PubMed Health⌕ Search

Biomedical subjects

I Schulz

Publications and source records attributed to I Schulz.

At least 37 records · Page 2Linked to original sources
PubMed1998

Pervanadate stimulates amylase release and protein tyrosine phosphorylation of paxillin and p125(FAK) in differentiated AR4-2J pancreatic acinar cells.

We have studied the role of protein tyrosine phosphorylation in amylase secretion from differentiated AR4-2J cells. The secretagogue bombesin, the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), and the protein-tyrosine phosphatase inhibitor pervanadate induced tyrosine phosphorylation of different proteins, including paxillin and p125(FAK), which was reduced or blocked by the tyrosine kinase inhibitors genistein and tyrphostin B56, respectively. Both PMA and pervanadate continuously increased amylase secretion with a similar time course, reaching the level of bombesin-induced amylase release after 60 min. Their effects were not additive and could be inhibited by preincubation of AR4-2J cells with genistein or tyrphostin B56, respectively. Inhibition of protein kinase C with Ro 31-8220 nearly abolished the effects of PMA, but had no effect on either pervanadate-induced protein tyrosine phosphorylation or amylase secretion. An increase in cytosolic free Ca2+ concentration by thapsigargin or A23187 caused a rapid increase in amylase release within the initial 5 min. In the presence of PMA or pervanadate, amylase secretion was further stimulated to levels comparable to those induced by bombesin after 30 min of stimulation. Inhibition of PMA-induced amylase secretion by Ro 31-8220 was less at elevated cytosolic free Ca2+ concentrations than without Ca2+. Furthermore, an increase in cytosolic free Ca2+ concentration had no effect on protein tyrosine phosphorylation in either the absence or presence of PMA or pervanadate. We therefore conclude that in the cascade of events that lead to bombesin-induced protein secretion from AR4-2J cells, protein tyrosine phosphorylation occurs downstream of protein kinase C activation. A further step in secretion that is Ca2+-dependent occurs distal to protein tyrosine phosphorylation.

Amylases↗
PubMed1998

LOE 908 blocks delayed rectifier type potassium channels in PC12 cells and cortical neurons in culture.

The effects of (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2- phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908) were studied on K+ currents in undifferentiated cells from a phaeochromocytoma cell line (PC12), in cortical neurons from rat in primary culture, in a rat blood lymphoma cell line (RBL-1) and in a kidney cell line (BHK21). In PC12 cells delayed rectifier K+ currents measured in the whole-cell mode of the patch clamp technique were almost completely blocked by 10 microM LOE 908. The IC50 value was 0.7 microM and the Hill coefficient 0.8. After washout of the inhibitor about 80% of the current recovered. In rat cortical neurons in primary culture LOE 908 inhibited tetraethylammonium (TEA, 10 mM)-sensitive delayed rectifying K+ currents (LOE 908: 1 microM, 61 +/- 25% inhibition; 10 microM 103 +/- 19% inhibition). In contrast to the inhibitory action of LOE 908 on delayed rectifying K+ currents, Ca(2+)-activated potassium currents in BHK21 cells were only inhibited by 25 +/- 5% (10 microM LOE 908, n = 5) and no effect of LOE 908 was found on inward-rectifying K+ currents in RBL-1 cells. We conclude that LOE 908 is a K+ channel blocker with selectivity for delayed outward rectifying K+ channels.

Acetamides↗
PubMed1998

Control of Ca2+ wave propagation in mouse pancreatic acinar cells.

We have investigated control mechanisms involved in the propagation of agonist-induced Ca2+ waves in isolated mouse pancreatic acinar cells. Using a confocal laser-scanning microscope, we were able to show that maximal stimulation of cells with acetylcholine (ACh, 500 nM) or bombesin (1 nM) caused an initial Ca2+ release of comparable amounts with both agonists at the luminal cell pole. Subsequent Ca2+ spreading to the basolateral membrane was faster with ACh (17.3 +/- 5.4 microns/s) than with bombesin (8.0 +/- 2.2 microns/s). The speed of bombesin-induced Ca2+ waves could be increased up to the speed of ACh-induced Ca2+ waves by inhibition of protein kinase C (PKC). Activation of PKC significantly decreased the speed of ACh-induced Ca2+ waves but had only little effect on bombesin-evoked Ca2+ waves. Within 3 s after stimulation, production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was higher in the presence of ACh compared with bombesin, whereas bombesin induced higher levels of diacylglycerol (DAG) than ACh. These data suggest that the slower propagation speed of bombesin-induced Ca2+ waves is due to higher activation of PKC in the presence of bombesin compared with ACh. The higher increase in bombesin-compared with ACh-induced DAG production is probably due to activation of phospholipase D (PLD). Inhibition of the PLD-dependent DAG production by preincubation with 0.3% butanol led to an acceleration of the bombesin-induced Ca2+ wave. In further experiments, we could show that ruthenium red (100 microM), an inhibitor of Ca(2+)-induced Ca2+ release in skeletal muscle, also decreased the speed of ACh-induced Ca2+ waves. The effect of ruthenium red was not additive to the effect of PKC activation. From the data, we conclude that, following Ins(1,4,5)P3-induced Ca2+ release in the luminal cell pole, secondary Ca2+ release from stores, which are located in series between the luminal and the basal plasma membrane, modifies Ca2+ spreading toward the basolateral cell side by Ca(2+)-induced Ca2+ release. Activation of PKC leads to a reduction in Ca2+ release from these stores and therefore could explain the slower propagation of Ca2+ waves in the presence of bombesin compared with ACh.

Acetylcholine↗
PubMed1998

Effect of intracellular pH on acetylcholine-induced Ca2+ waves in mouse pancreatic acinar cells.

We have used fluo 3-loaded mouse pancreatic acinar cells to investigate the relationship between Ca2+ mobilization and intracellular pH (pHi). The Ca2+-mobilizing agonist ACh (500 nM) induced a Ca2+ release in the luminal cell pole followed by spreading of the Ca2+ signal toward the basolateral side with a mean speed of 16.1 +/- 0.3 micron/s. In the presence of an acidic pHi, achieved by blockade of the Na+/H+ exchanger or by incubation of the cells in a Na+-free buffer, a slower spreading of ACh-evoked Ca2+ waves was observed (7.2 +/- 0.6 micron/s and 7.5 +/- 0.3 micron/s, respectively). The effects of cytosolic acidification on the propagation rate of ACh-evoked Ca2+ waves were largely reversible and were not dependent on the presence of extracellular Ca2+. A reduction in the spreading speed of Ca2+ waves could also be observed by inhibition of the vacuolar H+-ATPase with bafilomycin A1 (11.1 +/- 0.6 micron/s), which did not lead to cytosolic acidification. In contrast, inhibition of the endoplasmic reticulum Ca2+-ATPase by 2,5-di-tert-butylhydroquinone led to faster spreading of the ACh-evoked Ca2+ signals (25.6 +/- 1.8 micron/s), which was also reduced by cytosolic acidification or treatment of the cells with bafilomycin A1. Cytosolic alkalinization had no effect on the spreading speed of the Ca2+ signals. The data suggest that the propagation rate of ACh-induced Ca2+ waves is decreased by inhibition of Ca2+ release from intracellular stores due to cytosolic acidification or to Ca2+ pool alkalinization and/or to a decrease in the proton gradient directed from the inositol 1,4, 5-trisphosphate-sensitive Ca2+ pool to the cytosol.

Acetylcholine↗
PubMed1998

Immunolocalization of vacuolar-type H+-ATPase in rat submandibular gland and adaptive changes induced by acid-base disturbances.

Using antibodies against the 31-kD and 70-kD subunits of vacuolar type H+-ATPase (V-ATPase) and light microscopic immunocytochemistry, we have demonstrated the presence of this V-ATPase in rat submandibular gland. We have also investigated the adaptive changes of this transporter during acid-base disturbances such as acute and chronic metabolic acidosis or alkalosis. Our results show intracellularly distributed V-ATPase in striated, granular, and main excretory duct cells in controls, but no V-ATPase immunoreaction in acinar cells. Both acute and chronic metabolic acidosis caused a shift in V-ATPase away from diffuse distribution towards apical localization in striated and granular duct cells, suggesting that a V-ATPase could be involved in the regulation of acid-base homeostasis. In contrast, during acidosis the main excretory duct cells showed no changes in the V-ATPase distribution compared to controls. With acute and chronic metabolic alkalosis, no changes in the V-ATPase distribution occurred. (J Histochem Cytochem 46:91-100, 1998)

Acid-Base Imbalance↗
PubMed1997

Expression and characterization of a trpl homolog from rat.

Mammalian homologues of the Drosophila trp/trpl-gene-family code for "Ca(2+)-store-operated" channels. Here we describe the cloning and expression of a trp/trpl homologous gene from rat brain. The clone is named Rtrp3 because of its high homology to the recently described Htrp3 (Zhu et al (1996) Cell 85:661-671). Expression of Rtrp3 in the mammalian COS-1 cell line leads to 100% increase of capacitative Ca2+ entry as compared to the controls. This capacitative calcium entry can be completely blocked with La3+ (10 microM). We conclude that Rtrp3, a new member of the growing family of trp/trpl homologues in mammalians, is involved in "Ca(2+)-store-operated" Ca2+ entry into the cell.

Amino Acid Sequence↗
PubMed1997

Inwardly rectifying, voltage-dependent and resting potassium currents in rat pancreatic acinar cells in primary culture.

1. In exocrine pancreatic acinar cells in primary culture an inwardly rectifying, a voltage-dependent and a permanent resting K+ current were characterized. 2. Inwardly rectifying K+ currents could be elicited by elevation of the extracellular K+ concentration. The K+ inward currents were almost completely blocked by 5 mM Ba2+, whereas 10 mM TEA+ had only a partial effect. 3. Depolarizing voltage steps from negative clamp potentials evoked transient activation of a voltage-dependent K+ current. This voltage-dependent current could be blocked by 10 mM TEA+ and 1 mM 4-aminopyridine, but not by 5 mM Ba2+. 4. Neither the K+ inward rectifier nor the voltage-dependent K+ conductance produced a significant negative cell potential. Stable membrane potentials (-38.7 +/- 2.3 mV, n = 38) could only be recorded on cell clusters (> or = 5 cells). 5. Cell clusters, in contrast to single cells, had a permanent resting K+ conductance in addition to the inward rectifier and the voltage-dependent current. This resting K+ conductance was not blocked by TEA+, Ba2+, 4-aminopyridine or by the chromanol 293B. 6. Cytosolic alkalization by addition of NH4Cl to the bath solution decreased the resting K+ current. In parallel, electrical uncoupling of the cells and breakdown of the resting potential could be observed. The same effects could be produced when the cells were uncoupled by 0.2-1.0 mM n-octanol. It can be concluded that cell coupling is essential for maintenance of stable resting membrane potentials in pancreatic acinar cells.

4-Aminopyridine↗
PubMed1996

Depletion of intracellular calcium stores activates a calcium conducting nonselective cation current in mouse pancreatic acinar cells.

Receptor-mediated Ca2+ release from inositol (1,4,5)-trisphosphate (IP3)-sensitive Ca2+ stores causes "capacitative calcium entry" in many cell types (Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12; Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624). We used patch-clamp and fluorescence techniques in isolated mouse pancreatic acinar cells to identify ion currents and cytosolic calcium concentrations under conditions in which intracellular Ca2+ stores were emptied. We found that depletion of Ca2+ stores activated a calcium-release-activated nonselective cation current (ICRANC) which did not discriminate between monovalent cations. ICRANC possessed a significant conductance for Ca2+ and Ba2+. It was not inhibited by La3+, Gd3+, Co2+, or Cd2+ but was completely abolished by flufenamic acid or genistein. In whole cell and cell-attached recordings, a 40-45 pS nonselective cation channel was identified which was activated by Ca2+ store depletion. Calcium entry as detected by single cell fluorescence measurements with fluo-3 or fura-2, showed the same pharmacological properties as ICRANC. We conclude that in mouse pancreatic acinar cells 40-45 pS nonselective cation channels serve as a pathway for capacitative Ca2+ entry. This entry pathway differs from the previously described ICRAC (Hoth, M., and Penner, R. (1992) Nature 355, 353-356) in its ion-selectivity, pharmacological profile, and single-channel conductance.

Acetylcholine↗
PubMed1996

Tmp21 and p24A, two type I proteins enriched in pancreatic microsomal membranes, are members of a protein family involved in vesicular trafficking.

We report here on the isolation, cloning, and expression of two Mr 21,000 proteins from rat pancreatic acinar cells, the rat-Tmp21 (transmembrane protein, Mr 21,000) and the rat-p24A. Both proteins are transmembrane proteins with type I topology and share weak but significant homology to one another (23% identity). We further show the cloning and characterization of the human homologs, hum-Tmp21, which is expressed in two variants (Tmp21-I and Tmp21-II), and hum-p24A. Tmp21 proteins and p24A have highly conserved COOH-terminal tails, which contain motifs related to the endoplasmic reticulum retention and retrieval consensus sequence KKXX. The rat-p24 sequence is identical to the hamster CHOp24, a recently characterized component of coatomer-coated transport vesicles, which defines a family of proteins (called the p24 family) proposed to be involved in vesicular transport processes (Stamnes, M. A., Craighead, M. W., Hoe, M. H., Lampen, N., Geromanos, S., Tempst, P., and Rothman, J. E.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8011-8015). Sequence alignment and structural features identify the Tmp21 protein as a new member of this p24 family. Northern analysis of various tissues indicates that the Tmp21 proteins and the p24A protein are ubiquitously expressed. The integral membrane components Tmp21 and p24A are localized in microsomal membranes, zymogen granule membranes, and the plasma membrane and are absent from the cytosol. Both p24A and Tmp21 show weak homology to the yeast protein Emp24p, which recently has been shown to be involved in secretory protein transport from the endoplasmic reticulum to the Golgi apparatus. This leads us to conclude that the receptor-like Tmp21 and p24A are involved in vesicular targeting and protein transport.

Amino Acid Sequence↗
PubMed1996

Chloride and potassium conductances of mouse pancreatic zymogen granules are inversely regulated by a approximately 80-kDa mdr1a gene product.

Cl- and cation conductances were characterized in zymogen granules (ZG) isolated from the pancreas of wild-type mice (+/+) or mice with a homozygous disruption of the multidrug resistance P-glycoprotein gene mdr1a (-/-). Cl- conductance of ZG was assayed in isotonic KCl buffer by measuring osmotic lysis, which was induced by maximal permeabilization of ZG membranes (ZGM) for K+ with valinomycin due to influx of K+ through the artificial pathway and of Cl- through endogenous channels. To measure cation conductances, ZG (pHi 6.0-6.5) were suspended in buffered isotonic monovalent cation acetate solutions (pH 7.0). The pH gradient was converted into an outside-directed H+ diffusion potential by maximally increasing H+ conductance of ZGM with carbonyl cyanide m-chlorophenylhydrazone. Osmotic lysis of ZG was induced by H+ diffusion potential-driven influx of monovalent cations through endogenous channels and nonionic diffusion of the counterion acetate. ZGM Cl- conductances were not different in (-/-) and (+/+) mice (2.6 +/- 0.3 h-1 versus 3.1 +/- 0.2 h-1 (relative rate constant)). The nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) (0.5 mM) activated the Cl- conductance both in (+/+) and (-/-) mice. However, activation of Cl- conductance by AMP-PCP was reduced in (-/-) mice as compared with (+/+) mice (5.0 +/- 0.4 h-1 versus 7.6 +/- 0.7 h-1; p < 0. 005). In contrast, ZGM K+ conductance was increased in (-/-) mice as compared with (+/+) mice (14.2 +/- 2.0 h-1 versus 8.5 +/- 1.2 h-1; p < 0.03). In the presence of 0.5 mm AMP-PCP, which completely blocks K+ conductance but leaves a nonselective cation conductance unaffected, there was no difference between (-/-) and (+/+) mice (5.3 +/- 0.7 h-1 versus 3.2 +/- 0.5 h-1). In Western blots of ZGM from wild-type mice, a polyclonal MDR1 specific antibody labeled a protein band of approximately 80 kDa. In mdr1a-deficient mice, the intensity of this band was reduced to 39 +/- 7% of the wild-type signal. This indicates that a mdr1a gene product of approximately 80 kDa enhances the AMP-PCP-activated fraction of mouse ZGM Cl- conductance and reduces AMP-PCP-sensitive K+ conductance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Different time courses of GTP[gamma-S]-induced exocytosis and current oscillations in isolated mouse pancreatic acinar cells.

Exocytosis in isolated mouse pancreatic acinar cells was investigated using the dual-frequency method for measuring membrane capacitance and ionic conductances. Under control conditions, single exo- and endocytotic events could be resolved. The total cell capacitance slightly decreased to 98.7 +/- 0.9% of the initial cell capacitance within 10 min after establishing the whole-cell configuration. When guanosine 5'-O-(3-thiophosphate) (GTP[gamma-S] was added to the patch pipette, stepwise elevations in membrane capacitance occurred and the cell capacitance increased to 106.7 +/- 1.6% within 10 min. Exocytosis was also stimulated by GTP[gamma-S] when a Ca2+-free pipette solution supplemented with 1 to 10 mM ethylenebis(oxonitrilo) tetraacetate (EGTA) was used. Measurement of the DC current component in parallel with AC current analysis was used to isolate components of the Ca2+-dependent Cl- and monovalent cation conductances from the whole-cell conductance. These experiments demonstrate that in GTP[gamma-S]-stimulated pancreatic acinar cells: (1) activation of Cl- currents precedes that of cation currents, and (2) fusion of the zymogen granule membrane with the plasma membrane does not lead to incorporation of active Cl- or nonselective cation channels (>/= 10 pS).

Animals↗
PubMed1996

Quantification of intraluminal free [Ca] in the agonist-sensitive internal calcium store using compartmentalized fluorescent indicators: some considerations.

Many fluorescent Ca indicators, particularly those loaded as acetoxymethyl (AM)-ester derivatives, are known to become compartmentalized into organelles. This property can be exploited to measure changes in free [Ca] in subcellular compartments, including the inositol (1,4,5)-trisphosphate-sensitive store. However, quantitative measurement of free [Ca] within a particular compartment is complicated by the fact that dye may accumulate in a variety of organelles and, in many cases, by the Mg sensitivity of the indicator. Here the issue of the quantification of free [Ca] within the thapsigargin-sensitive store in BHK-21 fibroblasts using the low affinity Ca indicator, Mag-Fura-2, has been re-examined. At least 88 +/- 1.3% (SEM) of the compartmentalized dye was determined to be confined to the thapsigargin-sensitive store, with the remaining fraction accounted for by other compartments where [Ca] was below the detection limit for the dye (< 5 microM). In situ calibrations with ionophores indicated that the apparent free resting intraluminal [Ca] was 260 +/- 43 microM (SEM). Our analysis shows, however, that dye reporting from regions of low [Ca] contributes disproportionately to the Mag-Fura-2 ratio measured over the whole cell, potentially resulting in large underestimations of intraluminal [Ca] in agonist-sensitive stores. Free [Ca] in the agonist-sensitive store was calculated to be as high as 539 +/- 92 microM, assuming 12% of the Mag-Fura-2 to be in compartments where [Ca] was below 5 microM. In comparison, perturbations arising from the presence of Mg in stores are predicted to be relatively minor.

Animals↗
PubMed1996

ATP regulates calcium leak from agonist-sensitive internal calcium stores.

Under resting conditions, steady-state [Ca] in agonist-sensitive Ca stores reflects a balance between active uptake (usually mediated by a thapsigargin-sensitive Ca-ATPase of the SERCA family) and passive efflux of Ca. Even though this pump-leak cycle appears to be a common property of Ca-storing organelles, little is known about the nature of the leak pathway. Ca homeostasis in thapsigargin-sensitive internal Ca stores of single permeabilized BHK-21 fibroblasts was examined using digital image processing of compartmentalized mag-fura-2 (a low-affinity Ca indicator). It is shown here that the leak of Ca from internal stores is regulated specifically by the cytosolic ATP concentration. The rate of leak was 3.6 times slower in 0.375 mM[ATP] than in 4 mM [ATP] (Na or Mg salt). These effects were observed in the presence of 0 Ca/EGTA, thapsigargin, heparin, and ruthenium red, and therefore appear to be independent of the Ca-ATPase, the InsP(3) receptor and the ryanodine receptor. The ATP-stimulated leak was seen in a variety of cell types, including rat basophilic leukemia cells and mouse pancreatic acinar cells. Other nucleotides (ADP, GTP, CTP, and UTP) and nonhydrolyzable ATP analogs (AMP-PNP and ATPgammaS) did not reproduce the action of ATP. Changes in cellular metabolism and ensuing alterations in [ATP] will be expected to influence the filling state of internal Ca stores through effects on the passive leak pathway, potentially leading to modulation of Ca signaling and organellar function.

Adenosine Triphosphate↗
PubMed1995

Characterization of single potassium channels in mouse pancreatic acinar cells.

1. Single K(+)-selective channels with a conductance of about 48 pS (pipette, 145 mM KCl; bath, 140 mM NaCl + 4.7 mM KCl) were recorded in the patch-clamp whole-cell configuration in isolated mouse pancreatic acinar cells. 2. Neither application of the secretagogues acetylcholine (second messenger, inositol 1,4,5-trisphosphate) or secretin (second messenger, cAMP), nor addition of the catalytic subunit of protein kinase A to the pipette solution changed the activity of the 48 pS K+ channel. 3. Intracellular acidification with sodium propionate (20 mM) diminished activity of the 48 pS channel, whereas channel open probability was increased by cytosolic alkalization with 20 mM NH4Cl. 4. BaCl2 (5 mM), TEA (10 mM) or apamin (1 microM) added to the bath solution had no obvious effect on the kinetics of the 48 pS channel. Similarly, glibenclamide and diazoxide failed to influence the channel activity. 5. When extracellular NaCl was replaced by KCl, whole-cell recordings revealed an inwardly rectifying K+ current carried by a 17 pS K+ channel. 6. The inwardly rectifying K+ current was not pH dependent and could largely be blocked by Ba2+ but not by TEA. 7. Since the 48 pS K+ channel is neither Ca2+ nor cAMP regulated, we suggest that this channel could play a role in the maintenance of the negative cell resting potential.

Acetylcholine↗
PubMed1995

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells.

We have examined the effect of the Ca2+ (Mg2+)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent 45Ca2+ uptake into IP3-sensitive Ca2+ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40-50% of total Ca2+ uptake was inhibited by TG up to 10 nM (apparent Ki approximately 4.2 nM, Ca2+ pool I). An additional increase of inhibition up to 85-90% of total Ca2+ uptake could be achieved at 15 to 20 nM of TG (apparent Ki approximately 12.1 nM, Ca2+ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent Ki approximately 10 microM). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca2+ uptake. About 30-40% of total Ca2+ uptake was inhibited by 100 microM of vanadate (apparent Ki approximately 18 microM, Ca2+ pool II). The remaining 60-70% could be inhibited either by vanadate at concentrations up to 1 mM (apparent Ki approximately 300 microM) or by TG up to 10 nM (Ca2+ pool I). The amount of IP3-induced Ca2+ release was constant at approximately 25% over a wide range of Ca2+ filling. About 10-20% remained unreleasable by IP3. Reduction of IP3-releasable Ca2+ in the presence of inhibitors showed similar dose-response curves as Ca2+ uptake (apparent Ki approximately 3.0 nM for IP3-induced Ca2+ release as compared to approximately 4.2 nM for Ca2+ uptake at TG up to 10 nM) indicating that the highly TG-sensitive Ca2+ pump fills the IP3-sensitive Ca2+ pool I. At TG concentrations > 10 nM which blocked Ca2+ pool II the apparent Ki values were approximately 11.3 and approximately 12.1 nM, respectively. For inhibition by vanadate up to 100 microM the apparent Ki values were approximately 18 microM for Ca2+ uptake and approximately 7 microM for Ca2+ release (Ca2+ pool II). At vanadate concentrations up to 1 mM the apparent Ki values were approximately 300 and approximately 200 microM, respectively (Ca2+ pool I). Both Ca2+ pools I and II also showed different sensitivities to IP3. Dose-response curves for IP3 in the absence of inhibitors (control) showed an apparent Km value for IP3 at 0.6 microM. In the presence of TG (inhibition of Ca2+ pool I) the curve was shifted to the left with an apparent Km for IP3 at 0.08 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Capacitative Ca2+ influx and a Ca2+-dependent nonselective cation pathway are discriminated by genistein in mouse pancreatic acinar cells.

We have investigated the effect of genistein on the hormone-stimulated Ca2+ influx and on a 28pS nonselective cation channel in mouse pancreatic acinar cells using the Ca2+ indicator fluo3 and the patch-clamp technique. The identity of the Ca2+ influx pathway has not been established in this cell type so far. Therefore we have investigated the Ca2+-dependent nonselective cation channel as a potential pathway for Ca2+ influx. Capacitative Ca2+ entry was induced by depletion of intracellular Ca2+ stores with 500nM acetylcholine or with the Ca2+ ATPase inhibitor 2,5di-tert- butylhydroquinone. In the presence of 100microM genistein, Ca2+ release was unimpaired, whereas Ca2+ influx was reversibly suppressed. Patch-clamp experiments demonstrated that genistein had no effect on Ca2+-activated nonselective cation channels, the activity of which was measured in excised membrane patches (inside/out) or in the whole-cell configuration. Therefore we conclude that this 28pS nonselective cation channel does not contribute to Ca2+ influx into mouse exocrine pancreatic cells. With the exception of genistein and tyrphostin 25, other tyrosine kinase inhibitors such as methyl-2,5-dihydroxycinnamate, lavendustin A, herbimycin A, and tyrphostin B56 were without effect on Ca2+ signalling. Thus, the involvement of tyrosine phosphorylation in the activation of the Ca2+ entry mechanism in mouse pancreatic acinar cells is unclear.

Animals↗
PubMed1994

Monoclonal antibodies against MDR1 P-glycoprotein inhibit chloride conductance and label a 65-kDa protein in pancreatic zymogen granule membranes.

The regulation of Cl- and cation conductances by the nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) was characterized in isolated zymogen granules (ZG) from pancreatic acinar cells. ZG were purified from rat pancreas homogenate by Percoll gradient centrifugation. Cl- conductance was assayed by suspending ZG in isotonic KCl buffer and measuring osmotic lysis induced by maximal permeabilization of ZG membranes (ZGM) for K+ with the K+ ionophore valinomycin (Val). This resulted in influx of K+ through the artificial pathway and of Cl- through endogenous channels. To measure cation conductances ZG (pHi approximately 6) were suspended in pH 7 buffered isotonic monovalent cation acetate salts. The pH gradient was converted into an outside-directed H+ diffusion potential by maximally increasing H+ conductance of ZGM with the protonophore carbonyl cyanide p-chlorophenylhydrazone. Osmotic lysis of ZG was induced by H+ diffusion potential driven influx of monovalent cations through endogenous channels and non-ionic diffusion of the counterion acetate. In the absence of Val, ZG were stable in KCl buffer up to 2 h. AMP-PCP enhanced osmotic lysis approximately 4-fold compared to control, due to activation of Cl- conductance by AMP-PCP and K+ influx through an AMP-PCP-insensitive nonselective cation pathway, which could be blocked by 0.1 mM Ba2+, 0.5 mM quinine, or 0.2 mM flufenamate. In addition, a K+ and Rb+ selective cation conductance was found which was completely blocked by 0.5 mM AMP-PCP or 0.5 mM quinine. AMP-PCP induced Cl- conductance was strongly inhibited by two monoclonal antibodies against MDR1 P-glycoprotein (JSB-1 and C219; 5-10 micrograms/ml), but not by a monoclonal antibody against the cystic fibrosis transmembrane conductance regulator (M3A7; 5 micrograms/ml) or by mouse IgG. The AMP-PCP insensitive nonselective cation conductance was not blocked by monoclonal antibodies against MDR1 P-glycoprotein (MDR1). Immunoblot studies of ZG membranes revealed the presence of a major immunoreactive protein band of approximately 65 kDa with both monoclonal antibodies against MDR1, but no protein of the approximate size of MDR1 (approximately 170 kDa) was detected. We propose that the Cl- channel or a regulator of the channel, that is activated by the non-hydrolyzable ATP analog AMP-PCP in ZG membranes, is a member of the ATP binding cassette superfamily of transporters and may have homology to MDR1 P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗