J Nutr Biochem 2001 Jun;12(6):338-345

Protective effect of long term high fiber diet consumption on rat exocrine pancreatic function after chronic ethanol intake.

Bragado MJ, Garcia LJ, Lopez MA, Calvo JJ.

Departamento de Fisiologia y Farmacologia, Universidad de Salamanca, Caceres, Spain

The effects of ethanol administration on exocrine pancreas have been widely studied, but little is known about the effect of dietary fiber in combination with chronic ethanol on exocrine pancreatic function. The aim of this work was to examine the chronic effects of a high fiber diet, ethanol ingestion, and a combination of both on the function of the rat exocrine pancreas. Four groups of rats were fed for six months the following diets: 1.- NW: standard laboratory diet; 2.- FW: high fiber diet (15% cellulose); 3.- NE: standard laboratory diet and 20% ethanol in the drinking water; and 4.- FE: high fiber diet and 20% ethanol. Cholecystokinin (CCK) and acetylcholine (Ach) effects on amylase release and intracellular calcium mobilization in pancreatic acini were studied. In rats fed a 20% ethanol (NE), both the basal amylase release and the basal [Ca(2+)](i) were significantly increased; nonetheless, CCK and Ach-induced amylase release were significantly reduced compared with control rats. Ach- but not CCK-stimulated [Ca(2+)](i) increase in NE rats was significantly decreased compared with NW. In rats fed a combination of ethanol and a high fiber diet (FE) all the parameters under study were not significantly affected compared to control rats (NW). In conclusion, high fiber consumption does not alter the function of the exocrine pancreas. However, it ameliorates the deleterious effect of chronic ethanol consumption on pancreatic amylase secretion and, at least partially, reverses the ethanol-induced alterations on [Ca(2+)](i) in the rat exocrine pancreas.

Arch Biochem Biophys 2000 May 1;377(1):85-94

Activation of m3 muscarinic receptors induces rapid tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin in rat pancreatic acini.

Rosado JA, Salido GM, Garcia LJ

Department of Physiology, University of Extremadura, 10071-Caceres, Spain.

Tyrosine phosphorylation plays a key role in transmembrane and cytoplasmic signal transduction mechanisms stimulated by oncogenes, integrins, growth factors, neuropeptides, and bioactive lipids. Moreover, recent studies show that stimulation of odd-numbered muscarinic receptors increases the tyrosine phosphorylation of several proteins in different cellular types. The present study was aimed at examining whether activation of m3 muscarinic receptors in rat pancreatic acini evokes tyrosine phosphorylation of p125(FAK), and its substrates, p130(cas) and paxillin. Results show that stimulation of pancreatic acini with carbachol resulted in a rapid and transient increase in tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin. Tyrosine phosphorylation of these proteins occurred in a time- and concentration-dependent manner. Simultaneous blockage of both PKC activation and increases in [Ca(2+)](i) partially decreased p125(FAK), p130(cas), and paxillin tyrosine phosphorylation stimulated by carbachol. Pretreatment of pancreatic acini with Clostridium botulinum C3 transferase, which specifically inactivates p21(rho), partially inhibited carbachol-induced p125(FAK), p130(cas), and paxillin tyrosine phosphorylation. In contrast, this treatment had no effect on amylase release stimulated by carbachol. Cytochalasin D, which disrupts actin microfilaments network, completely inhibited carbachol stimulated tyrosine phosphorylation of these proteins without having significant effects in carbachol-stimulated amylase secretion. These results dissociate tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin from amylase secretion after m3 muscarinic receptors occupation in rat pancreatic acini. Taken together, these data suggest that (a) activation of m3 muscarinic receptors in rat pancreatic acini increases tyrosine phosphorylation of p125(FAK) and its substrates, p130(cas) and paxillin by diacylglycerol-activated PKC- and calcium- dependent, and independent pathways, (b) these responses require activation of p21(rho) and an intact actin cytoskeleton, and (c) p125(FAK), p130(cas), and paxillin are unlikely related to secretion in rat pancreatic acinar cells.

Biochem Pharmacol 2000 May 1;59(9):1077-89

Activation of tyrosine kinase pathway by vanadate in gallbladder smooth muscle.

Alcon S, Camello PJ, Garcia LJ, Pozo MJ

Department of Physiology, University of Extremadura, 10071, Caceres, Spain.

Vanadate, an inhibitor of tyrosine phosphatase activity, might induce gallbladder contraction through the stimulation of the tyrosine kinase pathway. The aim of this study was to characterize the effects of vanadate in the guinea pig gallbladder smooth muscle. Vanadate exerts contractile effects which are not mediated by neurotransmitter release. The tyrosine kinase inhibitor genistein nearly abolished vanadate contraction, suggesting that an increase in protein tyrosine phosphorylation mediates the actions of vanadate. This suggestion was confirmed by Western blot analysis. Vanadate contractions were reduced in the presence of methoxyverapamil or in Ca(2+)-free medium, suggesting that vanadate may induce Ca(2+) influx. Neither inactivation of the Na(+)/K(+) pump nor reversal of the Na(+)/Ca(2+) exchanger can account for vanadate's actions. Vanadate contractile effects were reduced by indomethacin, as well as mepacrine, the inhibitor of phospholipase A(2), but were not affected by phospholipase C inhibitors. Neither inhibitors of diacylglycerol lipase nor protein kinase C reduced the response induced by vanadate. These data indicate that the effects of vanadate on smooth muscle are mainly mediated by protein tyrosine phosphorylation and reveal a new link between tyrosine phosphorylation and arachidonic acid metabolism in the control of gallbladder smooth muscle contraction.

Cell Signal 2000 Mar 1;12(3):173-182

A role for phosphoinositides in tyrosine phosphorylation of p125 focal adhesion kinase in rat pancreatic acini.

Rosado JA, Salido GM, Garcia LJ

Department of Physiology, University of Extremadura, 10080-, Caceres, Spain

Previous studies have shown that different agonists increase tyrosine phosphorylation of the focal adhesion related proteins p125(FAK), p130(Cas), and paxillin in different cell types and that tyrosine phosphorylation depends on the integrity of the actin cytoskeleton. Because phosphoinositides are important for the maintenance of the cytoskeleton, the role of phosphoinositides in the tyrosine phosphorylation of these proteins in response to occupancy of m3 muscarinic and CCK(A) receptors has been investigated in pancreatic acini. Addition of carbachol or CCK-8 to pancreatic acini resulted in rapid increases in the tyrosine phosphorylation of p125(FAK), p130(Cas), and paxillin. Pretreatment of pancreatic acini with LY294002 or wortmannin resulted in a concentration-dependent inhibition of tyrosine phosphorylation of p125(FAK), p130(Cas), and paxillin stimulated by carbachol or CCK-8. Carbachol- or CCK-8-stimulated tyrosine phosphorylation of these proteins was not inhibited by rapamycin, PD 98059 or SB 203580, and thus it was dissociated from the activation of p70 S6 or MAP kinases. These results indicate that m3 muscarinic and CCK(A) receptor-mediated increase in p125(FAK), p130(Cas), and paxillin tyrosine phosphorylation in pancreatic acini depends on the ability of these cells to synthesise phosphoinositides.

J Biol Chem 1999 Oct 29;274(44):31261-71

Cholecystokinin activates PYK2/CAKbeta by a phospholipase C-dependent mechanism and its association with the mitogen-activated protein kinase signaling pathway in pancreatic acinar cells.

Tapia JA, Ferris HA, Jensen RT, Garcia LJ

Departamento de Fisiologia, Universidad de Extremadura, Caceres 10071, Spain.

PYK2/CAKbeta is a recently described cytoplasmic tyrosine kinase related to p125 focal adhesion kinase (p125(FAK)) that can be activated by a number of stimuli including growth factors, lipids, and some G protein-coupled receptors. Studies suggest PYK2/CAKbeta may be important for coupling various G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) cascade. The hormone neurotransmitter cholecystokinin (CCK) is known to activate both phospholipase C-dependent cascades and MAPK signaling pathways; however, the relationship between these remain unclear. In rat pancreatic acini, CCK-8 (10 nM) rapidly stimulated tyrosine phosphorylation and activation of PYK2/CAKbeta by both activation of high affinity and low affinity CCK(A) receptor states. Blockage of CCK-stimulated increases in protein kinase C activity or CCK-stimulated increases in [Ca(2+)](i), inhibited by 40-50% PYK2/CAKbeta but not p125(FAK) tyrosine phosphorylation. Simultaneous blockage of both phospholipase C cascades inhibited PYK2/CAKbeta tyrosine phosphorylation completely and p125(FAK) tyrosine phosphorylation by 50%. CCK-8 stimulated a rapid increase in PYK2/CAKbeta kinase activity assessed by both an in vitro kinase assay and autophosphorylation. Total PYK2/CAKbeta under basal conditions was largely localized (77 +/- 7%) in the membrane fraction, whereas total p125(FAK) was largely localized (86 +/- 3%) in the cytosolic fraction. With CCK stimulation, both p125(FAK) and PYK2/CAKbeta translocated to the plasma membrane. Moreover CCK stimulation causes a rapid formation of both PYK2/CAKbeta-Grb2 and PYK2/CAKbeta-Crk complexes. These results demonstrate that PYK2/CAKbeta and p125(FAK) are regulated differently by CCK(A) receptor stimulation and that PYK2/CAKbeta is probably an important mediator of downstream signals by CCK-8, especially in its ability to activate the MAPK signaling pathway, which possibly mediates CCK growth effects in normal and neoplastic tissues.

BBA - Molecular Cell Research 1999; 1448 (3):486-499

EGF stimulates tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin in rat pancreatic acini by a phospholipase C-independent process that depends on phosphatidylinositol 3-kinase, the small GTP-binding protein, p21rho, and the integrity of the actin cytoskeleton

Tapia JA, Camello C, Jensen RT, García LJ

Department of Physiology, University of Extremadura, Avda. de la Universidad, s/n, Cáceres 10074, Spain, and Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD 20892, USA

Epidermal growth factor (EGF) is a potent mitogen in many cell types including pancreatic cells. Recent studies show that the effects of some growth factors on growth and cell migration are mediated by tyrosine phosphorylation of the cytosolic tyrosine kinase p125 focal adhesion kinase (p125FAK) and the cytoskeletal protein, paxillin. The aim of the present study was to determine whether EGF activates this pathway in rat pancreatic acini and causes tyrosine phosphorylation of each of these proteins, and to examine the intracellular pathways involved. Treatment of pancreatic acini with EGF induced a rapid, concentration-dependent increase in p125FAK and paxillin tyrosine phosphorylation. Depletion of the intracellular calcium pool or inhibition of PKC activation had no effect on the response to EGF. However, inhibition of the phosphatidylinositol 3-kinase (PI3-kinase) or inactivation of p21rho inhibited EGF-stimulated phosphorylation of p125FAK and paxillin by more than 70%. Finally, cytochalasin D, a selective disrupter of the actin filament network, completely inhibited EGF-stimulated tyrosine phosphorylation of both proteins. All these treatments did not modify EGF receptor autophosphorylation in response to EGF. These results identify p125FAK and paxillin as components of the intracellular pathways stimulated after EGF receptor occupation in rat pancreatic acini. Activation of this cascade requires activation of PI3-kinase and participation of p21rho, but not PKC activation and calcium mobilization.

Biochemistry 1999;38(5):1497-1508

CCKA Receptor Activation Stimulates p130(Cas) Tyrosine Phosphorylation, Translocation, and Association with Crk in Rat Pancreatic Acinar Cells

Ferris HA, Tapia JA, Garcia LJ, Jensen RT

Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, and Department of Physiology, University of Extremadura, Caceres 10074, Spain.

p130(Cas) (Crk-associated substrate), because of its structure as an adapter protein, can interact when tyrosine-phosphorylated with a large number of cellular proteins and therefore be an important modulator of downstream signals. A number of growth factors, lipids, and a few G protein-coupled receptors can stimulate p130(Cas) tyrosine phosphorylation. Recent studies show that tyrosine phosohorylation of intracellular proteins by the hormone/neurotransmitter cholecystokinin (CCK) in rat pancreatic acinar cells may be an important signaling cascade. In this study, we show in rat dispersed pancreatic acini CCK-8 rapidly stimulates tyrosine phosphorylation of p130(Cas), reaching a maximum (6.6 +/- 1.4)-fold increase with a half-maximal effect at 0.3 nM. Activation of protein kinase C by TPA or increases in [Ca2+]i by the calcium ionophore A23187 stimulated p130(Cas) phosphorylation. Blockade of CCK increases in [Ca2+]i or PKC activity did not alter CCK-8-stimulated p130(Cas) phosphorylation; however, simultaneous blockage of both cascades caused a 50% inhibition. Partial inactivation by C. botulinum toxin of the small GTP-binding protein Rho caused a 41 +/- 12% decrease in the CCK-stimulated p130(Cas) phosphorylation. Disruption of the actin cytoskeleton with cytochalasin D, but not the microtubule network with colchicine, completely inhibited CCK-8-stimulated p130(Cas) phosphorylation. Total p130(Cas) under basal conditions was largely localized (70 +/- 2%) in the membrane fraction, and stimulation with CCK-8 induced total p130(Cas) translocation from the cytosolic fraction. CCK stimulation also caused a (5 +/- 1)-fold increase in p130(Cas) tyrosine phosphorylated in the plasma membrane. Treatment with tyrphostin B44 inhibited CCK-8-stimulated p130(Cas) phosphorylation, but it had no effect on p130(Cas) translocation. CCK-8 caused rapid formation of a p130(Cas)-Crk complex. In conclusion, our results demonstrate CCKA receptor activation causes rapid tyrosine phosphorylation of p130(Cas) through PLC-dependent and -independent mechanisms that require the participation of the small GTP-binding protein Rho and the integrity of the actin cytoskeleton, but not the microtubule network. Moreover, CCKA receptor activation causes translocation of p130(Cas) to the membrane and an increase in membrane tyrosine-phosphorylated p130(Cas). The translocation to the membrane does not require antecedent tyrosine phosphorylation. CCKA activation promotes the rapid formation of a p130(Cas)-Crk complex. These results suggest that p130(Cas) is likely an important modulator of downstream signals activated by CCK-8, possibly involved in regulating numerous cellular effects, such as effects on cell growth or cell shape.

Biochim Biophys Acta 1998 Sep 16;1404(3):412-26

Are tyrosine phosphorylation of p125(FAK) and paxillin or the small GTP binding protein, rho, needed for CCK-stimulated pancreatic amylase secretion?

Rosado JA, Salido GM, Jensen RT, Garcia LJ

Department of Physiology, University of Extremadura, 10071 Caceres, Spain.

Studies of a possible role of tyrosine phosphorylation in the secretory process in rat pancreatic acinar cells provide conflicting conclusions. Recent studies show that tyrosine phosphorylation of the focal adhesion kinase, p125FAK and the cytoskeletal protein, paxillin, may mediate a number of cellular changes and this phosphorylation is dependent on the activation of the small GTP binding protein, p21Rho (Rho). In this work we have investigated the role of tyrosine phosphorylation of each of these proteins and of the activation of Rho in pancreatic enzyme secretion. Pretreatment with genistein, a tyrosine kinase inhibitor, decreased CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin and CCK-8-stimulated amylase secretion by more than 60%, raising the possibility that tyrosine phosphorylation of these two proteins could be important in the ability of CCK-8 to stimulate amylase release. However, genistein did not alter the amylase release stimulated by TPA but inhibited TPA-stimulated p125FAK and paxillin tyrosine phosphorylation by 70%. Pretreatment with C3 transferase, which specifically inactivates Rho, causes a decrease in CCK-8-induced maximal amylase release by 33%. Moreover, C3 transferase pretreatment causes a 48% and a 38% decrease in the tyrosine phosphorylation of p125FAK and paxillin by CCK-8, respectively. Pretreatment with different concentrations of cytochalasin D, an actin cytoskeleton assembly inhibitor, completely inhibited CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin without having any effect on either the potency or efficacy of CCK-8 at stimulating amylase release. Furthermore, cytochalasin D completely inhibited TPA-stimulated tyrosine phosphorylation of both proteins without affecting TPA-stimulated amylase release. These results show that tyrosine phosphorylation of p125FAK and paxillin is not required for CCK-8 stimulation of enzyme secretion. However, our results suggest Rho is involved in the CCK-8 stimulation of amylase release by a parallel pathway to its involvement in the CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin.

Biochem J 1997 Oct 15;327( Pt 2):461-472

Cholecystokinin-stimulated tyrosine phosphorylation of p125FAK and paxillin is mediated by phospholipase C-dependent and -independent mechanisms and requires the integrity of the actin cytoskeleton and participation of p21rho.

Garcia LJ, Rosado JA, Gonzalez A, Jensen RT

Department of Physiology, University of Extremadura, Caceres 10080, Spain.

Recent studies show that the effects of some oncogenes, integrins, growth factors and neuropeptides are mediated by tyrosine phosphorylation of the cytosolic kinase p125 focal adhesion kinase (p125(FAK)) and the cytoskeletal protein paxillin. Recently we demonstrated that cholecystokinin (CCK) C-terminal octapeptide (CCK-8) causes tyrosine phosphorylation of p125(FAK) and paxillin in rat pancreatic acini. The present study was aimed at examining whether protein kinase C (PKC) activation, calcium mobilization, cytoskeletal organization and small G-protein p21(rho) activation play a role in mediating the stimulation of tyrosine phosphorylation by CCK-8 in acini. CCK-8-stimulated phosphorylation of p125(FAK) and paxillin reached a maximum within 2.5 min. The CCK-8 dose response for causing changes in the cytosolic calcium concentration ([Ca2+]i) was similar to that for p125(FAK) and paxillin phosphorylation, and both were to the left of that for receptor occupation and inositol phosphate production. PMA increased tyrosine phosphorylation of both proteins. The calcium ionophore A23187 caused only 25% of the maximal stimulation caused by CCK-8. GF109203X, a PKC inhibitor, completely inhibited phosphorylation with PMA but had no effect on the response to CCK-8. Depletion of [Ca2+]i by thapsigargin had no effect on CCK-8-stimulated phosphorylation. Pretreatment with both GF109203X and thapsigargin decreased CCK-8-stimulated phosphorylation of both proteins by 50%. Cytochalasin D, but not colchicine, completely inhibited CCK-8- and PMA-induced p125(FAK) and paxillin phosphorylation. Treatment with Clostridium botulinum C3 transferase, which inactivates p21(rho), caused significant inhibition of CCK-8-stimulated p125(FAK) and paxillin phosphorylation. These results demonstrate that, in pancreatic acini, CCK-8 causes rapid p125(FAK) and paxillin phosphorylation that is mediated by both phospholipase C-dependent and -independent mechanisms. For this tyrosine phosphorylation to occur, the integrity of the actin, but not the microtubule, cytoskeleton is essential as well as the activation of p21(rho).

Biochim Biophys Acta 1997 Sep 11;1358(2):189-199

CCK causes rapid tyrosine phosphorylation of p125FAK focal adhesion kinase and paxillin in rat pancreatic acini.

Garcia LJ, Rosado JA, Tsuda T, Jensen RT

Department of Physiology, University of Extremadura, Caceres, Spain.

Recent studies show CCK stimulates tyrosine phosphorylation (TYR PHOSP) of a number of proteins and evidence from the pancreas and other cellular systems suggest this could be important in mediating some of CCK's growth and secretory effects. In other tissues various neuropeptides such as bombesin can cause tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) and paxillin which are important in mediating their growth effects. The purpose of the present study was to determine the effects of CCK in rat pancreatic acini on the TYR PHOSP of these latter proteins. In dispersed rat pancreatic acini, cell lysates were incubated with an anti-phosphotyrosine mAb (PY20) which was immunoprecipitated and then analyzed by Western blotting with anti-phosphotyrosine mAb (4G10), anti-p125FAK mAb or anti-paxillin mAb. CCK-8 at 5 min increased TYR PHOSP of five proteins of molecular weight > 60,000 including a broad M(r) 110-130,000 and M(r) 70-80,000. An increase in TYR PHOSP of both p125FAK and paxillin was detected within 1 min of adding CCK and reached a maximum at 2.5 min with a 9.1 +/- 1.9-fold increase for p125FAK and 3.6 +/- 0.6-fold for paxillin. CCK-8 caused a half-maximal increase in TYR PHOSP of p125FAK at 0.1 nM and paxillin at 0.03 nM. CCK-JMV also stimulated an increase in TYR PHOSP of both proteins, but was only 50% as efficacious as CCK-8. CCK-JMV caused a half-maximal increase at 10 nM and maximal at 1 microM for both proteins. To investigate whether the low affinity CCK receptor state also caused TYR PHOSP of both proteins, increasing concentrations of CCK-JMV were added to a maximally effective CCK-8 concentration (1 nM). Detectable inhibition of CCK-8-stimulated TYR PHOSP occurred with 1 microM CCK-JMV and with 3 microM CCK-JMV the CCK-8-stimulated response was inhibited 50% and was the same as that seen with CCK-JMV alone. These studies demonstrate that in rat pancreatic acini, CCK causes rapid TYR PHOSP of both p125FAK and paxillin. This stimulation is mediated by both the high affinity and low affinity CCK receptor states. This phosphorylation of these proteins could be important in mediating CCK's effect on the cytoskeleton or growth effects as shown for a number of other agents (oncogenes, neuropeptides, integrins).

Rev Esp Fisiol 1997 Jun;53(2):231-237

Ionic requirements in histamine-evoked potassium efflux in guinea pig pancreas.

Rosado JA, Garcia LJ, Salido GM

Departamento de Fisiologia, Facultad de Veterinaria, Universidad de Extremadura, Caceres, Spain.

Guinea pig pancreatic segments were superfused during 10 min with physiological saline solutions containing 10(-6) M acetylcholine (ACh) or histamine (10(-3)-10(-6) M) and the potassium concentration in the effluent [K+]o) was measured by flame photometry. Histamine evoked a transient increase in [K/]o. The removal of calcium from the superfusing solution and addition of 10(-4) M EGTA caused a significant reduction in the histamine-evoked potassium outflow. Replacement of chloride (Cl-) in the physiological salt solution by nitrate (NO3-) caused a significant reduction in the histamine-evoked potassium release. However, when Cl- was replaced by bromide (Br-) the response to histamine was unaffected. Pre-treatment of pancreatic segments with furosemide (10(-4 M) or ouabain (10(-3) M) caused a marked reduction in the histamine-induced potassium release. The results suggest that ionic requirements in histamine-evoked potassium release are the same as those in acetylcholine-evoked potassium efflux.

Biochim Biophys Acta 1997 May 27;1356(3):343-354

The gastrin-releasing peptide receptor is differentially coupled to adenylate cyclase and phospholipase C in different tissues.

Garcia LJ, Pradhan TK, Weber HC, Moody TW, Jensen RT

Department of Physiology, University of Extremadura, Caceres, Spain.

Recent studies suggest that in some tissues GRP receptor activation can both stimulate phospholipase C and the adenylate cyclase pathway and that activation of the latter pathway may be important in mediating some of its well-described growth effects. However, other studies suggest GRP-R may not be coupled to adenylate cyclase. To investigate this possibility, in the present study we determined the coupling of the GRP receptors to each pathway in mouse, rat, and guinea pig pancreatic acini and compared it to that in mouse Swiss 3T3 cells and human SCLC cells, all of which possess well-characterized GRP receptors. Moreover, we tested the effect of PKC activation on the ability of GRP-related peptides to increase cAMP accumulation in these tissues. Changes in cAMP levels were determined with or without IBMX present, with or without forskolin, or both to amplify small increases in cAMP. In mouse, rat and guinea pig pancreatic acini, murine Swiss 3T3 cells and human SCLC cells, GRP-related peptides caused a 600%, 500%, 250%, 300% and 60% increase, respectively, in [3H]IP with 1-3 nM causing a half-maximal effect. In murine Swiss 3T3 cells, IBMX, forskolin, andIBMX plus forskolin caused a 300%, 3500% and 10500% increase in cAMP, respectivel y. GRP-related peptides and VIP caused an additional 70% increase in cAMP with GRP causing a half-maximal (EC50) increase in cAMP at 2.1 +/- 0.5 nM, which was not significantly different from the EC50 of 3.1 +/- 0.9 nM for increasing [3H]IP in these cells. GRP-related peptides did not stimulate increases in cAMP in mouse, rat or guinea pig pancreatic acini or in SCLC cells either alone, with IBMX or forskolin or both. However, in pancreatic acini IBMX, forskolin or both increased cAMP 3 to 8-, 10 to 500-, and 100 to 1000-fold increase and the addition of VIP caused an additional 20-, 2-, and3-fold increase in cAMP in the different species. In mouse pancreatic acini with TPA alone o r IBMX plus TPA, neither bombesin nor GRP increased cAMP. Furthermore, in mouse pancreatic acini,neither TPA nor TPA plus IBMX altered basal or VIP-stimulated increases in cAMP. In mouse Swiss 3T3 cells TPA significantly increased cAMP stimulated by Bn, GRP or VIP. These results demonstrated that GRP receptor activation in normal tissues from three different species and a human tumoral cell line do not result in adenylate cyclase activation, whereas in Swiss 3T3 cells it causes suchactivation. The results suggest that the difference in coupling to adenylate cyclase is likely a t leastpartially due to a difference in coupling to an adenylate cyclase subtype whose activation is regulated by PKC. Therefore, the possible growth effects mediated by this receptor in different embryonic or tumoral cells through activation of adenylate cyclase are not likely to be an important intracellular pathway for these effects in normal tissues.

Exp Physiol 1997 Jan;82(1):149-159

Acetylcholine-evoked potassium transport in the isolated guinea-pig pancreas.

Rosado JA, Singh J, Salido GM, Garcia LJ

Department of Physiology, Faculty of Veterinary Sciences, University of Extremadura, Caceres, Spain.

In this study, K+ concentration was measured in effluent samples from superfused guinea-pig pancreatic pieces in control conditions and during stimulation with ACh, employing the technique of flame photometry. ACh (10(-7)-10(-5) M) evoked a dose-dependent and sustained increase in K+ concentration in the effluent (K+ release). The removal of Ca2+ from the superfusing medium and the addition of 10(-4) M EGTA caused a significant (P < 0.05) reduction in the ACh-evoked K+ efflux.Replacement of extracellular Cl- in the superfusing physiological salt solution with NO3- abo lished the ACh-induced K+ efflux. In contrast, when Cl- was replaced with Br-, ACh still evoked marked K+ release. Pretreatment of pancreatic segments with the loop diuretic furosemide (10(-4) M) resulted in an inhibition of K+ efflux elicited by ACh. Stimulation of pancreatic segments with the Na(+)-K(+)-ATPase inhibitor ouabain (10(-3) M) caused a large efflux of K+. In the continuous presence of ouabain, ACh application elicited no further change in the K+ release. The results indicate that ACh-evoked K+ release from guinea-pig pancreatic segments is sensitive to ouabain, Cl-, furosemide and extracellular Ca2+ and that only the basal efflux is augmented by ouabain. The findings provide further evidence that a diuretic-sensitive coupled Na(+)-K(+)-Cl- cotransport system operates in the guinea-pig pancreas, as it does in other similar transporting epithelia, to bring about K+ mobilization.

Can J Physiol Pharmacol 1996 Dec;74(12):1342-1350

Secretin potentiates guinea pig pancreatic response to cholecystokinin by a cholinergic mechanism.

Alcon S, Rosado JA, Garcia LJ, Pariente JA, Salido GM, Pozo MJ

Department of Nursing, University of Extremadura, Caceres, Spain.

The effects of secretin and cholecystokinin on exocrine pancreas secretion in the guinea pig were investigated. The putative potentiating effect of these two hormones was studied in various settings to elucidate the effect of cholinergic stimuli in such interaction. In anesthetized guinea pig, intravenous infusion of cholecystokinin (0.75 pmol.kg-1.min-1) or secretin (0.5 pmol.kg-1.min-1) resulted in a marked and rapid increase of pancreatic juice flow and protein output. When cholecystokinin was combined with secretin, there was a significant increase in pancreatic, compared with cholecystokinin alone. This increase in pancreatic juice secretion and protein output was significantly suppressed by the prior administration of 100 micrograms/kg atropine. Similar results were obtained when trypsinogen release from pancreatic segments was measured in response to cholecystokinin (32 nM-32 pM) and (or) secretin (1 microM-32 nM). When we assayed the hormonal interaction on amylase release from dispersed pancreatic acini, we found that secretin (32 nM) failed to influence the secretory response to cholecystokinin (1 pM-10 nM). Thus we conclude that a combination of cholecystokinin and secretin resulted in a marked potentiation of the secretory responses in the exocrine guinea pig pancreas by a mechanism that involves cholinergic interactions present at the tissue level but not at the dispersed secretory cell level.

Clin Sci (Colch) 1996 Sep;91(3):365-369

Impairment of intracellular calcium homoeostasis in the exocrine pancreas after caerulein-induced acute pancreatitis in the rat.

Bragado MJ, San Roman JI, Gonzalez A, Garcia LJ, Lopez MA, Calvo JJ

Departamento de Fisiologia y Farmacologia, Universidad de Salamanca, Spain.

1. We have measured intracellular calcium concentrations in basal conditions and in response to cholecystokinin-octapeptide and acetylcholine in pancreatic acini isolated from rats with caerulein-induced acute pancreatitis and compared them with those in control rats. 2. We also measured amylase secretion in basal conditions and in response to cholecystokinin-octapeptide in both groups. 3. In pancreatic acini from rats with pancreatitis the basal intracellular calcium concentration was significantly increased (134.9 +/- 7.1 nmol/l compared with 71.8 +/- 2.9 nmol/l, P < 0.001). Moreover, the maximum values of intracellular calcium attained during the stimulation period were equivalent in acini from control and pancreatitic rats with no statistically significant differences. 4. In acini from control rats the differences between the resting levels of intracellular calcium and the maximum intracellular calcium values (delta[Ca2+]i) in response to several concentrations of cholecystokinin-octapeptide showed a clear dose-response relationship, with a half-maximal increase at 0.1 nmol/l and a maximal difference (delta[Ca2+]i = 259 +/- 50 nmol/l) at 1 nmol/l. In contrast, a right-shifted response, with a statistically significant smaller increase, was observed in acini from pancreatitic rats. 5. Basal amylase release was significantly higher in acini from rats with pancreatitis (11.7 +/- 1.0% of total compared with 5.9 +/- 1.1% of total, P < 0.001). In contrast, cholecystokinin-octapeptide and acetyl-choline-evoked amylase secretion was reduced by more than 85% in acini from pancreatitic rats. 6. In conclusion, calcium homoeostasis in pancreatic acinar cells from rats with caerulein-induced pancreatitis seems to be impaired. These results suggest excessive release of acinar free ionized calcium, or damage to the integrity of mechanisms that restore low resting levels of intracellular free ionized calcium, and the consequent calcium toxicity could be the key trigger in caerulein-induced acute pancreatitis.

Pancreas 1996 May;12(4):396-400

Histamine-evoked potassium release in the mouse and guinea pig pancreas.

Rosado JA, Tapia JA, Garcia LJ, Salido GM

Department of Physiology, Faculty of Veterinary, University of Extremadura, Caceres, Spain.

An investigation was made of the effects of histamine on the K+ concentration in the effluent in superfused guinea pig and mouse pancreatic segments. The effect of acetylcholine (ACh) was examined for comparison. Histamine evoked a dose-dependent and transient increase in the K+ concentration in the effluent (K+ release) but is less potent compared to the cholinergic agonist, ACh. At the same doses histamine and ACh evoke a much larger K+ release from mouse superfused pancreatic segments followed in the poststimulus period by a reuptake of K+. However, this reuptake of K+ was not observed in guinea pig superfused pancreatic segments. On the other hand, the cholinergic antagonist, atropine, completely abolished the K+ release in response to ACh and histamine from mouse and guinea pig pancreatic segments. Our results show the involvement of histamine in the control of K+ release in pancreatic tissue, with significant differences in the observed responses between species.

Anal Biochem 1995 Nov 20;232(1):129-132

Description of an automated method for the in vitro measurement of trypsinogen secretion from pancreatic segments.

San Roman JI, Tapia JA, Calvo JJ, Garcia LJ

Department of Physiology, University of Extremadura, Caceres, Spain.

The characterization of trypsinogen output from superfused pancreatic tissue by an automated spectrophotometric method is described. To test the method we investigated the time-course and the dose-response curve for acetylcholine-induced trypsinogen release from superfused pancreatic segments. We have demonstrated that this method allows the on-line detection and estimation of trypsinogen release with suitability, stability, and sensitivity.

Rev Esp Fisiol 1995 Mar;51(1):29-36

Dose-response effects of VIP on the rabbit exocrine pancreatic secretion. Comparison with PACAP-27 actions.

Rodriguez-Lopez AM, De Dios I, Garcia LJ, Lopez MA, Calvo JJ

Departamento de Fisiologia y Farmacologia, Universidad de Salamanca, Spain.

A dose-response study of the effects of vasoactive intestinal peptide (VIP) on the exocrine pancreatic secretion of the rabbit has been made. Furthermore, the actions of VIP and pituitary adenylate cyclase activating peptide (PACAP) on the exocrine pancreatic secretion were compared at a similar molar dose. After the infusion of VIP a linear dose-response relationship for pancreatic flow rate and bicarbonate output, up to the dose of 4 micrograms/kg, was observed. VIP acts as a partial agonist of secretin, the rabbit pancreas being less sensitive to VIP compared with other mammals. Moreover, VIP did not significantly stimulate the pancreatic protein output. PACAP stimulated the hydroelectrolyte fraction of the exocrine pancreatic secretion in a similar manner to that of VIP. Unlike what was observed with VIP, PACAP, on the same molar basis, significantly stimulated the protein and amylase outputs. Furthermore, PACAP releases VIP, so that the action of PACAP on the hydroelectrolyte fraction may be partially mediated by VIP; on the other hand, VIP is not involved in the effect of PACAP on the pancreatic enzyme secretion of this species.

Pancreas 1995 Jan;10(1):93-99

Cholinergic pathways are involved in secretin and VIP release and the exocrine pancreatic response after intraduodenally perfused acetic and lactic acids in the rat.

Sanchez-Vicente C, Rodriguez-Nodal F, Minguela A, Garcia LJ, San Roman JI, Calvo JJ, Lopez MA

Department of Physiology and Pharmacology, University of Salamanca, Spain.

The response of the exocrine pancreas to intraduodenal perfusion of acetic and lactic acids in normal and previously atropinized rats was studied. Secretin and vasoactive intestinal peptide (VIP) plasma levels in portal plasma were also measured. Intraduodenal perfusion of both acetic and lactic acids significantly stimulated flow rate (from 0.29 +/- 0.03 microliters/min to a maximum of 1.06 +/- 0.08 microliters/min after acetic and from 0.35 +/- 0.05 microliters/min to a maximum of 1.13 +/- 0.12 microliters/min after lactic acid perfusion) and protein output (from 11.16 +/- 2.33 micrograms/min to a maximum of 35.1 +/- 7.4 micrograms/min after acetic and from 8.98 +/- 0.95 micrograms/min to a maximum of 22.5 +/- 1.3 micrograms/min after lactic acid perfusion). Atropine treatment significantly inhibited pancreatic flow rate and protein output after acetic acid perfusion, but no inhibition of flow rate and a slight decrease in the protein output after lactic acid perfusion were seen. With respect to plasma peptide concentrations, significant increases in secretin and VIP levels were found after perfusion of both organic acids; atropine administration significantly decreased plasma secretin levelsafter acetic acid administration although it did not affect plasma VIP concentrations. By contrast, atropine significantly increased plasma secretin levels, but significantly lower values of plasma VIP concentrations were observed after lactic acid perfusion. Therefore, cholinergic mechanisms areinvolved in the release of secretin and VIP and different types of control of exocrine pancreatic secretion occur, depending on the features of the intraduodenal stimulant.

Clin Sci (Colch) 1994 Jul;87(1):85-90

Platelet-activating factor mediates pancreatic function derangement in caerulein-induced pancreatitis in rats.

Alonso R, Montero A, Arevalo M, Garcia LJ, Sanchez-Vicente C, Rodriguez-Nodal F, Lopez-Novoa JM, Calvo JJ

Departamento de Fisiologia y Farmacologia, Universidad de Salamanca, Spain.

1. We have assessed the role of platelet-activating factor in caerulein-induced acute pancreatitis (four subcutaneous injections of caerulein at a dose of 20 micrograms/kg) by measuring platelet-activating factor levels in portal blood, pancreatic tissue and peritoneal exudate in rats with and without pancreatitis. 2. We have also observed the effect of the platelet-activating factor antagonist, BN-52021, on the hyperamylasaemia and exocrine pancreatic secretion impairment associated with pancreatitis. 3. In rats with pancreatitis the basal pancreatic flow rate was increased (1.63 +/- 0.41 versus 0.25 +/- 0.03 microliters/min). Total protein output was similar in both untreated (5.98 +/- 1.93micrograms/min) and caerulein-injected (6.5 +/- 2.0 micrograms/min) animals. Amylase out put was lower in rats with pancreatitis (19.6 +/- 4.8 mu-units/min) than in controls (39.4 +/- 16.6 mu-units/min).4. Caerulein-treated animals had significantly higher serum amylase levels than untreated anim als. BN-52021 significantly reduced the caerulein-induced hyperamylasaemia. 5. Portal blood platelet-activating factor levels increased in rats with pancreatitis and in rats infused with cholecystokinin. Rats injected with caerulein and BN-52021 had portal blood levels of platelet-activating factor that were lower than those with pancreatitis. 6. Morphological derangements associated with pancreatitis (inflammatory infiltration and cell vacuolization) were also markedly reduced in BN-52021-treated animals. 7. The results of this study suggest that platelet-activating factor is involved in the development of caerulein-induced acute pancreatitis in rats.

Rev Esp Fisiol 1994 Jun;50(2):81-87

Effects of acute intravenous ethanol on basal exocrine pancreatic secretion in rat: cholinergic involvement.

Alonso RM, Alvarez MC, San Roman JI, Garcia LJ, Calvo JJ, Lopez MA

Departamento de Fisiologia y Farmacologia Universidad de Salamanca, Salamanca, Spain.

The effect of intravenous infusion of ethanol on the basal exocrine pancreatic secretion of anesthetized rats was studied. The cholinergic involvement on the actions of ethanol was also studied using previously atropinized animals. During the stimulation period, pancreatic flow rate was significantly increased by intravenous ethanol in both un-atropinized (199% compared with basal) and atropinized rats (195% compared with basal). Pancreatic protein output was also increased during ethanol administration in both groups of animals (171% and 165% compared with basal in, respectively, un-atropinized and atropinized rats). After the administration of ethanol, in the poststimulation period, pancreatic flow rate was further increased only in the atropinized group of rats (290% compared with basal), whose values were significantly higher than those of ethanol-treated un-atropinized animals (195% compared with basal). A similar profile of response was observed in pancreatic protein output. Since intravenous ethanol did not stimulate either secretin or VIP release to portal plasma, the present results point to a direct effect of this substance on the exocrine pancreas. Furthermore, atropine revealed the existence of an inhibitory cholinergic effect of ethanol on the exocrine pancreas. In summary, results show that the effect of intravenous ethanol on the basal exocrine pancreatic secretion is dual and antagonistic.

Pancreas 1994 Jan;9(1):123-128

Comparison between the effects of VIP and the novel peptide PACAP on the exocrine pancreatic secretion of the rat.

Alonso RM, Rodriguez AM, Garcia LJ, Lopez MA, Calvo JJ

Departamento de Fisiologia y Farmacologia, Universidad de Salamanca, Spain.

The effect of intravenous infusion of pituitary adenylate cyclase-activating peptide (PACAP) 27, a novel regulatory peptide that shows a close structural and chemical similarity to vasoactive intestinal peptide (VIP), on the rat exocrine pancreatic secretion was studied. PACAP and VIP stimulated the flow rate of exocrine pancreatic secretion (p < 0.05). However, protein output and amylase secretion were mainly stimulated by PACAP. Intravenous infusion of VIP increased the plasma levels of secretin (p < 0.05). On the other hand, PACAP released neither secretin nor VIP. Our results show: (a) both PACAP and VIP stimulate exocrine pancreatic secretion, (b) PACAP stimulation of pancreatic amylase and protein secretion is greater than that induced by VIP, and (c) PACAP probably exerts a direct effect on exocrine pancreas whereas some of the actions of VIP might be mediated by secretin.