Effects of External Osmotic Pressure on Vesicular Secretion from Bovine Adrenal Medullary Cells

Abstract

Secretion of catecholamines from individual vesicles of bovine adrenal medullary cells was studied with amperometry in media of various osmolarities and compared with results obtained in isotonic physiological buffer (315 mosM). Hypotonic solutions caused an increase in the number of amperometric spikes evoked by brief exposure to 5 mM Ba 2؉ . Under moderate hypertonic conditions (630 mosM), individual vesicular events were decreased in frequency, and lower amounts were secreted per event. Furthermore, the events were temporally broadened relative to those observed during release in isotonic conditions. At 970 mosM, exposure to 5 mM Ba 2؉ evoked even smaller secretory events that resemble the prespike feature that has been attributed to the initial opening of the fusion pore. The lack of large spikes is not due to failure of Ba 2؉ entry because fura-2 fluorescence reveals an increase in intracellular divalent ions. After exposure to Ba 2؉ in hypertonic solution, spikes could be induced with isotonic solution transiently directed onto the cell, but this process was not accompanied by a change in the concentration of intracellular divalent ions. Thus, this procedure provides an unique opportunity to temporally separate exocytotic secretion from entry of divalent ions.Secretory vesicles in chromaffin cells of the adrenal medulla sequester a remarkably high concentration of molecules. The major component, catecholamines, is present at a concentration of 550 mM, but the other vesicular constituents such as ATP (122 mM), Ca 2ϩ (17-30 mM), Mg 2ϩ (5 mM), ascorbate (22 mM), and the acidic protein chromogranin A are also present at high concentrations (1). Because the total soluble concentration of intravesicular components is more than 750 mM, it has long been of interest how these vesicles are able to form stable entities that are isotonic with 300 mosM solutions. It seems from nuclear magnetic resonance studies of intact vesicles that the contents form a dynamic viscous solution that is stabilized by ternary complex formation (2). Solution studies have revealed that catecholamines and ATP associate, resulting in a lowered osmotic pressure (3). Similarly, chromogranin A may play an important role in lowering osmolarity through interactions with Ca 2ϩ and catecholamines (4 -7). Such association significantly lowers apparent free concentration, as demonstrated for Ca 2ϩ ions in adrenal medullary vesicles (8). Whereas intravesicular association stabilizes vesicular storage, upon exocytosis the vesicular contents must dissociate to be extruded from the cell in their soluble form. The osmotic gradient that exists immediately upon cell vesicle fusion between the isotonic extracellular milieu and the vesicle contents certainly plays a role in the overall exocytotic process. Indeed, Knight and Baker (9) showed that sucrose-based hypertonic solutions inhibit the amount of Ca 2ϩ -dependent catecholamine secretion, whereas incubation with hypotonic media increases it. Similar results were obtained in other laboratories with cultu...

The Effects of Vesicular Volume on Secretion through the Fusion Pore in Exocytotic Release from PC12 Cells

“…Electrodes were held at ϩ 0.65 V versus a locally constructed sodium-saturated calomel reference electrode using a commercially available patch-clamp instrument (Axopatch 200B; Axon Instruments, Foster City, CA) configured as described previously (Borges et al, 1997). The output was digitized at 5 kHz and filtered at 2 kHz using an internal four-pole low-pass Bessel filter.…”

VMAT-Mediated Changes in Quantal Size and Vesicular Volume

“…Electrodes were held at ϩ0.65 V versus a locally constructed sodium-saturated calomel reference electrode that used a commercially available patch-clamp instrument (Axopatch 200B; Axon Instruments, Foster C ity, CA) configured as described previously (Borges et al, 1997). The output was digitized at 5 kHz and filtered at 2 kHz by using an internal four-pole low-pass Bessel filter.…”

“…The area of each amperometric spike is directly proportional to the number of molecules detected from a single vesicle by the relationship Q ϭ nNF, where Q is the charge of each current transient, N is the number of moles, F is Faraday's constant (96,485 C/equiv), and n is the number of electrons transferred per oxidized molecule (this was assumed to be 2 for catecholamines). The values I max and t 1/2 have been shown to be dependent on several factors, including the number of molecules released (Pothos et al, 1996(Pothos et al, , 1998a, the degranulation and extrusion of transmitter from the vesicle lumen (Wightman et al, 1995;Borges et al, 1997), and the type and magnitude of filtering used during the recording.…”

Phospholipid mediated plasticity in exocytosis observed in PC12 cells

“…were held at +700 mV vs. a locally constructed sodium-saturated calomel reference electrode using a commercially available patch-clamp instrument (Axopatch 200B; Axon Instruments, Foster City, CA) configured as described previously (Borges et al, 1997). Cells were prepared for experiments as described previously (Colliver et al, 2000a).…”