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1

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Large-scale purification of calf pancreatic zymogen granule membranes

Thevenod, F. ; Haase, W. ; Hopfer, U.

Amsterdam : Elsevier

Analytical Biochemistry 202 (1992), S. 54-60

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ISSN: 0003-2697

Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002

Topics: Biology , Chemistry and Pharmacology

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1016/0003-2697(92)90205-L

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Characterization of MgATP-Driven H+ uptake in to a microsomal vesicle franction from rat pancreatic acinar cells (1989)

Thévenod, F. ; Kemmer, T. P. ; Christian, A. L. ; [et al.]

Springer

The journal of membrane biology 107 (1989), S. 263-275

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ISSN: 1432-1424

Keywords: H+ ATPase ; endoplasmic reticulum ; proton gradient ; Cl− transport ; protonophores ; acridine orange

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Summary In microsomal vesicles, as isolated from exocrine pancreas cells, MgATP-driven H+ transport was evaluated by measuring H+-dependent accumulation of acridine orange (AO). Active H+ uptake showed an absolute requirement for ATP with simple Michaelis-Menten kinetics (K m for ATP 0.43 mmol/liter) with a Hill coefficient of 0.99. H+ transport was maximal at an external pH of 6.7, generating an intravesicular pH of 4.8. MgATP-dependent H+ accumulatioin was abolished by protonophores. such as nigericin (10−6 mol/liter) or CCCP (10−5 mol/liter), and by inhibitors of nonmitochondria H+ ATPase, such as NEM or NBD-Cl, at a concentration of 10−5 mol/liter. Inhibitors of both mitochondrial and nonmitochondrial H+ pumps, such as DCCD (10−5 mol/liter) or Dio 9 (0.25 mg/ml), reduced microsomal H+ transport by about 90%. Vanadate (2×10−3 mol/liter). a blocker of those ATPases, which form a phosphorylated intermediate, did not inhibit H+ transport. The stilbene derivative DIDS (10−4 mol/liter), which inhibits anion transport systems, abolished H+ transport completely. MgATP-dependent H+ transport was found to be anion dependernt in the sequence Cl−〉Br−〉gluconate−; in the presence of SO 4 −2 . CH3COO− or No 3 − , no H+ transport was observed. MgATP-dependent H+ accumulation was also cation dependent in the sequence K+〉Li+〉Na+=choline+, As shown by dissipation experiments in the presence of different ion gradients and ionophores, both a Cl− and a K+ conductance, as well as a small H+ conductance. were found in the microsomal membranes. When membranes containing the H+ pump wer further purified by Percoll gradient centrifugatin (ninefold enrichment comparad to homogenate), no correlation with markers for endoplasmic reticulum., mitochondria, plasma membranes, zymogen graules or Golgi membranes was found. The present data indicate that the H+ pump located in microsomes from rat exocrine pancreas is a vacuolar-or “V”-type H+ ATPase and has most similarities to that described in endoplasmic reticulum. Golgi apparatus or endosomes.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF01871941

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Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells (1995)

Ozawa, T. ; Thévenod, F. ; Schulz, I.

Springer

The journal of membrane biology 144 (1995), S. 111-120

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ISSN: 1432-1424

Keywords: Thapsigargin ; Vanadate ; Ca2+ pump ; Ca2+ ATPase ; SERCA ; Endoplasmic reticulum

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Abstract 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≈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≈12.1 nm, Ca2+ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent Ki≈10 μm). 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 μm of vanadate (apparent Ki≈18 μm, Ca2+ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent Ki≈300 μm) or by TG up to 10 nm (Ca2+ pool I). The amount of IP3-induced Ca2+ release was constant at ≈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≈ 3.0 nm for IP3-induced Ca2+ release as compared to ≈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 ≈11.3 and ≈12.1 nm, respectively. For inhibition by vanadate up to 100 μm the apparent Ki values were ≈18 μm for Ca2+ uptake and ≈7 μm for Ca2+ release (Ca2+ pool II). At vanadate concentrations up to 1 mm the apparent Ki values were ≈300 and ≈200 μm, 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 μm. 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 μm. In the presence of vanadate (inhibition of Ca2+ pool II), the apparent Km for IP3 was 2.1 μm. These data allow the conclusion that there are at least three different Ca2+ uptake mechanisms present in pancreatic acinar cells: TG- and IP3 insensitive but highly vanadate-sensitive Ca2+ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca2+ pools with different TG-, vanadate- and IP3-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00232797

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4

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Evidence for Involvement of a Zymogen Granule Na+/H+ Exchanger in Enzyme Secretion from Rat Pancreatic Acinar Cells (1996)

Anderie, I. ; Thévenod, F.

Springer

The journal of membrane biology 152 (1996), S. 195-205

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ISSN: 1432-1424

Keywords: Key words: Sodium/hydrogen exchange — Amiloride — Ethylisopropylamiloride — Calmodulin dependent protein kinase — Calmodulin antagonists — Calmidazolium

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Abstract. We have characterized a Na+/H+ exchanger in the membrane of isolated zymogen granules (ZG) from rat exocrine pancreas and investigated its role in secretagogue-induced enzyme secretion. ZG Na+/H+ exchanger activity was estimated by measuring Na+ or Li+ influx and consequent osmotic swelling and lysis of ZG incubated in Na- or Li-acetate. Alternatively, intragranule pH was investigated by measuring absorbance changes in ZG which had been preloaded with the weak base acridine orange. Na+- or Li+-dependent ZG lysis was enhanced by increasing inward to outward directed H+ gradients. Na+-dependent ZG lysis was not prevented by an inside-positive K+ diffusion potential generated by valinomycin which argues against parallel operation of separate electrogenic Na+ and H+ permeabilities and for coupled Na+/H+ exchange through an electroneutral carrier. Na+- and Li+-dependent ZG lysis was inhibited by EIPA (EC50∼25 μm) and benzamil (EC50∼100 μm), but only weakly by amiloride. Similarly, absorbance changes due to release of acridine orange from acidic granules into the medium were obtained with Na+ and Li+ salts only, and were inhibited by EIPA, suggesting the presence of a Na+/H+ exchanger in the membrane. Na+ dependent lysis of ZG was inhibited by 0.5 mm MgATP and MgATP-γ-S by about 60% and 35%, respectively. Inhibition by MgATP was prevented by incubation of ZG with alkaline phosphatase (100 U/ml), or by the calmodulin antagonists calmidazolium (0.75 μm), trifluoperazine (100 μm) and W-7 (500 μm), suggesting that the ZG Na+/H+ exchanger is regulated by a ZG membrane-bound calmodulin-dependent protein kinase. Na+ dependence of secretagogue (CCK-OP)-stimulated amylase secretion was investigated in digitonin permeabilized rat pancreatic acini and was higher in acini incubated in Na+ containing buffer (30 mm NaCl/105 mm KCl buffer; 6.4 ± 0.4% of total amylase above basal) compared to buffer without Na+ (0 mm NaCl/135 mm KCl buffer; 4.7 ± 0.4% of total amylase above basal, P 〈 0.03). EIPA (50 μm) reduced CCK-OP-induced amylase secretion in Na+ containing buffer from 7.5 ± 0.6% to 4.1 ± 0.8% (P 〈 0.02). In the absence of Na+ in the buffer, CCK-OP-stimulated amylase release was not inhibited by 50 μm EIPA. The data suggest that an amiloride insensitive, EIPA inhibitable Na+/H+ exchanger is present in ZG membranes, which is stimulated by calmodulin antagonists and could be involved in secretagogue-induced enzyme secretion from rat pancreatic acini.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002329900097

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