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M Gratzl

Publications and source records attributed to M Gratzl.

At least 91 records · Page 5Linked to original sources
PubMed1989

Expression of the neural cell adhesion molecule NCAM in endocrine cells.

We examined the expression of the neural cell adhesion molecule NCAM in a number of endocrine tissues of adult rat and in an endocrine tumor cell line. NCAM was found by immunoelectron microscopy to be present on the surface of all endocrine cells in the three lobes of the hypophysis, although staining was relatively less intense in the intermediate lobe, and in pancreatic islets. Pituicytes, hypophyseal glial cells, were also labeled for NCAM. A rat insulinoma cell line (RIN A2) also expressed NCAM as judged by immunocytochemistry. Analysis of NCAM antigenic determinants (Mr 180, 140, and 120 KD) revealed large variations in the relative proportions of NCAM polypeptides present in the different tissues. Although all tissues and cell lines expressed NCAM-140, NCAM-180 was not detected in the adenohypophysis, pancreas, or adrenal medulla, and NCAM-120 was found in none of the endocrine tissues or cell lines except at low levels in the neurohypophysis. The tumor cell line expressed significant levels of NCAM-180, which was most abundant in the neurohypophysis. These results show that NCAM expression appears to be a general property of endocrine cells, although the antigenic composition differs markedly from that in brain tissue. These data are discussed with regard to the embryological origins of the different endocrine tissues, and possible functional implications are suggested.

Animals↗
PubMed1988

Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles.

Chromaffin vesicles were first purified by differential and density gradient centrifugation in isotonic (Percoll) gradients. In subsequent sucrose gradients p38/synaptophysin exhibited the same distribution as established marker substances of chromaffin vesicles. Quantification of immunoblots revealed that 750 ng p38/synaptophysin per mg of protein were present in the chromaffin vesicles recovered from the sucrose gradient. Thus the amount of p38/synaptophysin per mg protein of chromaffin vesicles is about 100 times lower than that observed in clear (synaptic) vesicles. However, because of the large difference in surface area and protein content, the amount of p38/synaptophysin per single vesicle is the same in both types of organelles.

Adrenal Medulla↗
PubMed1988

Cellular distribution and amount of chromogranin A in bovine endocrine pancreas.

We determined the cellular distribution and the amount of chromogranin A in endocrine cells of bovine pancreas using a polyclonal antibody against bovine adrenomedullary chromogranin A. The relative amounts of chromogranin A in the different cells of the endocrine pancreas were determined by computer-assisted analyses of the optical densities of the immunoreactivities in the stained sections. More than 80% of the immunoreactive chromogranin A was located in the pancreatic B-cells. In immunoblots of acid tissue extracts, only one chromogranin A band (MW 74 KD) was observed. Quantification of the immunoblots revealed that 3 micrograms of chromogranin A and 918 micrograms of insulin were present per gram pancreas (wet weight), equivalent to a molar ratio of 460 mumol chromogranin A per mol insulin.

Animals↗
PubMed1987

Ca2+-stimulated catecholamine release from alpha-toxin-permeabilized PC12 cells: biochemical evidence for exocytosis and its modulation by protein kinase C and G proteins.

Two possible cellular pathways of catecholamines from the chromaffin vesicles of PC12 cells to the surrounding medium are explored in this study. The direct one circumventing the cytoplasm can be activated in alpha-toxin-permeabilized cells with micromolar levels of free Ca2+. Catecholamine metabolites formed in the cytoplasm (i.e., 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylethanol) are neither formed nor released from the cells under these conditions. However, when vesicular catecholamines were discharged into the cytoplasm by addition of the ionophore nigericin, such metabolites are formed and released into the medium independent of Ca2+. Both types of experiments provide direct evidence for the operation of Ca2+-induced exocytosis of dopamine and noradrenaline in permeabilized PC12 cells. The Ca2+ dependence of dopamine or noradrenaline release, as measured by the determination of the endogenous catecholamines using the high-performance liquid chromatography technique, exhibits two different phases. One is already activated below 1 microM free Ca2+ and plateaus at 1-5 microM free Ca2+, while a second occurs in the presence of larger amounts of free Ca2+ (10-100 microM). Ca2+-induced catecholamine release from the permeabilized cells can be modulated in different ways: It is enhanced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate and the diacylglycerol 1-oleyl-2-acetylglycerol provided Mg2+/ATP is present, and it is inhibited by guanosine 5'-O-(3-thiotriphosphate). The latter effect is abolished by pretreatment of the cells with pertussis toxin but not by cholera toxin. Thus, it appears that Ca2+-induced exocytosis can be modulated via the protein kinase C system, as well as via GTP binding proteins.

Adrenal Gland Neoplasms↗
PubMed1987

Endocrine cells share expression of N-CAM with neurones.

The expression of the neural cell adhesion molecule, N-CAM, was examined in the anterior lobe of rat hypophysis by immunocytochemistry at light and electron microscope levels. In addition, N-CAM antigenic determinants present in adrenal medulla, anterior hypophysis and PC12 cells were compared by immunoblotting with those found in cerebellum. All secretory cells in the anterior hypophysis were found to be N-CAM positive on their surfaces, but not all of the three polypeptide determinants typical of cerebellum were present in the endocrine tissues or cell line tested. In addition, a new N-CAM determinant of 49 kDa not present in cerebellum was found in adrenal medulla and hypophysis, although it was absent from PC12 cells. The possible implications of these data are discussed.

Adrenal Medulla↗
PubMed1987

Immunological characterization of chromogranins A and B and secretogranin II in the bovine pancreatic islet.

Antisera against chromogranin A and B and secretogranin II were used for analysing the bovine pancreas by immunoblotting and immunohistochemistry. All three antigens were found in extracts of fetal pancreas by one dimensional immunoblotting. A comparison with the soluble proteins of chromaffin granules revealed that in adrenal medulla and in pancreas antigens which migrated identically in electrophoresis were present. In immunohistochemistry, chromogranin A was found in all pancreatic endocrine cell types with the exception of most pancreatic polypeptide-(PP-) producing cells. For chromogranin B, only a faint immunostaining was obtained. For secretogranin II, A- and B-cells were faintly positive, whereas the majority of PP-cells exhibited a strong immunostaining for this antigen. These results establish that chromogranins A and B and secretogranin II are present in the endocrine pancreas, but that they exhibit a distinct cellular localization.

Adrenal Medulla↗
PubMed1987

Purification of alpha-toxin from Staphylococcus aureus and application to cell permeabilization.

Crude alpha-toxin was produced by Staphylococcus aureus, strain Wood 46. The amount of exotoxin was monitored during growth and all subsequent purification steps by determination of its hemolytic activity against rabbit erythrocytes. The culture supernatant was treated with ammonium sulfate (75% saturation). The resulting precipitate was dialyzed and subjected to cation-exchange chromatography. The fractions containing the hemolytic activity were further purified by gel chromatography. The final product was enriched by a factor of 8.5 compared to the crude toxin. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified toxin exhibited one major band. It caused the release of 86Rb+ and ATP from rat insulinoma (RIN A2) as well as pheochromocytoma cells (PC12) in culture, indicating efficient permeabilization of their plasma membranes for small molecules.

Adenosine Triphosphate↗
PubMed1987

Further characterization of dopamine release by permeabilized PC12 cells.

Rat pheochromocytoma cells (PC12) permeabilized with staphylococcal alpha-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC12 cells. Permeabilization with alpha-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC12 cells.

Adenosine Triphosphate↗
PubMed1986

Ca2+ binding to chromaffin vesicle matrix proteins: effect of pH, Mg2+, and ionic strength.

Recently we found that Ca2+ within chromaffin vesicles is largely bound [Bulenda, D., & Gratzl, M. (1985) Biochemistry 24, 7760-7765]. In order to explore the nature of these bonds, we analyzed the binding of Ca2+ to the vesicle matrix proteins as well as to ATP, the main nucleotide present in these vesicles. The dissociation constant at pH 7 is 50 microM (number of binding sites, n = 180 nmol/mg of protein) for Ca2+-protein bonds and 15 microM (n = 0.8 mumol/mumol) for Ca2+-ATP bonds. When the pH is decreased to more physiological values (pH 6), the number of binding sites remains the same. However, the affinity of Ca2+ for the proteins decreases much less than its affinity for ATP (dissociation constant of 90 vs. 70 microM). At pH 6 monovalent cations (30-50 mM) as well as Mg2+ (0.1-0.5 mM), which are also present within chromaffin vesicles, do not affect the number of binding sites for Ca2+ but cause a decrease in the affinity of Ca2+ for both proteins and ATP. For Ca2+ binding to ATP in the presence of 0.5 mM Mg2+ we found a dissociation constant of 340 microM and after addition of 35 mM K+ a dissociation constant of 170 microM. Ca2+ binding to the chromaffin vesicle matrix proteins in the presence of 0.5 mM Mg2+ is characterized by a Kd of 240 microM and after addition of 15 mM Na+ by a Kd of 340 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1986

Characterization of hormone and protein release from alpha-toxin-permeabilized chromaffin cells in primary culture.

Addition of Staphylococcus aureus alpha-toxin to adult bovine chromaffin cells maintained in primary culture causes permeabilization of cell membrane as shown by the release of intracellular 86Rb+. The alpha-toxin does not provoke a spontaneous release of either catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However the addition of micromolar free Ca2+ concentration induced the co-release of noradrenaline and chromogranin A. In alpha-toxin-treated cells, the released chromogranin A could not be sedimented and lactate dehydrogenase was still associated within cells, which provides direct evidence that secretory product is liberated by exocytosis. By contrast, permeabilization of cells with digitonin caused a Ca2+-dependent but also a Ca2+-independent release of secretory product, a dramatic loss of lactate dehydrogenase, as well as release of secretory product in a sedimentable form. Ca2+-dependent exocytosis from alpha-toxin-permeabilized cells required Mg2+-ATP and did not occur in the presence of other nucleotides. Thus alpha-toxin is a convenient tool to permeabilize chromaffin cells, and has the advantage of keeping intracellular structures, specifically the exocytotic machinery, intact.

Adenosine Triphosphate↗
PubMed1986

Na+/Ca2+ exchange in coated microvesicles.

Coated microvesicles isolated from bovine neurohypophyses could be loaded with Ca2+ in two different ways, either by incubation in the presence of ATP or by imposition of an outwardly directed Na+ gradient. Na+, but not K+, was able to release Ca2+ accumulated by the coated microvesicles. These results suggest the existence of an ATP-dependent Ca2+-transport system as well as of a Na+/Ca2+ carrier in the membrane of coated microvesicles similar to that present in the membranes of secretory vesicles from the neurohypophysis. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ of the ATP-dependent uptake was 0.8 microM. The average Vmax. was 2 nmol of Ca2+/5 min per mg of protein. The total capacity of microvesicles for Ca2+ uptake was 3.7 nmol/mg of protein. Both nifedipine (10 microM) and NH4Cl (50 mM) inhibited Ca2+ uptake. The ATPase activity in purified coated-microvesicles fractions from brain and neurohypophysis was characterized. Micromolar concentrations of Ca2+ in the presence of millimolar concentrations of Mg2+ did not change enzyme activity. Ionophores increasing the proton permeability across membranes activated the ATPase activity in preparations of coated microvesicles from brain as well as from the neurohypophysis. Thus the enzyme exhibits properties of a proton-transporting ATPase. This enzyme seems to be linked to the ion accumulation by coated microvesicles, although the precise coupling of the proton transport to Ca2+ and Na+ fluxes remains to be determined.

Animals↗
PubMed1986

Chromogranins, widespread in endocrine and nervous tissue, bind Ca2+.

The proteinaceous components of the secretory vesicle contents isolated from bovine adrenal medulla bind Ca2+ (number of binding sites, 152 +/- 52 nmol Ca2+ per mg protein; dissociation constant, 54 +/- 8 microM (n = 5)). SDS-polyacrylamide gel electrophoresis and 45Ca2+ binding of the proteins following their separation and blotting on nitrocellulose revealed that Ca2+ binds to chromogranins. Moreover, it was shown that the chromogranins, like other known Ca2+-binding proteins, can be specifically stained with a cationic carbocyanine dye. The Ca2+-binding function of the chromogranins described here, in conjunction with recent findings concerning Ca2+ transport across chromaffin vesicle membranes and the widespread distribution findings concerning Ca2+ transport across chromaffin vesicle membranes and the widespread distribution of chromogranins in many different endocrine and nerve cells, points to the general importance of these proteins in the metabolism of Ca2+.

Adrenal Medulla↗
PubMed1986

Chromogranin A in the pancreatic islet: cellular and subcellular distribution.

Chromogranin A (CGA) is the major soluble protein within secretory vesicles of chromaffin cells. A polyclonal antiserum was raised against bovine CGA and characterized in two-dimensional immunoblots. Cellular and subcellular distribution of CGA in bovine pancreatic islet was investigated by immunocytochemistry. At the light microscopic level, CGA-like immunoreactivity was found in the same cells that react with antibodies against insulin, glucagon, and somatostatin. A minority of cells containing pancreatic polypeptide also showed faint immunostaining. At the ultrastructural level (protein A-gold technique), CGA-like immunoreactivity was confined exclusively to the secretory vesicles. Whereas the hormones were localized mainly in the central part of the secretory vesicles, CGA was present predominantly in the periphery. These findings indicate that a CGA-like protein is a regular constituent of the matrix of secretory vesicles in pancreatic endocrine cells.

Animals↗
PubMed1985

Matrix free Ca2+ in isolated chromaffin vesicles.

Isolated secretory vesicles from bovine adrenal medulla contain 80 nmol of Ca2+ and 25 nmol of Mg2+ per milligram of protein. As determined with a Ca2+-selective electrode, a further accumulation of about 160 nmol of Ca2+/mg of protein can be attained upon addition of the Ca2+ ionophore A23187. During this process protons are released from the vesicles, in exchange for Ca2+ ions, as indicated by the decrease of the pH in the incubation medium or the release of 9-aminoacridine previously taken up by the vesicles. Intravesicular Mg2+ is not released from the vesicles by A23187, as determined by atomic emission spectroscopy. In the presence of NH4Cl, which causes the collapse of the secretory vesicle transmembrane proton gradient (delta pH), Ca2+ uptake decreases. Under these conditions A23187-mediated influx of Ca2+ and efflux of H+ cease at Ca2+ concentrations of about 4 microM. Below this concentration Ca2+ is even released from the vesicles. At the Ca2+ concentration at which no net flux of ions occurs the intravesicular matrix free Ca2+ equals the extravesicular free Ca2+. In the absence of NH4Cl we determined an intravesicular pH of 6.2. Under these conditions the Ca2+ influx ceases around 0.15 microM. From this value and the known pH across the vesicular membrane an intravesicular matrix free Ca2+ concentration of about 24 microM was calculated. This is within the same order of magnitude as the concentration of free Ca2+ in the vesicles determined in the presence of NH4Cl.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗
PubMed1985

Minimal requirements for exocytosis. A study using PC 12 cells permeabilized with staphylococcal alpha-toxin.

The membrane-permeabilizing effects of streptolysin O, staphylococcal alpha-toxin, and digitonin on cultured rat pheochromocytoma cells were studied. All three agents perturbed the plasma membrane, causing release of intracellular 86Rb+ and uptake of trypan blue. In addition, streptolysin O and digitonin also damaged the membranes of secretory vesicles, including a parallel release of dopamine. In contrast, the effects of alpha-toxin appeared to be strictly confined to the plasma membrane, and no dopamine release was observed with this agent. The exocytotic machinery, however, remained intact and could be triggered by subsequent introduction of micromolar concentrations of Ca2+ into the medium. Dopamine release was entirely Ca2+ specific and occurred independent of the presence or absence of other cations or anions including K+ glutamate, K+ acetate, or Na+ chloride. Ca2+-induced exocytosis did not require the presence of Mg2+-ATP in the medium. The process was insensitive to pH alterations in the range pH 6.6-7.2, and appeared optimal at an osmolarity of 300 mosm/kg. Toxin permeabilization seems to be an excellent method for studying the minimal requirements for exocytosis.

Adrenal Gland Neoplasms↗
PubMed1985

Alpha-toxin permeabilized rat pheochromocytoma cells: a new approach to investigate stimulus-secretion coupling.

The channel forming alpha-toxin of Staphylococcus aureus (about 50 micrograms/ml) markedly reduces the Ca2+ requirement for dopamine release by the rat pheochromocytoma cell line (PC 12). Maximal secretion by intact cells requires approximately 1 mM Ca2+, whereas release by alpha-toxin-permeabilized cells can already be triggered with microM concentrations of Ca2+. The latter process reaches a plateau at about 1 microM free Ca2+ and increases again with 10-20 microM free Ca2+. The sensitivity to low concentrations of Ca2+ indicates that the toxin, as a selective cell membrane permeabilizing agent, can be used as a powerful instrument to study stimulus-secretion coupling.

Adrenal Gland Neoplasms↗
PubMed1984

Distribution of chromaffin secretory vesicles, acetylcholinesterase, and lysosomal enzymes in sucrose and Percoll gradients.

Crude chromaffin secretory vesicles, obtained by differential centrifugation, were further purified on isotonic (Percoll) gradients. The chromaffin vesicle fractions recovered from the gradients contain acetylcholinesterase as well as lysosomal enzymes. With the aid of a subsequent sucrose gradient lysosomal enzymes could be removed from chromaffin vesicle fractions, but not acetylcholinesterase. This suggests that lysosomal enzymes do not pass through the chromaffin vesicles during the biogenesis of lysosomes but acetylcholinesterase does.

Acetylcholinesterase↗