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U Hopfer

Publications and source records attributed to U Hopfer.

At least 73 records · Page 4Linked to original sources
PubMed1990

Chloride transport across the membrane of parotid secretory granules.

The Cl- transport pathways in secretory granules isolated from the parotid glands of rats were characterized by the technique of ionophore-induced lysis in defined salt solutions. The granules were shown to possess a Cl- conductance that exhibited a distinct anion selectivity with a sequence I- greater than Br- greater than Cl- greater than F- greater than SO4(2-) much greater than gluconate-. This conductance could be reduced approximately 40% by the stilbene 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) from the cytoplasmic side; the half-maximal concentration for inhibition was 50 microM. Furthermore, the apparent Cl- conductance was reduced by outwardly directed granule H+ gradients and stimulated by inwardly directed gradients. An outwardly directed H+ gradient mimics the in vivo environment and may serve in a regulatory capacity, providing for a tonic inhibition of transport until the granule fuses with the luminal membrane. The granules also possessed a Cl(-)-HCO3- exchange based on electroneutrality of Cl- uptake and stimulation of this uptake by HCO3-. This pathway displayed a different anion selectivity, I- greater than Br- greater than F- greater than Cl- much greater than SO4(2-) much greater than gluconate-, and was not inhibited by SITS on the cytoplasmic side. The presence of these electrolyte transport pathways in the granule membrane is consistent with the production of primary fluid by parotid acinar cells after fusion of granules with the luminal plasma membrane.

Animals↗
PubMed1989

Properties of rabbit pepsinogen granules.

Pepsinogen granules were isolated from the rabbit stomach using isoosmotic Percoll density gradients, low free calcium (10(-7) M), and conditions that minimize physical damage. These granules were enriched approximately eightfold with respect to pepsinogen and were free from contamination by mitochondria and endoplasmic reticulum. Electrophoretic examination shows pepsinogen to account for approximately 80% of the Coomassie Blue-stainable intragranular protein and the granule membrane to yield a simple spectrum of proteins similar to other granule systems. In addition to purity, the isolated granules displayed a high degree of osmotic stability at physiologic conditions of pH, temperature, and ionic strength. This stability suggests strict regulation of the granule electrolyte transport pathways, which are shown to include a Cl- conductance, Cl-/anion exchange, and a K+ conductance. These transport systems in the granule membrane are consistent with the promotion of primary fluid secretion. Furthermore, granule-mediated ion transport would allow the chief cell to couple fluid secretion directly to exocytotic pepsinogen secretion and flush the enzyme from the base of oxyntic glands.

Animals↗
PubMed1988

Separation of cell organelles in density gradients based on their permeability characteristics.

The buoyant density of intracellular organelles is dependent in part on the nature of the buffer composition of the density gradient and the permeability characteristics of the organelle membrane to the constituents of this buffer. Therefore, knowledge of the transport properties of different organelles allows the design of density gradients useful for their purification. We have used this approach to significantly decrease mitochondrial contamination of pancreatic zymogen granules in a one-step purification procedure on a 40% Percoll density gradient. These gradients, prepared with isoosmotic sucrose, yield a narrow band of zymogen granules and mitochondria. However, by substitution of sucrose with salts to which mitochondria but not zymogen granules are permeable, the densities of mitochondria are altered to give a significant separation. For example, the incorporation of 100 mM sodium succinate in the Percoll gradient can produce a 70% reduction in mitochondrial contamination. The increased ionic strength has an additional beneficial effect on zymogen granule yield by 5-10%. The recognition and utilization of transport pathways in organelle membranes is the principal feature of this technique and should prove to be widely applicable to other isolation procedures.

Amylases↗
PubMed1988

Potassium transport by pancreatic and parotid zymogen granule membranes.

Zymogen granules that were stable at physiological conditions of pH, ionic strength, and temperature were isolated from the rat pancreas and parotid. The cation permeability of these granules was evaluated to characterize the mechanism of secretagogue-stimulated fluid secretion by acinar cells. Granule swelling and lysis provide a measure of the rate of cation transport, since the use of ionophore combinations such as tripropyltin and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) will render cation conductance the rate-limiting step for salt influx. This technique supplies evidence for the existence of K+ conductance in the granule membrane. The pancreatic and parotid granules have a K+-selective conductance that is not inhibited by the K+ channel blockers barium, tetraethylammonium, quinidine, cesium, or 4-aminopyridine. Furthermore, the intragranular pH of pancreatic zymogen granules was measured to be approximately 6.5 and was identified as a factor that modulates the K+ conductance. Although the pancreatic and parotid granules were qualitatively identical, quantitatively the relative K+ transport rate constant was over twofold higher for the parotid than for the pancreatic granules. The zymogen granule K+ conductance may have an important role in active K+ secretion by exocrine glands, which is prominent in the parotid after stimulation with beta-adrenergic agents.

Animals↗
PubMed1988

Secretagogues activate chloride transport pathways in pancreatic zymogen granules.

The membrane permeability of pancreatic zymogen granules was evaluated in vitro with granules isolated from rats in different secretory states: 1) untreated, 2) pretreated with a muscarinic antagonist, 3) pretreated with a muscarinic and an adrenergic antagonist, 4) pretreated as in 3 and then stimulated with the secretagogue cholecystokinin 4 min before death, and 5) pretreated as in 3 and then stimulated with the secretagogue secretin 4 min before death. Granules isolated from untreated rats had variable ionic permeabilities but in general possessed both chloride conductance and electroneutral exchange pathways with low permeabilities to alkali metal ions. In contrast, granules from animals pretreated with secretory antagonists had very low ion permeabilities to both inorganic anions, such as chloride, and alkali metal ions. Injection of the peptide secretagogues cholecystokinin or secretin resulted in a relatively fast (within 4 min) activation or induction of high chloride permeabilities through both chloride conductance and chloride/hydroxide (or chloride/bicarbonate) exchange pathways. In addition, the secretagogues increased the cation permeability of the granule membrane, which exhibited a distinct potassium selectivity. Chloride conductance has been postulated to play a major role in fluid secretion coupled to exocytosis of macromolecules [R. C. DeLisle and U. Hopfer, Am. J. Physiol. 250 (Gastrointest. Liver Physiol. 13): G489-G496, 1986]. These results demonstrate that granules may actively participate in the secretory process and suggest that some of the physiological targets in the cascade of events leading to secretion are anion and cation transporters in the zymogen granule membrane.

Acetylcholine↗
PubMed1986

Electrolyte permeabilities of pancreatic zymogen granules: implications for pancreatic secretion.

Zymogen granules from rat pancreas were prepared on a 40% Percoll gradient at free calcium levels less than 0.2 microM. We have previously shown [Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9)] that zymogen granules prepared by this method are stable in vitro for more than 1 h in "physiological buffers." The electrolyte permeabilities of the zymogen granule membrane were investigated to determine the basis for this stability. Ionic permeabilities were estimated from rates of osmotic lysis and measured as decrease in optical density (OD) of granule suspensions. OD correlated linearly with lysis, as indicated by release of amylase, except for the highest and lowest 10% of the OD of intact granules. Lysis of freshly isolated granules was slow in Na+ or K+ salt solutions (e.g., t1/2 approximately 3 h for Cl-) but was accelerated 5- to 50-fold when cation ionophores were present simultaneously. This behavior indicates that zymogen granules have low endogenous permeabilities to the cations Na+ and K+, but are highly permeable to a variety of anions. Both anion conductance and anion-exchange pathways were found. The relative selectivity of the anion conductance pathway was SCN- greater than Br- approximately NO-3 greater than SO2-(4) greater than acetate- approximately Cl- greater than isethionate-. The relative selectivity sequence for anion/-OH- exchange was acetate- greater than SCN- greater than Br- approximately NO-3 approximately Cl- much greater than isethionate- greater than SO2-(4). The anion transport blocker DIDS blocked the electrogenic pathway with a half-maximal effectiveness at approximately 2 microM. DIDS had little effect on the anion-exchange pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

Regulation of amiloride-sensitive electrogenic sodium transport in the rat colon by steroid hormones.

The role of steroids in the regulation of colonic sodium transport was examined by infusing steroids into adrenalectomized (ADX) rats and evaluating the short-circuit current (ISC) in vitro. Amiloride-sensitive ISC was induced by aldosterone and corticosterone with half-maximal doses (ED50) of 2 and 260 micrograms X kg-1 X h-1), respectively. Synthetic glucocorticoids such as methylprednisolone (33 mg/kg) and dexamethasone (ED50 = 30 micrograms X kg-1 X h-1) were also effective. Supramaximal doses of aldosterone (7.5 times ED50) for 24 h increased the total ISC (7-fold), the amiloride-sensitive ISC (366-fold), and the conductance (2-fold), as well as the potassium-stimulated phosphatase activity (2-fold) (reported previously). Compared with aldosterone, supramaximal doses of dexamethasone (4 times ED50) produced greater increases in the total ISC (15-fold) and the amiloride-sensitive ISC (674-fold). In contrast to aldosterone, dexamethasone also increased the amiloride-insensitive ISC (3-fold). Glucocorticoid action was not mediated by insulin since the ISC from diabetic ADX rats was increased by dexamethasone to a similar extent (11-fold) as in nondiabetic rats. Estradiol, progesterone, and testosterone did not stimulate the colonic ISC of ADX rats. The ED50 values of corticosterone and aldosterone, measured in terms of amiloride-sensitive sodium transport, produced serum levels that were slightly above those of unstressed, adrenal-intact animals and thus must be considered physiological. It is concluded that at physiological levels both steroids may mediate amiloride-sensitive sodium transport in the rat colon. However, as judged from changes in serum steroid levels, aldosterone is the physiological regulator of elevated sodium absorption in sodium deficiency.

Adrenalectomy↗
PubMed1985

Amiloride-sensitive salt and fluid absorption in small intestine of sodium-depleted rats.

Secondary hyperaldosteronism produced by Na+ depletion was associated with increases in salt and fluid absorption in both the small intestine and the distal colon but not in the cecum and the proximal colon. Because these changes had not been documented for the small intestine, this study focused on the regulation of this tissue. Increased NaCl and water absorption was expressed in vitro by increases in short-circuit current and transepithelial potential and in vivo by increased fluid absorption and a decreased luminal content of Na+ and water. For example, the short-circuit current in the ileum of Na+-depleted rats was 2-fold that of adrenalectomized and 1.3-fold that of adrenal-intact control animals. The short-circuit current was inhibitable 24 +/- 14% by micromolar concentrations of amiloride in Na+-deficient animals compared with 1 +/- 3% in control animals. Similarly, ileal fluid absorption in vivo was 2.3-fold higher in Na+-deficient relative to control animals. The additional fluid absorption was sensitive to 50 microM amiloride, whereas amiloride had no effect in control animals. Furthermore, the Na+ content of the chyme from the ileum of Na+-deficient animals was about half that of controls. These results suggest that mineralocorticoids can induce the amiloride-sensitive Na+ transporter in the small intestine and that this type of epithelial salt transport can become a major pathway for salt retention by the small intestine.

Adrenalectomy↗
PubMed1985

Regional specificity of iron uptake by small intestinal brush-border membranes from normal and iron-deficient mice.

Fe(II)-ascorbate uptake by purified small intestinal brush-border membrane vesicles prepared from proximal and distal segments was studied in normal and iron-deficient mice. Iron was maintained in a reduced, soluble form by a 20-fold excess of ascorbate at a physiological pH of 7.2-7.4. In normal mice, iron uptake by proximal membrane vesicles was three- to fourfold greater (approximately 1,700 pmol/mg prot) than from distal segments (approximately 500 pmol/mg prot). In iron-deficient mice, uptake of Fe(II) was also greater in proximal membranes (approximately 3,200 pmol/mg prot) than uptake from distal segments (approximately 350 pmol/mg prot), and the regional difference was almost 10-fold, without any change in distal segmental iron uptake. These results are consistent with the pattern of intestinal iron absorption in iron-replete and iron-deficient animals and indicate that regulatory changes in proximal intestinal brush-border membranes may account for the increased iron absorption known to occur in iron deficiency.

Animals↗
PubMed1985

Secondary active nutrient transport in membrane vesicles: theoretical basis for use of isotope exchange at equilibrium and contributions to transport mechanisms.

A detailed and quantitative analysis of secondary active transport mechanisms in membrane vesicles is complicated by heterogeneity of the vesicles. Functional heterogeneity can be demonstrated by the time-dependence of isotope exchange of any solute at equilibrium. The need for more than one rate constant in the fit proves functional heterogeneity. To treat the heterogeneity quantitatively, it is suggested to subject entire time curves of exchange to inverse Laplace transformations that yield the corresponding distribution of rate constants. The computer program CONTIN by Provencher (1982a, b, c) can be used to carry out such a transformation. The distribution of rate constants under a particular set of conditions can be used to calculate a highly reliable initial rate. In addition, for spherical vesicles a mean, surface area-averaged permeability constant can be calculated if the size distribution of the vesicle population is known by other measurements and this size distribution is independent of the permeability distribution. Kinetic measurements under equilibrium conditions on the rabbit intestinal Na-glucose transporter indicate (using Cleland's nomenclature) an ordered iso-bi-bi mechanism with glide symmetry for substrate and co-substrate binding to the transporter at one interface and release at the other (first-in-first-out) (Hopfer & Groseclose, 1980). The kinetics are consistent with a gated pore mechanism of coupled Na-glucose cotransport. A similar mechanism seems to hold for renal Na-lactate cotransport (Mengual et al., 1983).

Animals↗
PubMed1984

Iron transport across brush-border membranes from normal and iron-deficient mouse upper small intestine.

We have studied Fe(III)-citrate and Fe(II)-ascorbate uptake by purified intestinal brush-border membrane vesicles from normal (iron-replete) and iron-deficient mice. In iron-replete mice using a final Fe(III) concentration of 1.43 microM, 25-30 pmol of Fe(III)/mg of protein were bound to the membranes versus 65-70 pmol in iron-deficient mice. Fe(II) uptake in normal mice using a final Fe(II) concentration of 1.79 microM was 1600-1800 pmol/mg of protein versus 3600-4000 pmol in iron-deficient mice. Evidence that Fe(II) was transported into the vesicles by a membrane carrier-mediated process was obtained by observing saturation kinetics under conditions of isotope exchange at equilibrium in mice rendered iron-deficient, but not in iron-replete mice. Eighty per cent of the transported Fe(II) could be removed by strong chelating agents. The remainder was exchangeable with Fe(II) in the medium when measured under equilibrium conditions. We can explain these results by the following model; iron uptake appears to be a 2-fold process. The first step is the transport of Fe(II) across the membrane by a carrier-mediated process which is biologically regulated. The second step is the subsequent binding of iron on the inside of the membrane. The number of binding sites is also regulated by the iron status of the mouse. The membrane binding affinity for Fe(II) appears to be weaker than that for dithiothreitol but stronger than for ascorbate.

Animals↗
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↗