PubMed Health⌕ Search

Biomedical subjects

K Rosenheck

Publications and source records attributed to K Rosenheck.

At least 19 recordsLinked to original sources
PubMed1998

Evaluation of the electrostatic field strength at the site of exocytosis in adrenal chromaffin cells.

Exocytosis in secretory cells consists of release from intracellular storage granules directly into the extracellular space via fusion of the granule membrane with the plasma membrane of the cell. It is considered here as comprising two distinct processes. One is the close apposition of granule and plasma membranes. The other arises from interactions between the two membranes during the process of apposition, leading to the formation of a fusion pore. In the following it is shown for the case of the adrenal medullary chromaffin cell that the fusion pore can be ascribed to electroporation of the granule membrane, triggered by the strong electric field existing at the site of exocytosis. Based on an electric surface charge model of the cytoplasmic side of the plasma membrane, resulting from the negatively charged phosphatidylserine groups, it is found that the electrostatic field strength at the site of exocytosis reaches values on the order of 10(8) V/m at small intermembrane distances of 3 nm and lower. The field strength increases with the size of the disc-shaped plasma membrane region generating the electric field, reaching an approximate limit for a radius of 10 nm, at a surface charge density of 5.4 x 10(-2) C/m2. According to previous experimental evaluations of threshold field strength, this field is sufficiently strong to cause membrane electroporation. This step is a precondition for the subsequent membrane fusion during the ongoing process of apposition, leading to secretion.

Animals↗
PubMed1993

Stimulus-induced association of Ca(2+)-binding proteins with the plasma membrane detected in situ by photolabeling of intact chromaffin and PC12 cells.

To investigate the involvement of cytosolic proteins in exocytosis, a system with high temporal and spatial resolution has been developed that allows us to detect the interaction of Ca(2+)- and membrane-binding proteins with the plasma membrane during stimulation of intact chromaffin and PC12 (rat pheochromocytoma) cells. We used 5-iodonaphthalene-1-azide (INA), a hydrophobic label that rapidly partitions into the lipid bilayer of biological membranes. Upon photolysis the label covalently attaches to membrane-embedded domains of proteins. Cells, preincubated with INA in the dark, were stimulated by either 300 microM carbamoylcholine or 60 mM K+ and irradiated (20 s) at various time intervals after stimulation. Subsequently, the cytosolic Ca(2+)- and membrane-binding proteins were isolated in the presence of EGTA (EGTA extract). Of the approximately 40 proteins in the EGTA extract, 15 (15-100 kDa) are labeled in both cell types. Upon stimulation, labeling is increased up to 3-fold in some of the proteins compared to cells labeled under basal conditions. In the absence of external Ca2+, no increase is observed. The rate of label incorporation is similar to the rate of exocytosis in several of these proteins. These results indicate that in the event of triggered exocytosis some of the Ca(2+)-binding proteins interact with the plasma membrane and temporarily embed in the lipid bilayer. Our findings support the hypothesis according to which stimulus-induced alterations in the structure of the Ca(2+)-binding proteins lead to their transient insertion into the membrane and thereby to membrane fusion.

Adrenal Medulla↗
PubMed1989

(+)-PN200-110 and ouabain binding sites in purified bovine adrenomedullary plasma membranes and chromaffin cells.

Bovine adrenal medulla plasma membranes were purified by a differential centrifugation procedure using sucrose and Urografin discontinuous density gradients; the membranes were enriched 10-12-fold in acetylcholinesterase activity and [3H]ouabain binding sites. Specific (+)-[3H]PN200-110 binding to these membranes amounted to 90% of total binding and was saturable and of high affinity (KD = 41 pM; Bmax = 119 fmol/mg of protein) with a Hill coefficient close to 1, a result suggesting the presence of a single, homogeneous population of dihydropyridine receptors. The association and dissociation rate constants were, respectively, 7.5 X 108 M-1 min-1 and 0.023 min-1. Unlabeled (+)-PN200-110 displaced (+)-[3H]PN200-110 binding with a potency 100-fold higher than (-)-PN200-110 (IC50,0.5 and 45nM, respectively). Although the two enantiomers of BAY K 8644 completely displaced (+)-[3H]PN200-110 binding, they exhibited no stereoselectivity (IC50, 69 and 83 nM,respectively). Whereas ( +/- )-nitrendipine very potently displaced (+)-[3H]PN200-110 binding (IC50 = 1.3 nM) verapamil and cinnarizine displaced the binding by only 30 and 40% at 1 microM, and diltiazem increased it by 20% at 10 microM. [3H]Ouabain bound to plasma membranes with a KD of 34 nM and a Bmax of 9.75 pmol/mg of protein, a figure 80-fold higher than the Bmax for (+)-PN200-110. [3H]Ouabain also bound to intact chromaffin cells with a Bmax of 244 fmol/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗
PubMed1986

Destabilization of actin filaments as a requirement for the secretion of catecholamines from permeabilized chromaffin cells.

In the search for a functional role of cytoskeletal proteins in the mechanism(s) of stimulus-secretion coupling, we have previously demonstrated that the actomyosin system might be involved in the transport of cations across the plasma membrane of bovine adrenal chromaffin cells [(1986) J. Biol. Chem. 261, 5745-5750]. To establish whether actin and myosin might also be involved in later stages of the cellular response, we have examined the possible effects of various actin-specific reagents on the calcium-mediated secretion of catecholamines from digitonin-permeabilized cells. F-Actin-destabilizing agents, such as cytochalasin D or DNase 1, were found to promote Ca2+-stimulated (as well as basal) secretion. By contrast, stabilizers, like phalloidin, produced the opposite effect. It is concluded that stimulus-secretion coupling in chromaffin cells might require the reorganization of actin for modulating both ion transport across the plasma membrane and exocytotic secretion per se.

Actin Cytoskeleton↗
PubMed1986

Phospholipase C and diacylglycerol lipase activities associated with plasma membranes of chromaffin cells isolated from bovine adrenal medulla.

The plasma membranes of bovine adrenal chromaffin cells were isolated and the activities of enzymes involved in arachidonic acid liberation were investigated. Only a minute activity of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) could be detected using externally added phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as substrate. When membranes were treated with exogenous phospholipase C (orthophosphoric acid diester phosphohydrolase, EC 3.1.4.1) there was a liberation of free fatty acids from the sn-2 position of PC. The enzyme responsible for this effect could be demonstrated to be a diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) localized in the plasma membrane. Using phosphatidylinositol (PI) as a substrate, it was found that an endogenous phospholipase C exists which co-purifies with the membrane preparation. The produced diacylglycerol is subsequently hydrolyzed by diacylglycerol lipase liberating arachidonic acid. The two enzymes, phospholipase C and diacylglycerol lipase were characterized. Phospholipase C was found to be calcium dependent and PI specific, showing an activity of 60 pmol/micrograms protein per h (1.2 mM Ca2+), whereas the diacylglycerol lipase was calcium independent hydrolyzing diacylglycerol at a rate of 7.2 pmol/micrograms protein per h. The lipase but not the phospholipase C was inhibited 50% by 1.7 mM para-bromophenacylbromide.

Adrenal Medulla↗
PubMed1986

Control of stimulus-secretion coupling in adrenal medullary chromaffin cells by microfilament-specific macromolecules.

We have incorporated the myosin fragment heavy meromyosin (HMM), which is known to interact mechanochemically and enzymatically with actin filaments, into intact chromaffin cells of the bovine adrenal medulla, in order to study the possible involvement of actin and myosin in stimulus-secretion coupling. HMM was found to stimulate secretion of catecholamines, to cause depolarization of the plasma membrane, and to enhance 22Na+ uptake. HMM-stimulated catecholamine secretion was dependent on the presence of extracellular Na+. The Na+ uptake caused by HMM was inhibited by 10 microM amiloride. Acetylcholine-stimulated catecholamine secretion and 22Na+ uptake were both enhanced by HMM incorporation. A Na+/H+ antiporter, activated by the interaction of HMM with the cells' microfilaments, seems to be involved in HMM action and could possibly also be a component of stimulus-secretion coupling in chromaffin cells, induced by regular agonists.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Ultrastructural and cytochemical characterization of adrenal medullary plasma membrane vesicles and their interaction with chromaffin granules.

Plasma membrane vesicles obtained by density gradient centrifugation of bovine adrenal medullary homogenates were analyzed by electron microscopic methods, including negative staining, ultrathin sections and freeze-fracture replicas. Rapid freezing showed the intramembrane structure of plasma membrane vesicles to be distinct from that of other organelle membranes, such as chromaffin granules. Cytochemical demonstration of acetylcholinesterase (EC 3.1.1.7) activity on most membrane profiles confirmed that plasma membrane vesicles are derived predominantly from plasma membranes. About half of the plasma membrane vesicles were smaller than 0.15 micron and almost none larger than 0.55 micron. Practically all were composed of single shells. Most vesicles were impermeable to cytochemical markers of the size of Ruthenium red (Mr 800) and none were permeable to markers larger than 40 kDa. Surface charge probes, concanavalin A binding and endogenous actin decoration with heavy meromyosin indicated that the major fraction of plasma membrane vesicles is oriented right-side-out. A minor population with opposite orientation could also be detected. Isotonic ionic media caused vesicle aggregation in suspensions of plasma membrane vesicles and chromaffin granules. Freeze-fracturing always revealed clusters of membrane-intercalated particles at the sites of contact between aggregated membranes.

Adrenal Medulla↗
PubMed1985

Direct fluorometric assay of catecholamine secretion from isolated bovine adrenal chromaffin cells.

The intrinsic fluorescence of catecholamines can be exploited for a simple and speedy fluorescence assay of secretion from bovine adrenal chromaffin cells. Catecholamines are separated from concomitantly secreted proteins by centrifugation in 0.4 M perchloric acid, and their fluorescence is measured directly in a conventional spectrofluorometer without chemical derivatization. Catecholamine release, assayed by this technique, is virtually identical to data obtained in parallel by the classical trihydroxyindole method. Intrinsic catecholamine fluorescence does not differentiate between epinephrine and norepinephrine. Interferences from small molecular weight compounds not precipitated in perchloric acid are rare, and can be accounted for using appropriate calibration curves.

Adrenal Medulla↗
PubMed1985

Membrane potential and catecholamine secretion by bovine adrenal chromaffin cells: use of tetraphenylphosphonium distribution and carbocyanine dye fluorescence.

Changes in plasma membrane potential of isolated bovine adrenal chromaffin cells were measured independently by two chemical probe methods and related to corresponding effects on catecholamine secretion. The lipophilic cation tetraphenylphosphonium (TPP+) and the carbocyanine dye 3,3'-dipropylthiadicarbocyanine [DiS-C3-(5)] were used. The necessity of evaluating the subcellular distribution of TPP+ among cytoplasmic, mitochondrial, secretory granule, and bound compartments was demonstrated and the resting plasma membrane potential determined to be -55 mV. The relationship between membrane potential and catecholamine secretion was determined in response to variations in extracellular K+ and to the presence of several secretagogues including cholinergic receptor ligands, veratridine, and ionophores for Na+ and K+. The dependence of potential on K+ concentration fit the Goldman constant field equation with a Na/K permeability ratio of 0.1. The dependence of both K+- and veratridine-evoked catecholamine secretion on membrane potential exhibited a potential threshold of about -40 mV before a significant rise in secretion occurred. This is likely related to the threshold for opening of voltage-sensitive Ca2+ channels. Acetylcholine and nicotine evoked a large secretory response without a sufficiently sustained depolarization to be detectable by the relatively slow potential sensitive chemical probes. Decamethonium induced a detectable depolarization of the chromaffin cells. Veratridine and gramicidin evoked both membrane depolarization and catecholamine release. By contrast the K ionophore valinomycin evoked significant levels of secretion without any depolarization. This is consistent with its utilization of an intracellular source of Ca2+ and the independence of its measured secretory response on extracellular Ca2+.

Acetylcholine↗
PubMed1984

Possible involvement of actin and myosin in Ca2+ transport through the plasma membrane of chromaffin cells.

The exocytosis of catecholamines by chromaffin cells following stimulation (e.g. by acetylcholine) is accompanied by a rise in the level of intracellular free Ca2+. Actually, secretion can be induced merely by making the cells leaky to Ca2+ from the external medium. We have recently demonstrated that secretion can be increased by the introduction of DNase-I, the F-actin depolymerizing agent, or of heavy meromyosin, the enzymatically active fragment of myosin. Suspecting that these changes might be associated with a higher intracellular level of Ca2+, we now have measured the influx of 45Ca2+ into chromaffin cells which have undergone fusion with DNase-I- or with heavy meromyosin-loaded liposomes. In both cases, a marked increase in Ca2+ uptake has been observed, which could be abolished by Co2+ ions (a Ca2+ channel blocker), suggesting an intimate involvement of the cellular actomyosin system in the process of Ca2+ ions transport through the Ca2+ channels of the plasma membrane.

Actins↗
PubMed1981

Stimulus-secretion coupling in isolated adrenal chromaffin cells: calcium channel activation and possible role of cytoskeletal elements.

The catecholamine secretory function of a preparation of isolated bovine adrenal chromaffin cells has been further characterized under conditions designed to elucidate the mechanism of calcium channel activation and the possible role of cytoskeletal elements in stimulus-secretion coupling. Three related sets of data were obtained: (1) Differences in kinetics, Ca dependence, strength, and additivity of the secretory response to acetylcholine (ACh) versus excess K; (2) the effects on secretion of the Ca channel-blocking agents, Ni, Mg, and verapamil; and (3) the Ca dependence of vinblastine action on ACh- and K-evoked secretion. The results suggest that a major portion of the Ca influx required for catecholamine release enters the cell via voltage-dependent Ca channels with some additional Ca influx via the ACh receptor channel. Comparison of the present secretion data with corresponding known electrophysiological properties of isolated chromaffin cells provides added evidence for a role of chromaffin cell action potentials in regulation of Ca influx and the secretory response. Elevated Ca concentrations enhanced K-evoked secretion to levels comparable to that of Ach but did not induce a vinblastine block of K-evoked release. This provides further evidence against a role of microtubules in the common exocytosis event per se. However, a role of cytoskeletal elements in directing the movement of secretory granules, or an action of vinblastine at cholinergic receptors, remain distinct possibilities.

Acetylcholine↗
PubMed1979

Cholinergic stimulants and excess potassium ion increase the fluidity of plasma membranes isolated from adrenal chromaffin cells.

Chromaffin cell membranes from the bovine adrenal medulla were labelled with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene, and the fluorescence polarization (P) of the membrane suspensions was measured as a function of temperature. The P versus t profiles, between 20 and 37 degrees C, showed two linear regions separated by a break in the vicinity of 30 degrees C, reflecting a change in the phase behaviour of the constitutent lipids. Decreases in P values at higher temperature indicated progressive fluidization of the lipid bilayer. Previous incubation with either acetylcholine (0.5 mM) or nicotine (50 microM) produced further fluidization, the extent of which depended on the presence of added Ca2+ (2.2 mM). Thus, the flow activation energy, delta E, between approx. 30 and 37 degrees C was 9.1 kcal/mol for acetylcholine and 8.8 kcal/mol for acetylcholine plus Ca2+, as compared to 7.9 kcal/mol in the absence of acetylcholine and Ca2+. In the presence of nicotine, delta E was 11.4 kcal/mol when Ca2+ was absent and 9.5 kcal/mol when it was present. The cholinergic blocker, hexamethonium (0.5 mM), abolished the acetylcholine- or nicotine-induced changes. 65 mM K+ produced a similar fluidization, which was reversed by addition of Ca2+. An additive effect was observed when the membranes were incubated with both nicotine and K+, with delta E = 16.6 kcal/mol in the presence of Cas2+. These results indicate a receptor-mediated modulation of the lipid distribution between rigid and fluid regions in the membrane, which could be of importance for stimulated catecholamine secretion in the intact cell.

Acetylcholine↗
PubMed1979

Differential scanning calorimetry of chromaffin granule membranes.

Differential scanning calorimetry thermograms of native chromaffin granule membranes exhibit several peaks in the 15-35 degrees C region. Extraction of cholesterol increases the size of the melting peaks. Addition of Ca2+ ions does not seem to influence the lipid transitions.

Animals↗
PubMed1978

Proteolysis and flash photolysis of bacteriorhodopsin in purple membrane fragments.

Pronase treatment of aqueous suspensions of purple membrane fragments from H.halobium leads to the cleavage of bacteriorhodopsin. The protein fragments remaining in the membrane after treatment with relatively small concentrations of enzyme (2% w/w) in normal daylight range in molecular weight from 20,000--21,000 daltons, indicating that cleavage occurs mainly near the extremities of the protein chain. At higher enzyme concentrations the relative amounts of protein fragments having smaller molecular weight increase. Generally, the relative loss of retinal chromophore is larger than that of protein and thus the retinal binding site seems to be located near one of the chain ends that is cleaved off by enzyme. Irradiation with white light during the time of proteolysis (at both low and high enzyme concentrations) results in extensive cleavage, so that under certain conditions no high molecular weight components can be detected in SDS-polyacrylamide gels. It, therefore, appears that parts of the bacteriorhodopsin chain become more exposed to enzyme digestion when the purple membrane is illuminated. Enzyme treated aqueous purple membrane fragment suspensions still show photocycle activity. The main consequence of proteolysis is a pronounced appearance of biphasicity in the decay of M412 and the regeneration of bR570. Simultaneously the yield of O660 is reduced. As with untreated purple membrane, the correlation between the rates of decay of M412 and regeneration of bR570 is greatest when the yield of O660 is lowest.

Bacterial Proteins↗