Kurt Rosenheck

Permeability Changes Induced by Electric Impulses in Vesicular Membranes

SummaryElectric impulses were found to cause transient permeability changes in the membranes of vesicles storing biogenic amines. Release of catecholamines induced by electric fields (of the order of 20 kV/cm and decaying exponentially with a decay time of about 150 μsec) was studied, using the chromaffin granules of bovine adrenomedullary cells as a vesicular model system. Far-UV-absorption spectroscopy was applied to determine the amount of catecholamines released from suspended vesicles. A polarization mechanism is suggested for the induction of short-lived permeability changes caused by electric fields. Such transient changes in permeability may possibly represent a part of the sequence of events leading to stimulated neurohumoral secretion.

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.

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

Abstract: 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.

Kinetics of permeability changes induced by electric impulses in chromaffin granules

SummaryElectric field pulses, ranging in intensity from 20 to 50 kV/cm and in duration from 10 to 40 μsec, caused a transient increase in the membrane permeability of chromaffin granules from the bovine adrenal medulla, that led to partial release of granule soluble constituents. This transient permeability change was long-lived, as compared to the pulse duration, and the main part of material efflux occurred after the termination of the pulse. During the latter phase the temporarily increased permeability decayed to its original value, in the absence of the electric field. This indicated that the structural perturbation induced in the membrane was transient and apparently reversible. The release event was characterized by a field-dependent permeability coefficient ranging from 2×10−4 cm/sec at 30 kV/cm to 3×10−3 cm/sec at 50 kV/cm. The resealing process of the membrane could be described by two relaxation times, both of which decreased with increasing field strength. τ1 varied from about 3.0 msec at 30 kV/cm to less than 2.0 msec at 50 kV/cm, while τ2 varied from about 100 to about 40 msec in the same interval of field strength. The distribution in the degree of filling of granules that had been partially depleted by an electric field pulse indicated that the population could be considered homogeneous with respect to release.

Effect of electric fields on light-scattering and fluorescence of chromaffin granules

SummaryElectric field pulses (in the 5 to 25 kV/cm range) were found to cause a transient increase in the intensity of scattered light (wavelength=369 nm, scattering angle=90°) from aqueous sucrose suspensions of chromaffin granules. Similar observations were made with the membranes of osmotically lysed chromaffin granules. Under the same experimental conditions the degree of polarization of the scattered light changed only very slightly. The fluorescence of the hydrophobic probe diphenyl-hexatriene, incorporated into the membrane of intact chromaffin granules, showed similar transient changes in the intensity. The calculated relaxation times for these changes in optical properties were approximately 150 μsec for the rising phase, and approximately 1 msec for the early stage of the decay. A further relaxation time of about 30 msec was also observed by using this probe. Essentially, all of these signals originated from the granule membrane, and could be attributed to rather small changes in particle size, membrane thickness or refractive index. Moreover, these signals were found to be completely reversible. Catecholamine release from intact granules, pulsed at voltages of 25 kV/cm, occurs already during the first few milliseconds of the transient membrane change.

The circular dichroism of bacteriorhodopsin: Asymmetry and light-scattering distortions

The location of the binding site for retina/in bacteriorhodopsin and the orientation of the retinal chromophore with respect to the membrane components are still not known. CD can, in principle, give information on intermolecular arrangement and sitesymmetry. The CD-spectrum of bacteriorhodopsin in the visible spectral region is composed of negative and positive components of unequal strength [ 1 ]. This asymmetry in the CD-spectrum of purple membrane suspensions in water has been attributed [1 ] to a superposition of a symmetric pair of negative and positive bands arising from exciton-interaction between neighbouring chromophores in a regular array, and another, positive band that is due to the interaction between each single chromophore and the protein site to which it is bound. In the analysis of the CD-spectra of suspensions of large particles distorting effects due to light-scattering, on band shape, as well as intensity, should be taken into consideration [2-4]. It is shown in this work both experimentally and theoretically that the asymmetry in the CD-spectrum of the purple membrane in water suspensions can be attributed to such scattering distortions.

On the role of ATP and divalent metal ions in the storage of catecholamines

The storage vesicles of catecholamines in the adrenal medulla, i.e., the chromaffln granules, contain a remarkably high content of catecholamine (> 0.5 M) and nucleotides (mainly ATP, -0.1 M), as well as substantial amounts of acidic protein (chromogranin A) and divalent metal ions [ 1,2]. The mechanism by which such high concentrations are stored is unknown. Suggestions have been made that catecholamines participate in non-diffusible storage complexes with ATP [3,4] or with ATP together with divalent metal ions [S], or that catecholamines form aggregates with ATP which may play a role in their storage [6]. The possible formation of either binary catecholamineATP complexes or ternary complexes with metal ions, in aqueous solution, has been demonstrated by several spectroscopic studies [7-l 51. Recently, on the basis of potentiometric and ultraviolet absorption measurements of ternary catecholamine-metal ion-ATP complexes, a ‘metal-coordination hypothesis’ [ 161 was proposed in which the storage of catecholamine is attributed to the formation of multinuclear structures which contain catecholamine, ATP, divalent metal ions, and possibly phospholipids. It suggests that catecholamine chelate divalent metal ions either through ionized ring hydroxyls or through the amine and the &hydroxyl of the side chain. However, examination of this hypothesis reveals several incon-

The possible implication of membrane-associated actin in stimulus-secretion coupling in adrenal chromaffin cells

Chromaffin cells of the bovine adrenal medulla were fused with liposomes containing DNAaseI. Resting membrane potential measurements, obtained by the use of the cyanine dye diS-C3 (5), showed that DNAaseI incorporation causes depolarization from −56 to −31 mV, which is similar to that induced by ouabain. The level of basal secretion which occurs after the introduction of DNAaseI is increased several fold. Actin filaments, which can be depolymerized by DNAaseI, thus appear to be involved in stimulus-secretion coupling in chromaffin cells. It is suggested that plasma membrane-associated microfilaments control the Na+-K+-pump and/or the permeability of Ca2+ ions thus affecting the membrane potential as well as secretion.

New Method of Measuring Linear Dichroism in the Ultraviolet: Application to Helical Polymers

A new method of measuring linear dichroism is described wherein the determination is carried out with a single spectral run without moving either sample or polarizer. It makes possible, for the first time, reliable quantitative measurements of dichroism. As examples, spectra (down to 180 mμ) of dichroic films of poly‐L‐lysine and of deoxyribonucleic acid are presented.

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.