Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. I. Use of chlorotetracycline as fluorescent probe

Stimulus-secretion coupling in pancreatic exocrine cells was studied using dissociated acini, prepared from mouse pancreas, and chlorotetracycline (CTC), a fluorescent probe which forms highly fluorescent complexes with Ca2+ and Mg2+ ions bound to membranes. Acini, preloaded by incubation with CTC (100 microM), displayed a fluorescence having spectral properties like that of CTC complexed to calcium (excitation and emission maxima at 398 and 527 nm, respectively). Stimulation with either bethanechol or caerulein resulted in a rapid loss of fluorescence intensity and an increase in outflux of CTC from the acini. After 5 min of stimulation, acini fluorescence had been reduced by 40% and appeared to be that of CTC complexed to Mg2+ (excitation and emission maxima at 393 and 521 nm, respectively). The fluorescence loss induced by bethanechol was blocked by atropine and was seen at all agonist concentrations that elicited amylase release. Maximal fluorescence loss, however, required a bethanechol concentration three times greater than that needed for maximal amylase release. In contrast, acini preloaded with ANS or oxytetracycline, probes that are relatively insensitive to membrane-bound divalent cations, displayed no secretagogue-induced fluorescence changes. These results are consistent with the hypothesis that CTC is able to probe some set of intracellular membranes which release calcium during secretory stimulation and that this release results in dissociation of Ca(2+)-complexed CTC.     

Crambene induces pancreatic acinar cell apoptosis via the activation of mitochondrial pathway

We investigated the apoptotic pathway activated by crambene (1-cyano-2-hydroxy-3-butene), a plant nitrile, on pancreatic acinar cells. As evidenced by annexin V-FITC staining, crambene treatment for 3 h induced the apoptosis but not necrosis of pancreatic acini. Caspase-3, -8, and -9 activities in acini treated with crambene were significantly higher than in untreated acini. Treatment with caspase-3, -8, and -9 inhibitors inhibited annexin V staining, as well as caspase-3 activity, pointing to an important role of these caspases in crambene-induced acinar cell apoptosis. The mitochondrial membrane potential was collapsed, and cytochrome c was released from the mitochondria in crambene-treated acini. Neither TNF-alpha nor Fas ligand levels were changed in pancreatic acinar cells after crambene treatment. These results provide evidence for the induction of pancreatic acinar cell apoptosis in vitro by crambene and suggest the involvement of mitochondrial pathway in pancreatic acinar cell apoptosis.     

Mechanism of induction of pancreatic acinar cell apoptosis by hydrogen sulfide

The present study investigated the mechanism of mouse pancreatic acinar cell apoptosis induced by H₂S in an in vitro system, using isolated pancreatic acini. Treatment of pancreatic acini with 10 µM NaHS (a donor of H₂S) for 3 h caused phosphatidylserine externalization as shown by annexin V binding, an indicator of early stages of apoptosis. This treatment also resulted in the activation of the caspase cascade and major changes at the mitochondrial level. Caspase-3, -8, and -9 activities were stimulated by H₂S treatment. Treatment with inhibitors of caspase-3, -8, and -9 significantly inhibited H₂S-induced phosphatidylserine externalization as shown by reduced annexin V staining. The mitochondrial membrane potential was collapsed in H₂S-treated acini as evidenced by fluorescence microscopy and quantitative analysis. Furthermore, the treatment of acini with H₂S caused the release of cytochrome c by the mitochondria. To investigate the mechanism underlying pancreatic acinar cell apoptosis, we also characterized the protein expression of a range of molecules that are each known to influence the apoptotic pathway. Among proapoptotic proteins, Bax expression was activated in H₂S-treated cells but not Bid, and the antiapoptotic proteins Bcl-X_L and Bcl-2 did not show any activation in pancreatic acinar cell apoptosis. The death effector domain-containing protein Flip is downregulated in H₂S-treated acini. These results demonstrate the induction of pancreatic acinar cell apoptosis in vitro by H₂S and the involvement of both mitochondrial and death receptor pathways in the process of apoptosis. caspase; H₂S; mitochondria; Bcl-2; Flip     

Effects of 1-oleoyl-2-acetyl glycerol are distinct from those of phorbol ester in rat pancreatic acini

1-oleoyl-2-acetyl glycerol (OAG), a potent activator of protein kinase C, inhibited the binding of 125I-labelled epidermal growth factor (EGF) in isolated rat pancreatic acini. Unlike cholecystokinin-octapeptide (CCK8) and the C-kinase activator 12-O-tetradecanoyl phorbol-13-acetate (TPA), two inhibitors of 125I-EGF endocytosis in the pancreas, OAG had no effect on the distribution of bound ligand between the cell surface and intracellular compartments. Unlike TPA, OAG failed to potentiate the inhibitory effects of the calcium ionophore A23187 on 125I-EGF cell-associated radioactivity and had no effect on either basal or carbachol-stimulated amylase release in acini. These data suggest that the actions of the synthetic diacyl-glycerol OAG are not fully equivalent with the action of other known activators of protein kinase C in the pancreatic acinar cell.     

Non-uniform distribution of mitochondria in pancreatic acinar cells

The distribution of mitochondria in pancreatic acinar cells was investigated using confocal fluorescence microscopy and transmission electron microscopy (EM). Acinar cells were studied either after enzymatic isolation or in small segments of undisassociated pancreatic tissue. Loading of isolated acinar cells with Mito Tracker Green or Red, a fluorescence mitochondrial probe, showed that mitochondria are predominantly situated in the perigranular, subplasmalemmal and perinuclear regions. Subsequent applications of EM fixatives induced a leak of the fluorescent indicator to the cytosol but did not change the distribution of mitochondria. EM was then performed on isolated acinar cells and on acinar cells of pancreatic tissue segments. The intracellular distribution of mitochondria was quantified by calculating the percentage of the cross-sectional area that was occupied by mitochondria. In isolated acinar cells the highest density of mitochondria was seen in the perigranular region, where mitochondria occupied 25.69±1.58% of the area, then the subplasmalemmal region with 12.61±0.77% and the perinuclear region with 9.07±0.97% (n=26). Similar results were obtained from acinar cells of pancreatic tissue segments: the perigranular 22.9±1.95%, subplasmalemmal 12.45±0.78% and perinuclear regions 9.07±0.97% (n=26). The outer mitochondrial membranes were frequently positioned close to membranes of the ER, which followed the outer contour of mitochondria. Mitochondria were never found in direct contact with the nuclear envelope: there were usually layers of ER between the mitochondrial and nuclear membranes. Subplasmalemmal mitochondria were found in a very close proximity to the plasma membrane with no ER layers between the mitochondrial and the corresponding plasma membranes. We conclude that in pancreatic acinar cells mitochondria are preferentially distributed to perigranular, subplasmalemmal and perinuclear regions and this distribution is not affected by isolation or fixation procedures.P.R. Johnson and N.J. Dolman contributed equally to the study. This work was supported by a Medical Research Council programme grant. P.R.J. is a Medical Research Council PhD student and N.J.D. is a Wellcome Trust PhD student.     

Evidence of a direct TRH effect on the rat exocrine pancreas.

In the present study, the effect of TRH on amylase secretion was determined both in vivo, by cannulating the pancreatic duct of rats, as well as in vitro, by using isolated lobules and dissociated acini. The results show that TRH inhibited both basal and stimulated in vivo amylase secretion. Nevertheless, the in vitro experiments failed to show a TRH-related inhibitory effect when TRH was used alone, although the hormone did blunt the secretion elicited by CCK8 and bethanechol from isolated lobules and dissociated acini. Results suggest that TRH can inhibit stimulated amylase secretion in rats through a direct effect on acinar cells.     

Estrogens influence cholecystokinin stimulated pancreatic amylase release and acinar cell membrane cholecystokinin receptors in rat.

This study examines the influence of ovariectomy and administration of a pharmacologic dose of estradiol on amylase release from isolated-dispersed rat pancreatic acini and cholecystokinin receptors on rat acinar cell membranes. Rats were sham ovariectomized (intact) or ovariectomized (Ovx) and 21 day timed release pellets containing either estradiol (2.5 mg) or vehicle, were implanted subcutaneously. Eighteen days later, pancreatic acini were isolated from rats by collagenase digestion and differential centrifugation. Total cellular amylase, basal and cholecystokinin octapeptide (CCK8) stimulated amylase release and CCK membrane receptors were measured. Acini isolated from estradiol treated Ovx rats had significantly greater total cellular amylase, compared to acini isolated from either intact or Ovx rats. The amplitude of both total stimulated amylase release and percent total stimulated amylase release were significantly greater for acini isolated from vehicle treated Ovx rats, than acini isolated from either intact or estradiol treated Ovx rats. The magnitude of percent total amylase release of acini isolated from estradiol treated Ovx rats was significantly lower than that of acini isolated from intact rats. Cholecystokinin receptor concentration was significantly greater on membranes prepared from vehicle treated Ovx rats, compared to membranes prepared from either intact or estradiol treated Ovx rats. These data indicate that ovariectomy is associated with increased responsiveness of pancreatic acini to CCK stimulation, while chronic estradiol treatment of ovariectomized rats is associated with increased total cellular amylase and decreased acinar cell responsiveness to CCK8. Estrogen mediated alterations in acinar cell amylase content and amylase release may play a role in estrogen related pancreatitis.     

Fine structural modifications of liver, pancreas and brown adipose tissue mitochondria from hibernating, arousing and euthermic dormice

An ultrastructural and morphometric study was performed on mitochondria of euthermic, hibernating and arousing hazel dormice (Muscardinus avellanarius), in order to investigate possible modifications during the seasonal cycle. Hepatocytes, pancreatic acinar cells and brown adipocytes were considered. Our results demonstrated that: (1) the general morphology of mitochondria of all cell types shows slight modifications during the seasonal cycle; (2) mitochondrial size and inner membrane length significantly increase from euthermia to hibernation and decrease upon arousal in all cell types; (3) mitochondrial matrix granules drastically increase in number during hibernation and decrease upon arousal in hepatocytes and pancreatic acinar cells, whereas they do not change in brown adipocytes. These structural modifications are probably related to the changes in cellular energy needs during the euthermia-hibernation-arousal cycle.     

Stimulation of a membrane tyrosine phosphatase activity by somatostatin analogues in rat pancreatic acinar cells.

A phosphoryl protein tyrosine phosphatase (PTPase) activity has been characterized in rat pancreatic acinar membranes using 32P-labeled poly(Glu,Tyr) as substrate. Acinar membranes exhibited a high affinity for the substrate, with an apparent Km of 0.46 microM and an apparent Vmax of 0.9 nmol.mg protein-1.min-1. Acinar membrane PTPase activity displayed specific characteristics of other PTPases; it was inhibited by the inhibitors Zn2+, orthovanadate and by the divalent cations Mn2+ and Mg2+, and was stimulated by the reducing-agent dithiothreitol. It was also inhibited by soybean trypsin inhibitor and stimulated by trypsin. Gel permeation of pancreatic acinar membranes gave a single peak of enzyme activity with an apparent molecular mass of 70 000 Da. Further purification by HPLC on DEAE revealed two peaks of PTPase activity at 120 mM and 180 mM NaCl. These two peaks reacted in a Western-blot procedure with anti-(peptide) serum directed towards conserved domain of PTPase as a common 67-kDa form associated with lower-molecular-mass proteolytic fragments (31-56 kDa). Incubation of pancreatic acini with somatostatin analogues, SMS 201-995 or BIM 23014, resulted in a stimulation of membrane PTPase activity. The stimulation was rapid and transient, with a maximal level reached within 15 min of addition. The two analogs stimulated PTPase activity in a dose-dependent manner with half-maximal activation occurring at 7 pM and 37 pM and maximal activation at 0.1 nM and 0.1-1 nM for SMS 201-995 and BIM 23014, respectively. The stimulated-membrane PTPase activity also eluted at an apparent molecular mass of 70 kDa in gel-permeation chromatography. The two analogs inhibited the binding of [125I-Tyr3]SMS 201-995 to pancreatic acinar membranes with similar relative potencies to that observed on stimulation of PTPase activity. We conclude that pancreatic acinar membranes possess a low-molecular-mass PTPase which is stimulated by somatostatin analogs at concentrations involving activation of membrane somatostatin receptors.     

Effects of cholecystokinin on cytosolic calcium in human pancreatic cancer cells.

Cholecystokinin (CCK) has been shown to increase cytosolic calcium and stimulate enzyme release from pancreatic acinar cells and a rat acinar cell line, AR42J. CCK is also trophic to normal pancreas and pancreatic cancer; however, the cellular mechanisms which regulate CCK-stimulated growth are unknown. The effect of CCK on intracellular calcium was evaluated in four human pancreatic cancer cell lines known to grow in response to CCK but not secrete enzymes (SW-1990, MIA PaCa-2, BXPC-3 and PANC-1) and a rat acinar cell line (AR42J) shown to secrete enzymes but not grow with CCK. By using single cell fluorescence microscopy in fura-2 loaded cells, intracellular calcium [Ca2+]i was measured. After obtaining baseline fluorescent cell images, synthetic CCK-octapeptide (CCK8) was added to the cells and images of cell fluorescence captured. [Ca2+]i of the rat acinar cells increased (603%) over the baseline within the first minute after the addition of CCK (4.10(-13) M to 4.10(-10) M) in 77% of cells tested. In contrast [Ca2+]i failed to significantly change in the human cancer cells treated with CCK. To further localize the defect in hormone signal transduction in cancer cells, cells were suspended in low calcium media and the plasma membranes were selectively permeabilized with digitonin. Media free calcium concentration was continuously monitored by fura-2 fluorescence. Addition of inositol 1,4,5-trisphosphate (IP3) resulted in a marked increase in medium calcium concentration indicating IP3 was capable of releasing calcium from intracellular stores in both the AR42J rat acinar cell line and in the human pancreas cancer cell lines. In conclusion, CCK does not increase cytosolic calcium in human pancreatic cancer cells in contrast to rat acinar cells although all contain IP3-sensitive intracellular Ca2+ pools. Our results suggest that growth promoting and secretory effects of CCK on pancreatic cells may occur via two independent signalling pathways.     

Monitoring of membrane-bound divalent cations in plant mitochondria using chlorotetracycline fluorescence

Chlorotetracycline (CTC) shows a strongly enhanced fluorescence upon addition of mitochondria isolated from Jerusalem artichoke ( Helianthus tuberosus L.) tubers in a low-cation medium. This indicates the presence of membrane-bound divalent cations. The chelation by CTC of the membrane-bound divalent cations does not affect the oxidation of exogenous NADH significantly. The removal of the bound divalent cations using ethyleneglycol-bis-(β-aminoethylether)-N,N'-tetraacetic acid (EGTA) and EDTA causes an 80% decrease in CTC fluorescence. Titration of CTC fluorescence (a direct measure of bound divalent cations) and 9-aminoacridine fluorescence (a measure of surface potential) with EGTA and EDTA gives similar curves, although CTC fluorescence responds more slowly to the addition of chelators. The same bound divalent cations appear to be monitored by CTC fluorescence or by 9-aminoacridine fluorescence.     

Mitochondrial and extramitochondrial Ca2+ pools in the perfused rat liver. Mitochondria are not the origin of calcium mobilized by vasopressin

A nondisruptive technique developed by Bellomo et al. (Bellomo, G., Jewell, S. A., Thor, H., and Orrenius, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6842-6846) has been used to examine the distribution of calcium ions between mitochondrial and extramitochondrial compartments in the perfused rat liver. The amount of calcium released by the uncoupler 2,4-dinitrophenol from the mitochondrial compartment was 19 +/- 2 nmol X g-1, wet weight, which is equivalent to a total calcium concentration of 3.5 X 10(-4) M in the mitochondria and is by several orders of magnitude smaller than the concentration thought to be present in these organelles. The amount of calcium released from the liver in the presence of the divalent cation ionophore A 23187 was 96 +/- 7 nmol X g-1, wet weight, which is of the same order of magnitude as the amount released by the calcium-dependent hormone vasopressin (97 +/- 11 nmol X g-1, wet weight). Experiments with different sequential combinations of hormone with uncoupler or ionophore reveal that in the perfused liver, in contrast to isolated hepatocytes or isolated mitochondria, the amount of calcium attributable to the mitochondria is too small to account for the calcium released during hormonal stimulation. Consequently extramitochondrial calcium stores are the main source of cellular calcium mobilized under this condition. In addition these findings imply that in the liver several mitochondrial enzymes, e.g. alpha-oxoglutarate dehydrogenase, can be effectively regulated by calcium and that the role of mitochondria in buffering the cytosolic free calcium in vivo has to be reconsidered.     

The effects of chlorotetracycline on calcium movements in isolated rat liver mitochondria

Chlorotetracycline was used as a fluorescent chelate probe for visualizing calcium movements in rat liver mitochondria. It was demonstrated that under specified conditions, chlorotetracycline-associated fluorescence may be employed as a monitor of calcium uptake by mitochondrial membranes, e.g., at low calcium and Chlorotetracycline concentrations and in the absence of exogenous phosphate or acetate. However, at elevated calcium concentrations, e.g., >0.05 mm, a transient fluorescence response was observed upon addition of calcium to energized mitochondria. This transient or cyclic behavior of the chlorotetracycline-associated fluorescence was minimized by increasing the chlorotetracycline concentration, the mitochondrial protein concentration, or by including magnesium in the incubation. Also, it was demonstrated that chlorotetracycline addition to mitochondria which had been loaded previously with ⁴⁵Ca resulted in a rapid efflux of the accumulated ⁴⁵Ca. Because of the various effects of chlorotetracycline on the ability of the mitochondria to accumulate and to retain calcium, caution must be exercised in the interpretation of experimental results when this fluorescent chelate probe is utilized to monitor the association of divalent metal cations with biological membranes.     

Cholecystokinin induces caspase activation and mitochondrial dysfunction in pancreatic acinar cells. Roles in cell injury processes of pancreatitis

Apoptosis and necrosis are critical parameters of pancreatitis, the mechanisms of which remain unknown. Many characteristics of pancreatitis can be studied in vitro in pancreatic acini treated with high doses of cholecystokinin (CCK). We show here that CCK stimulates apoptosis and death signaling pathways in rat pancreatic acinar cells, including caspase activation, cytochrome c release, and mitochondrial depolarization. The mitochondrial dysfunction is mediated by upstream caspases (possibly caspase-8) and, in turn, leads to activation of caspase-3. CCK causes mitochondrial alterations through both permeability transition pore-dependent (cytochrome c release) and permeability transition pore-independent (mitochondrial depolarization) mechanisms. Caspase activation and mitochondrial alterations also occur in untreated pancreatic acinar cells; however, the underlying mechanisms are different. In particular, caspases protect untreated acinar cells from mitochondrial damage. We found that caspases not only mediate apoptosis but also regulate other parameters of CCK-induced acinar cell injury that are characteristic of pancreatitis; in particular, caspases negatively regulate necrosis and trypsin activation in acinar cells. The results suggest that the observed signaling pathways regulate parenchymal cell injury and death in CCK-induced pancreatitis. Protection against necrosis and trypsin activation by caspases can explain why the severity of pancreatitis in experimental models correlates inversely with the extent of apoptosis.     

Changes in the Distribution of Calcium-Sequestering Membranes during the First Cell Cycle of the Sea Urchin, Lytechinus variegatus

Chlortetracycline (CTC) has been used to study sequential changes in the distribution of calcium-sequestering membranes during the first cell cycle of fertilized sea urchin eggs CTC staining patterns first appear as a diffuse ring around the centered zygote nucleus at the time of syngamy. As development proceeds, the ring becomes brighter and then elongates concurrently with the formation of the streak apparatus. Fluorescence subsequently accumulates in the centrospheres of the developing mitotic apparatus and is present in mitotic asters throughout mitosis. When the mitotic apparatus disappears, the fluorescence associated with each aster condenses into a bright ring surrounding each daughter nucleus. Ultrastructural studies show that CTC-fluorescent areas are rich in membranes while experiments with rhodamine 123, a mitochondrion-specific laser dye, indicate that mitochondria are excluded from areas in which membranes accumulate. Microtubule inhibitors prevent the initial accumulation of fluorescence around the zygote nucleus and arrest the development of existing fluorescence patterns when applied at later stages. In contrast, changes in fluorescence patterns are unaffected by the microfilament inhibitor cytochalasin D. These observations show that calcium-sequestering membranes are associated consecutively with the sperm aster, streak, and mitotic apparatus and that the continual reorganization of these membranes during the first cell cycle depends on the assembly and disassembly of microtubules.     

An implication of novel methodology to study pancreatic acinar mitochondria under in situ conditions

Mitochondria maintain numerous energy‐consuming processes in pancreatic acinar cells, yet characteristics of pancreatic mitochondrial oxidative phosphorylation in native conditions are poorly studied. Besides, it is not known which type of solution is most adequate to preserve functions of pancreatic mitochondria in situ. Here we propose a novel experimental protocol suitable for in situ analysis of pancreatic mitochondria metabolic states. Isolated rat pancreatic acini were permeabilized with low doses of digitonin. Different metabolic states of mitochondria were examined in KCl‐ and sucrose‐based solutions using Clark oxygen electrode. Respiration of digitonin‐treated, unlike of intact, acini was substantially intensified by succinate or mixture of pyruvate plus malate. Substrate‐stimulated respiration rate did not depend on solution composition. In sucrose‐based solution, oligomycin inhibited State 3 respiration at succinate oxidation by 65.4% and at pyruvate plus malate oxidation by 60.2%, whereas in KCl‐based solution, by 32.0% and 36.1%, respectively. Apparent respiratory control indices were considerably higher in sucrose‐based solution. Rotenone or thenoyltrifluoroacetone severely inhibited respiration, stimulated by pyruvate plus malate or succinate, respectively. This revealed low levels of non‐mitochondrial oxygen consumption of permeabilized acinar cells. These results suggest a stronger coupling between respiration and oxidative phosphorylation in sucrose‐based solution. Copyright © 2012 John Wiley & Sons, Ltd.     

H2O2-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (psi m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+],) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas.     

Calcium signal compartmentalization

Cytosolic calcium signals are produced by suddenly increasing the concentration of free calcium ions (Ca2+). This can occur by opening channels permeable to Ca2+ either in the surface cell membrane or in the membranes of intracellular organelles containing high Ca2+ concentrations. Ca2+ signals can control several different processes, even in the same cell. In pancreatic acinar cells, for example, Ca2+ signals do not only control the normal secretion of digestive enzymes, but can also activate autodigestion and programmed cell death. Recent technical advances have shown that different patterns of Ca2+ signals can be created, in space and time, which allow specific cellular responses to be elicited. The mechanisms responsible for Ca2+ signal compartmentalization are now largely known and will be described on the basis of recent studies of Ca2+ transport pathways and their regulation in pancreatic acinar cells. It turns out that the Ca2+ handling as well as the structural characteristics of the endoplasmic reticulum (ER) and the mitochondria are of particular importance. Using a variety of Ca(2+)-sensitive fluorescent probes placed in different sub-cellular compartments in combination with local uncaging of caged Ca2+, many new insights into Ca2+ signal generation, compartmentalization and termination have recently been obtained.     

CHLOROTETRACYCLINE-ASSOCIATED FLUORESCENCE CHANGES DURING CALCIUM UPTAKE AND RELEASE BY RAT BRAIN SYNAPTOSOMES1

Abstract– The fluorescent divalent metal chelate-probe, chlorotetracycline (CTC), was used as a dynamic monitor of calcium association with rat brain snynaptosomes. The determined fluorescence excitation and emission maxima, 412 nm and 522 nm respectively, were used to monitor membrane-calcium interactions as a function of various parameters. Positive correlations were observed between increased or decreased fluorescence quantum yield and the uptake of both CTC and ⁴⁵Ca by synpatosomes. The divalent metal ionophore A23187 enhanced fluorescence as well as probe and ⁴⁵Ca uptake. Whereas, the polar chelator, EGTA, markedly reduced fluorescence, and the synaptosomal bound CTC and ⁴⁵Ca. The CTC fluorescence changes also demonstrated the saturable manner in which ⁴⁵Ca bound synaptosomes. At concentrations greater than 100μg/ml, CTC bound to the synaptosomes in a manner which quenched fluorescence at 522 nm. Also, CTC, at concentrations above 15 μg/ml, enhanced the uptake of ⁴⁵Ca. At CTC concentrations between 10 and 15 μg/ml the quenching and iono-phoretic properties of the probe were minimized without affecting the capability of using the probe to visualize calcium interactions with synaptosomal membranes. Also, at a low CTC concentration (12.5 μg/ml) the inhibition of calcium uptake by increasing monovalent ion concentrations was clearly demonstrated.     

Monoclonal antibodies as probes for plasma membrane domains in the exocrine pancreas

Monoclonal antibodies (mAb) were generated as probes for the plasma membrane domains of pancreatic acinar cells. Primary monolayer cultures of mouse pancreatic acinar cells, which have an expanded apical surface relative to normal pancreas, were used to immunize rats. With conventional immunization and fusion protocols, 3% of the hybridomas were positive against the acinar lumen by indirect immunofluorescence of mouse pancreas cryosections. Culturing of spleen cells from an immunized rat on the apical surface of acinar cell monolayer cultures before fusion with the myeloma (an in vitro boost) doubled the percentage of hybridomas producing apical membrane-specific mAb. Monoclonal antibodies were characterized by immunofluorescence, ultrastructural immunoperoxidase cytochemistry, immunoprecipitation, and immunoblotting. One antibody, acinar-1 (IgG2a), labeled the apical membranes of pancreatic acinar cells, hepatocytes, salivary and lacrimal gland acinar cells, and the brush border of small intestine enterocytes. This mAb precipitated and blotted a protein of 94 KD. Acinar-2 (IgM) also labeled pancreatic acinar cell apical membranes but did not label other tissues and did not precipitate or blot. Acinar-3 labeled pancreatic acinar cell lateral membranes. Duct-1 (IgM) labeled pancreatic duct apical membrane and ducts in liver and salivary glands but did not precipitate or blot. These domain-specific mAb demonstrate that common antigenic determinants occur in the apical surfaces of several exocrine epithelia and may be important in secretion.