Search PubMed⌕ Search

Biomedical subjects

I Schulz

Publications and source records attributed to I Schulz.

At least 91 records · Page 5Linked to original sources
PubMed1986

Ca2+-, phorbol ester-, and cAMP-stimulated enzyme secretion from permeabilized rat pancreatic acini.

Enzyme secretion from the exocrine pancreas is stimulated by receptor-activated breakdown of phosphatidylinositol 4,5-bisphosphate and consequent rise of both inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, which leads to Ca2+ release and to activation of protein kinase C, respectively. Another way involves receptor-mediated stimulation of adenylate cyclase and consequent rise of cAMP and activation of protein kinase A. In the present work we have studied direct stimulation, inhibition, and mutual interaction of these pathways on enzyme secretion from isolated rat pancreatic acini that had been permeabilized by treatment with saponin or digitonin. The data were compared with those obtained in isolated intact acini. The data show that with increasing free Ca2+ concentrations greater than 10(-6) M protein release increases in "leaky" but not in "intact" cells and is maximal at approximately 10(-3) M, increasing about twofold compared with that in the absence of Ca2+. In the presence of the acetylcholine analogue carbachol, this effect of Ca2+ is enhanced by about threefold in leaky cells and is also present in intact cells to a similar extent. cAMP and its analogues, dibutyryl cAMP (dbcAMP) and 8-bromo-cAMP stimulate protein release by about twofold in the presence of Ca2+ in leaky cells. In intact acini cAMP has no effect, and cAMP analogues stimulate enzyme secretion by about twofold in some but not all experiments. Similarly, forskolin, an activator of adenylate cyclases and inhibitors of cyclic nucleotide-dependent phosphodiesterases, such as 3-isobutyl-1-methylxanthine (IBMX) and R0 201724, stimulate protein release in permeabilized acini. The Ca2+-binding protein calmodulin has no effect on enzyme secretion, whereas the calmodulin antagonist trifluoperazine dihydrochloride stimulates protein release in leaky but not in intact acini. The activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates protein release in a Ca2+-dependent manner and enhances cAMP-induced secretion. The effects of carbachol, TPA, cAMP, and a combination of both TPA and cAMP are inhibited by the polyamine spermine in permeabilized cells. Spermine has no effect on carbachol-induced enzyme secretion in intact cells. The data suggest that enzyme secretion from pancreatic acinar cells is mediated by cAMP protein kinase A and by Ca2+ phospholipid protein kinase C in a Ca2+-dependent way and that interaction occurs between both pathways.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Phosphorylated intermediate of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in endoplasmic reticulum from rat pancreatic acinar cells.

Formation and decomposition of the phosphorylated intermediate of endoplasmic reticulum (Ca2+ + Mg2+)-ATPase from pancreatic acinar cells have been studied using lithium dodecyl sulfate- and tetradecyltrimethylammonium bromide-polyacrylamide gel electrophoresis. Incorporation of 32P from [gamma-32P]ATP is Ca2+-dependent (approximate Km for free [Ca2+] = 2-3 X 10(-8) mol/liter). Formation of the 100-kDa phosphoprotein is rapid, reaching maximal 32Pi incorporation within 1 s at room temperature. At 4 degrees C, phosphorylation is slower and dephosphorylation is drastically decreased. For dephosphorylation, Mg2+ and monovalent cations such as K+ or Na+ are necessary. Vanadate inhibits both 32P incorporation and 32P liberation dose dependently (Km = 3 X 10(-6) mol/liter), whereas mitochondrial inhibitors and ouabain have no effect. The phosphoprotein is stable at pH 2 and destabilizes with increasing pH being completely decomposed at pH 9. Reduction of 32P incorporation in the presence of high concentrations of cold ATP and hydroxylamine suggests formation of acylphosphate present in the ATPase intermediate. The characteristics of Ca2+, cation, and pH dependencies of the ATPase activity are similar to those previously described for MgATP-dependent Ca2+ transport into rough endoplasmic reticulum from pancreatic acinar cells (Bayerdörffer, E., Streb, H., Eckhardt, L., Haase, W., and Schulz, I. (1984) J. Membr. Biol. 81, 69-82). The data suggest that the 100-kDa phosphoprotein as described in this study is the intermediate of this Ca2+ transport ATPase.

Adenosine Triphosphatases↗
PubMed1985

Relationship between secretagogue-induced Ca2+ release and inositol polyphosphate production in permeabilized pancreatic acinar cells.

We have previously shown that inositol trisphosphate (IP3) releases Ca2+ from a nonmitochondrial pool of permeabilized rat pancreatic acinar cells (Streb, H., Irvine, R. F., Berridge, M. J., and Schulz, I. (1984) Nature 306, 67-69). This pool was later identified as endoplasmic reticulum (Streb, H., Bayerdorffer, E., Haase, W., Irvine, R. F., and Schulz, I. (1984) J. Membr. Biol. 81, 241-253). As IP3 is produced by hydrolysis of phosphatidylinositol bisphosphate on activation of many "Ca2+-mobilizing receptors," our observation supported the proposal that IP3 functions as a second messenger to release Ca2+ from the endoplasmic reticulum. We have here used the same preparation of permeabilized acinar cells to study the relationship of secretagogue-induced Ca2+ release and IP3 production. We show that: 1) secretagogue-induced Ca2+ release in permeabilized cells is accompanied by a parallel production of inositol trisphosphate. 2) When the secretagogue-induced increase in intracellular free Ca2+ concentration was abolished by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering, secretagogue-induced IP3 production was unimpaired. 3) When secretagogue-induced IP3 production was reduced by inhibiting phospholipase C with neomycin, secretagogue-induced Ca2+ release was also abolished. 4) When the IP3 breakdown was reduced either by lowering the free Mg2+ concentration of the incubation medium or by adding 2.3-diphosphoglyceric acid, the rise in IP3 and the release of Ca2+ induced by secretagogues were both increased. These results further support the role of IP3 as a second messenger to induce Ca2+ mobilization.

2,3-Diphosphoglycerate↗
PubMed1985

Na+/Ca2+ countertransport in plasma membrane of rat pancreatic acinar cells.

The presence of a coupled Na+/Ca2+ exchange system has been demonstrated in plasma membrane vesicles from rat pancreatic acinar cells. Na+/Ca2+ exchange was investigated by measuring 45Ca2+ uptake and 45Ca2+ efflux in the presence of sodium gradients and at different electrical potential differences across the membrane (= delta phi) in the presence of sodium. Plasma membranes were prepared by a MgCl2 precipitation method and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the plasma membrane, (Na+ + K+)-ATPase was enriched by 23-fold. Markers for the endoplasmic reticulum, such as RNA and NADPH cytochrome c reductase, as well as for mitochondria, the cytochrome c oxidase, were reduced by twofold, threefold and 10-fold, respectively. For the Na+/Ca2+ countertransport system, the Ca2+ uptake after 1 min of incubation was half-maximal at 0.62 mumol/liter Ca2+ and at 20 mmol/liter Na+ concentration and maximal at 10 mumol/liter Ca2+ and 150 mmol/liter Na+ concentration, respectively. When Na+ was replaced by Li+, maximal Ca2+ uptake was 75% as compared to that in the presence of Na+. Amiloride (10(-3) mol/liter) at 200 mmol/liter Na+ did not inhibit Na+/Ca2+ countertransport, whereas at low Na+ concentration (25 mmol/liter) amiloride exhibited dose-dependent inhibition to be 62% at 10(-2) mol/liter. CFCCP (10(-5) mol/liter) did not influence Na+/Ca2+ countertransport. Monensin inhibited dose dependently; at a concentration of 5 X 10(-6) mol/liter inhibition was 80%. A SCN- or K+ diffusion potential (= delta phi), being positive at the vesicle inside, stimulated calcium uptake in the presence of sodium suggesting that Na+/Ca2+ countertransport operates electrogenically, i.e. with a stoichiometry higher than 2 Na+ for 1 Ca2+. In the absence of Na+, delta phi did not promote Ca2+ uptake. We conclude that in addition to ATP-dependent Ca2+ outward transport as characterized previously (E. Bayerdörffer, L. Eckhardt, W. Haase & I. Schulz, 1985, J. Membrane Biol. 84:45-60) the Na+/Ca2+ countertransport system, as characterized in this study, represents a second transport system for the extrusion of calcium from the cell. Furthermore, the high affinity for calcium suggests that this system might participate in the regulation of the cytosolic free Ca2+ level.

Adenosine Triphosphatases↗
PubMed1985

Electrogenic calcium transport in plasma membrane of rat pancreatic acinar cells.

ATP-dependent 45Ca2+ 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 mumol/liter free Ca2+, and half-maximal with 0.9 and 0.5 mumol/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+ greater than Rb+ greater than Na+ greater than Li+ greater than choline+ in plasma membranes and Rb+ greater than or equal to K+ greater than or equal to Na+ greater than Li+ greater than choline+ for rough endoplasmic reticulum. The anion sequence was Cl greater than or equal to Br greater than or equal to 1 greater than SCN greater than NO3 greater than isethionate greater than cyclamate greater than gluconate greater than SO2(4) greater than or equal to glutarate and Cl- greater than Br greater than gluconate greater than SO2(4) greater than NO3 greater than 1 greater than cyclamate greater than or equal to 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.

Adenosine Triphosphate↗
PubMed1985

Coupled Na+-H+ exchange in isolated acinar cells from rat exocrine pancreas.

Isolated acinar cells from the rat exocrine pancreas were loaded with 6-carboxyfluorescein diacetate (CFDA), and the intracellular pH (pHi) was estimated from the pH-dependent fluorescence intensity of trapped 6-carboxyfluorescein liberated from CFDA by intracellular esterases. The intracellular fluorescence intensity was calibrated by equilibrating the internal and external pH with nigericin in K+ buffers. In the absence of Na+ (130 mmol/l K+) a pHi of 6.86 +/- 0.04 was found; in its presence (130 mmol/l Na+) a pHi of 7.17. Acute addition of Na+ increased intracellular pH with increasing Na+ concentrations, reaching a maximum at 150 mmol/l with an apparent Km of approximately 40 mmol/l. Of the different cations tested on pHi, such as Li+, K+, Rb+, and Cs+, only Li+ showed an effect on pHi similar to that of Na+. Amiloride dose dependently inhibited both Na+- and Li+-induced alkalinization (apparent Km approximately 10(-5) mol/l). In the presence of ouabain pHi was decreased by 0.2 pH units. Intracellular acidification induced by permeable buffers such as acetic acid-acetate or CO2-HCO3- was dissipated more rapidly in the presence of Na+ compared with K+ or with Na+ and amiloride in the medium. In Li+-preincubated cells intracellular acidification was higher in the absence of Li+ in the extracellular medium than in its presence. This Li+ gradient-induced acidification was dependent on the extracellular pH, was highest at an extracellular pH of 7.05, and decreased with increasing pH to 7.5. The results allow the conclusion that a coupled Na+-H+ exchange is present in pancreatic acinar cells and that the intracellular pH rather than the extracellular Na+ concentration regulates this transport mechanism.

Amiloride↗
PubMed1985

Stimulus-secretion coupling in exocrine glands: the role of inositol-1,4,5-trisphosphate, calcium and cAMP.

Enzyme, electrolyte and fluid secretion from exocrine glands is stimulated by neurotransmitters and peptide hormones. Whereas for some of these secretagogues calcium is an important intracellular messenger, for others it is cyclic AMP. Regulation of steady state free Ca2+ concentration at rest and at stimulation have been studied in isolated permeabilized acinar cells from pancreas, parotid and lacrimal glands by measuring the free Ca2+ concentration of the surrounding incubation medium with a Ca2+-specific macroelectrode. Ca2+ transport mechanisms have been further characterized in subcellular membrane fractions by measuring 45Ca2+ uptake into membrane vesicles from rough endoplasmic reticulum (RER) and plasma membranes (PM). The data show that the intracellular messenger for secretagogue-induced Ca2+ release from RER is inositol-1,4,5-trisphosphate (IP3) which is produced during stimulation by phospholipase C mediated hydrolysis of phosphatidylinositol-bisphosphate. At rest both Ca2+ uptake into RER and Ca2+ extrusion from the cell is promoted by (Ca2+ + Mg2+)-ATPases with different characteristics in both types of membranes and by a coupled Na+/Ca2+ countertransport in the PM which keep cytosolic free Ca2+ concentration at a low level of approximately 2 - 4 X 10(-7) mol/l. During stimulation the Ca2+ permeability of endoplasmic reticulum membrane increases via IP3 and that of the PM by a yet unknown "receptor-operated" mechanism. These events lead to increase in cytosolic free Ca2+ concentration that is a trigger for enzyme, electrolyte and fluid secretion.

Animals↗
PubMed1984

Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas.

We have previously shown that inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983, Nature (London) 306:67-69). This observation suggests that IP3 might provide the missing link between activation of the muscarinic receptor and Ca2+ release from intracellular stores during stimulation. In order to localize the intracellular IP3-sensitive calcium pool, IP3-induced Ca2+ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl2. IP3-induced Ca2+ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000 X g, sucrose-density centrifugation, or MgCl2 precipitation there was a close correlation of Ip3-induced Ca2+ release with the endoplasmic reticulum markers ribonucleic acid (r = 0.96, 1.00, 0.91, respectively) and NADPH cytochrome c reductase (r = 0.63, 0.98, 0.90, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochrome c oxidase (r = -0.64) and glutamate dehydrogenase (r = -0.75) and with the plasma membrane markers (Na+ + K+)-ATPase (r = -0.81) and alkaline phosphatase (r = -0.77) in all fractions analyzed. IP3-induced Ca2+ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca2+ from endoplasmic reticulum in pancreatic acinar cells.

Animals↗
PubMed1984

Characterization of calcium uptake into rough endoplasmic reticulum of rat pancreas.

ATP-dependent Ca2+ uptake into isolated pancreatic acinar cells with permeabilized plasma membranes, as well as into isolated endoplasmic reticulum prepared from these cells, was measured using a Ca2+ -specific electrode and 45Ca2+. Endoplasmic reticulum was purified on an isopycnic Percoll gradient and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the rough endoplasmic reticulum RNA was enriched threefold and the typical marker for the plasma membrane Na+,K+(Mg2+)ATPase was decreased 20-fold. When different fractions of the Percoll gradient were compared, 45Ca2+ uptake correlated with the RNA content and not with the Na+,K+(Mg2+)ATPase activity. The characteristics of nonmitochondrial Ca2+ uptake into leaky isolated cells and 45Ca2+ uptake into isolated endoplasmic reticulum were very similar: Calcium uptake was maximal at 0.3 and 0.2 mmol/liter free Mg2+, at 1 and 1 mmol/liter ATP, at pH 6.0 and 6.5, and free Ca2+ concentration of 2 and 2 mumol/liter, respectively. Calcium uptake decreased at higher free Ca2+ concentration. 45Ca2+ uptake was dependent on monovalent cations (Rb+ greater than K+ greater than Na+ greater than Li+ greater than choline+) and different anions (Cl- greater than Br- greater than SO4(2-) greater than NO3- greater than I- greater than cyclamate- greater than SCN-) in both preparations. Twenty mmol/liter oxalate enhanced 45Ca2+ uptake in permeabilized cells 10-fold and in vesicles of endoplasmic reticulum, fivefold. Calcium oxalate precipitates in the endoplasmic reticulum of both preparations could be demonstrated by electron microscopy. The nonmitochondrial Ca2+ pool in permeabilized cells characterized in this study has been previously shown to regulate the cytosolic free Ca2+ concentration to 0.4 mumol/liter. Our results provide firm evidence that the endoplasmic reticulum plays an important role in the regulation of the cytosolic free Ca2+ concentration in pancreatic acinar cells.

Adenosine Triphosphate↗
PubMed1984

Isolation of stable pancreatic zymogen granules.

Isolated pancreatic zymogen granules have been reported to lyse in common electrolyte solutions such as NaCl or KCl or at pH values above 5.5. A new method, based on an isosmotic Percoll density gradient, was developed for the isolation of zymogen granules and applied to rat pancreas. The granules are highly purified as judged by electron microscopic appearance and specific amylase activity. These granules exhibit a high degree of stability at physiological pH and in isotonic NaCl or KCl. Zymogen granule diameters, determined with a Coulter Counter, were 1.0 +/- 0.2 micron in either isotonic NaCl and KCl. These size values, obtained in physiological solutions, are comparable with granule sizes determined in intact cells by microscopy. Amylase activity averaged 0.66 microU per granule and protein content averaged 0.31 pg per granule; these values were not significantly influenced by different conditions of pH between 5.5 and 7.0 and ionic strength from near 0 to 0.15. The granule density estimated from the protein content was 1.13 g/ml, which agrees well with the behavior of granules in a density gradient. The properties of zymogen granules from the new preparation rectify the apparent discrepancy between their role as a storage organelle and their previously reported in vitro instability.

Animals↗
PubMed1983

Characterization of Mg-ATP-dependent Ca2+ transport in cat pancreatic microsomes.

Mg-ATP-dependent 45Ca2+ uptake and Ca2+-ATPase activity have been examined in isolated microsomes obtained by differential centrifugation and in purified subcellular fractions obtained by Ficoll-sucrose density centrifugation in the presence of mitochondrial inhibitors. Mg-ATP-dependent 45Ca2+ uptake increased with increasing EGTA-buffered free [Ca2+], reaching a maximum of 2 nmol 45Ca2+ X 15 min-1 X mg prot-1 at 2 mumol/1 [Ca2+] in the incubation medium. Half-maximal 45Ca2+ uptake was at approximately 0.2 mumol/1 [Ca2+]. Maximal Ca2+ -Mg2+ -ATPase activity was 130 nmol X 15 min-1 X mg prot-1 at 2 mumol/l [Ca2+], with an apparent Km of approximately 0.3 mumol/l [Ca2+]. The Ca2+ ionophore A23187 (10(-6) mol/l), the mercurial compounds mersalyl (10(-5) mol/l) and CH3ClHg (10(-3) mol/l), as well as La3+ (10(-4) mol/l), vanadate (10(-4) mol/l), and saponin (50 micrograms/mg prot), abolished Mg-ATP-promoted 45Ca2+ uptake. In the absence of Mg2+, ATP did not provoke 45Ca2+ uptake. Using the purified smooth membrane fraction (F1) from the Ficoll-sucrose density gradient (enrichment of Na+-K+-ATPase specific activity by ninefold and of NADH-cytochrome c reductase by threefold as compared with total tissue homogenate), Mg-ATP-dependent 45Ca2+ uptake correlated better with Na+-K+-ATPase (r = 0.97) than with the smooth endoplasmic marker NADH-cytochrome c reductase (r = 0.52). No correlation was found with RNA, the marker for rough endoplasmic reticulum. We conclude that pancreatic plasma membranes contain a Ca2+-Mg2+-ATPase that represents the Ca2+ extrusion system from acinar cells. It is also possible that vesicular membrane structures associated with the plasma membrane, or endocytotic plasma membrane vesicles, take up Ca2+ and represent an intracellular Ca2+ pool.

Adenosine Triphosphate↗
PubMed1983

Regulation of cytosolic free Ca2+ concentration in acinar cells of rat pancreas.

Ca2+ uptake into isolated exocrine pancreatic cells with highly permeable plasma membrane was determined by measuring the decrease in free Ca2+ concentration of the surrounding incubation medium with a Ca2+-specific electrode. In the presence of Mg-ATP and respiratory substrates the free Ca2+ concentration of the incubation medium decreased rapidly after addition of leaky cells until a stable medium free Ca2+ concentration of 4.2 +/- 0.1 X 10(-7) mol/l was obtained. Changes in the medium free Ca2+ concentration at steady state by addition of Ca2+ or EGTA were buffered by cellular uptake or release, respectively, until the steady-state free Ca2+ concentration was reestablished. When nonmitochondrial Ca2+ uptake was determined in the presence of a combination of mitochondrial inhibitors (10(-5) mol/l antimycin, 5 X 10(-6) mol/l oligomycin, and 10(-2) mol/l azide), the rate of uptake was considerably reduced, while the steady-state concentration was unaltered. In contrast, mitochondrial uptake that could be observed in the presence of the ATPase inhibitor vanadate (2 X 10(-3) mol/l) proceeded at the same rate as the control, but the minimal medium free Ca2+ concentration reached was 2.4 +/- 0.1 X 10(-7) mol/l higher than the control. Addition of secretagogues at steady-state free Ca2+ concentration resulted in a Ca2+ release of 0.73 +/- 0.08 nmol/mg protein. The increase in medium free Ca2+ concentration was entirely transient and followed by reuptake to the prestimulation level. The data indicate that a cytosolic free Ca2+ concentration of 4 X 10(-7) mol/l can be regulated in pancreatic acinar cells by a nonmitochondrial Mg2+-dependent Ca2+ pool.

Animals↗