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Biomedical subjects

M Schramm

Publications and source records attributed to M Schramm.

At least 127 records · Page 7Linked to original sources
PubMed1977

[Local contractility and acidosis in the ischemic myocardium of the dog (author's transl)].

In open chest dogs up to 8 side branches of the R, circumflexus and descendens were embraced and could selectively be occluded. With a new developed method local myocardial length changes in the ischemic areas, and with H+-sensitive minielectrodes the interstitial H+-activities were measured. All changes which were observed during the 3-30 min lasting coronary artery occlusions (H+-increases to about 500 neq/1, increases of diastolic length, decreases of the contraction amplitude, ST-elevations) returned to preocclusion levels during reperfusion. LVP and dp/dt remained unchanged during ischemia and reperfusion.

Animals↗
PubMed1976

Coupling of catecholamine receptor from one cell with adenylate cyclase from another cell by cell fusion.

The experiments test the hypothesis that beta-adrenergic receptor is an independent unit that can be transferred from one adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4-6-1-1[ system to another. Turkey erythrocytes in which the catalytic activity of adenylate cyclase had been inactivated by N-ethylmaleimide or by heat contributed the beta-adrenergic receptor. Friend erythroleukemia cells (F cells) that possessed no measurable beta-adrenergic receptor contributed the adenylate cyclase. The erythrocytes in which the enzyme had been inactivated were fused with the F cells by Sendai virus. The cell ghosts of the fused preparation demonstrated adenylate cyclase activity which was strikingly enhanced by isoproterenol. Controls of fusion of F cells with each other or with human erythrocytes failed to show a response to isoproterenol. It was therefore concluded that the beta-adrenergic receptor of the turkey erythrocytes must have become functionally coupled to the adenylate cyclase of the mouse F cells. Activation by isoproterenol was demonstrable within a few minutes after fusion, and inhibitors of protein synthesis had no effect. Thus, coupling must have occurred between the preexisting components. The findings suggest that it may be possible in the future to confer on cells that possess an adenylate cyclase system new hormonal responses by inserting a receptor into their cell membrane. It is proposed that the procedure of massive heterologous cell fusion, as used in the present study, can be used to analyze the function of other cell membrane components.

Adenylyl Cyclases↗
PubMed1976

Secretion of old versus new exportable protein in rat parotid slics. Control by neurotransmitters.

The possibility that old and new secretory granules do not mix and that older exportable protein can be secreted preferentially was tested on parotid gland in vitro. Slices from fasted animals were pulse labeled for 3 min with L-[3H]leucine. Subcellular fractionstion showed that after 1 90-min chase period, the formation of new labeled secretory granules was mostly completed. The ratio of label in secretory granules to label in microsomes increased 250-fold during the period 5--90 min postpulse. After the 90-min chase, a submaximal rate of secretion was initiated by adding a low concentration of isoproterenol to the slices. Preferential secretion of old unlabeled exportable protein was evident from the finding that the percent of total amylase secreted was 3.5-fold greater than the percent of labeled protein secreted. Preferential secretion of old unlabeled exportable amylase was undiminished even when the chase period before addition of isoproterenol was extended to 240 min. Such long chase incubations were still meaningful due to the fact that the spontaneous rat of amylase release and radioactive protein release from the slices was negligibly low. A high isoproterenol concentration added to the slices after a 90-min chase produced the following results. An initial phase of preferential secretion of old unlabeled protein was soon replaced by secretion of a random mixture of new and old exportable protein. Electron micrographs indicated that high rates of secretion involved sequential fusion of secretory granules so that the lumen extended deep into the cell where the new labeled granules were presumably located. At low rates of secretion, the lumen showed no such deep extensions. Experiments were also conducted on slices from glands which had been largely depleted of old granules by prior injection of isoproterenol into the animals. Secretion of labeled protein from such slices stopped with the export of 80% of the labeled protein. This finding indicates that about 20% of the radioactive protein is cellular nonexportable protein and that the slices are capable of exporting the entire amount of secretory protein which was symthesized in vitrol. In addition to the beta-adrenergic receptor which mediates protein secretion, the parotid acinar cell also possesses an alpha-adrenergic and a cholinergic receptor both of which cause K+ release, vacuole formation, and water secretion. Activation of either of the latter two receptors in conjunction with the beta-adrenergic receptor increased randomization of the protein secreted. It is concluded that in the rat parotid acinar cell there is little spontaneous mixing between old granules near the luminal cell membrane and new granules coming up behind from the Golgi complex. The neurotransmitters which induce secretion produce the observed randomization.

Amylases↗
PubMed1975

The proteins of the content of the secretory granules of the rat parotid gland.

The proteins of the secretory granules of the rat parotid gland were characterized by sodium dodecylsulfate gel electrophoresis, by chromatography of [3-H]proline-labeled proteins on DEAE-cellulose and by amino acid analysis. Sodium dodecylsulfate gel electrophoresis of the secretory granule content showed five principal proteins and a limited number of minor components. Only two of the principal bands could be identified as known secretory enzymes of the parotid gland. One was identified as the alpha-amylase and one as deoxyribonuclease. Peroxidase and ribonuclease form minor portions of the secretory proteins. The other three major proteins constitute, together, about 60% by weight, of the secretory granule content proteins. Of these, one which represents more than 30% of the total granule protein was found to contain uniquely high amounts of leucine residues (21 mole%). Another one of these principal proteins was relatively rich in cysteine residues (7 mole%). The fifth principal protein was found to contain high amounts of proline (28 mole%) glutamic acid (17 mole%) and glycine (18 mole%) residues. Its amino acid composition was very similar to that of the proline-se granules. This protein, however, differed from the "membranous" proline-rich proteins by several criteria. Two minor glycoproteins of the secretory granule content were also found to be rich in proline residues (37 mole%). As with the other proline-rich proteins of the granule, they contained no sulphur-containing amino acids, stained faintly pink with Coomassie Blue and were underestimated by the Lowry method. They differ however, from all the other proline-rich proteins of the granule by having a significantly higher content of threonine, less glycine (9 mole%) and much less glutamic acid (3 mole%). Of the principal proteins, only the deoxyribonuclease and the half-cystine-rich proteins were positively stained by periodic acid Schiff staining. The possible functions of the leucine-rich, the half cystine-rich and the various proline-rich proteins are discussed.

Amino Acids↗
PubMed1975

A persistent active state of the adenylate cyclase system produced by the combined actions of isoproterenol and guanylyl imidodiphosphate in frog erythrocyte membranes.

Isoproterenol plus guanylyl imidodiphosphate (Gpp(NH)p) activate frog erythrocyte adenylate cyclase to a level much higher than the sum of the activities produced by the catecholamine and the synthetic nucleotide tested separately. Propranolol, the beta-receptor blocking agent, failed to inhibit activity when added after the enzyme system had been preincubated with both isoproterenol and Gpp(NH)p. However, if propranolol was added after only one of the two components had been added, it inhibited the effect of isoproterenol. Production of the propranolol-resistant state by treatment with isoproterenol and Gpp(NH)p did not require the presence of the productive substrate (MgATP). The activated propranolol-resistant state persisted following various treatments of the enzyme preparation including extensive washings of the membranes; considerable activity was retained even after sonication or treatment with the detergent Lubrol-PX, treatments which caused inactivation of the native enzyme. Extensive dilution of the membranes following pretreatment with isoproterenol and Gpp(NH)p did not significantly reduce to the activity of the enzyme. Readdition of isoproterenol after dilution caused some inhibition of adenylate cyclase activity, indicating apparently that the beta-receptor has not become inaccessible. In contrast, preincubation with isoproterenol alone failed to render the enzyme system refractive to propranolol, and dilution readily reduced the activity to negligibly low values. Preincubation with Gpp(NH)p alone also produced a persistent active state but the activity was much lower than that obtained throught the combined action of isoproterenol and Gpp(NH)p. The findings suggest that the hormone may be required only to facilitate the initial interaction of the enzyme with Gpp(NH)p. The differences, in this respect, between Gpp(NH)p and the more labile natural nucleotide, GTP, are discussed.

Adenosine Triphosphate↗
PubMed1975

Non-parallel transport of membrane proteins and content proteins during assembly of the secretory granule in rat parotid gland.

The insertion of newly synthesized protein molecules into the membrane of the secretory granule of the rat parotid gland was studied by in vivo labeling with [3-H]-proline and [3-H]leucine. 2 h after the injection of the amino acid into the rat, the membrane fraction isolated from the secretory granules was found to be highly labeled with proline but only slightly labeled with leucine. The ratio of proline label in the granule membrane to that in the granule's secretory content was roughly equivalent to the ratio of total proline in the proteins of these two fractions. In contrast the ratio of leucine label in the membrane to that in the secretory content was much less than would be expected from the relative amount of leucine in both fractions. Separation of the proteins of the granule membrane by gel electrophoresis in presence of sodium dodecylsulfate showed that a considerable amount of these proteins was unlabeled. The labeled proteins could be selectively extracted from the membrane by 0.15 M Nacl solution or by dilute buffer at pH 4.5. These extracted proteins were found to contain a high proportion of proline residues and a negligible amount of leucine residues. In the extract proline constituted 36 mole % of the total amino acids. Proline plus glycine plus glutamic acid constituted more than 80 mole % and leucine constituted about 1 mole% of the total amino acids. Further analyses by gel electrophoresis in presence of sodium dodecylsulfate showed that the fractions of secretory granule membrane and secretory granule content are relatively free of contamination by proteins from other subcellular structures. It is suggested that the proteins which will constitute the mature secretory granule are transported to the site of final assembly by two pathways. The proline-rich proteins are transported to the site of assembly in close coordination with all the exportable proteins. The other membrane proteins arrive by a different pathway. Two alternative mechanisms are suggested to explain the finding that a considerable part of the membrane proteins are not labeled. I. The pathway of the intracellular transport of the unlabeled membrane proteins is similar to that of the secretory proteins but the newly synthesized membrane protein molecules are diluted in a large intermediate pool--the GOLgi complex. II. The proteins that did not get labeled are derived by a process of reutilization, from membranes of granules which have previously discharged their content in the process of secretion.

Amino Acids↗
PubMed1975

Fatty acids as modulators of membrane functions: catecholamine-activated adenylate cyclase of the turkey erythrocyte.

Activation of the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1[ from turkey erythrocytes by isoproterenol decreased precipitously below 26 degrees. Certain unsaturated fatty acids enhanced the activation by isoproterenol up to 25-fold at reduced temperatures. The fatty acid also enhanced the formation of a persistent active state of the enzyme which was produced by preincubation with guanosine 5'-(beta,gamma-imino)triphosphate [Gpp(NH)p]. Once the enzyme had been activated by Gpp(NH)p plus isoproterenol the reaction rate was no longer as temperature sensitive and the fatty acid had little effect. The synthetic Gpp(NH)p apparently substituted for the natural GTP, which is known to play a regulatory role in the adenylate cyclase system. The findings suggest that the function of GTP which is mediated by the hormone is the temperature-sensitive event which is enhanced by the fatty acid. The use of free fatty acid to probe membrane-associated reactions in intact cells and in isolated membrane preparations is proposed.

Adenylyl Cyclases↗
PubMed1975

The catecholamine-responsive adenylate cyclase system and its modification by 5'guanylylimidodiphosphate.

The frog erythrocyte adenylate cyclase system was inhibited by addition of acidic phospholipids. The ratio of activity produced by isoproterenol to that produced by norepinephrine was not significantly changed by addition of various phospholipids and lipid extracts. Addition of isoproterenol plus Gpp(NH)p to the frog erythrocyte membranes produced an adenylate cyclase activity much higher than the sum of activities produced by these reagents when tested separately. The same high rate was still obtained after preincubation of the enzyme with isoproterenol plus Gpp(NH)p followed by exhaustive washing of the preparation. ATP and Mg++ were not required for the preincubation. When isoproterenol alone was preincubated with the enzyme, a persistent active state was not produced; dilution or washing readily reduced the activity to the low basal level. Formation of the persistent active state by preincubation with Gpp(NH)p plus isoproterenol was prevented by the beta-receptor blocking agent propranolol which reduced the activity to that obtained by Gpp(NH)p alone. However, propranolol completely failed to inhibit the activity if added after the enzyme had been preincubated with the catecholamine plus the nucleotide. Propranolol readily blocked the activity when added to enzyme which had been preincubated with isoproterenol alone. The findings lead to the tentative conclusion that the hormone acts by facilitating the action of Gpp(NH)p and that, having done so, the hormone perhaps plays no further role. It is further suggested that in the case of the natural nucleotide GTP, the hormone is required continuously for activation in order to maintain GTP at a regulatory site.

Adenylyl Cyclases↗
PubMed1975

The functions of cyclic AMP and calcium as alternative second messengers in parotid gland and pancreas.

Biochemical and ultrastructural studies of rat parotid gland slices have led to the identification of alpha- and beta-adrenergic receptors and a cholinergic receptor, all operating within the same secretory cell. While cyclic AMP serves as the second messenger in the beta-adrenergic response of enzyme secretion, Ca++ serves as the second messenger in the alpha-adrenergic and in the cholinergic responses which both lead to K+ release and water secretion. Ca++ also serves as a second messenger for the muscarinic cholinergic receptor in rat pancreas slices in which it causes enzyme secretion. Analysis of this information leads to the conclusion that neither the neurotransmitter, nor the receptor, nor the second messenger are unique for a certain type of response. The latter seems to be dictated by a component of the specific response pathway which is affected by the second or a subsequent messenger. By having different neurotransmitters operate the same response and a single neurotransmitter operate different responses diversity of control is achieved.

Animals↗
PubMed1974

A calcium ionophore simulating the action of epinephrine on the alpha-adrenergic receptor.

Calcium acting through the ionophore A-23187 caused massive K(+) release from rat parotid slices. The ionophore thus simulates the action of epinephrine on the alpha-adrenergic receptor in this system. The alpha-adrenergic and cholinergic receptors are, however, not involved in the action of the ionophore since phetolamine and atropine had no effect on the ionophore-induced K(+) release. Amylase secretion, which is induced by epinephrine through the beta-adrenergic receptor but does not require Ca(++) in the medium, was only slightly increased by the ionophore in the presence of Ca(++). The K(+) released by Ca(++) through the ionophore was regained by the cells upon chelation of Ca(++) with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Similar to the action of epinephrine on the alpha-adrenergic receptor, the K(+) release induced by the ionophore is specific for Ca(++), which cannot be substituted by 2.5 mM Sr(++), Mg(++), or Ba(++). The results suggest that epinephrine, like the ionophore, introduces Ca(++) into the cell to mediate K(+) release. It is proposed that the ionophore A-23187 may be an effective probe to test whether other Ca(++)-requiring hormones and neurotransmitters act in a similar fashion to produce their diverse physiological responses.

Acetates↗