([Ca2+]1) and cyclic AMP concentrations were measured in single ducts using fura-2 microspectrofluorimetry and radioimmunoassay techniques respectively. Changes in the levels of these intracellular messengers were correlated with fluid secretion.3. ACh stimulated ductal fluid secretion. The dose required for a half-maximal response was about 0 4 ,/M and maximal secretion was achieved with 10 /LM ACh.These effects of ACh were blocked by atropine and by removal of extracellular Ca2". 4. ACh was about four orders of magnitude less potent as an activator of ductal fluid transport than the hormone secretin; however, the maximal rates of fluid secretion evoked by these two agonists were similar.5. ACh caused a dose-dependent rise in duct cell [Ca2+]i, but had no effect on cyclic AMP. In contrast, secretin increased duct cell cyclic AMP, but had no effect on [Ca2+]1. tTo whom reprint request should be sent.

SUMMARYA technique is described, involving tissue dissociation and micro-dissection, for the isolation of interlobular ducts from the pancreas of copper-deficient rats. The average length and outside diameter of the isolated ducts were 589 0+ 18 6 and 78 1 + 1 6 ,um (mean +S.E.M., n = 425) respectively. Between twenty and fifty ducts could be obtained from each pancreas. Frequently, the smaller intralobular ducts, which had outside diameters of between 15 and 25 ,um, were observed as branches of the interlobular ducts. Light and electron microscopy showed that the isolated ducts were structurally intact, and that the epithelial cells possessed all the typical ultrastructural features of duct cells within the gland of copper-replete rats. The isolated ducts consumed oxygen at a rate of 2 27 + 0 55 ml 02/min. 100 g wet weight duct epithelium (n = 6). The concentrations of ATP, ADP and AMP in the ducts were 3-78+0 81, 0 68+0-19 and 0-41 + 013 mmol/l duct epithelium (n = 8) respectively. These data give values for ATP: ADP and ATP:AMP ratios of 5 6:1 and 9-2:1 respectively, and an energy charge of 0 85+0 01 (n = 8) suggesting that the epithelial cells are healthy and in a stable metabolic state. In the presence of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (0-67 mM), the basal concentration ofcyclic AMP in the isolated ducts was 17 4 + 0-7 ,tmol/l duct epithelium (n = 3).Secretin (0 1 nM-1 /M) caused a dose-related increase in cyclic AMP content up to a maximum of 3760 + 85 3 ,umol/l duct epithelium (n = 4). This indicates that the epithelial cells possess secretin receptors, and that these receptors can be functionally linked to adenylate cyclase.

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SUMMARY1. In the isolated cat pancreas, stimulated maximally with secretin, increasing the perfusate potassium concentration (at the expense of sodium ions) caused a copious secretion of amylase from the gland, reduced the volume rate of secretion and caused vasoconstriction.2. Rubidium and caesium had similar effects to potassium: lithium, though depressing secretary rate, had no effect on enzyme secretion or vasoconstrictor action.3. Amylase secretion was detected at potassium concentrations of 30 mm and was maximal at 80-90 mm, output declining as the concentration was raised to 120 mM.4. Amylase secretion was maximal during the first few minutes of exposure to excess potassium, but remained above basal levels throughout the test period. Secretory rate was depressed by a constant amount during the test period.5. Atropine sulphate blocked the effect on enzyme secretion without affecting the reduction in secretary rate.6. During perfusion with excess potassium a vasodepressor material with the properties of acetylcholine was detected in the effluent from the gland. 7. The reduction in secretary rate, when perfusate sodium was replaced by potassium, was equal to that obtained when sodium was replaced by sucrose.8. It is concluded that potassium stimulates amylase secretion indirectly * Part of this work was carried out during tenure of a Luccock Research Studentship, University of Newcastle upon Tyne. 612B. E. ARGENT, R. M. CASE AND T. SCRATCHERD by releasing acetylcholine from nerve terminals in the gland, and that the reduction in secretary rate is due not to excess potassium but to sodium deficiency.