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H B Pollard

Publications and source records attributed to H B Pollard.

At least 163 records · Page 9Linked to original sources
PubMed1982

Further characterization of the aggregation and fusion of chromaffin granules by synexin as a model for compound exocytosis.

Synexin was isolated from bovine liver and found to aggregate adrenal chromaffin granules in the same Ca2+-dependent manner as previously described for adrenal synexin. The chromaffin granule aggregating activity of liver synexin was blocked in vitro by the addition of an antibody prepared to the 47,000 molecular weight band extracted from an SDS gel of an adrenal medullary synexin preparation. Chromaffin granules aggregated by synexin fused when exposed to cis-unsaturated fatty acids at concentrations comparable to those released from phospholipids by stimulated secretory cells. The synexin-induced aggregation reaction was blocked by Erythrosin B, a common food coloring, and by the phenothiazine antipsychotic trifluoperazine and promethazine. The aggregation and fusion of chromaffin granules thus appears to be a useful model system for studying synexin from diverse tissues and for testing pharmacologically or toxicologically active substances for effects on secretory systems.

Adrenal Medulla↗
PubMed1982

In vitro reconstitution of chromaffin granule-cytoskeleton interactions: ionic factors influencing the association of F-actin with purified chromaffin granule membranes.

Chromaffin granules are the secretory vesicles directly involved in exocytosis of catecholamines, enkephalins, and other components from adrenal medullary cells. The granules occupy a large portion of the cytoplasmic volume and thus may interact extensively with cytoskeletal elements such as actin. Indeed, using both sedimentation techniques and falling ball viscometry [Fowler et al: J Cell Biol 88: 388, 1981] to measure actin binding by membranes, we were able to show that chromaffin granules bind F-actin via a protein site on the membrane, and that these interactions are reversibly inhibited by raising the free calcium ion concentration to micromolar levels ([Ca++]free for half-maximal inhibition approximately 2.6 x 10(-7)M)[Fowler and Pollard: Nature 295:336, 1982]. Here, we show that F-actin-chromaffin granule interactions are unaffected by changes in pH between about pH 6.4 and 7.4 but are about 50% inhibited by raising the pH from 7.5 to 8.0. They are also 50% inhibited by increasing the KCl concentration to about 200 mM but are not significantly affected by increasing concentrations of K-glutamate up to 500 mM or by varying the MgCl2 concentration between 0 and 6 mM. The interactions between chromaffin granule membranes and F-actin are also reduced in the presence of ATP, AMP-PNP, or free pyrophosphate; cAMP and AMP are without effect. The ability of chromaffin granule membranes to interact with F-actin under conditions that may approximate the resting intracellular environment (neutral pH, low KCl, 1-2 mM MgCl2, 1 mM ATP, [Ca++]free less than 10(-7)M, 30 degrees C) suggests that these interactions may partially reconstitute naturally occurring associations between chromaffin granules and the cytoskeleton. Further, regulation of chromaffin granule membrane-actin interactions by ionic factors (pH, calcium, chloride ions, nucleotides) that vary intracellularly leads us to propose that associations between actin and the chromaffin granule membrane could influence the location and dislocation of these organelles in the cytoplasm.

Actins↗
PubMed1982

Induction of ion-permeable channels by the venom of the fanged bloodworm Glycera dibranchiata.

Venom from the poison glands of the polychaete annelid Glycera convoluta has been reported to dramatically increase the frequency of miniature end-plate potentials at the frog and crayfish neuromuscular junctions, without causing detectable ultrastructural changes. We report here that addition of venom from the related annelid Glycera dibranchiata to one side of a lipid bilayer results in the formation of ion-permeable channels in the membrane. The channel forming activity was found in the void volume of a Sephadex G-25 column (mol. wt. greater than 5000). The conductance of a single channel is about 350 pmho in 0.1 M NaCl and is ohmic. The channels exhibit moderate (but not ideal) cation selectivity in NaCl or KCl gradients. Other selectivity measurements suggest that Ca2+ and Mg2+ are also permeable. The channels show a slight voltage sensitivity. The steady state conductance at--70 mV (side opposite venom) is about 5 times the conductance at + 70 mV. We suggest that these channels in the venom may evoke transmitter release at neuromuscular junctions either by (1) depolarizing the pre-synaptic terminal and thus opening voltage-dependent Ca2+ channels, or (2) directly allowing Ca2+ to enter the terminal. Black widow spider venom is known to produce similar effects on neuromuscular junctions and lipid bilayers. The single channel conductances and ionic selectivities of the channels found in the venoms of Glycera and Latrodectus are strikingly similar. Taken together, these results suggest that channel formation can explain the electrophysiologic effects of these two different venoms.

Animals↗
PubMed1982

Granule fusion and fission (discharge) are biochemically dissociable events of exocytotic hormone release.

Granule fusion and the subsequent fission leading to hormone discharge are distinct and separable events in exocytosis. As an index of fusion, we followed the recruitment of granule-bound somatostatin receptors to the islet surface, an event which accompanies secretion vesicle migration and insulin secretion. Granule fission was monitored by measuring insulin release. Substitution of the impermeant salt sodium isethionate for NaCl led to a 90% decrease in glucose-stimulated insulin release with no inhibition of somatostatin receptor recruitment. The phenothiazine drugs, trifluoperazine and promethazine, believed to inhibit calcium-sensitive proteins involved with stimulus-secretion coupling blocked insulin release and somatostatin receptor recruitment in parallel. This suggests that these agents suppress intracellular events promoting fusion of the secretion granule with the plasma membrane. IBMX appears to stimulate specifically granule fission since IBMX-induced insulin release occurs acutely without an increase in somatostatin receptor recruitment. Sodium isethionate, which inhibits granule lysis, blocked IBMX-stimulated insulin release.

1-Methyl-3-isobutylxanthine↗
PubMed1982

ATP-activated exchange of catecholamines by isolated intact chromaffin granules.

Isolated chromaffin granules were found to accumulate exogenous (R)-[3H]-norepinephrine at 37 C in the presence of Mg2+-ATP by a process involving one-to-one exchange for endogenous epinephrine. By contrast, omission of Mg2+-ATP from the incubation medium resulted in a substantial unidirectional efflux of the endogenous epinephrine independent of external catecholamine concentrations. However, the effects of ATP on the spontaneous efflux and the exchange process were found to occur with different kinetic parameters and differential sensitivity to a stilbene disulfonate inhibitor of the granule ATPase, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. These data lead us to conclude that ATP catalyzes bidirectional catecholamine flux across the granule membrane (not net accumulation) by an ATPase-dependent mechanism. In addition, ATP has the ability to suppress unidirectional efflux of catecholamines from granules by a mechanism independent of the ATPase or the exchange mechanism. The data also allow us to distinguish among three possible models for how these processes might be occurring.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1980

A biophysical model of the chromaffin granule. Accurate description of the kinetics of ATP and Cl- dependent granule lysis.

A model is constructed to describe the behavior of isolated chromaffin granules (secretory vesicles of the adrenal medulla) when they are induced to release their contents by incubation with MgATP and Cl-. The model is based on the assumption that the release event is osmotic lysis due to the ATPase dependent influx of protons and osmotically active Cl- ions. The consequences of this influx of osmotically active particles are predicted from osmotic fragility curves determined by suspending granules in hypotonic media. Turbidity measurements on granule suspensions undergoing the ATP and Cl- dependent release reaction are used to fit the parameters of the model. The model then successfully described the time course, Cl- dependence, ATP dependence, and osmotic strength suppression of the release event as monitored either by measurement of turbidity changes or of epinephrine release. The degree of suppression of release predicted in hypertonic media is also in agreement with published data on hypertonic suppression of exocytosis from several cell types: chromaffin cells, blood platelets, and parathyroid cells. Therefore, the model may also provide an accurate description of some of the events occurring during exocytosis.

Adenosine Triphosphatases↗
PubMed1980

Role of intracellular proteins in the regulation of calcium action and transmitter release during exocytosis.

A central problem in cellular neurobiology is how the process of exocytosis of transmitters and hormones is regulated at the molecular level. Calcium is important in the process and may act by initiating the formation of fusion complexes of secretory granules to each other and to plasma membranes. We have discovered and isolated a new 47,000 MW protein (synexin) from adrenal medulla tissue that fuses chromaffin granule membranes only in the presence of calcium. Synexin activity was detected in a number of secretory tissues including human platelets and bovine brain, and the synexin molecule was found by immunofluorescent cytochemistry to be localized to the cytoplasm of chromaffin cells. Purified synexin molecules were found to self-associate in the presence of Ca++ to form paracrystalline arrays of 50 X 150 A rods, and the association was dependent on [Ca++] in an identical fashion to the Ca++ dependence of granule membrane fusion. On the basis of these data we suggest that synexin may be the intracellular receptor for calcium during exocytosis. However, the actual release event of 'fission' of the secretory vesicle-plasma membrane complex did not appear to be related to synexin action, and we have considered the hypothesis that the chemiosmotic mechanism for ATP, Cl--dependent chromaffin granule lysis might provide the necessary localized force. We have now shown that the granule model successfully predicts the secretory properties of human platelets, bovine parathyroid cells, and bovine chromaffin cells. Like the granule lysis system, secretion from these cells required specific anions in the medium was inhibited by anion transport blocking drugs and proton ionophores, and was suppressed by elevated osmotic strength. We suggest that secretory granules fused to plasma membranes in secreting cells, perhaps by synexin, may experience net solute uptake and subsequently undergo local, outwardly directed osmotic lysis, or exocytosis.

Adrenal Medulla↗