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H B Pollard

Publications and source records attributed to H B Pollard.

195 records · Page 11Linked to original sources
PubMed1976

Regulation of the transmembrane potential of isolated chromaffin granules by ATP, ATP analogs, and external pH.

The transmembrane potential of isolated chromaffin granules has been measured using the permeant ions [14C]methylamine and [35S]thiocyanate, as well as the fluorescent probe, 9-aminoacridine. At pH 7.0, the granule membrane had a Nernst proton potential of -45mV, inside negative. This potential was sensitive to the external pH, but was unaffected by K+,Na+, Ca2+, Mg2+, or other cations. The pH of zero potential was 6.25 for both methylamine and thiocyanate. Thiocyanate also had a Nernst potential of similar magnitude and sign to that of methylamine at pH 7.0, and was also sensitive to variation in external pH. Mg2+ATP was found to depolarize the granule membrane by a saturable mechanism with a K 1/2 for ATP of 40 muM. Ca2+ was only 30% as effective as Mg2+ in supporting the ATP effect. The pH optimum for this process was 6.25 and appeared to be accompanied by a marked alkalinization of the granule interior. The specificity for ATP was further tested with structural analogs of ATP and GTP. The rate of change of membrane potential in response to changes in external pH or Mg2+ATP was estimated using the fluorescent probe 9-aminoacridine. Changes came to completion in less than 1 s. This suggested that the ATP effects were not dependent on an enzymatic transformation but on an ATP-induced conformational change in the membrane. We conclude that the chromaffin granule exists in at least two proton permeability states, corresponding to the presence or absence of Mg2+ATP. These states may be related to hormone release from granules and regulation of secretion in vivo.

Adenosine Triphosphate↗
PubMed1976

Regulation of secretion from the adrenal medulla. Evidence for adenylate cyclase activity in secretory vesicle membranes.

Adenylate cyclase activity has been found in purified secretory vesicle membranes from the adrenal medulla. Activity was detected both by formation of radioactive cAMP from [alpha-32P]ATP and by the competitive protein binding assay for cAMP. Activity was highest at pH 8.0 to 8.5, and was stimulated by sodium fluoride and GppNHp, a GTP analogue known to stimulate adenylate cyclase activity in plasma membrane preparations. The reaction rate was strongly dependent on the molar ratio of Mg2+:ATP in the system. This is the first demonstration of adenylate cyclase in a secretory vesicle membrane.

Adenylyl Cyclases↗
PubMed1976

ATP-stimulated transmitter release and cyclic AMP synthesis in isolated chromaffin granules.

ATP stimulates chromaffin granules from the bovine adrenal medulla to release epinephrine and specific soluble proteins. ATP analogs substituted in the beta-gamma position with either nitrogen or carbon were also found to be effective at inducing release from isolated chromaffin granules. However, an ATP analog substituted at the alpha-beta position with carbon was strongly inhibitory. Cyclic AMP was also found to be synthesized by isolated chromaffin granules under release conditions. ATP analogs were effective as substrates for adenylate cyclase in the same order as their efficiency for inducing release from vesicles. Hydrolysis at the beta-gamma linkage of ATP therefore is probably not necessary for release; however, hydrolysis at the alpha-beta position may be important in the release process. Cyclic AMP may be produced and play a regulatory role in this event.

Adenosine Triphosphate↗
PubMed1976

Control of adenylate cyclase from secretory vesicle membranes by beta-adrenergic agents and nerve growth factor.

Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity of purified secretory vesicle membranes from the adrenal medulla is inhibited by I-isoproterenol and I-epinephrine, as well as by nerve growth factor (NGF). The effect of these agents was found to be dose-dependent and, in the case of the catecholamines, saturable. NGF was active at concentrations as low as 10(-8) M. Oxidized NGF was only minimally active, and insulin was completely inactive. Neither dopamine nor phenylephrine had activity. Inhibition of cyclase by either isoproterenol or epinephrine was blocked by I-propranolol, a specific beta-antagonist, but propranolol by itself had no effect on adenylate cyclase activity. The data indicate that the secretory vesicle membrane has beta-adrenergic receptors coupled to the adenylate cyclase. Propranolol was also found to block the NGF-induced inhibition of cyclase. We conclude that the granule membrane has beta-adrenergic receptors as well as NGF-reactive sites, and that the two may be functionally linked.

Adenylyl Cyclase Inhibitors↗
PubMedDate not supplied

Multiple potassium and chloride channels in the human colon carcinoma cell line SW1116.

SW1116 cells have a profound capacity for secreting mucin molecules bearing the Lewisa epitope. Mucin molecules with the same epitope have been found to be elevated in the serum of patients with cystic fibrosis, a disease with defective ion channels. We therefore decided to study ion channels in this cell line. In the present work, we report the presence of two K(+)-channels and two Cl(-)-channels in the apical membrane of SW1116 cells. One of the K(+)-channels has a large conductance (approximately 278 pS), anomalous rectifying properties, and is inactivated rapidly. The second type exhibited a linear I/V curve (19 pS), was voltage insensitive and inactivation was not observed. In cell-attached patches, spontaneous openings of chloride channels were seen with higher frequency than previously reported in other colon carcinoma cell lines or airway epithelial cells. Inside-out experiments allowed identification of two different Cl(-)-channels (Cl(-)-1 and Cl(-)-2). Both exhibited rectification, but in opposite directions, and both were insensitive to NIPAB.

Chloride Channels↗
PubMedDate not supplied

Mechanism of acquired resistance to methotrexate in P388 murine leukemia cells and in their doxorubicin-resistant subline.

The mechanisms of acquired resistance to MTX were studied in P388 murine leukemia cell lines that were sensitive or resistant to ADR. The rate of MTX accumulation in ADR-sensitive cells that have acquired resistance to MTX was found to be lower than that measured in cells that were sensitive to both drugs. Furthermore, in contrast to drug-sensitive cells, in the ADR-sensitive MTX-resistant cells, most of the intracellular MTX (86.2%) was bound and MTX polyglutamation was not detected. The initial rate of MTX accumulation in cells that were resistant to both drugs was comparable to that measured in cells that were sensitive to both drugs or that were resistant only to ADR. However, in the cells that were resistant to both drugs, the rate of MTX accumulation was maintained at its initial level for a period that was considerably longer than that found in the other cell lines. After 3 h of exposure to MTX, the accumulation of MTX in cells that were resistant to both drugs was fourfold higher than that measured in cells that were sensitive to both drugs. Furthermore, while 65 to 70% of the intracellular MTX was free, in cells sensitive to both drugs, or resistant only to ADR, the corresponding value in cells that were resistant to both drugs was less than 1.5%, and a much lower proportion of the MTX was polyglutamated. The sensitivity to TMQ of ADR-sensitive, MTX-resistant cells was similar to that found in cells that were sensitive to ADR and MTX. However, ADR-resistant cells, sensitive or resistant to MTX, were markedly resistant to TMQ. The sensitivity of ADR-resistant MTX-sensitive cells to TMQ was restored by the presence of 10 microM verapamil. Such an effect was not observed in cells resistant to both drugs. It is suggested that P388 cells that have previously acquired resistance to ADR, when now selected by MTX, retain the MTX-transport system (in contrast to ADR-sensitive, MTX-resistant cells) and become resistant to MTX by increasing the activity of DHFR. The results obtained in ADR-resistant cells also suggested that resistance to TMQ was part of the multidrug resistance phenomenon.

Animals↗