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Biomedical subjects

H B Pollard

Publications and source records attributed to H B Pollard.

At least 145 records · Page 8Linked to original sources
PubMed1985

Purification of synexin by pH step elution from chromatofocusing media in the absence of ampholytes.

Synexin can be purified to virtual homogeneity by a modification of the conventional chromatofocusing technique. Ampholytes are omitted from the procedure altogether and the protein is eluted by a specific pH step chosen on the basis of the pI of the protein. This modification of the conventional method eliminates the separation of ampholytes from the purified protein, an insurmountable problem in our case, and reduces the cost of purification profoundly.

Adrenal Medulla↗
PubMed1985

Real-time measurements of acetylcholine-induced release of ATP from bovine medullary chromaffin cells.

The luminescent oxidation of luciferin has been used to monitor acetylcholine-induced ATP release from cultured bovine chromaffin cells. Acetylcholine (1-100 microM) evoked ATP release of up to 30% of the total cellular ATP. This secretion required external free calcium and could also be elicited by K+-induced membrane depolarization. The size of the cytosolic ATP compartment was estimated as 5% of the ATP in the cell by solubilising the cell membrane using digitonin (20 microM) or by application to the cells of brief pulses (2 microseconds) of high electric field (2000 V/cm). Blockers of the voltage-gated Ca2+ channel effectively blocked K+-induced ATP release, while the acetylcholine antagonists d-tubocurarine and beta-bungarotoxin inhibited the acetylcholine-induced release of ATP. These data support the concept that ATP is released together with the catecholamines by exocytosis of chromaffin granule contents.

Acetylcholine↗
PubMed1985

The phorbol ester TPA enhances A23187--but not carbachol- and high K+-induced catecholamine secretion from cultured bovine adrenal chromaffin cells.

The effect of the phorbol ester TPA on catecholamine secretion was studied in cultured bovine adrenal chromaffin cells. The pretreatment of chromaffin cells with TPA caused the enhancement of catecholamine secretion induced by the calcium ionophore, A23187. By contrast, neither carbachol- nor high K+-induced secretion was changed by TPA pretreatment. These results support the concept that protein kinase C plays an important role as a factor transducing the Ca2+ signal to the exocytotic process of catecholamine secretion in bovine adrenal chromaffin cells.

Animals↗
PubMed1985

Characterization of protein kinase C and its role in catecholamine secretion from bovine adrenal-medullary cells.

Protein kinase C activity towards exogenous histone was detected in a cytosolic fraction of bovine adrenal medulla. The enzyme was dependent on Ca2+ and phosphatidylserine for its activity, with half-maximal activation being achieved at approx. 18 microM free Ca2+ and 8 micrograms of phosphatidylserine/ml. Both diolein and 4 beta-phorbol 12-myristate 13-acetate (TPA) decreased the Ca2+ requirement of the enzyme, half-maximal activation being obtained at approx. 12 microM and 9 microM free Ca2+ respectively in the presence of these agents. Many endogenous proteins in the adrenal-medullary cytosolic fraction were detected whose phosphorylation was dependent on the presence of both Ca2+ and phosphatidylserine. TPA stimulated catecholamine release from cultured bovine adrenal-chromaffin cells in a Ca2+-dependent manner. A23187 also stimulated catecholamine secretion, and at sub-optimal concentrations of TPA and A23187 a synergistic secretory response was obtained. These results are consistent with protein kinase C having a regulatory role in exocytosis in bovine adrenal chromaffin cells.

Adrenal Medulla↗
PubMed1985

Enhancement of Ca2+-induced catecholamine release by the phorbol ester TPA in digitonin-permeabilized cultured bovine adrenal chromaffin cells.

The phorbol ester, 4 beta-phorbol 12-myristate acetate (TPA), increased the extent of catecholamine release induced by Ca2+, without affecting the basal release response in digitonin-permeabilized chromaffin cells. This finding is consistent with the hypothesis that protein kinase C has a role to play in stimulus-secretion coupling in the bovine adrenal medullary chromaffin cell.

Adrenal Medulla↗
PubMed1985

Chromaffin granule-cytoskeleton interaction. Stabilization by F-actin of ATPase in purified chromaffin granule membranes.

The influence of cytoskeletal elements on the chromaffin granule function was studied using a model system consisting of purified granule membranes and F-actin. The membrane ATPase was partially inactivated by incubation at 37 degrees C, and this inactivation was prevented by adding F-actin. The stabilizing action of F-actin on the ATPase was abolished by adding DNase I. Detergent-solubilized ATPase was more rapidly and profoundly inactivated, but was not stabilized by F-actin. The stabilization of ATPase by F-actin may be due to the cross-linking of granule membranes with F-actin and the native structure of the granule membrane may be required for preserving the stability of membrane ATPase. These findings thus suggest the possibility that the interaction of microfilaments with chromaffin granules may influence the function of chromaffin granules within the cell.

Actins↗
PubMed1985

Synexin binds in a calcium-dependent fashion to oriented chromaffin cell plasma membranes.

Oriented plasma membrane fragments from chromaffin cells, isolated on polylysine-coated polyacrylamide beads, bind synexin in a calcium dependent manner. Synexin binding was also detected on beads coated with chromaffin granule membranes, but not to beads coated with erythrocyte membranes or to uncoated beads. Synexin binding to plasma membrane coated beads showed a specific requirement for calcium (K1 2 = 200 microM) and was insensitive to other divalent cations such as magnesium, strontium and barium. Synexin binding to either plasma membrane or granule membrane coated beads was saturable, was partially reversible by EGTA and was directly observed by SDS-polyacrylamide gel electrophoresis.

Adrenal Medulla↗
PubMed1985

Endothelial cells from bovine adrenal medulla develop capillary-like growth patterns in culture.

The endocrine barrier between chromaffin cells and the blood stream in the adrenal medulla is made of capillary endothelial cells. We have now succeeded in isolating endothelial cells from adrenal medullary tissue, which are probably derived from this barrier. These cells grow on plastic surfaces in the absence of special growth factors or collagen overlays and differentiate into organized structures quite similar to true capillaries. The cells contain factor VIII:R, a marker for endothelial cells, and form intercellular junctions characteristic of capillary endothelial cells. They also synthesize and secrete basal lamina structures and engage in transcytosis, a characteristic ultrastructural and functional combination of exocytosis and endocytosis across the thin endothelial cell processes. These endothelial cells can take up and deaminate catecholamines by A-type monoamine oxidase, an enzyme functionally distinct from the B-type monoamine oxidase found in chromaffin cells. These data indicate that the chromaffin cell and its endothelial cell neighbor may constitute the functional unit of catecholamine metabolism in the adrenal medulla.

Adrenal Medulla↗
PubMed1985

Glucocorticoid receptors and regulation of phenylethanolamine-N-methyltransferase activity in cultured chromaffin cells.

Glucocorticoids are known to regulate the enzyme phenylethanolamine-N-methyltransferase (PNMT) in the adrenal medulla of the rat and are thereby thought to control the synthesis of epinephrine. We have examined the details of this relationship in a simplified system, chromaffin cell primary cultures derived from bovine adrenal medulla. Cultured chromaffin cells were found to have a cytosolic, high affinity, saturable glucocorticoid-binding protein with the steroid specificity of a classical glucocorticoid receptor and a Kd of approximately 1 nM. Treatment of cultured cells with dexamethasone or hydrocortisone at any time up to 21 days in culture increased PNMT activity in the soluble fraction of the cell. The concentration of hormone required to produce a half-maximal response was 10 nM dexamethasone when cells were cultured in the presence of 5% fetal calf serum, or 1 nM in a defined serum-free medium. These dose-response relationships are consistent with mediation of this effect by the glucocorticoid receptor. Unexpectedly, however, the glucocorticoid-induced increment in PNMT activity was not inhibited by cycloheximide at concentrations up to 50 microM, and an acceleration of protein synthesis by insulin treatment did not augment the glucocorticoid effect on PNMT. Treatment of the cells with dexamethasone (100 microM) prevented the decline in the epinephrine-to-norepinephrine ratio seen over time in culture, an effect consistent with increased PNMT activity. However, there was no effect of dexamethasone on the ability of the cells to secrete catecholamines in response to stimulation with high KCl or 30 microM nicotine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

A rapid and simplified assay method for tyrosine hydroxylase.

Tyrosine hydroxylase can be measured by release of tritiated water from labeled tyrosine, and the assay method has now been modified to allow recovery of 3H2O from the reaction mixture in a much more rapid and less tedious manner than previously possible. In the new method, the tyrosine hydroxylase reaction is stopped with sodium carbonate, pH 11.6. At this pH the tritium in 3H2O, but not other 3H species, is extracted into an organic scintillant containing 25% isoamyl alcohol, toluene, 2,5-diphenyloxazole, and p-bis-[2-(5-phenyloxazolyl)]benzene. The selective extraction occurs by means of exchange of tritium in 3H2O with the hydroxyl proton of isoamyl alcohol. It is the [3H]isoamyl alcohol that is then extracted into the scintillant and quantified by liquid scintillation spectrometry. Although the organic extraction method is somewhat less sensitive than the more frequently used ion-exchange method for isolating the 3H2O formed in the tyrosine hydroxylase reaction, it is much more rapid, as well as cost effective, since the enzyme reaction, extraction, and counting are carried out within the same vial.

Adrenal Gland Neoplasms↗
PubMed1984

Isolated chromaffin cells from adrenal medulla contain primarily monoamine oxidase B.

Cultured chromaffin cells from bovine adrenal medulla were found to contain primarily the B form of monoamine oxidase. This monoamine oxidase B enzyme was somewhat distinct from B enzymes from other sources, in that noradrenaline was a much poorer substrate than serotonin. Nonetheless, studies with selective inhibitors of the A form (clorgyline) and the B form [(-)-deprenyl] confirmed that chromaffin cell monoamine oxidase was the B form. The observation that chromaffin cell monoamine oxidase has poor affinity for catecholamines is consistent with physiological needs that require the cell to synthesize and store large amounts of catecholamines.

Adrenal Medulla↗
PubMed1984

An osmotic mechanism for exocytosis from dissociated chromaffin cells.

Dissociated chromaffin cells from bovine adrenal medulla were stimulated to secrete epinephrine and dopamine beta-hydroxylase with a variety of secretagogues in a study designed to test the hypothesis that the chemiosmotic lysis reaction of isolated chromaffin granules might in some way be related to the mechanism of release during exocytosis. Increasing the osmotic strength of the incubation medium with either NaCl or sucrose led to suppression of secretion of epinephrine from the cells regardless of whether secretion was induced with veratridine or acetylcholine. Suppression of secretion was approximately exponential with respect to osmotic strength. Epinephrine secretion occurred only if the medium contained a permeant anion such as chloride, and secretion induced by veratridine was suppressed when Na isethionate replaced NaCl in the medium. In an extensive study with different monovalent anions veratridine supported epinephrine secretion according to the following activity series: Br-, I-, NO3- greater than methylsulfate, SCN- greater than Cl greater than acetate much greater than isethionate. A similar series, except for the potency of NO3-, was observed with A23187 as agonist. In general, the anion series for granule lysis was analogous. However, there was a poor quantitative correlation between the anion dependence of chemiosmotic granule lysis and the anion dependence of cell secretion. Anion transport inhibitors such as probenecid and pyridoxal phosphate also inhibited secretion while the stilbene disulfonates were inactive. The ineffectiveness of the stilbene disulfonates further distinguished chemiosmotic granule lysis from cell secretion. Secretion of catecholamines, induced by veratridine or nicotine, a cholinergic agonist, was suppressed when NaCl in the medium was replaced by isosmotic sucrose and unexpectedly low levels of dopamine beta-hydroxylase were observed in some cases. In sum, these properties of secreting chromaffin cells resembled some properties of isolated chromaffin granules incubated in ATP and Cl-, but were different in a number of instances. We, therefore, have interpreted our data to indicate that while some mechanistic relationships may indeed exist between the release event in exocytosis from chromaffin cells and the chemiosmotic lysis reaction characteristic of isolated chromaffin granules, an understanding of the energetics of exocytosis awaits the discovery of reasons for the quantitative differences between the two systems.

Adrenal Medulla↗
PubMed1983

Differential control of insulin secretion and somatostatin-receptor recruitment in isolated pancreatic islets.

Somatostatin receptors appear to be localized to secretory granules in pancreatic islet homogenates. Recruitment of these receptors to the islet-cell surfaces may mark the contact event between secretory granules and plasma membranes before release of insulin by fission. Isethionate, an impermeant anionic replacement for chloride, blocks the release step but does not affect receptor recruitment. By contrast, low concentrations of phenothiazine drugs, such as trifluoperazine and promethazine, inhibit both receptor recruitment and secretion. Scatchard analysis of phenothiazine effects on somatostatin receptors reveals that these drugs reduce the number of receptors but do not affect the affinity of the receptor for somatostatin. These data indicate that membrane contact and fission steps during exocytosis can be biochemically separated.

Animals↗
PubMed1983

Hydrocortisone inhibition of ascorbic acid transport by chromaffin cells.

Adrenal chromaffin cells have been found to accumulate ascorbic acid by a saturable high affinity mechanism that is inhibited by hydrocortisone. The rate of ascorbic acid transport into cells was linear for at least 1 h and had a Km of 103 microM, a value approaching the reported concentration of ascorbic acid in the adrenal vein during stress. The uptake process itself, representing net accumulation rather than exchange, was inhibited by 0 degrees C, lack of sodium, ouabain, and by dinitrophenol and iodoacetate. Hydrocortisone but not the inactive analogue hydrocortisone hemisuccinate was found to inhibit ascorbic acid uptake in a reversible manner, with an ID50 of 62 microM. This value was within the reported steroid concentration in the adrenal portal system during a significant stress. Both ascorbic acid and hydrocortisone are secreted from cortical cells during stress into the adrenal portal system and thus contact medullary chromaffin cells. We suggest that the control of ascorbic acid uptake by hydrocortisone indicates the existence of a heretofore unanticipated biochemical aspect of the adrenal stress response.

Ascorbic Acid↗
PubMed1983

Inhibition of synexin activity and exocytosis from chromaffin cells by phenothiazine drugs.

Synexin activity was found to be inhibited by low concentrations of the phenothiazine drugs, trifluoperazine and promethazine. Both drugs were also similarly effective at blocking nicotine and veratridine-induced catecholamine secretion from cultured chromaffin cells. These data thus indicate that synexin may be a target of phenothiazine drugs when they block secretion from chromaffin cells.

Adrenal Medulla↗