Effect of dopamine on amylase secretion from guinea pig pancreatic acinar cells in vitro

Dopamine has been shown to effect pancreatic flow, protein output and amylase secretion in a variety of species. However, there is conflicting evidence regarding the role of dopamine on amylase release in vitro. Specific studies were conducted to evaluate the effect of dopamine and to compare its effects with other substances on basal- and secretagogue-stimulated amylase secretion in a guinea pig dispersed pancreatic acinar cells preparation. Dopamine (10(-6) M) induced a small, but significant (P less than 0.05) increase of amylase secretion. Established secretagogues (10(-6) M) including bombesin, cholecystokinin-octapeptide (CCK-8) and carbachol as anticipated induced significantly larger responses. Other substances tested (10(-6) M) including thyrotropin-releasing hormone (TRH) and muscimol were without effect. Complete dose-response studies (10(-11)-10(-3) M) in the presence of bombesin, CCK-8 and carbachol revealed that dopamine does not affect amylase release in response to these secretagogues. These findings suggest that dopamine is a weak stimulant of amylase secretion in vitro, and that it may therefore play a minor role in regulation of pancreatic enzyme secretion. Several factors including vascular, hormonal and neural have been implicated in regulation of pancreatic exocrine secretion. In particular, autonomic nervous system activity, notably cholinergic, has been shown to affect the secretory status of the pancreatic acinar cell. In addition, several biologically active peptides including bombesin, cholecystokinin (CCK), secretin, vasoactive intestinal peptide (VIP), substance P, gastrin and stimulation of cholinergic (muscarinic) receptors with carbachol have been shown to stimulate pancreatic enzyme secretion both in vivo and in vitro. Certain controversy regarding the role of the sympathetic nervous system in regulation of pancreatic exocrine secretion does exist. For example, several studies with agonists and antagonists of noradrenergic and dopaminergic receptor subtypes suggest a stimulatory effect on pancreatic fluid, electrolyte and enzyme secretion. However, these responses are species-specific and variations inherent to the model have been described. Dopamine administration has been shown to stimulate pancreatic bicarbonate and enzyme secretion in a variety of species including mice, dogs, and man. Radioligand binding studies with 3H-dopamine have revealed the presence of high- and low-affinity dopamine binding sites in dog pancreatic acinar cells. Stimulation of these receptors has been correlated with dose-dependent increases in intracellular cAMP levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modelling mechanism of calcium oscillations in pancreatic acinar cells

We present a simple model for calcium oscillations in the pancreatic acinar cells. This model is based on the calcium release from two receptors, inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) through the process of calcium induced calcium release (CICR). In pancreatic acinar cells, when the Ca²⁺ concentration increases, the mitochondria uptake it very fast to restrict Ca²⁺ response in the cell. Afterwards, a much slower release of Ca²⁺ from the mitochondria serves as a calcium supply in the cytosol which causes calcium oscillations. In this paper we discuss a possible mechanism for calcium oscillations based on the interplay among the three calcium stores in the cell: the endoplasmic reticulum (ER), mitochondria and cytosol. Our model predicts that calcium shuttling between ER and mitochondria is a pacemaker role in the generation of Ca²⁺oscillations. We also consider the calcium dependent production and degradation of (1,4,5) inositol-trisphosphate (IP₃), which is a key source of intracellular calcium oscillations in pancreatic acinar cells. In this study we are able to predict the different patterns of calcium oscillations in the cell from sinusoidal to raised-baseline, high frequency and low-frequency baseline spiking.     

Cyclic AMP accelerates calcium waves in pancreatic acinar cells

Cytosolic Ca(2+) (Ca(i)(2+)) flux within the pancreatic acinar cell is important both physiologically and pathologically. We examined the role of cAMP in shaping the apical-to-basal Ca(2+) wave generated by the Ca(2+)-activating agonist carbachol. We hypothesized that cAMP modulates intra-acinar Ca(2+) channel opening by affecting either cAMP-dependent protein kinase (PKA) or exchange protein directly activated by cAMP (Epac). Isolated pancreatic acinar cells from rats were stimulated with carbachol (1 muM) with or without vasoactive intestinal polypeptide (VIP) or 8-bromo-cAMP (8-Br-cAMP), and then Ca(i)(2+) was monitored by confocal laser-scanning microscopy. The apical-to-basal carbachol (1 muM)-stimulated Ca(2+) wave was 8.63 +/- 0.68 microm/s; it increased to 19.66 +/- 2.22 microm/s (*P < 0.0005) with VIP (100 nM), and similar increases were observed with 8-Br-cAMP (100 microM). The Ca(2+) rise time after carbachol stimulation was reduced in both regions but to a greater degree in the basal. Lag time and maximal Ca(2+) elevation were not significantly affected by cAMP. The effect of cAMP on Ca(2+) waves also did not appear to depend on extracellular Ca(2+). However, the ryanodine receptor (RyR) inhibitor dantrolene (100 microM) reduced the cAMP-enhancement of wave speed. It was also reduced by the PKA inhibitor PKI (1 microM). 8-(4-chloro-phenylthio)-2'-O-Me-cAMP, a specific agonist of Epac, caused a similar increase as 8-Br-cAMP or VIP. These data suggest that cAMP accelerates the speed of the Ca(2+) wave in pancreatic acinar cells. A likely target of this modulation is the RyR, and these effects are mediated independently by PKA and Epac pathways.     

Secretagogue induced calcium mobilization in single pancreatic acinar cells

Microspectrofluorometry of fura-2 was utilized to monitor [Ca2+]i in single acinar cells stimulated with a cholinergic agonist and cholecystokinin. A similar amplitude of agonist induced Ca mobilization between single cell and populational approaches was observed. New findings in single cells not observable in populations of cells include: 1) the maintenance of a sustained elevation in [Ca2+]i above basal levels throughout agonist application, 2) the reloading of the agonist-sensitive Ca pool only following removal of the agonist and 3) the presence of oscillations of [Ca2+]i in response to agonist application which is enhanced at lower agonist concentrations.     

Agonist-stimulated pathways of calcium signaling in pancreatic acinar cells.

In pancreatic acinar cells stimulation of different intracellular pathways leads to different patterns of Ca2+ signaling. Bombesin induces activation of both phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and phospholipase D (PLD). The latter leads to generation of diacylglycerol (DAG) in addition to that produced by activation of PIP2-PLC. Strong activation of protein kinase C (PKC) results in inhibition of Ca(2+)-induced Ca2+ release from Ca2+ pools arranged in sequence to the luminally located IP3-sensitive Ca2+ pools. Consequently the Ca2+ wave which starts in the luminal cell pole is slower in the presence of bombesin (5 microm/s) as compared to that in the presence of acetylcholine (17 microm/s) which activates PIP2-PLC but not PLD. Activation of high-affinity CCK-receptors triggers a Ca2+ wave with slow propagation (5 microm/s) due to stimulation of phospholipase A2 (PLA2) and generation of arachidonic acid, which in turn leads to inhibition of Ca(2+)-induced Ca2+ release. Low-affinity CCK-receptors are coupled to both PIP2-PLC and PLD.     

Vanadate inhibits the calcium extrusion in rat pancreatic acinar cells

Our objective was to evaluate the role of vanadate on calcium extrusion in Fura-2-loaded rat pancreatic acinar cells by digital microscopic fluorimetry and spectrofluorimetry. In the absence of extracellular calcium, perfusion of pancreatic acinar cells with 1 nM CCK-8 and 1 mM vanadate did not significantly affect the typical transient calcium spike induced by CCK-8, but the plateau phase of calcium in response to CCK-8 remained elevated. In addition, vanadate was able to inhibit calcium efflux evoked by CCK-8 when we determined directly calcium transport across plasma membrane using Calcium Green-5N hexapotassium salt (cell impermeant form) in cell populations. The effect of vanadate on calcium extrusion was strongly blocked by the sulfhydryl-reducing agent dithiothreitol (DTT). The present results demonstrate that vanadate is able to irreversibly inhibit the calcium extrusion. This effect of vanadate can be blocked using DTT, indicating that its action is probably mediated by oxidation of sulfhydryl groups of Ca2+-ATPases.     

Ca++-dependent disassembly and reassembly of occluding junctions in guinea pig pancreatic acinar cells. Effect of drugs

Incubation of guinea pig pancreatic lobules in Ca++-free Krebs-Ringer bicarbonate solution (KRB) containing 0.5 mM ethylene glycol-bis(beta- aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) results in the progressive fragmentation of the occluding zonulae (ZO) with formation of multiple discrete junctions (fasciae occludentes) localized in the lateral and lumenal plasmalemma. After 1--2 h of such incubation, most ZO appear completely disassembled. This results in the disappearance of the heterogeneity in density of intramembrane particles on the P- fracture faces of the basolateral and lumenal plasmalemma. If Ca++ ions are reintroduced into the incubation fluid at this point, continous zonulae reform around the apices of the cells; in contrast, the density of intramembrane particles (imp) at the lumenal plasmalemma remains the same as in the basolateral region, at least for 3 h after Ca++ reintroduction. When added to the incubation fluid, cycloheximide (at a dose known to inhibit protein synthesis greater than 95%) and cytochalasin B (at doses which disrupt microfilaments and modify the cell shape) had no effect on the organization of ZO, on their disassembly in Ca++-free, EGTA medium, or on their Ca++-dependent reformation. Likewise, the organization and disassembly of ZO were unaffected by colchicine; however, after treatment with the latter drug the reassembly was defective, with formation of strand networks on the lateral surface and incomplete segregation of the lumenal region. Antimycin A, on the other hand, when added to the Ca++-EGTA medium, induced a large proliferation of long, infrequently anastomosed junctional strands, usually arranged to form ribbons, festoons, and other bizarre arrays. The possible relationship of these in vitro findings to the in vivo biogenesis and turnover of occluding junctions is discussed. It is suggested that the impairment of reassembly of zonulae by colchicine might be correlated with the disorder induced by the drug on the general organization of pancreatic exocrine cells. Moreover, antimycin A could act by promoting the aggregation of a pool of free junctional strand components (or precursors) that might exist normally in pancreatic exocrine cells.     

Na+ transport processes in isolated guinea pig nasal gland acinar cells

In the dispersed acinar cells of the submucosal nasal gland in the guinea pig, intracellular Na⁺ concentration ([Na⁺]i) was measured with a microfluorimetric imaging method and the cytosolic indicator dye, sodium-binding benzofuran isophthalate, under HCO₃^?-free conditions. In the unstimulated condition, the [Na⁺]i was averaged to 12.8 ± 5.2 mM. Addition of 100 μM ouabain or removal of external K⁺ caused an increase in [Na⁺]i. Replacement of external Cl^? with NO₃^? or addition of 0.5 mM furosemide reversibly decreased the [Na⁺]i. The recovery process from the reduced [Na⁺]i was inhibited by removal of either K⁺ or Cl^? in the bath solution. These findings indicate the presence of a continuous influx of Na⁺ coupled with K⁺ and Cl^? movement. Application of acetylcholine (ACh, 1 μM) caused an increase in [Na⁺]i by about 15–20 mM, which was completely inhibited by addition of 10 μM atropine. Increased cytosolic Na⁺ induced by ACh was extruded by the Na⁺-K⁺ pump. Removal of external Cl^? and addition of 50 μM dimethylamiloride inhibited ACh-induced increase in [Na⁺]i by about 66% and 19%, respectively. In both unstimulated and stimulated state, Na⁺-K⁺ pump, Na-K-Cl cotransport, and Na⁺-H⁺ exchange play a critical role in maintaining intracellular electrolyte environment and in controlling a continuous secretion of nasal fluids. © 1995 Wiley-Liss, Inc.     

A model of calcium waves in pancreatic and parotid acinar cells

We construct a mathematical model of Ca(2+) wave propagation in pancreatic and parotid acinar cells. Ca(2+) release is via inositol trisphosphate receptors and ryanodine receptors that are distributed heterogeneously through the cell. The apical and basal regions are separated by a region containing the mitochondria. In response to a whole-cell, homogeneous application of inositol trisphosphate (IP(3)), the model predicts that 1), at lower concentrations of IP(3), the intracellular waves in pancreatic cells begin in the apical region and are actively propagated across the basal region by Ca(2+) release through ryanodine receptors; 2), at higher [IP(3)], the waves in pancreatic and parotid cells are not true waves but rather apparent waves, formed as the result of sequential activation of inositol trisphosphate receptors in the apical and basal regions; 3), the differences in wave propagation in pancreatic and parotid cells can be explained in part by differences in inositol trisphosphate receptor density; 4), in pancreatic cells, increased Ca(2+) uptake by the mitochondria is capable of restricting Ca(2+) responses to the apical region, but that this happens only for a relatively narrow range of [IP(3)]; and 5), at higher [IP(3)], the apical and basal regions of the cell act as coupled Ca(2+) oscillators, with the basal region partially entrained to the apical region.     

Electrogenic calcium transport in plasma membrane of rat pancreatic acinar cells

Summary ATP-dependent⁴⁵Ca²⁺ uptake was investigated in purified plasma membranes from rat pancreatic acinar cells. Plasma membranes were purified by four subsequent precipitations with MgCl₂ and characterized by marker enzyme distribution. When compared to the total homogenate, typical marker enzymes for the plasma membrane, (Na⁺,K⁺)-ATPase, basal adenylate cyclase and CCK-OP-stimulated adenylate cyclase were enriched by 43-fold, 44-fold, and 45-fold, respectively. The marker for the rough endoplasmic reticulum was decreased by fourfold compared to the total homogenate. Comparing plasma membranes with rough endoplasmic reticulum, Ca²⁺ uptake was maximal with 10 and 2 mol/liter free Ca²⁺, and half-maximal with 0.9 and 0.5 mol/liter free Ca²⁺. It was maximal at 3 and 0.2 mmol/liter free Mg²⁺ concentration, at an ATP concentration of 5 and 1 mmol/liter, respectively, and at pH 7 for both preparations. When Mg²⁺ was replaced by Mn²⁺ or Zn²⁺ ATP-dependent Ca²⁺ uptake was 63 and 11%, respectively, in plasma membranes; in rough endoplasmic reticulum only Mn²⁺ could replace Mg²⁺ for Ca²⁺ uptake by 20%. Other divalent cations such as Ba²⁺ and Sr²⁺ could not replace Mg²⁺ in Ca²⁺ uptake. Ca²⁺ uptake into plasma membranes was not enhanced by oxalate in contrast to Ca²⁺ uptake in rough endoplasmic reticulum which was stimulated by 7.3-fold. Both plasma membranes and rough endoplasmic reticulum showed cation and anion dependencies of Ca²⁺ uptake. The sequence was K⁺>Rb⁺>Na⁺>Li⁺>choline⁺ in plasma membranes and Rb⁺K⁺Na⁺>Li⁺>choline⁺ for rough endoplasmic reticulum. The anion sequence was Cl^–Br^–I^–>SCN^–>NO ₃ ^– >isethionate^– >cyclamate^–>gluconate^–>SO ₄ ^2– glutarate^– and Cl^–>Br>gluconate>SO ₄ ^2– >NO ₃ ^– >I^–>cyclamate^–SCN^–, respectively. Ca²⁺ uptake into plasma membranes appeared to be electrogenic since it was stimulated by an inside-negative K⁺ and SCN diffusion potential and inhibited by an inside-positive diffusion potential. Ca²⁺ uptake into rough endoplasmic reticulum was not affected by diffusion potentials. We assume that the Ca²⁺ transport mechanism in plasma membranes as characterized in this study represents the extrusion system for Ca²⁺ from the cell that might be involved in the regulation of the cytosolic Ca²⁺ level.     

Oscillations of cytosolic calcium in single pancreatic acinar cells stimulated by acetylcholine

The changes in cytosolic free calcium concentration [( Ca2+]i) were monitored (fura-2) in single, isolated, mouse pancreatic acinar cells stimulated by acetylcholine (ACh). Responses to ACh at concentrations between 10(-7) and 5 x 10(-7) M are marked by the appearance of regular, sinusoidal, oscillations in [Ca2+]i. At 37 degrees C the oscillations are transient, being seen only in the initial rising phase of the calcium signal. At 30 degrees C regular oscillations can be maintained throughout the period of ACh application. This study reports that release of intracellular calcium and influx of extracellular calcium are both involved in the generation of these oscillatory calcium signals.     

Extracellular calcium and the organization of tight junctions in pancreatic acinar cells

In pancreatic lobules incubated in Ca²⁺-free Krebs-Ringer bicarbonate solution +0.5 mM EGTA tight junctions are first disarrayed and then break up into fasciae occludentes and small fibrillar fragments, which move laterally in the plane of the plasmalemma and often wind up around the gap junctions. The interruption of the continuity of tight junctions results in the disappearance of the difference in intramembranous particle density between the lateral and luminal regions of the plasmalemma. These results are consistent with the interpretation of tight junctions as dynamic structures, probably resulting from a specific polymerization of intramembranous particles and confirm that tight junctions might have a role in establishing and maintaining the regional differences of the plasmalemma.     

TRPM4 links calcium signaling to membrane potential in pancreatic acinar cells

Transient receptor potential cation channel subfamily M member 4 (TRPM4) is a Ca²⁺-activated nonselective cation channel that mediates membrane depolarization. Although, a current with the hallmarks of a TRPM4-mediated current has been previously reported in pancreatic acinar cells (PACs), the role of TRPM4 in the regulation of acinar cell function has not yet been explored. In the present study, we identify this TRPM4 current and describe its role in context of Ca²⁺ signaling of PACs using pharmacological tools and TRPM4-deficient mice. We found a significant Ca²⁺-activated cation current in PACs that was sensitive to the TRPM4 inhibitors 9-phenanthrol and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). We demonstrated that the CBA-sensitive current was responsible for a Ca²⁺-dependent depolarization of PACs from a resting membrane potential of −44.4 ± 2.9 to −27.7 ± 3 mV. Furthermore, we showed that Ca²⁺ influx was higher in the TRPM4 KO- and CBA-treated PACs than in control cells. As hormone-induced repetitive Ca²⁺ transients partially rely on Ca²⁺ influx in PACs, the role of TRPM4 was also assessed on Ca²⁺ oscillations elicited by physiologically relevant concentrations of the cholecystokinin analog cerulein. These data show that the amplitude of Ca²⁺ signals was significantly higher in TRPM4 KO than in control PACs. Our results suggest that PACs are depolarized by TRPM4 currents to an extent that results in a significant reduction of the inward driving force for Ca²⁺. In conclusion, TRPM4 links intracellular Ca²⁺ signaling to membrane potential as a negative feedback regulator of Ca²⁺ entry in PACs.     

Isolation and monolayer culture of guinea pig pancreatic duct epithelial cells

Summary Monolayers of cultured epithelial cells have been prepared from fragments of guinea pig pancreatic excretory ducts isolated by a simple procedure employing collagenase digestion and manual selection, through which virtually all of the ductal system can be recovered. The isolated fragments were cultured in enriched Waymouth's medium on extracellular matrices of various composition and thickness, including: thin (<5 μm) and thick (0.5 mm) layers of rat tail collagen; thin layers of human placental collagen; thin layers of Matrigel (a reconstituted basement membrane material); uncoated tissue culture plastic; and the cellulose ester membranes of Millipore Millicells. Cells spread rapidly from duct fragments cultured on uncoated plastic or on plastic coated with thin layers of rat tail collagen or human placental collagen and formed epithelial monolayers. However, these cells were squamous and lacked the abundant basolateral membrane amplification and apical microvilli characteristic of freshly isolated duct epithelial cells. Cells did not spread from duct fragments cultured on Matrigel. In contrast, when fragments of pancreatic ducts were explanted onto either a thick layer of rat tail collagen or onto Millicell membranes, cells readily spread and formed confluent monolayers of cuboidal epithelial cells characterized by abundant mitochondria, apical microvilli, and basolateral plasma membrane elaboration. These results demonstrate that different forms of extracellular matrix modulate the growth and differentiation of pancreatic duct epithelial cells, and that culture on a permeable substrate markedly enhances the maintenance of differentiated characteristics in this cell type. The monolayers formed on Millicell membranes should provide a useful model system for physiologic analysis of the regulation of electrolyte secretion by this epithelium. This research was supported by grants DK32994 and DK35912 from the National Institutes of Health, Bethesda, MD.     

Effects of antioxidants on calcium signal induced by cholecystokinin in mouse pancreatic acinar cells

Digital imaging fluorescence microscopy was used to study the effect of two antioxidants, N-acetyl-cysteine (NAC) and glutathione, on the cytosolic free calcium concentration ([Ca2+]i) induced by cholecystokinin-octapeptide (CCK-8) of mouse pancreatic acinar cells. When acinar cells were preincubated with either NAC or glutathione, subsequent stimulation with CCK-8 in the presence of each antioxidant had no significant effect on the typical pattern of [Ca2+]i transient evoked by the gastrointestinal hormone. However, application of NAC to acinar cells pretreated for 60 min with the same antioxidant, strongly blocked the oscillatory pattern initiated by CCK-8, inhibiting both amplitude and frequency of calcium oscillations. By contrast, glutathione had no effect on the oscillatory pattern evoked by CCK-8. The present results allow us to speculate that during [Ca2+]i oscillation there is a production of oxidants that facilitate oscillations by enhancing release of calcium from internal stores.     

Acetylcholine and cholecystokinin induce different patterns of oscillating calcium signals in pancreatic acinar cells

Receptor-activated cytoplasmic calcium (Ca2+) oscillations have been investigated in single pancreatic acinar cells by microfluorimetry (Fura-2 as indicator). At submaximal concentrations of the agonists acetylcholine (ACh) and cholecystokinin octapeptide (CCK-8), both give rise to oscillatory changes in the cytosolic free calcium concentration ([Ca2+]i). The patterns of oscillations are markedly and consistently different for each of these two agonists. The ACh induced oscillations are superimposed upon a median elevation in background [Ca2+]i. The CCK-8 induced oscillations are of longer duration with [Ca2+]i returning to prestimulus levels between the discrete spikes. The ACh induced oscillations are rapidly abolished upon removal of extracellular Ca2+ while the CCK-8 induced oscillations persist for many minutes in the absence of external Ca2+. The CCK-8, but not the ACh, induced oscillations are increased in duration by the protein kinase C (PKC) inhibitor staurosporine and abolished by the PKC activating phorbol ester PMA. It is clear that CCK-8 and ACh do not activate receptor transduction mechanisms in an identical manner to generate oscillating [Ca2+]i signals.