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H B Pollard

Publications and source records attributed to H B Pollard.

At least 181 records · Page 10Linked to original sources
PubMed1979

Self-association of synexin in the presence of calcium. Correlation with synexin-induced membrane fusion and examination of the structure of synexin aggregates.

It has been proposed (Creutz, C. E., Pazoles, C. J., and Pollard, H. B. (1978) J. Biol. Chem. 253, 2858-2866) that synexin, an adrenal medullary protein that causes Ca2+-dependent aggregation of isolated chromaffin granules, might be the intracellular receptor for Ca2+ in the process of exocytosis. We now report that Ca2+ interacts directly with isolated synexin, inducing rapid self-association. Mg2+ or Sr2+ cannot substitute for Ca2+, and Ba2+ is only weakly effective at stimulating self-association. We have analyzed 90 degree light scattering data to determine a titration curve for the activation of synexin by Ca2+ in the self-association reaction. The curve has a Hill coefficient of 2.3 and is half-maximal at 200 micron Ca2+, which correlates exactly with the Ca2+ titration curve for the aggregation of chromaffin granules by synexin. Electron microscopy of negatively stained samples reveals that synexin monomers associate to form 50 A by 150 A rods which in turn associate side to side and end to end to form bundles of parallel rods. We suggest that synexin exists as a soluble monomer in the cytoplasm of the resting chromaffin cell and that when the cell is stimulated to secrete, the increase in the intracellular concentrations of Ca2+ causes synexin to form rods, similar to those formed in vitro, which bind to chromaffin granule and plasma membranes, joining them together to form the "pentalaminar" fusion complexes characteristic of the first step in exocytosis.

Adrenal Medulla↗
PubMed1979

Phosphorylation of specific, distinct proteins in synaptosomes and axons from squid nervous system.

Synaptosomes and axons from squid were incubated with [gamma-(32)P]ATP or [(32)P]orthophosphate and specific, distinct proteins were found to be labeled in each preparation. In axoplasm, only the major 200,000 M(r) neurofilament protein and a specific protein of approximately 400,000 M(r) were labeled, as reported previously [Pant, H. C., Shecket, G., Gainer, H. & Lasek, R. J. (1978) J. Cell Biol. 78, R23-R27]. These results were independent of whether the cosubstrates were (32)PO(4) (2-) or [gamma-(32)P]ATP. However, synaptosomes lacked the 200,000 M(r) neurofilament protein and several lower molecular weight proteins were labeled instead, the most prominent being a 47,000 M(r) species. [gamma-(32)P]ATP was much more effective in labeling the 47,000 M(r) species than (32)PO(4) (2-). Synaptosomes also contained a distinct 250,000 M(r) protein species which, however, was not labeled. The protein kinase activity in synaptosomes was sensitive to various pharmacological agents, depending on whether the labeled phosphate came directly from ATP or orthophosphate. Carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, a mitochondrial H(+) uncoupler, almost completely inhibited incorporation of (32)P into protein with (32)PO(4) (2-) as cosubstrate, as expected, but produced only 32% inhibition with [gamma-(32)P]ATP as cosubstrate. The activity could be augmented by incubating synaptosomes in a calcium-free medium and could be suppressed by increasing intrasynaptosomal Ca(2+) with A23187, a Ca(2+) ionophore. The latter effect was more prominent with (32)PO(4) (2-) than with [gamma-(32)P]ATP as cosubstrate. Depolarizing agents such as veratridine and high K(+) also suppressed activity, and the veratridine effect was completely reversed by tetrodotoxin or by omission of Ca(2+) when [gamma-(32)P]ATP was used, and partially reversed when (32)PO(4) (2-) was used. We conclude that the morphological transformation of an axon into a terminal is accompanied by significant changes in protein and phospho-protein composition that may be related to synaptic transmission.

Animals↗
PubMed1978

Role of anions in parathyroid hormone release from dispersed bovine parathyroid cells.

It is known that permeant anions are required for the release of epinephrine from isolated chromaffin granules and of serotonin from intact platelets. We have now investigated the role of anions in the release of a polypeptide hormone, parathyroid hormone, from dispersed bovine parathyroid cells. The release is inhibited 60%-80% by decreasing either [Cl-] or [OH-] and 60%-70% by replacement of NaCl with the impermeant anion isethionate. By contrast, substitution of various monovalent cations in the medium had no effect on the release. Disodium 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) and probenecid, which are known to block anion transport in the erythrocyte, also cause a dose-dependent 90%-100% inhibition of release. Moreover, kinetic analysis of inhibition by probenecid suggests that it is competitive with respect to either OH- or Cl-. These results suggest that anions and the anion transport system may play a role in exocytosis of a polypeptide hormone. The proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone was was also found to block hormone release, and the possibility is discussed of a "chemosmotic" mechanism for exocytosis in this system similar to that previously postulated for chromaffin granules and platelets.

Animals↗
PubMed1978

Dopamine-beta-hydroxylase-induced changes in the electrical conductance of bimolecular lipid membranes.

Dopamine-beta-hydroxylase (DBH), an enzyme that catalyzes the conversion of dopamine (DA) to norepinephrine (NE) in adrenal medullary chromaffin granules, increases the electrical conductance of bimolecular lipid membranes. The conductances increase requires both DA and Ca2+ and occurs in discrete steps. The conductance, which increases as the square of the DBH concentration, is nonselective for cations over anions and requires the native conformation of DBH. NE cannot replace DA.

Adrenal Glands↗
PubMed1977

Evidence for control of serotonin secretion from human platelets by hydroxyl ion transport and osmotic lysis.

Serotonin secretion from human platelets, stimulated either by thrombin or the calcium ionophore A23187, was found to be inhibited by anion transport blocking drugs such as 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), pyridoxal phosphate, probenecid, and suramin. These drugs have previously been shown to inhibit ATP-evoked release of epinephrine from isolated chromaffin granules by blocking chloride uptake and subsequent osmotic lysis. However, in contrast to granule release, platelet secretion was insensitive to chloride and, instead, was dependent on OH-. Platelet release was suppressed by low pH, and inhibition by the transport blocking drugs was competitive only with respect to OH-. Serotonin release from platelets was also suppressed by increasing extracellular osmotic strength, and the relationship between suppression and external osmotic strength was quantitatively similar to that observed in the case of chromaffin granules. We conclude that platelet exocytosis could occur when serotonergic granules are closely juxtaposed to the plasma membrane, thus exposing the granule anion transport site to the more alkaline medium. Secretion of serotonin could occur as a consequence of OH- transport and osmotic lysis of the granule-plasma membrane complex, analogous to the chemiosmotic mechanism of chloride-dependent epinephrine release from isolated chromaffin granules.

Anions↗
PubMed1977

Regulation of release from isolated adrenergic secretory vesicles by ATP-mediated changes in transmembrane potential and anion permeability.

Isolated chromaffin granules release their contents when exposed to calcium, magnesium, ATP, and high levels of chloride ions. The mechanism of release is not well-understood, but changes in anion permeability may be involved. We found that another anion, thiocyanate (SCN-), also activated release in a fashion similar to chloride, while isethionate (HO-CH2-CH2SO3-) an impermeant anion, was inactive. Mg++-ATP was found to activate the uptake of 36Cl and 14C-SCN, leading us to conclude that activation of anion uptake might be involved in the release process. The 36Cl and the 14C-SCN compartments were then compared by studying displacement of the trace anions by excess cold mass. Chloride and SCN displaced large amounts of both 36Cl and 14C-SCN, while isethionate displaced little of either tracer anion. We suggest, on the basis of these data, that ATP-mediated anion uptake may be the basis for the release mechanism. Release may occur as a consequence of anion and subsequent water uptake into granules, resulting in osmotic imbalance and osmotic shock. This may also be of physiologic importance, and we propose a cellular model for secretion based on the biochemical properties of the isolated chromaffin granule.

Adenosine Triphosphate↗