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.     

Role of microtubule assembly in lysosomal enzyme secretion from human polymorphonuclear leukocytes. A reevaluation

The dose-related inhibition by colchicine of both lysosomal enzyme release and microtubule assembly was studied in human polymorphonuclear leukocytes (PMN) exposed to the nonphagocytic stimulus, zymosan-treated serum (ZTS). Cells were pretreated with colchicine (60 min, 37 degrees C) with or without cytochalasin B (5 microng/ml, 10 min) and then stimulated with ZTS (10%). Microtubule numbers in both cytochalasin B- treated and untreated PMN were increased by stimulation and depressed below resting levels in a dose-response fashion by colchicine concentrations above 10(-7) M. These concentrations also inhibited enzyme release in a dose-response fashion although the inhibition of microtubule assembly was proportionately greater than the inhibition of enzyme release. Other aspects of PMN morphology were affected by colchicine. Cytochalasin B-treated PMN were rounded, and in thin sections the retracted plasma membrane appeared as invaginations oriented toward centrally located centrioles. Membrane invaginations were restricted to the cell periphery in cells treated with inhibitory concentrations of colchicine, and the centrioles and Golgi apparatus were displaced from their usual position. After stimulation and subsequent degranulation, the size and number of membrane invaginations greatly increased. They remained peripheral in cells pretreated with greater than 10(-7) M colchicine but were numerous in the pericentriolar region in cells treated with less than 10(-7) M. Similarly, untreated PMN that were permitted to phagocytose immune precipitates had many phagosomes adjacent to the centriole. After colchicine treatment, phagosomes were distributed randomly, without any preferential association with the centrioles. These data suggest that microtubules are involved in maintaining the internal organization of cells and the topologic relationships between organelles and the plasma membrane.     

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.     

Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line

ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 +/- 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 +/- 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Cai2+) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Cai2+ was unaffected, but the initial response and overall time to Cai2+ peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.     

Simultaneous capacitance and amperometric measurements of exocytosis: a comparison.

We measured the exocytotic response induced by flash photolysis of caged compounds in isolated mast cells and chromaffin cells. Vesicle fusion was measured by monitoring the cell membrane capacitance. The release of vesicular contents was followed by amperometry. In response to a GTP gamma S stimulus we found that the time integral of the amperometric current could be superimposed on the capacitance trace. This shows that the integrated amperometric signal provides an alternative method of measuring the extent and kinetics of the secretory response. Very different results were obtained when photolysis of caged Ca2+ (DM-nitrophen) was used to stimulate secretion. In mast cells, there was an immediate, graded increase in membrane capacitance that was followed by step increases (indicative of granule fusion). During the initial phase of the capacitance increases, no release of oxidizable secretory products was detected. In chromaffin cells we also observed a considerable delay between increases in capacitance, triggered by uncaging Ca2+, and the release of oxidizable secretory products. Here we demonstrate that there can be large increases in the membrane capacitance of a secretory cell, triggered by flash photolysis of DM-nitrophen, which indicate events that are not due to the fusion of granules containing oxidizable substances. These results show that increases in capacitance that are not resolved as steps cannot be readily interpreted as secretory events unless they are confirmed independently.     

Astrocyte swelling leads to membrane unfolding, not membrane insertion

The mechanisms mediating the release of chemical transmitters from astrocytes are the subject of intense research. Recent experiments have shown that hypotonic conditions stimulate the release of glutamate and ATP from astrocytes, but a mechanistic understanding of this process is not available. To determine whether hypotonicity activates the process of regulated exocytosis, we monitored membrane capacitance by the whole-cell patch-clamp technique whilst a hypotonic medium was applied to cultured astrocytes. If exocytosis is triggered under hypotonic conditions, as it is following increases in cytosolic calcium, a net increase in membrane surface area, monitored by measuring the whole-cell membrane capacitance, is expected. Simultaneous measurements of cell size and whole-cell membrane conductance and surface area demonstrated that hypotonic medium (210 mOsm for 200 s) resulted in an increase in membrane conductance and in the swelling of cultured astrocytes by an average of 40%, as monitored by cell cross-sectional area, but without any corresponding change in membrane surface area. As we have demonstrated that capacitance measurements have the sensitivity to detect increases in cell surface area as small as 0.5%, we conclude that cell swelling occurs via an exocytosis-independent mechanism, probably involving the unfolding of the plasma membrane.     

Optocapacitance: physical basis and its application

The observation that membrane capacitance increases with temperature has led to the development of new methods of neuronal stimulation using light. The optocapacitive effect refers to a light-induced change in capacitance produced by the heating of the membrane through a photothermal effect. This change in capacitance manifests as a current, named optocapacitive current that depolarizes cells and therefore can be used to stimulate excitable tissues. Here, we discuss how optocapacitance arises from basic membrane properties, the characteristics of the optocapacitive current, its use for neuronal stimulation, and the challenges for its application in vivo.     

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.     

Structure and Development of Viruses as Observed in the Electron Microscope: X. Entry and Uncoating of Adenovirus

Stages in the direct penetration of adenovirus through the cell membrane are illustrated. Phagocytosis with rupture of the vacuole and release of virus into the cytoplasm may also account for entry of some particles. Uncoating by digestion within phagosomes was not observed. Rather, alteration of capsid and core occurred to virions free in the cytoplasm. Nucleoprotein released from virus close to the nucleus was transported to the nuclear matrix by a unique mechanism. These events were not prevented by puromycin and hence were not dependent upon the synthesis of new enzymes. They were, however, energy-dependent.     

Cytochalasin D attenuates the desensitisation of pressure-stimulated vesicle fusion in guard cell protoplasts

Fusion of vesicular membranes with the plasma membrane during pressure-driven swelling of guard cell protoplasts was studied using patch clamp capacitance measurements. Hydrostatic pressure pulses were applied via the patch pipette and resulted in an immediate and linear increase in membrane capacitance, a parameter proportional to the surface area. In any given protoplast, pressure-stimulated increases in membrane capacitance could be provoked repetitively. However, the rate of rise in capacitance upon the same strength of stimulation decreased exponentially with time (tau = 4 min) for subsequent pressure stimuli. This process was the result of a desensitisation of the plasma membrane to mechanical forces. Incubation of guard cell protoplasts in cytochalasin D, which depolymerises actin filaments, nearly abolished this desensitisation process. These results suggest that membrane stretch initiates a reactive process that may fortify or stabilise the plasma membrane of guard cell protoplasts.     

Focal exocytosis by eosinophils--compound exocytosis and cumulative fusion.

We have investigated the granule fusion events during exocytosis in horse eosinophils by time-resolved patch-clamp capacitance measurements. Stimulation with intracellular GTP gamma S leads to a stepwise capacitance increase by 4.0 +/- 0.9 pF. At GTP gamma S concentrations < 20 microM the step size distribution is in agreement with the granule size distribution in resting cells. Above 80 microM the number of steps is reduced and very large steps occur. The total capacitance increase, however, is unaffected. These results show that at high GTP gamma S concentrations granule--granule fusion occurs inside the cell forming large compound granules, which then fuse with the plasma membrane (compound exocytosis). The electrical equivalent circuit of the cell during degranulation indicates the formation of a degranulation sac by cumulative fusion events. Fusion of the first granule with the plasma membrane induces fusion of further granules with this granule directing the release of all the granular material to the first fusion pore. The physiological function of eosinophils is the killing of parasites. Compound exocytosis and cumulative fusion enable the cells to focus the release of cytotoxic proteins to well defined target regions and prevent uncontrolled diffusion of this material, which would damage intact host cells.     

Sperm factor initiates capacitance and conductance changes in mouse eggs that are more similar to fertilization than IP(3)- or Ca(2+)-induced changes

We used patch clamp electrophysiology and concurrent imaging with the Ca(2+)-sensitive dye, fura-2, to study the temporal relationship between membrane capacitance and conductance and intracellular free Ca(2+) concentration ([Ca(2+)](i)) during mouse egg fertilization. We found an approximately 2 pF step increase in egg membrane capacitance and a minor increase in conductance with no change in [Ca(2+)](i) at sperm fusion. This was followed approximately 1 min later by a rise in [Ca(2+)](i) that led to larger changes in capacitance and conductance. The most common pattern for these later capacitance changes was an initial capacitance decrease, followed by a larger increase and eventual return to the approximate starting value. There was some variation in this pattern, and sub-microM peak [Ca(2+)](i) favored capacitance decrease, while higher [Ca(2+)](i) favored capacitance increase. The magnitude of accompanying conductance increases was variable and did not correlate well with peak [Ca(2+)](i). The intracellular introduction of porcine sperm factor reproduced the postfusion capacitance and conductance changes with a similar [Ca(2+)](i) dependence. Raising [Ca(2+)](i) by the intracellular introduction of IP(3) initiated fertilization-like capacitance changes, but the conductance changes were slower to activate. Capacitance decrease could be induced when [Ca(2+)](i) was increased modestly by activation of an endogenous Ca(2+) current, with little effect on resting conductance. These results suggest that net turnover of the mouse egg surface membrane is sensitive to [Ca(2+)](i) and that sperm and the active component of sperm factor may be doing more than initiating the IP(3)-mediated release of intracellular Ca(2+).     

Novel insight into current models of NADPH oxidase regulation, assembly and localization in human polymorphonuclear leukocytes.

We review herein the definition of the NADPH oxidase-activating site in human neutrophils and eosinophils, together with the new biochemical findings of the assembly of NADPH oxidase components and the signal transduction for the activation of NADPH oxidase. The activation of this enzyme is associated with multiple interrelated signaling pathways. Upon cell stimulation, the second messengers act on the assembly of NADPH oxidase components. The cytosolic components are first phosphorylated, and then associated with the membrane components. Small GTP-binding proteins and cytoskeletal components also participate in the activation of the NADPH oxidase. The cytochemical findings demonstrate that the superoxide generated by NADPH oxidase activity is initially localized in distinct types of intracellular granules, and not at the plasma membrane as previously believed. Thus, the assembly of NADPH oxidase components possibly occurs at the limiting membrane of the intracellular compartments. The oxidant-producing compartments mobilize and become associated with the plasma membrane upon cell stimulation with soluble stimulants, or fuse to phagosomes upon stimulation with particulate stimulants. Accordingly, superoxide is released to the extracellular space and into phagosomes in proportion to the oxidant-producing intracellular granule association with the plasma membrane and with the phagosomal membrane, respectively.     

Membrane dielectric responses of human T-lymphocytes following mitogenic stimulation

Human peripheral blood T-lymphocytes, normally resting at the G0 phase, were stimulated with phytohemagglutinin (PHA) and interleukin-2 (IL-2) to induce the cell division cycle. The cells were examined at 24-h intervals for up to 96 h by flow cytometry to determine cell cycle distributions and by electrorotation to determine dielectric properties. The average membrane specific capacitance was found to vary from 12 (+/-1.5) mF/m2 prior to stimulation to 10 (+/-1.5) and 16 (+/-3.5) mF/m2 at 24 and 48 h after stimulation, respectively, and to remain unchanged up to 96 h after stimulation. Scanning electron microscopy studies of the cells revealed an increased complexity in cell membrane morphology following stimulation, suggesting that the observed change in the membrane capacitance was dominated by the alteration of cell surface structures. The average electrical conductivity of the cell interior decreased from approximately 1.1 S/m prior to stimulation to approximately 0.8 S/m at 24 h after stimulation and showed little change thereafter. The average dielectric permittivity of the cell interior remained almost unchanged throughout the course of the cell stimulation. The percentage of T-lymphocytes in the S and G2/M phases increased from approximately 4% prior to stimulation to approximately 11 and approximately 34% at 24 and 48 h after stimulation, respectively. The large change in membrane specific capacitance between the 24 and 48 h time period coincided with the large alteration in the cell cycle distribution where the S and G2/M populations increased by approximately 23%. These data, together with an analysis of the variation of the membrane capacitance during the cell cycle based on the cell cycle-dependent membrane lipid accumulation, show that there is a correlation between membrane capacitance and cell cycle phases that reflects alterations in the cell plasma membrane.     

Endocytic membrane traffic with respect to phagosomes in macrophages infected with non-pathogenic bacteria: phagosomal membrane acquires the same composition as lysosomal membrane

A morphometric analysis was made to study membrane traffic in bone marrow-derived macrophages, containing phagosomes with partially degraded Bacillus subtilis. Cell surface glycoproteins, labeled with radioactive galactose by terminal glycosylation, provided a covalent autoradiographic membrane marker. Membrane compartments were characterized in terms of cytochemical staining for horseradish peroxidase taken up by receptor-mediated endocytosis. The area, composition, and exchange rates of endocytic membrane compartments were measured as in a previous analysis for non-infected macrophages, devoid of phagosomes. In direct comparison with this earlier study, the present data allowed an assessment of the involvement of phagosomes in the interactions between endocytic membrane compartments. The presence of phagosomes led to a 30% reduction of lysosomal membrane area. The rate at which cell surface-derived label flowed into the lysosomal membrane pool was reduced by the same fractional amount. This suggested a linear relationship between flow rate and membrane area. The initial flow rate of label into phagosomes was higher than expected, based on their membrane area being only about 60% that of lysosomes. This rate could only be measured during the early phase of the experiments when phagosomes were younger, therefore displaying a fast exchange rate, reminiscent of the endosome compartment. However, steady-state conditions, at late times, strongly suggested that phagosomes with degraded contents finally acquire membrane of lysosomal origin. First, the composition of phagosome membrane became the same as that of lysosomes, remaining unchanged as compared to non-infected cells. Second, the membrane area of phagosomes amounted to the loss of lysosomal membrane area in infected cells.     

Hormone-stimulated polyphosphoinositide breakdown in rat liver plasma membranes. Roles of guanine nucleotides and calcium

Calcium-sensitive inositide release in a purified rat liver plasma membrane preparation is increased by calcium-mobilizing hormones in the presence of guanine nucleotides. Vasopressin-stimulated inositide release is evident in the presence of GTP or its nonhydrolyzable analogs guanyl-5'-yl imidodiphosphate and guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). The stimulation of inositide release by (-)-epinephrine (alpha 1), angiotensin II, or vasopressin in the presence of either 1 microM or 10 microM GTP gamma S correlates with the number of receptors present for each hormone. The guanine nucleotide and hormonal stimulation is evident on both inositol trisphosphate production and phosphatidylinositol bisphosphate degradation. Ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (1 mM) completely abolishes stimulation by guanine nucleotides and hormone. Prior treatment of plasma membranes with cholera toxin or islet activating protein or prior injection of animals with islet activating protein does not affect stimulation of inositide release by GTP gamma S or GTP gamma S plus vasopressin. Stimulation by GTP gamma S is dependent upon magnesium and is inhibitable by guanosine 5'-(2-O-thio) diphosphate. Inositide release from the plasma membrane exhibits half-maximal stimulation by calcium at approximately 100 nM free calcium in the presence of 1.5 mM MgCl2 and at approximately 10 microM free calcium in the presence of 10 mM MgCl2. Addition of guanine nucleotides decreases the requirement for calcium and also increases the activity at saturating calcium. The results presented suggest that calcium-mobilizing hormones stimulate polyphosphoinositide breakdown in rat liver plasma membranes through a novel guanine nucleotide binding protein.     

Growth factor- and heparin-dependent regulation of constitutive and agonist-mediated human endothelial barrier function

We report functional differences in constitutive and agonist-mediated endothelial barrier function between cultured primary and Clonetics human umbilical vein endothelial cells (pHUVEC and cHUVEC) grown in soluble growth factors and heparin. Basal transendothelial resistance (TER) was much lower in pHUVEC than in cHUVEC grown in medium supplemented with growth factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and human epithelial growth factor (EGF), and heparin. On the basis of a numerical model of TER, the increased basal TER in cHUVEC was due to effects on cell-matrix adhesion and membrane capacitance. Heparin and bFGF increased constitutive TER in cultured pHUVEC, and heparin mediated additional increases in constitutive TER in pHUVEC supplemented with bFGF. EGF attenuated bFGF-mediated increases in TER. On the basis of the numerical model, in contrast to cHUVEC, heparin and bFGF augmented TER through effects on cell-cell adhesion and membrane capacitance in pHUVEC. Thrombin mediated quantitatively greater amplitude and a more sustained decline in TER in cultured cHUVEC than pHUVEC. Thrombin-mediated barrier dysfunction was attenuated in pHUVEC conditioned in EGF in the presence or absence of heparin. Thrombin-mediated barrier dysfunction was also attenuated when monolayers were exposed to low concentrations of heparin and further attenuated in the presence of bFGF. cAMP stimulation mediated differential attenuation of thrombin-mediated barrier dysfunction between pHUVEC and cHUVEC. VEGF displayed differential effects in TER in serum-free medium. Taken together, these data demonstrate marked differential regulation of constitutive and agonist-mediated endothelial barrier function in response to mitogens and heparin stimulation.     

Secretory phospholipase A2 is released from pancreatic beta-cells and stimulates insulin secretion via inhibition of ATP-dependent K+ channels

The release of sPLA(2) from single mouse pancreatic beta-cells was monitored using a fluorescent substrate of the enzyme incorporated in the outer leaflet of the plasma membrane. Stimulation of beta-cells with agents that increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) induced a rapid release of sPLA(2) to the extracellular medium. Exogenous sPLA(2) strongly stimulated insulin secretion in mouse pancreatic islets at both basal and elevated glucose concentrations. The stimulation of insulin secretion by sPLA(2) was mediated via inhibition of ATP-dependent K(+) channels and an increase in [Ca(2+)](i). Measurements of cell capacitance in single beta-cells revealed that sPLA(2) did not modify depolarisation-induced exocytosis. Our data suggest that a positive feedback regulation of insulin secretion by co-released sPLA(2) is operational in pancreatic beta-cells and point to this enzyme as an autocrine regulator of insulin secretion.     

Exocytotic events unrelated to regulation of water permeability in amphibian tight epithelia: effects of oxytocin, PMA and insulin on membrane capacitance, water and Na+ transport

We measured the effects of oxytocin on capacitance and hydroosmotic water flow in the urinary bladder of the toad Bufo marinus and the skins of Rana pipiens and Rana temporaria. Oxytocin increased capacitance in all these tissues but stimulated hydroosmotic water flow only in the urinary bladder. We also measured the effects of oxytocin and PMA on the capacitance and hydroosmotic water flow of the toad urinary bladder. Both agents produced increases in membrane capacitance that were additive, however, PMA produced a stimulation of water flow that was only a fraction of that caused by oxytocin. Comparison of the effects of PMA and insulin in toad urinary bladder showed that in contrast with PMA, insulin did not increase membrane capacitance in this tissue. Moreover, insulin stimulated Isc in the urinary bladder while PMA produced an inhibition of variable magnitude. These results suggest that: (1) oxytocin can promote the fusion with the apical membrane of cytoplasmic membranes with or without water channels; (2) oxytocin and PMA stimulate the fusion with the apical membrane of cytoplasmic membranes originating in different pools; membranes in each pool have different water permeabilities and their insertion is controlled by different signals; (3) PMA and insulin act through different mechanisms in the toad urinary bladder.     

大鼠嗜铬细胞分泌的膜电容和微碳纤电极检测

在原代培养的大鼠肾上腺嗜铬细胞上,综合运用细胞内钙测定法和全细胞膜片钳法,以检测膜电容变化为手段测定单一肾上腺嗜铬细胞的胞吐过程。－７０ｍＶ到＋２０ｍＶ去极化引起的钙电流和细胞膜电容的变化以及吹加６０ｍｍｏｌ／ＬＫＣｌ时,细胞内游离钙离子浓度［Ｃａ２＋］ｉ和细胞膜电容变化的同时检测,表明了Ｃａ２＋对细胞胞吐的控制作用。而用微碳纤电极则能检测到吹加６０ｍｍｏｌ／ＬＫＣｌ导致嗜铬细胞胞吐时儿茶酚胺的量子化释放。细胞膜电容检测和微碳纤电极检测从不同侧面动态的反映了细胞胞吐过程与Ｃａ２＋的相关性