Potentiating role of cyclic AMP in pancreatic enzyme secretion, demonstrated by means of forskolin.

The role of cyclic AMP in the regulation of enzyme secretion by the rabbit pancreas has been investigated by means of forskolin, an activator of the catalytic subunit of adenylate cyclase. Forskolin increases the cyclic AMP level in isolated pancreatic acini in a dose-dependent way. Basal amylase release, however, remains unchanged. Forskolin potentiates the increase in amylase release induced by the C-terminal octapeptide of cholecystokinin (CCK-8). Potentiation is already apparent at hormone concentrations which are only marginally effective in stimulating amylase secretion. CCK-8 alone does not raise the cellular cAMP level, but it potentiates the forskolin-induced increase. In relative terms, potentiation is higher with decreasing concentration of forskolin. These results indicate that cAMP alone does not play a direct role in CCK-stimulated pancreatic enzyme secretion in the rabbit, but it potentiates enzyme secretion already stimulated through a cAMP-independent process.

Amiloride is a cholinergic antagonist in the rabbit pancreas.

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na+), or in a medium containing only 25 mM Na+. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin ( PzO ) is not affected. Amiloride also inhibits the carbachol-induced 45Ca efflux from rabbit pancreatic acini, but again not that induced by PzO . The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and 45Ca efflux are 40 and 80 microM, respectively. Amiloride also competitively inhibits the specific binding of [3H]quinuclidinyl benzylate ( [3H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.

Synergistic effect of A23187 and a phorbol ester on amylase secretion from rabbit pancreatic acini.

The combination of the ionophore A23187 and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulates amylase secretion from rabbit pancreatic acini up to a level equal to, or slightly higher than when carbachol is used as stimulant. Each of the two compounds alone gives only a minor stimulation. This synergistic effect of A23187 and TPA supports a role of protein kinase C in pancreatic enzyme secretion.

Studies on (K+ + H+)-ATPase V. Chemical composition and molecular weight of the catalytic subunit.

(1) A (K+ + H+)-ATPase preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The Detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH2 terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, n = 6) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, N = 6) for protein only. (8) In combination with the molecular mass of 444 kDa (S. E. 10, n = 4) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.

Studies on (Na+ + K+)-activated ATPase. XLIX. Content and role of cholesterol and other neutral lipids in highly purified rabbit kidney enzyme preparation.

(1) The neutral lipids and the free and bound fatty acids of a highly purified (Na+ + K+)-ATPase preparation from rabbit kidney outer medulla have been analysed. (2) On a dry weight basis, the total lipid content is nearly the same as the total protein content, and consists for 66% of phospholipids and for 34% of neutral lipids and free fatty acids. In the latter category cholesterol is the main component (71%). (3) On a molar basis the enzyme preparation contains 382 mol phospholipids, 67 mol free fatty acids, 9, 16 and 12 mol mono-, di- and triacylglycerols, 249 and 19 mol free and esterified cholesterol per mol enzyme. (4) The fatty acid composition of each lipid and of the free fatty acid fraction, present in the enzyme preparation, is reported. (5) All cholesterol and part of the phospholipids can be removed by hexane extraction, leaving 66% of the (Na+ + K+)-ATPase activity. Oxidation of all cholesterol to cholest-4-en-3-one by cholesterol oxidase leaves 85% of the (Na+ + K+)-ATPase activity. These results indicate that cholesterol is not essential for (Na+ + K+)-ATPase activity.

The reflexion coefficient as a measure of transepithelial permeability in the isolated rabbit pancreas.

1. The reflexion coefficients of a number of non-electrolytes and electrolytes have been determined in the isolated rabbit pancreas. 2. The reflexion coefficients of the following non-electrolytes were: urea, -0.02; glycerol, 0.06; erythritol, 0.11; sorbitol, 0.41; mannitol, 0.42; arabinose, 0.72; xylose, 0.74, assuming a value of 1.00 for sucrose. 3. These values are equal within the experimental error to values previously obtained with a tracer technique for the same preparation, but they are significantly lower than those reported by other investigators for the isolated perfused cat pancreas. 4. Addition of 100 mM-sucrose to the bathing medium resulted in proportionally increased Na+ and K+ concentrations in the secreted fluid. The secreted fluid remained isotonic with the bathing medium under all circumstances. 5. Addition of 10(-5) M-carbachol to the bathing medium led to a reduction in the reflexion coefficient of sucrose from 1 to 0.85, but only when 25 mM-sucrose was used. 6. The reflexion coefficients of electrolytes were: NaCl, 0.50; KCl, 0.51; NaHCO3, 0.52 and choline chloride, 1.02. 7. It is concluded that the isolated rabbit pancreas is highly permeable, both to electrolytes and to small non-electrolytes, probably being more leaky than any other epithelium studied so far.

Role of calcium in exocrine pancreatic secreation. VI. Characteristics of the paracellular pathway for divalent cations.

(1) The transepithelial permeability for Ca2+ and Mg2+ in the isolated rabbit pancreas has been studied. (2) Values for the permeability of the unstimulated pancreas were obtained either by adding radioactive tracers to the bathing medium and measuring their concentration in the secreted fluid under steady-state conditions, or by analysis of the Ca2+ and Mg2+ concentrations in the secreted fluid after correction for protein-bound divalent cations. (3) Both methods give almost the same results: 27 and 26% for Ca2+ and 21 and 18% for Mg2+, respectively; both values being expressed as the percentage of the concentrations in the bathing medium. (4) The amounts of Ca2+ and Mg2+, appearing in the secretory fluid after correction for protein-bound cations, are linearly related to the extracellular Ca2+ and Mg2+ concentrations in the bathing medium, which indicates passive permeation. The two cations appear to pass through the paracellular route in their hydrated form. (5) Stimulation with carbachol or pancreozymin causes an increase in the paracellular permeability. This increase is approximately equal for the two divalent cations. Its time dependence and magnitude depend on the concentration of the stimulant rather than on the type of stimulant.

Blocking by 2,4,6-triaminopyrimidine of increased tight junction permeability induced by acetylcholine in the pancreas.

1. The permeability of the paracellular pathway in the isolated rabbit pancreas has been studied with the aid of 2,4,6-triaminopyrimidine. 2. Addition of 2,4,6-triaminopyrimidine (1--10 mM) to the bathing medium has no effect on the rate of fluid secretion or on protein, Na+, K+, Ca2+ and sucrose concentrations in the secreted fluid. 3. When 1 x 10(-5) M carbachol is also added to the 2,4,6-triaminopyrimidine-containing bathing medium, there is a marked reduction in the increase of the paracellular permeability for sucrose and Ca2+ found upon addition of carbachol alone. The enzyme secretion, induced by carbachol, is not affected. 4. The minimal concentration of 2,4,6-triaminopyrimidine in the bathing medium required to reach its maximal effect on the paracellular permeability is approx. 0.55 mM at pH 7.4. 5. The effect of 2,4,6-triaminopyrimidine on the paracellular permeability after carbachol stimulation is also present when 2,4,6-triaminopyrimidine is added 5 min after the addition of 1 x 10(-5) M carbachol. 6. 2,4,6-Triaminopyrimidine has no effect on the increases in enzyme secretion and sucrose permeability caused by 1 x 10(-8) pancreozymin C octapeptide. 7. 2,4,6-Triaminopyrimidine appears in the secreted fluid at a concentration of 50% of that in the bathing medium. Upon addition of 1 x 10(5) M carbachol this concentration increases up to 80%. 8. These results indicate that: (a) the increased paracellular permeability upon stimulation with carbachol is not caused by the enzyme secretion as such and (b) addition of 2,4,6-triaminopyrimidine prevents the carbachol-induced increase in permeability of a channel in the tight junction complex.