Swelling-activated pathways in human T-lymphocytes studied by cell volumetry and electrorotation

Small organic solutes, including sugar derivatives, amino acids, etc., contribute significantly to the osmoregulation of mammalian cells. The present study explores the mechanisms of swelling-activated membrane permeability for electrolytes and neutral carbohydrates in Jurkat cells. Electrorotation was used to analyze the relationship between the hypotonically induced changes in the electrically accessible surface area of the plasma membrane (probed by the capacitance) and its permeability to the monomeric sugar alcohol sorbitol, the disaccharide trehalose, and electrolyte. Time-resolved capacitance and volumetric measurements were performed in parallel using media of different osmolalities containing either sorbitol or trehalose as the major solute. Under mild hypotonic stress in 200 mOsm sorbitol or trehalose solutions, the cells accomplished regulatory volume decrease by releasing cytosolic electrolytes presumably through pathways activated by the swelling-mediated retraction of microvilli. This is suggested by a rapid decrease of the area-specific membrane capacitance C(m) (microF/cm2). The cell membrane was impermeable to both carbohydrates in 200 mOsm media. Whereas trehalose permeability remained also very poor in 100 mOsm medium, extreme swelling of cells in a strongly hypotonic solution (100 mOsm) led to a dramatic increase in sorbitol permeability as evidenced by regulatory volume decrease inhibition. The different osmotic thresholds for activation of electrolyte release and sorbitol influx suggest the involvement of separate swelling-activated pathways. Whereas the electrolyte efflux seemed to utilize pathways preexisting in the plasma membrane, putative sorbitol channels might be inserted into the membrane from cytosolic vesicles via swelling-mediated exocytosis, as indicated by a substantial increase in the whole-cell capacitance C(C) (pF) in strongly hypotonic solutions.     

Membrane capacitance and conductance changes parallel mucin secretion in the human airway epithelium

Measurement of the magnitude and kinetics of exocytosis from intact epithelia has historically been difficult. Using well-differentiated cultures of human bronchial epithelial cells, we describe the use of transepithelial impedance analysis to enable the real-time quantification of mucin secretagogue-induced changes in membrane capacitance (surface area) and conductance. ATPgammaS, UTP, ionomycin, and PMA induced robust increases in total cellular capacitance that were demonstrated to be dominated by a specific increase in apical membrane surface area. The UTP-induced increase in capacitance occurred in parallel with goblet cell emptying and the secretion of mucin and was associated with decreases in apical and basolateral membrane resistances. The magnitude and kinetics of the capacitance increases were dependent on the agonist and the sidedness of the stimulation. The peak increase in capacitance induced by UTP was approximately 30 mucin granule fusions per goblet cell. Secretagogue-induced decreases in apical membrane resistance were independent of exocytosis, although each of the secretagogues induced profound reductions in basolateral membrane resistance. Transepithelial impedance analysis offers the potential to study morphological and conductance changes in cultured human bronchial epithelial cells.     

Osmotic swelling of hepatocytes increases membrane conductance but not membrane capacitance.

We have used the whole-cell patch-clamp technique to study changes in membrane conductance and membrane capacitance after osmotic swelling in rat hepatocytes. Hypoosmotic solutions induced an instantaneous increase in the volume of patch-clamped cells that was followed by a slow decline reminiscent of regulatory volume decrease as seen in intact cells. These morphological changes were associated with a transient increase in membrane conductance. The rise in conductance was not correlated with changes in capacitance, neither in time after the initiation of cell swelling nor in magnitude. Therefore we conclude that an osmotically induced increase in conductance is probably a result of the activation of existent channels in the plasmalemma and not a result of the fusion of vesicle membrane containing ionic channels.     

Membrane chloride conductance and capacitance in Jurkat T lymphocytes during osmotic swelling.

Video microscopy and whole-cell patch-clamp recording were used to monitor changes in relative cell volume (V/Vo), chloride conductance (gCl), and membrane capacitance (Cm) during osmotically induced swelling in Jurkat T lymphocytes. Cellular swelling was initiated with hyperosmotic pipette solutions. Simultaneous evaluation of V/Vo and gCl revealed a 59-s delay between the inception of swelling and the activation of outwardly rectifying, ATP-dependent Cl- channels. Following the delay, increases in V/Vo and gCl progressed in parallel. In contrast, Cm, a measure of cell surface area, fell gradually at a rate of approximately 150 fF/min after whole-cell access was achieved. The decline in Cm lasted 200 s and was followed by a rapid rise (approximately 750 fF/min). The rise in Cm coincided with a variable increase in "leak" current, gCl increased at a slower rate and reached lower peak values in experiments performed without ATP; ATP had no effect on the biphasic Cm time course. The temporal separation of conductance and capacitance during swelling suggests that gCl and Cm vary independently, supporting the hypothesis that a large portion, if not all, of the whole-cell Cl- conductance activated during swelling is provided by volume-sensitive Cl- channels preexisting in the plasma membrane.     

The noise of membrane capacitance measurements in the whole-cell recording configuration

High-resolution measurement of membrane capacitance in the whole-cell-recording configuration can be used to detect small changes in membrane surface area that accompany exocytosis and endocytosis. We have investigated the noise of membrane capacitance measurements to determine the fundamental limits of resolution in actual cells in the whole-cell mode. Two previously overlooked sources of noise are particularly evident at low frequencies. The first noise source is accompanied by a correlation between capacitance estimates, whereas the second noise source is due to "1/f-like" current noise. An analytic expression that summarizes the noise from thermal and 1/f sources is derived, which agrees with experimental measurements from actual cells over a large frequency range. Our results demonstrate that the optimal frequencies for capacitance measurements are higher than previously believed. Finally, we demonstrate that the capacitance noise at high frequencies can be reduced by compensating for the voltage drop of the sine wave across the series resistance.     

The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane

The mechanism whereby cAMP stimulates Cl(-) flux through CFTR ion channels in secretory epithelia remains controversial. It is generally accepted that phosphorylation by cAMP-dependent protein kinase increases the open probability of the CFTR channel. A more controversial hypothesis is that cAMP triggers the translocation of CFTR from an intracellular pool to the cell surface. We have monitored membrane turnover in Calu-3 cells, a cell line derived from human airway submucosal glands that expresses high levels of CFTR using membrane capacitance and FM1-43 fluorescence measurements. Using a conventional capacitance measurement technique, we observe an apparent increase in membrane capacitance in most cells that exhibit an increase in Cl(-) current. However, after we carefully correct our recordings for changes in membrane conductance, the apparent changes in capacitance are eliminated. Measurements using the fluorescent membrane marker FM1-43 also indicate that no changes in membrane turnover accompany the activation of CFTR. Robust membrane insertion can be triggered with photorelease of caged Ca(2)+ in Calu-3 cells. However, no increase in Cl(-) current accompanies Ca(2)+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca(2)+ lead to transport of CFTR to the plasma membrane in Calu-3 cells. In addition, we conclude that membrane capacitance measurements must be interpreted with caution when large changes in membrane conductance occur.     

ATP can stimulate exocytosis in rat brown adipocytes without apparent increases in cytosolic Ca2+ or G protein activation

Extracellular ATP activates large increases in cell surface area and membrane turnover in rat brown adipocytes (Pappone, P. A., and Lee, S. C. 1996. J. Gen. Physiol. 108:393-404). We used whole-cell patch clamp membrane capacitance measurements of membrane surface area concurrently with fura-2 ratio imaging of intracellular calcium to test whether these purinergic membrane responses are triggered by cytosolic calcium increases or G protein activation. Increasing cytosolic calcium with adrenergic stimulation, calcium ionophore, or calcium-containing pipette solutions did not cause exocytosis. Extracellular ATP increased membrane capacitance in the absence of extracellular calcium with internal calcium strongly buffered to near resting levels. Purinergic stimulation still activated exocytosis and endocytosis in the complete absence of intracellular and extracellular free calcium, but endocytosis predominated. Modulators of G protein function neither triggered nor inhibited the initial ATP-elicited capacitance changes, but GTPgammaS or cytosolic nucleotide depletion did reduce the cells' capacity to mount multiple purinergic responses. These results suggest that calcium modulates purinergically-stimulated membrane trafficking in brown adipocytes, but that ATP responses are initiated by some other signal that remains to be identified.     

Endocytosis and exocytosis protect cells against severe membrane tension variations

The ability of cells to regulate their shape and volume is critical for many cell functions. How endocytosis and exocytosis, as important ways of membrane trafficking, affect cellular volume regulation is still unclear. Here, we develop a theoretical framework to study the dynamics of cell volume, endocytosis, and exocytosis in response to osmotic shocks and mechanical loadings. This model can not only explain observed dynamics of endocytosis and exocytosis during osmotic shocks but also predict the dynamics of endocytosis and exocytosis during cell compressions. We find that a hypotonic shock stimulates exocytosis, while a hypertonic shock stimulates endocytosis; and exocytosis in turn allows cells to have a dramatic change in cell volume but a small change in membrane tension during hyposmotic swelling, protecting cells from rupture under high tension. In addition, we find that cell compressions with various loading speeds induce three distinct dynamic modes of endocytosis and exocytosis. Finally, we show that increasing endocytosis and exocytosis rates reduce the changes in cell volume and membrane tension under fast cell compression, whereas they enhance the changes in cell volume and membrane tension under slow cell compression. Together, our findings reveal critical roles of endocytosis and exocytosis in regulating cell volume and membrane tension.     

Kinetics of erythrocyte swelling and membrane hole formation in hypotonic media.

Red blood cell (RBC) swelling and membrane hole formation in hypotonic external media were studied by measuring the time-dependent capacitance, C, and the conductance, G, in the beginning of the beta-dispersion range. At high and moderate osmolarities of the external solution the capacitance reaches a steady-state whereas at low osmolarities it reveals a biphasic kinetics. Examination of RBC suspensions exposed to different concentrations of HgCl(2) demonstrates that water transport through mercury-sensitive water channel controls RBC swelling. Unlike the capacitance, an increase in the conductance to a stationary level is observed after a certain delay. A comparison of G(t) curves recorded for the suspensions of the intact cells and those treated with cytochalasin B or glutaraldehyde demonstrates the significant effect of the membrane viscoelasticity on the pore formation. It is shown that the stretched membrane of completely swollen RBC retains its integrity for a certain time, termed as the membrane lifetime, t(memb). Therefore, the resistivity of RBCs to a certain osmotic shock may be quantified by the distribution function of RBC(t(memb)).     

Calcium regulates the mode of exocytosis induced by hypotonic shock in isolated neuronal presynaptic endings

A decrease in the osmolarity of incubation medium is accompanied by calcium influx in neuronal presynaptic endings. We studied the influence of Ca2+ on exocytosis induced by hypotonic shock using the hydrophilic fluorescent dye acridine orange and the hydrophobic fluorescent dye FM2-10. It was shown using acridine orange that lowering of osmolarity to 230 mOsm/l induces exocytosis both in calcium-containing and calcium-free medium. By contrast, we were able to demonstrate calcium-dependence of exocytosis using styryl dye FM2-10. Lowering of osmolarity leads to increase of [3H]D-aspartate and [3H]GABA release in calcium-free medium. Addition of calcium inhibits hypotonic-induced neurotransmitter release. Decreasing of NaCl concentration to 92 mM in isotonic medium is able to induce d-aspartate and GABA release. Thus, our data suggest that hypotonic swelling induces calcium-independent exocytosis possibly by a "kiss and run" mechanism. Calcium influx mediated by stretch channels is able to provoke full fusion between plasma membrane and synaptic vesicles. [3H]D-aspartate and [3H]GABA released by hypotonic shock is determined by sodium lowering rather than by osmolarity decreasing itself.     

Pharmacological characterization of swelling-induced D-[3H]aspartate release from primary astrocyte cultures

During stroke orhead trauma, extracellular K⁺concentration increases, which can cause astrocytes to swell. In vitro,such swelling causes astrocytes to release excitatory amino acids, which may contribute to excitotoxicity in vivo. Several putative swelling-activated channels have been identified through which suchanionic organic cellular osmolytes can be released. In the presentstudy, we sought to identify the swelling-activated channel(s) responsible forD-[³H]aspartaterelease from primary cultured astrocytes exposed to either KCl orhypotonic medium. KCl-inducedD-[³H]aspartaterelease was inhibited by the anion channel inhibitors 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), dideoxyforskolin, L-644711, ATP, ITP, 3'-azido-3'-deoxythymidine, DIDS, andtamoxifen but not by cAMP. The cell swelling caused by raised KCl wasnot inhibited by extracellular ATP or tamoxifen as measured by an electrical impedance method, which suggests that these anion channel inhibitors directly blocked the channel responsible for efflux. Extracellular nucleotides and DIDS, however, had no or only partial effects onD-[³H]aspartaterelease from cells swollen by hypotonic medium, but such release wasinhibited by NPPB, dideoxyforskolin, and tamoxifen. Of theswelling-activated channels so far identified, our data suggest that avolume-sensitive outwardly rectifying channel is responsible forD-[³H]aspartaterelease from primary cultured astrocytes during raised extracellularK⁺ and possibly during hypotonicmedium-induced release.

     

Adipocyte cell size enlargement involves plasma membrane area increase

The adipocyte enlargement is associated with an increase in the cytoplasmic lipid content, but how the plasma membrane area follows this increase is poorly understood. We monitored single-cell membrane surface area fluctuations, which mirror the dynamics of exocytosis and endocytosis. We employed the patch-clamp technique to measure membrane capacitance (C(m)), a parameter linearly related to the plasma membrane area. Specifically, we studied whether insulin affects membrane area dynamics in adipocytes. A five-minute cell exposure to insulin increased resting C(m) by 12 ± 4%; in controls the change in C(m) was not different from zero. We measured cell diameter of isolated rat adipocytes microscopically. Twenty-four hour exposure of cells to insulin resulted in a significant increase in cell diameter by 5.1 ± 0.6%. We conclude that insulin induces membrane area increase, which may in chronic hyperinsulinemia promote the enlargement of plasma membrane area, acting in concert with other insulin-mediated metabolic effects on adipocytes.     

Purinergic receptor stimulation increases membrane trafficking in brown adipocytes

Stimulation of brown adipocytes by their sympathetic innervation plays a major role in body energy homeostasis by regulating the energy- wasting activity of the tissue. The norepinephrine released by sympathetic activity acts on adrenergic receptors to activate a variety of metabolic and membrane responses. Since sympathetic stimulation may also release vesicular ATP, we tested brown fat cells for ATP responses. We find that micromolar concentrations of extracellular ATP initiates profound changes in the membrane trafficking of brown adipocytes. ATP elicited substantial increases in total cell membrane capacitance, averaging approximately 30% over basal levels and occurring on a time scale of seconds to minutes. The membrane capacitance increase showed an agonist sensitivity of 2-methylthio-ATP > or = ATP > ADP > > adenosine, consistent with mediation by a P2r type purinergic receptor. Membrane capacitance increases were not seen when cytosolic calcium was increased by adrenergic stimulation, and capacitance responses to ATP were similar in the presence and absence of extracellular calcium. These results indicate that increases in cytosolic calcium alone do not mediate the membrane response to ATP. Photometric assessment of surface-accessible membrane using the dye FM1- 43 showed that ATP caused an approximate doubling of the amount of membrane actively trafficking with the cell surface. The discrepancy in the magnitudes of the capacitance and fluorescence changes suggests that ATP both activates exocytosis and alters other aspects of membrane handling. These findings suggest that secretion, mobilization of membrane transporters, and/or surface membrane expression of receptors may be regulated in brown adipocytes by P2r purinergic receptor activity.     

An optimized approach to membrane capacitance estimation using dual-frequency excitation.

We present an optimized solution to the problem of membrane impedance estimation when a patch-clamped cell is stimulated by a dual-frequency, sinusoidal excitation. The complete data set of raw whole-cell current samples is typically reduced, via digital lock-in detection, to measurements of the complex cell model admittance at the two stimulus frequencies. We describe a statistical model of both data sets and demonstrate that the admittance data adequately represent the essential features obtained from the raw data. The parameter estimates obtained by a nonlinear weighted least-squares solution (NWLS), which under normal recording conditions is equivalent to the maximum likelihood solution, essentially obtain the theoretical lower bound on variance established by the Cramér-Rao bound. Our software implementation of the NWLS solution produces estimates of the cell model parameters that are less noisy than other dual-frequency systems. Our system can be used 1) to measure slow changes in membrane capacitance-in the face of large, slow changes in membrane resistance, 2) to detect with confidence capacitance changes expected from the exocytosis of moderate-sized dense core granules, and 3) to reduce the cross-talk between transient changes in membrane conductance and membrane capacitance.     

Kinetics of erythrocyte swelling and membrane hole formation in hypotonic media

Red blood cell (RBC) swelling and membrane hole formation in hypotonic external media were studied by measuring the time-dependent capacitance, C, and the conductance, G, in the beginning of the β-dispersion range. At high and moderate osmolarities of the external solution the capacitance reaches a steady-state whereas at low osmolarities it reveals a biphasic kinetics. Examination of RBC suspensions exposed to different concentrations of HgCl₂ demonstrates that water transport through mercury-sensitive water channel controls RBC swelling. Unlike the capacitance, an increase in the conductance to a stationary level is observed after a certain delay. A comparison of G(t) curves recorded for the suspensions of the intact cells and those treated with cytochalasin B or glutaraldehyde demonstrates the significant effect of the membrane viscoelasticity on the pore formation. It is shown that the stretched membrane of completely swollen RBC retains its integrity for a certain time, termed as the membrane lifetime, t_memb. Therefore, the resistivity of RBCs to a certain osmotic shock may be quantified by the distribution function of RBC(t_memb).     

Exocytosis in bovine chromaffin cells: studies with patch-clamp capacitance and FM1-43 fluorescence

In response to physiological stimuli, neuroendocrine cells secrete neurotransmitters through a Ca(2+)-dependent fusion of secretory granules with the plasma membrane. We studied insertion of granules in bovine chromaffin cells using capacitance as a measure of plasma membrane area and fluorescence of a membrane marker FM1-43 as a measure of exocytosis. Intracellular dialysis with [Ca(2+)] (1.5-100 microM) evoked massive exocytosis that was sufficient to double plasma membrane area but did not swell cells. In principle, in the absence of endocytosis, the addition of granule membrane would be anticipated to produce similar increases in the capacitance and FM1-43 fluorescence responses. However, when endocytosis was minimal, the changes in capacitance were markedly larger than the corresponding changes in FM1-43 fluorescence. Moreover, the apparent differences between capacitance and FM1-43 fluorescence changes increased with larger exocytic responses, as more granules fused with the plasma membrane. In experiments in which exocytosis was suppressed, increasing membrane tension by osmotically induced cell swelling increased FM1-43 fluorescence, suggesting that FM1-43 fluorescence is sensitive to changes in the membrane tension. Thus, increasing membrane area through exocytosis does not swell chromaffin cells but may decrease membrane tension.     

Membrane reserves and hypotonic cell swelling

To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o(-), CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by swelling cells in extreme 98% hypotonic (approximately 6 mOsm) solution until membrane rupture; all cell types examined demonstrated surprisingly large membrane reserves and could increase their SA 3.6 +/- 0.2-fold and V 10.7 +/- 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10 degrees C) reduced SA and V increases of A549 cells to 1.7 +/- 0.3-fold and 4.4 +/- 0.9-fold, respectively. Interestingly, blocking exocytosis did not affect SA and V changes during moderate swelling in 50% hypotonicity. Thus, mammalian cells accommodate moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant endomembrane insertion is observed only during extreme swelling. Large membrane reserves may provide a simple mechanism to maintain membrane tension below the lytic level during various cellular processes or acute mechanical perturbations and may explain the difficulty in activating mechanogated channels in mammalian cells.     

Functional down-regulation of volume-regulated anion channels in AQP4 knockdown cultured rat cortical astrocytes

In the brain, the astroglial syncytium is crucially involved in the regulation of water homeostasis. Accumulating evidence indicates that a dysregulation of the astrocytic processes controlling water homeostasis has a pathogenetic role in several brain injuries. Here, we have analysed by RNA interference technology the functional interactions occurring between the most abundant water channel in the brain, aquaporin-4 (AQP4), and the swelling-activated Cl(-) current expressed by cultured rat cortical astrocytes. We show that in primary cultured rat cortical astrocytes transfected with control small interfering RNA (siRNA), hypotonic shock promotes an increase in cellular volume accompanied by augmented membrane conductance mediated by volume-regulated anion channels (VRAC). Conversely, astroglia in which AQP4 was knocked down (AQP4 KD) by transfection with AQP4 siRNA changed their morphology from polygonal to process-bearing, and displayed normal cell swelling but reduced VRAC activity. Pharmacological manipulations of actin cytoskeleton in rat astrocytes, and functional analysis in mouse astroglial cells, which retain their morphology upon knockdown of AQP4, suggest that stellation of AQP4 KD rat cortical astrocytes was not causally linked to reduction of VRAC current. Molecular analysis of possible candidates of swelling-activated Cl(-) current provided evidence that in AQP4 KD astrocytes, there was a down-regulation of chloride channel-2 (CIC-2), which, however, was not involved in VRAC conductance. Inclusion of ATP in the intracellular saline restored VRAC activity upon hypotonicity. Collectively, these results support the view that in cultured astroglial cells, plasma membrane proteins involved in cell volume homeostasis are assembled in a functional platform.     

cAMP directly facilitates Ca-induced exocytosis in bovine lactotrophs

We have used the whole cell patch clamp technique on single prolactin-secreting bovine lactotrophs to measure plasma membrane capacitance (Cm), an index of membrane surface area, under voltage-clamp during cytosol dialysis with Ca and cAMP. cAMP increased the magnitude and rate of Ca-induced exocytosis (Cm increase) without affecting membrane conductance; however, cAMP had no detectable effect on Cm when intracellular Ca was low. We thus report new evidence that cAMP can facilitate Ca-induced secretion in a synergistic fashion, by acting directly on the secretory apparatus, independently of membrane conductance activation.