A model for fluid secretion in the exocrine pancreas

Fluid secretion by the isolated rabbit pancreas is strongly dependent on the presence of Na+ in the bathing medium. Substitution of Na+ by another cation such as Li+ or K+ causes an inhibition of fluid secretion rate and a change in the composition of the secreted fluid which is dependent on the nature of the substituent cation. Stimulation of the pancreas by CCK-8 or carbachol increases paracellular ion permeability and, in some cases, also fluid secretion rate. We present a simple, quantitative model for ion and water secretion which accounts for the effects observed upon Na+ substitution and stimulation. The main features are active, Na+-dependent transcellular HCO3- transport and passive, paracellular cation and anion permeation. The activity of the HCO3- pump is dependent on the energy status of the cell and on the Na+ concentration in the bathing medium, and is competitively inhibited by K+. The paracellular ion permeabilities can be modulated by stimulatory agonists. We examine the extent to which, according to the model, fluid secretion is controlled by the various system parameters such as ion permeabilities and ion pump activity, and by external parameters such as the ion concentrations in the bathing medium. In addition, calculation of the effects of changes in these parameters are carried out in order to gain more insight in the mechanisms of secretion.     

Role of calcium in exocrine pancreatic secretion. I. Calcium movements in the rabbit pancreas.

1. Calcium movements in the isolated rabbit pancreas and in rabbit pancreas fragments have been studied with the aid of 4 5 Ca2+. 2. Addition of 4 5 Ca2+ to the incubation medium of the isolated rabbit pancreas results in an immediate appearance of isotope in the secreted fluid reaching a constant specific activity in 30 min. The absolute activity in the secreted fluid is 30-40% of that in the incubation medium. 3. Addition of 10(-5) M carbachol after 2 h preincubation with 4 5 Ca2+ results in enzyme secretion accompanied by calcium release. There is also an increase in 4 5 Ca2+ secretion, but this is maximal 10 min after the protein and total calcium peaks. 4. Partial removal of 4 5 Ca2+ from the bathing medium, before stimulation, reduces the increase in 4 5 Ca2+ secretion nearly proportionally. 5. [3H]Mannitol, added to the bathing medium, appears in the secreted fluid and behaves upon carbachol stimulation similarly to 4 5 Ca2+. 6. Upon repeated stimulation with 10(-5) M acetylcholine, a 4 5 Ca2+ peak appears, even in virtual absence of enzyme secretion. In this case the peak coincides with a small total calcium peak. 7. Efflux studies of rabbit pancreas fragments, preloaded with 4 5 Ca2+, show a carbachol-stimulated 4 5 Ca2+ efflux in addition to a release of amylase. 8. These studies indicate that there are three calcium movements in rabbit pancreas which can all be influenced by cholinergic agents: (a) an extracellular route for calcium and other small molecules and ions; (b) a calcium release across the apical membrane along with the enzymes, originating from a pool which does not freely exchange with 4 5 Ca2+ in the bath; (c) a calcium flux across the serosal membrane, which involves calcium exchanging freely with 4 5 Ca2+ from the bath. The third flux is thought to result from an increase in cytoplasmic calcium, which may be involved in the stimulus-secretion coupling of pancreatic enzyme secretion.     

Role of monovalent cations in fluid secretion from the exocrine rabbit pancreas

The role of Na+ in fluid secretion by the isolated rabbit pancreas was investigated. The fluid secretion rate is reduced upon replacement of Na+ in the bathing medium by Li+, K+ or choline. The inhibition depends on the nature of the substituting cation, and is largest with choline. Upon replacement, the substituent cation appears in the secreted fluid, and the Na+ concentration in the secreted fluid is decreased in a mirror-like fashion. When Na+ is replaced by Li+ or choline, the secretory Na+ concentration is decreased, although less than in the bathing medium, and the K+ concentration is increased. When Na+ is replaced by K+, the Na+ and the K+ concentration in the secreted fluid are approximately equal to their bathing medium concentrations. In the Li+ and choline medium, stimulation of the pancreas by carbachol or CCK-8 increases the fluid secretion rate. In addition, it increases the Li+ or choline concentration, and decreases the Na+ and K+ concentrations in the secreted fluid. In normal and K+ medium, stimulation causes only a slight increase in fluid secretion rate, with no change in the secretory Na+ concentration. In normal medium, stimulation leads to a decrease in the secretory K+ concentration. The effects of replacing Na+ appear to be the result of a direct inhibition of the active HCO3- transport underlying secretion, and an indirect inhibition related to the permeability of the pancreas for the various cations. The stimulants are likely to act by increasing the permeability of the tight junctions.     

Effects of ouabain on fluid transport and electrical properties of Necturus gallbladder. Evidence in favor of a neutral basolateral sodium transport mechanism

Net fluid transport (Jv) and electrical properties of the cell membranes and paracellular pathway of Necturus gallbladder epithelium were studied before and after the addition of ouabain (10(-4) M) to the serosal bathing medium. The glycoside inhibited Jv by 70% in 15 min and by 100% in 30 min. In contrast, the potentials across both cell membranes did not decrease significantly until 20 min of exposure to ouabain. At 30 min, the basolateral membrane potential (Vcs) fell only by ca 7 mV. If basolateral Na transport were electrogenic, with a coupling ratio (Na:K) of 3:2, the reductions of Vcs at 15 and 30 min should be 12--15 and 17--21 mV, respectively. Thus, we conclude that the mechanism of Na transport from the cells to the serosal bathing solution is not electrogenic under normal transport conditions. The slow depolarization observed in ouabain is caused by a fall of intracellular K concentration, and by a decrease in basolateral cell membrane K permeability. Prolonged exposure to ouabain results also in an increase in paracellular K selectivity, with no change of P Na/P Cl.     

Electrical parameters of the toad skin: effects of forskolin.

Forskolin stimulated short-circuit current (SCC) and transepitelial electrical conductance (G) in the isolated skin of the toad Bufo arenarum in a concentration-dependent manner, between 1.0 x 10(-6) and 2.4 x 10(-5) M. At the latter concentration, glandular secretion appeared to be stimulated also. The increase in G was considerably greater in skins bathed in Ringer solution than in solutions containing no chloride. The increased SCC was abolished by amiloride, a specific blocker of sodium transport in amphibian membranes, irrespective of the anion present in the solution bathing the skin. G was also decreased by amiloride to control values in skins bathed in solutions without chloride, but remained elevated in the presence of Cl-. The increase in SCC following exposure to forskolin, 4.4 x 10(-6) M, was not altered when furosemide, a specific blocker of chloride transport, was present in the Ringer solution bathing the dermal side of the skin. The response to forskolin, 2.4 x 10(-5) M, however, was significantly decreased by dermal furosemide; the inhibitor was ineffective in the absence of chloride. The data indicate that forskolin acts on at least two sites: stratum granulosum cells (the main pathway for sodium transport, and an alternate site, responsible for the increase in permeability to chloride. In addition, at high concentration of the agent, glandular secretion is also stimulated. The data suggest that the adenylate cyclase-cyclic AMP system is involved in the regulation of the permeability of the toad skin to sodium and chloride, probably by separate cell types.     

Transpancreatic transport of digestive enzyme

When porcine α-amylase or bovine chymotrypsinogen A was added to the medium bathing the rabbit pancreas in short-term organ culture, the secretion of these enzymes collected via the duct system increased greatly. To determine if it was indeed the amylase added to the bath that was recovered in secretion, endogenous enzyme stores were prelabeled during a 4 h incubation with [³H]- leucine and the specific radioactivity of amylase in secretion followed. The addition of unlabeled exogenous amylase to the bathing medium reduced the specific radioactivity of secreted amylase of about 90% suggesting that the response was due to the transpancreatic transport of the added enzyme. This inhibition was maintained over time, and was a result, not only of the increased secretion of unlabeled enzyme, but also of a 72% steady-state inhibition in the secretion of endogenous (labeled) amylase. This latter observation indicates that the exogenous enzyme crosses the acinar cell and mixes with endogenous cellular stores. A cellular route is also suggested by the observation that the addition of amylase to the bath increased the amylase concentration in ductal fluid relative to that in the bath by about 20 times; it did not reduce it as would be expected if paracellular shunts were involved. In addition, a cellular pathway is suggested by the observation that a 2 h prior incubation in bovine chymotrypsinogen resulted in a greatly augmented chymotrpsinogen response to a maximal cholinergic stimulus. In all, the data support the prediction of the equilibrium theory of digestive enzyme secretion that enzyme secretion should be responsive to mass action, and the prediction of the enteropancreatic circulation hypothesis that a capacity exists for a substantial transpancreatic flux of digestive enzyme.     

Inhibition of mucin secretion in a colonic adenocarcinoma cell line by DIDS and potassium channel blockers

The factors which influence the exocytosis of mucins are not well characterized. Since the physical properties of mucins may be affected significantly by the co-secretion of electrolytes and water, we studied the relationship between ion movement and mucin secretion in T84 cells, a human colonic adenocarcinoma cell line which has been well characterized with respect to apical chloride secretion. Secretion of mucin was assessed by immunoassay of mucin appearing in the medium within 30 min of stimulation. Cells were grown on plastic in DMEM/Ham's F12 medium and experiments were carried out at 70% confluence. Mucin secretion was stimulated by the calcium ionophore A23187, or A23187 plus vasoactive intestinal polypeptide. Stimulated mucin secretion was not affected by loop diuretics (furosemide (1 x 10(-3) M) or bumetanide (1 x 10(-4) M)), with or without the addition of ouabain (5 x 10(-5) M) and amiloride (1 x 10(-5) M), making it unlikely that transcellular chloride movements in necessary for mucin secretion. However, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; (1 x 10(-5) and 5 x 10(-5) M) and three potassium channel blockers BaCl2 (1 x 10(-3) and 5 x 10(-3) M), tetraethylammonium chloride (1 x 10(-2) M) and quinine (5 x 10(-4) M) inhibited mucin secretion. A DIDS-sensitive chloride channel or chloride/bicarbonate exchanger and a Ca2(+)-dependent potassium channel may play important roles in mucin secretion. Since plasma membranes are sparingly permeable to DIDS, the DIDS-sensitive site is likely to be on the apical plasma membrane, perhaps at an initiation locus for exocytosis.     

Tight junctional permeability of the resting and carbachol stimulated exocrine rabbit pancreas

Summary The permeability of the pancreatic epithelium to horseradish peroxidase is investigated in the resting and carbachol stimulated rabbit pancreas. Horse radish peroxidase administered to the bathing medium of the isolated rabbit pancreas appears in the secreted fluid of the pancreas in a relatively low concentration. Carbachol stimulates both protein secretion and the passage of horse radish peroxidase into the secretory fluid. Histochemical assessment shows that horseradish peroxidase enters the interstitial spaces of the pancreatic tissue and is present along basal and lateral plasma membranes of acinar and ductular cells. In the absence of carbachol, horseradish peroxidase is seen more frequently in the tight junctions of ductular cells than in those of acinar cells. However, in the carbachol stimulated gland horseradish peroxidase is observed in the junctions between adjacent acinar cells more frequently than in the unstimulated gland. Freeze-fracture of acinar cells shows that the number of tight junctional strands and the tight junction depth are slightly decreased upon carbachol stimulation. The findings suggest that cholinergic stimulation of the exocrine pancreas increases the permeability of the acinar cell junctions to moderately large molecules such as horseradish peroxidase. This may result in an increase of the concentration of the molecule in the secreted fluid.     

Tight junctional permeability of the resting and carbachol stimulated exocrine rabbit pancreas

The permeability of the pancreatic epithelium to horseradish peroxidase is investigated in the resting and carbachol stimulated rabbit pancreas. Horse radish peroxidase administered to the bathing medium of the isolated rabbit pancreas appears in the secreted fluid of the pancreas in a relatively low concentration. Carbachol stimulates both protein secretion and the passage of horse radish peroxidase into the secretory fluid. Histochemical assessment shows that horseradish peroxidase enters the interstitial spaces of the pancreatic tissue and is present along basal and lateral plasma membranes of acinar and ductular cells. In the absence of carbachol, horseradish peroxidase is seen more frequently in the tight junctions of ductular cells than in those of acinar cells. However, in the carbachol stimulated gland horseradish peroxidase is observed in the junctions between adjacent acinar cells more frequently than in the unstimulated gland. Freeze-fracture of acinar cells shows that the number of tight junctional strands and the tight junction depth are slightly decreased upon carbachol stimulation. The findings suggest that cholinergic stimulation of the exocrine pancreas increases the permeability of the acinar cell junctions to moderately large molecules such as horseradish peroxidase. This may result in an increase of the concentration of the molecule in the secreted fluid.     

Transpancreatic transport of digestive enzyme.

When porcine alpha-amylase or bovine chymotrypsinogen A was added to the medium bathing the rabbit pancreas in short-term organ culture, the secretion of these enzymes collected via the duct system increased greatly. To determine if it was indeed the amylase added to the bath that was recovered in secretion, endogenous enzyme stores were prelabeled during a 4 h incubation with [3H]-leucine and the specific radioactivity of amylase in secretion followed. The addition of unlabeled exogenous amylase to the bathing medium reduced the specific radioactivity of secreted amylase by about 90% suggesting that the response was due to the transpancreatic transport of the added enzyme. This inhibition was maintained over time, and was a result, not only of the increased secretion of unlabeled enzyme, but also of a 72% steady-state inhibition in the secretion of endogenous (labeled) amylase. This latter observation indicates that the exogenous enzyme crosses the acinar cell and mixes with endogenous cellular stores. A cellular route is also suggested by the observation that the addition of amylase to the bath increased the amylase concentration in ductal fluid relative to that in the bath by about 20 times; it did not reduce it as would be expected if paracellular shunts were involved. In addition, a cellular pathway is suggested by the observation that a 2 h prior incubation in bovine chymotrypsinogen resulted in a greatly augmented chymotrypsinogen response to a maximal cholinergic stimulus. In all, the data support the prediction of the equilibrium theory of digestive enzyme secretion that enzyme secretion should be responsive to mass action, and the prediction of the enteropancreatic circulation hypothesis that a capacity exists for a substantial transpancreatic flux of digestive enzyme.     

Inhibition of CCK or carbachol-stimulated amylase release by nicotine

This study was undertaken to investigate the mechanisms of action of nicotine on receptor mediated enzyme secretion in isolated rat pancreatic acini. Acinar cells were isolated from untreated and nicotine treated rats by collagenase digestion and differential centrifugation. Cells from the untreated animals were incubated with either varying concentrations of nicotine (range 10 microM to 30 mM) or with a fixed dose of 10 mM nicotine with varying concentrations of carbachol(10nM to 100 microM). Cells from the nicotine treated animals(16 weeks in drinking water) were incubated with either a fixed dose of CCK-8(10(-10) M) or carbachol(10(-5) M). All incubations were conducted at 37 C for 30 min. Amylase released in the media was measured by spectrophotometry. In pancreatic acinar cells isolated from control rats, amylase release stimulated by carbachol was inhibited by nicotine. Acinar cells isolated from rats treated with nicotine at nicotine concentrations of 1.23 mM also showed significant inhibition of amylase release in response to CCK-8 and carbachol compared to their identical controls. Nicotine induced inhibition curves of amylase release stimulated by carbachol were non-parallel suggesting that the effect of nicotine on acinar cells is regulated by mechanisms other than carbachol receptors. Nicotine may have a direct inhibitory effect on the intracellular mechanisms of pancreatic enzyme secretion. We conclude that the mechanism by which nicotine inhibits pancreatic enzyme secretion is complex.     

Evaluation of basement membrane degradation during TNF-alpha-induced increase in epithelial permeability

We evaluated whether tumor necrosis factor (TNF)-alpha induces an increase in permeability of an alveolar epithelial monolayer via gelatinase secretion and basement membrane degradation. Gelatinase secretion and epithelial permeability to radiolabeled albumin under unstimulated and TNF-alpha-stimulated conditions of an A549 human epithelial cell line were evaluated in vitro. TNF-alpha induced both upregulation of a 92-kDa gelatinolytic activity (pro form in cell supernatant and activated form in extracellular matrix) and an increase in the epithelial permeability coefficient compared with the unstimulated condition (control: 1.34 +/- 0.04 x 10(-6) cm/s; 1 microg/ml TNF-alpha: 1.47 +/- 0.05 x 10(-6) cm/s, P < 0.05). The permeability increase in the TNF-alpha-stimulated condition involved both paracellular permeability, with gap formation visualized by actin cytoskeleton staining, and basement membrane permeability, with an increase in the basement membrane permeability coefficient (determined after cell removal; control: 2.58 +/- 0.07 x 10(-6) cm/s; 1 microg/ml TNF-alpha: 2.82 +/- 0.02.10(-6) x cm/s, P < 0.05). Because addition of gelatinase inhibitors [tissue inhibitor of metalloproteinase (TIMP)-1 or BB-3103] to cell supernatants failed to inhibit the permeability increase, the gelatinase-inhibitor balance in the cellular microenvironment was further evaluated by cell culture on a radiolabeled collagen matrix. In the unstimulated condition, spontaneous collagenolytic activity inhibited by addition to the matrix of 1 microg/ml TIMP-1 or 10(-6) M BB-3103 was found. TNF-alpha failed to increase this collagenolytic activity because it was associated with dose-dependent upregulation of TIMP-1 secretion by alveolar epithelial cells. In conclusion, induction by TNF-alpha of upregulation of both the 92-kDa gelatinase and its inhibitor TIMP-1 results in maintenance of the gelatinase-inhibitor balance, indicating that basement membrane degradation does not mediate the TNF-alpha-induced increase in alveolar epithelial monolayer permeability.     

Control by calcium of protein discharge and membrane permeability to potassium in the rat lacrimal gland

Discharge of protein from slices of rat exorbital lacrimal gland was stimulated by 10^?5 M carbachol. This response was blocked by 10^?4 M atropine or by the omission of extracellular calcium. In the latter case, secretion could be restored by the reintroduction of calcium to the medium. Carbachol (10^?5 M) also stimulated the release of ⁸⁶Rb (a marker for potassium) from the slices. This effect was completely blocked by 10^?4 M atropine. The initial transient release of ⁸⁶Rb was only partially inhibited by Ca removal, but the later sustained phase of release was completely blocked. As with protein secretion, this effect of Ca removal could be reversed by re-introduction of Ca to the medium. It is concluded that activation of cholinergic receptors in the lacrimal gland stimulates protein discharge and increases potassium permeability by mechanisms utilizing extracellular calcium ions.     

Endothelium-derived relaxing factor contributes to the regulation of endothelial permeability.

To determine whether endothelium-derived relaxing factor (EDRF) contributes to the regulation of endothelial permeability, the transendothelial flux of 14C-sucrose, a marker for the paracellular pathway across endothelial monolayers (Oliver, J. Cell. Physiol. 145:536-548, 1990), was examined in monolayers of bovine aortic endothelial cells grown on collagen-coated filters. The permeability coefficient of 14C-sucrose was significantly decreased by 10(-3) M 8-Bromoguanosine 3',5'-cyclic monophosphate or by 5 x 10(-6) M glyceryl trinitrate, an activator of soluble guanylate cyclase. Depletion of L-arginine from endothelial monolayers increased 14C-sucrose permeability from 3.21 +/- 0.59 to 3.88 +/- 0.50 x 10(-5) cm.sec-1 (mean +/- SEM; n = 6; P < 0.05). The acute administration of 5 x 10(-4) M L-arginine to monolayers depleted of this amino acid decreased 14C-sucrose permeability from 2.91 +/- 0.27 to 2.52 +/- 0.26 x 10(-5) cm.sec-1 (n = 11; P < 0.05). 14C-sucrose permeability was increased by 10(-7) M bradykinin and this effect was enhanced by the presence of each one of the following compounds: 10(-5) M methylene blue, 4 x 10(-6) M oxyhemoglobin, 5 x 10(-4) M NG-methyl-L-arginine or 5 x 10(-4) M N omega-nitro-L-arginine. These results suggest that EDRF contributes to the sealing of the endothelial monolayer and that EDRF released by bradykinin acts as a feedback inhibitor attenuating the increase in endothelial permeability induced by this peptide. Because endothelial cells have the ability to contract and relax and possess guanylate cyclase responsive to nitric oxide, our results suggest that EDRF decreases 14C-sucrose permeability by relaxing endothelial cells, thereby narrowing the width of endothelial junctions.     

Proteinase-activated receptor-2-induced colonic inflammation in mice: possible involvement of afferent neurons,nitric oxide,and paracellular permeability

Activation of colonic proteinase-activated receptor-2 (PAR-2) provokes colonic inflammation and increases mucosal permeability in mice. The mechanism of inflammation is under debate and could be neurogenic and/or the consequence of tight-junction opening with passage of exogenous pathogens into the lamina propria. The present study aimed to further characterize the inflammatory effect of PAR-2 activation by investigating: 1) the role of NO, 2) the role of afferent neurons, and 3) a possible cause and effect relationship between colonic paracellular permeability changes and mucosal inflammation. Thus, intracolonic infusion to mice of the PAR-2-activating peptide, SLIGRL, increased both myeloperoxidase (MPO) activity and damage scores indicating colonic inflammation, and enhanced colonic permeability to (51)Cr-EDTA from 2 to 4 h after its infusion. NO synthase inhibitors, L-NAME and aminoguanidine, as well as the neurotoxin capsaicin and NK1, calcitonin gene-related peptide (CGRP) receptor antagonists, SR140333 and CGRP(8-37), prevented SLIGRL-induced MPO and damage score increases and permeability. In contrast, although the tight-junction blocker, 2,4,6-triaminopyrimidine, and the myosin L chain kinase inhibitor, ML-7, prevented SLIGRL-induced increase in permeability, they did not prevent MPO and damage score increases. Taken together our data show that both NO and capsaicin-sensitive afferent neurons are involved in PAR-2-mediated colonic inflammation and paracellular permeability increase. Nevertheless, the inflammation process is not a consequence of increased permeability which results at least in part from the activation of myosin L chain kinase.     

Evidence for a direct and indirect action of leucine enkephalin at the feline ileocecal sphincter

An in vitro whole organ bath preparation was used to examine the effects of leucine enkephalin on the cat ileocecal sphincter (ICS) intraluminal pressure and myoelectric activity. The bath allowed separation of the bathing media surrounding the ICS and the ileum. Leucine enkephalin (2 x 10(-7) M) when added to the ileal bathing medium caused a delayed increase in ICS spike activity and pressure which was blocked by tetrodotoxin (10(-5)M). In contrast, leucine enkephalin (2 x 10(-7)M) added directly to the ICS bathing medium caused an immediate spike-associated contractile response which was tetrodotoxin-resistant. Thus both an indirect and direct opiate action at the ICS was demonstrated.     

Comparative role of retinol, retinoic acid and beta-carotene on progesterone secretion by pig corpus luteum in vitro

Quadruplicate wells of pig luteal cells were incubated for 24 h in the presence of different concentrations of retinol, beta-carotene (0, 1 x 10(-5), 1 x 10(-6) and 1 x 10(-7) M) or retinoic acid (0, 1 x 10(-6), 1 x 10(-7) and 1 x 10(-8) M). In addition, the responsiveness of luteal cells to LH challenge was also evaluated. Progesterone was assayed in the media. Cell viability was estimated using trypan blue exclusion and showed over 95% viability. In the presence of LH, progesterone content in the medium was increased by 7-fold. As compared to their respective controls, all concentrations of retinoic acid and beta-carotene increased progesterone content in the media. The highest level of stimulation was observed with 1 x 10(-6) M-retinoic acid (5-fold increase) and 1 x 10(-7) M-beta-carotene (10-fold increase). Only 1 x 10(-5) M-retinol stimulated progesterone secretion (over 3-fold). Therefore, retinol, retinoic acid and beta-carotene stimulate progesterone secretion by pig luteal cells in vitro.