Different time courses of GTP[γ-S]-induced exocytosis and current oscillations in isolated mouse pancreatic acinar cells

Exocytosis in isolated mouse pancreatic acinar cells was investigated using the dual-frequency method for measuring membrane capacitance and ionic conductances. Under control conditions, single exo- and endocytotic events could be resolved. The total cell capacitance slightly decreased to 98.7 ± 0.9% of the initial cell capacitance within 10 min after establishing the whole-cell configuration. When guanosine 5′-O-(3-thiophosphate) (GTP[γ-S] was added to the patch pipette, stepwise elevations in membrane capacitance occurred and the cell capacitance increased to 106.7 ± 1.6% within 10 min. Exocytosis was also stimulated by GTP[γ-S] when a Ca²⁺-free pipette solution supplemented with 1 to 10 mM ethylenebis(oxonitrilo) tetraacetate (EGTA) was used. Measurement of the DC current component in parallel with AC current analysis was used to isolate components of the Ca²⁺-dependent Cl⁻ and monovalent cation conductances from the whole-cell conductance. These experiments demonstrate that in GTP[γ-S]-stimulated pancreatic acinar cells: (1) activation of Cl⁻ currents precedes that of cation currents, and (2) fusion of the zymogen granule membrane with the plasma membrane does not lead to incorporation of active Cl⁻ or nonselective cation channels (≥ 10 pS). Received: 11 March 1996/Accepted: 3 May 1996     

Calmodulin increases the initial rate of exocytosis in adrenal chromaffin cells

The effect of calmodulin on exocytosis in bovine adrenal medullary chromaffin cells was examined by the use of patch-clamp capacitance recording. Calmodulin was dialysed into cells via the patch-pipette and cells stimulated by depolarisation. Following a test stimulation, cells were dialysed with a control or with a calmodulin containing buffer for 10mins and then were stimulated at 2min intervals thereafter. The inclusion of calmodulin in the pipette did not increase Ca²⁺ currents which instead decreased during dialysis. The presence of calmodulin, however, resulted in a 2-fold increase in the initial rate of exocytosis during the 10min depolarisation step. These results demonstrate the utility of the patch-clamp capacitance technique for the examination of the effect of soluble proteins on exocytosis and in conjunction with previous work on permeabilised chromaffin cells suggest that calmodulin regulates late steps in Ca²⁺-dependent exocytosis.     

Ca2+-dependent capacitance increases in rat basophilic leukemia cells following activation of store-operated Ca2+ entry and dialysis with high-Ca2+-containing intracellular solution

 Ca²⁺-dependent vesicular fusion was studied in single whole-cell patch-clamped rat basophilic leukemia (RBL) cells using the capacitance technique. Dialysis of the cells with 10 μM free Ca²⁺ and 300 μM guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]) resulted in prominent capacitance increases. However, dialysis with either Ca²⁺ (225 nM to 10 μM) or GTP[γ-S] alone failed to induce a capacitance change. Under conditions of weak Ca²⁺ buffering (0.1 mM EGTA), activation of Ca²⁺-release-activated Ca²⁺ (CRAC) channels by dialysis with inositol 1,4,5-trisphosphate (InsP ₃) failed to induce a capacitance increase even in the presence of GTP[γ-S]. However, when Ca²⁺ATPases were inhibited by thapsigargin, InsP ₃ and GTP[γ-S] led to a pronounced elevation in membrane capacitance. This increase was dependent on a rise in intracellular Ca²⁺ because it was abolished when cells were dialysed with a high level of EGTA (10 mM) in the recording pipette. The increase was also dependent on Ca²⁺ influx because it was effectively suppressed when external Ca²⁺ was removed. Our results demonstrate that I _CRAC represents an important source of Ca²⁺ for triggering a secretory response. Received: 1 May 1998 / Received after revision: 15 June 1998 / Accepted: 2 July 1998     

Interdigitated microelectrode-based microchip for electrical impedance spectroscopic study of oral cancer cells

In this study, electric/electrochemical impedance spectroscopy and cyclic voltammetry were used to study the cellular activities of oral cancer cell line CAL 27, including the kinetics of cell adhesion, spreading, and cell proliferation on interdigitated microelectrodes (IMEs). Impedance spectra of CAL 27 cells on IMEs electrodes were obtained in cell growth medium and in 0.1 M PBS with 50 mM [Fe(CN)₆]^3−/4− as redox probe. Equivalent circuits were used to model both cases. In cell growth medium, impedance spectra allowed us to analyze the changes in capacitance and resistance due to cell attachment on the IMEs over the entire experiment period. It was found that cell spreading caused the most significant decrease in capacitance component and slight increase in resistance component. Impedance change at given frequencies (between 10 kHz to 100 kHz) was found to be linearly increased with increasing cell number of CAL 27 on the IMEs. In comparison with non-cancer oral epithelial cells (Het-1A), at equal cell number, cancer cells always generated impedance several folds higher than that of non-cancer cells. In the presence of [Fe(CN)₆]^3−/4−, impedance spectra allowed us to analyze the change in electron transfer resistance of IMEs due to cell attachment, in which an increase trend was observed at 24 h with increasing cell number from 2500 cells to 10000 cells on IMEs. Double layer capacitance was also affected by cell attachment, and a decrease in double layer capacitance was observed with increasing cell number on the electrodes. Cyclic voltammetric measurements correlated well with the impedance results. The results of this study demonstrated the use of electrochemical approaches to obtain and understand cellular behaviors/activities of oral cancer cells, potentially providing useful tools for cancer cell research.     

Passive electrical properties and voltage dependent membrane capacitance of single skeletal muscle fibers

The passive membrane capacitance and conductance of isolated single muscle fibers were investigated using a vaseline gap method. The results obtained with this method are consistent with those obtained using the microelectrode technique. It was confirmed that the membrane capacitance of skeletal muscle consisted of a large capacitance of tubular membrane (7–10 F/cm²) and a much smaller capacitance of surface membrane (1–2 F/cm²). The relative time constants of these two components vary from one sample to another, resulting in one time and two time constant behaviors.Secondly, the capacitance of isolated skeletal muscle fibers was investigated during hyper- and depolarizing pulses, using the transient bridge technique with the vaseline gap method. Measurements were performed at two frequencies, i.e. 500 Hz and 20 kHz. It was found that the membrane capacitance increased by 15–20% with depolarizations. The voltage dependent membrane capacitance was no affected by the addition of tetrodotoxin in bathing solution blocking sodium current and muscle contraction. Also, blocking both Na and K current did not have an appreciable effect on the non-linear behavior of membrane capacitance. The origin of voltage dependent capacitance in muscle membrane appears to be distributed among several non-linear ionic processes such as Na and K currents and the flux of Ca and Cl ions and their accumulation.     

Graded action potentials generated by differentiated human neuroblastoma cells

Stimulus-dependent impulses and resting membrane parameters of human SH-SY5Y neuroblastoma cells, induced to differentiate by retinoic acid, were investigated with tight-seal recording techniques. Mean resting potential was -53 mV, mean input resistance 2.1 GΩ, mean capacitance 14 pF, and mean time constant 30 ms. Rectangular current steps induced clearly stimulus-dependent impulses, with stronger current steps causing impulses of larger amplitude. The degree of impulse variability differed significantly among different cells. The current thresholds for impulse generation ranged from 35 to 100 pA for 10 ms current steps. With longer current steps, thresholds below 10 pA were recorded. In response to 0.5-1 s long current steps, most cells generated only a single impulse, but a few cells generated two impulses. When two impulses were generated, the interval between the impulses decreased with increasing stimulus strength. Whole-cell currents were recorded under voltage-clamp conditions. Voltage-activated, tetrodotoxin-sensitive Na⁺ currents and ‘delayed rectifier’ K⁺ currents were recorded. The degree of impulse variability was correlated to the maximum Na⁺ current density. Cells with large Na⁺ currents showed little impulse variability, while a marked variability was recorded in cells with intermediate or small Na⁺ currents. Cells which generated more than one impulse in response to prolonged stimuli belonged to the group with large Na⁺ currents. Spontaneous impulse-currents were recorded from cell-attached membrane patches on intact cells. Also these impulses showed a large variability in amplitude: In each of five cells analysed, the peak-to-peak amplitude varied by a factor larger than 1.7.     

Simulation framework for electrophysiological networks: Effect of syncytial properties on smooth-muscle synaptic potentials

A building block-based software framework was developed to simulate electrophysiological networks. The synaptic potentials generated during neurotrans-mission were simulated in an existing discrete bidomain model of smooth muscle, using cubic three-dimensional grids of varying sizes. The model is automatically derived and numerically solved and the results of the simulation agree with previous results obtained analytically. An enhanced model was also proposed, incorporating an additional (junctional) capacitance in the network. The correctness of the model was verified, and the effect of the extra capacitance on the synaptic potentials was explored. It was found that, with a junctional capacitance C_i of 1.4×10⁻¹⁰F incorporated, the peak amplitude of the spontaneous excitatory junction potential V_peak declined by ∼13% at node 0 and by ∼37% at node 3x for a system size of 9³. Similar results were obtained for different system sizes. V_peak also declined as the junctional capacitance C_i was increased. In a system of size 11³, a 200-fold increase in C_i induced a 55% reduction at node 0. It is suggested that the type of modular simulation framework developed here may find general applicability for simulations of other physiological systems.     

An examination of the role of intracellular ATP in the activation of store-operated Ca2+ influx and Ca2+-dependent capacitance increases in rat basophilic leukaemia cells

 The role of ATP in both the activation of store-operated Ca²⁺ current I _CRAC and in Ca²⁺-dependent vesicular fusion was examined in a study of rat basophilic leukaemia (RBL) cells using the whole-cell patch-clamp technique. Fusion was monitored via changes in plasma membrane capacitance. Following a decrease in the levels of intracellular ATP, achieved using the mitochondrial poison antimycin and the ATP synthase inhibitor oligomycin, as well as a reduction of glycolysis by removal of external glucose, I _CRAC activated in a manner similar to control cells when stores are depleted by dialysis with a pipette solution containing either inositol 1,4,5-trisphosphate (InsP ₃) or ionomycin together with a high concentration of EGTA. Dialysis of cells for 150 s with the non-hydrolysable ATP analogue 5′-adenylylimidodiphosphate (AMP-PNP) (2 mM) in addition to the mitochondrial inhibitors also failed to prevent activation of I _CRAC following external application of ionomycin and thapsigargin, when compared with control recordings obtained with 2 mM ATP instead. Ca²⁺-dependent vesicular fusion was triggered by dialysing cells with 10 μM Ca²⁺ and guanosine-5′-O-(3-thiotriphosphate (GTP[γ-S]). The capacitance increase was unaffected by inhibition of glycolysis, mitochondrial inhibitors or dialysis with either AMP-PNP or adenosine 5′-O-(3-thiotriphosphate) (ATP[γ-S]) instead of ATP. We conclude that ATP hydrolysis does not seem to be necessary for the activation of I _CRAC or for the capacitance increases elicited by high concentrations of intracellular Ca²⁺. Received: 1 May 1998 / Received after revision: 16 July 1998 / Accepted: 16 July 1998     

Potassium currents induced by hydrostatic pressure modulate membrane potential and transmitter release in vestibular type II hair cells

Vestibular type II hair cells respond to increases in the hydrostatic pressure with pressure-dependent K⁺ currents. We examined whether such currents may modulate transmitter release (assessed as membrane capacitance increments) by altering membrane potentials and voltage-gated Ca²⁺ currents. Capacitance increments were dependent on voltage-gated Ca²⁺ influx. Stimulating currents (0.7 nA) in current clamp induced depolarisations that were more negative by 8.7 ± 2.1 mV when the bath height was elevated from 0.2 to 0.5 cm. In voltage clamp, protocols were used that simulated the time course of the membrane potential in current clamp at either low (control) or high hydrostatic pressure (high bath). The low bath protocol induced significantly larger Ca²⁺ currents and increases in capacitance than the high bath protocol. We conclude that pressure-dependent K⁺ currents may alter the voltage response of vestibular hair cells to an extent critical for Ca²⁺ currents and transmitter release. This mechanism may contribute to vestibular dysfunction in Meniere’s disease. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Duong Dinh and Haasler contributed equally to this work.     

Transepithelial capacitance decrease reveals closure of lateral interspace in A6 epithelia

 A sine wave method was used to measure transepithelial capacitance (C _T) at 4.1 kHz (C ^HF _T ). Model calculations show that C ^HF _T reflects the equivalent capacitance of the series arrangement of apical and basolateral membrane capacitance. Cell swelling induced by reducing the basolateral osmolality from 260 to 140 mosmol/kg H₂O (NaCl or sucrose removal) transiently decreased C ^HF _T . The decrease in C ^HF _T (ΔC ^HF _T ) reached its maximum 30 s after the onset of cell swelling and a complete recovery of C ^HF _T was attained within 3–4 min. ΔC ^HF _T could be diminished by manoeuvres that reduced the rate or amplitude of cell swelling, i.e. lowering the temperature or treatment with low concentrations of glutaraldehyde (0.025%). ΔC ^HF _T increased with the magnitude of the osmotic perturbation but saturated at large volume expansions. ΔC ^HF _T increased with culture time. Electron micrographs showed a clear correlation between time course of C ^HF _T changes and the closure of the lateral interspace (LIS). A striking correlation between the occurrence of C ^HF _T recovery and the ability of the cells to develop a regulatory volume decrease (RVD) was found: Gd³⁺ (0.5 mM) inhibited both phenomena. The frequency dependence of C _T was obtained from impedance spectra recorded over the range of 4 Hz to 22 kHz. These data agree with model calculations in which the contribution of the access resistance to the lateral membrane was included. All observations are consistent with the idea that ΔC ^HF _T originates from the closure of the LIS during cell swelling. The latter phenomenon increases the access resistance to the lateral membrane, which results in a marked reduction of the basolateral membrane area detected at high frequencies with capacitance measurements. Received: 14 September 1998 / Received after revision and accepted: 23 November 1998     

The outer hair cell motor in membrane patches

 We have used patch-clamp techniques to record the charge movement associated with motility in patches of basolateral membrane from isolated outer hair cells. Charge movement has been measured from the voltage-dependent capacitance. Using 3 to 4 MΩ pipettes with tip diameters of 3 μm the measured maximum voltage-dependent capacitance was 56 ± 6 fF at –36 mV when the resting membrane potential was –20 mV. The calculated total charge movement was 5.6 ± 0.6 fC (n = 13) and the inferred density of univalent motor elements was 8400/μm². Negative pressure (applied via the pipette) increased membrane tension and shifted the capacitance peak to depolarised potentials. Under conditions of isotropic membrane stress there was no change in the peak measured capacitance in contrast to that measured in previous whole-cell recordings. Received: 19 December 1996 / Received after revision: 21 February 1997 / Accepted: 5 March 1997     

Effects of external calcium on acetylcholine-evoked increases in membrane capacitance in rat pancreatic acinar cells

Effects of external calcium on acetylcholine-induced increases in membrane capacitance and conductance were investigated with the patch-clamp technique in combination with the phase-sensitive detection method, in single dialysed pancreatic acinar cells of rats. Both increases depended on an increase in [Ca²⁺]_i, and a high concentration of EGTA in the cell-dialysing solution made ACh ineffective. In acinar cells exposed to a bathing solution containing the normal concentration of Ca²⁺ (2.5 mM CaCl₂), the increase in membrane capacitance was transient and synchronous with that in membrane conductance (current) in response to 0.2 M acetylcholine. However, in a bathing solution without CaCl₂ and with EGTA (0.2 mM), the increase in membrane capacitance was sustained after the membrane conductance recovered to the original level during the ACh-stimulation. The evidence suggests that external calcium facilitates either the resealing of the fusion- or fission-pores formed at the contact between the secretory granule and the luminal cell membrane, or the membrane retrieval (endocytosis) in Ca²⁺-dependent exocytosis.     

Cell-attached measurements of attofarad capacitance steps in rat melanotrophs

 Capacitance changes in cell-attached patches of rat melanotrophs were measured by a high-frequency lock-in amplifier. The background noise of around 30 aF allowed the detection of discrete steps due to fission (endocytosis) and fusion (exocytosis) of vesicles with diameters as small as 60 nm. The amplitude of both types of steps was similar with a modal value of around 300 aF. The frequency of these steps was not changed, if secretagougues such as ionomycin and/or dibutyril cAMP, were applied to the bathing solution. Moreover, this treatment did not result in an increased appearance of expected 2000 to 3000 aF steps due to exocytosis of secretory granules. We conclude that the likely explanation for recorded capacitance steps is that they represent the constitutive vesicle traffic. From the typical frequency and amplitude of these events (around 1 min^–1, 300 aF) in a membrane patch (26 fF) it is estimated that the whole membrane of a rat melanotroph may be ingested under our conditions in 1 to 2 h. Received: 10 February 1997 / Accepted: 3 April 1997     

Quantal ATP release in rat β-cells by exocytosis of insulin-containing LDCVs

Quantal release of adenosine triphosphate (ATP) was monitored in rat pancreatic β-cells expressing P2X₂ receptors. Stimulation of exocytosis evoked rapidly activating and deactivating ATP-dependent transient inward currents (TICs). The unitary charge (q) of the events recorded at 0.2 μM [Ca²⁺]_i averaged 4.3 pC. The distribution of the ³√q of these events could be described by a single Gaussian. The rise times averaged ∼5 ms over a wide range of TIC amplitudes. In β-cells preloaded with 5-hydroxytryptamine (5-HT; accumulating in insulin granules), ATP was coreleased with 5-HT during >90% of the release events. Following step elevation of [Ca²⁺]_i to ∼5 μM by photo release of caged Ca²⁺, an increase in membrane capacitance was observed after 33 ms, whereas ATP release first became detectable after 43 ms. The step increase in [Ca²⁺]_i produced an initial large TIC followed by a series of smaller events that echoed the changes in membrane capacitance (ΔC _m). Mathematical modeling suggests that the large initial TIC reflects the superimposition of many unitary events. Exocytosis, measured as ΔC _m or TICs, was complete within 2 s after elevation of [Ca²⁺]_i with no sign of endocytosis masking the capacitance increase. The relationship between total charge (Q) and ΔC _m was linear with a slope of ∼1.2 pC/fF. The latter value predicts a capacitance increase of 3.6 fF for the observed mean value of q, close to that expected for exocytosis of individual insulin granules. Our results indicate that measurements of ATP release and ΔC _m principally (≥85–95%) report exocytosis of insulin granules.     

Electrokinetic measurements of dielectric properties of membrane for apoptotic HL-60 cells on chip-based device

The specific membrane capacitance and conductance of mammalian cells reflect the surface morphological complexities and barrier functions of cell membrane, respectively, and could potentially respond to cell physiological and pathological changes in a measurable manner. In this study, an electrokinetic system was developed by using negative dielectrophoretic force (nDEP force) assisted positioning and electroroation (ROT) measurement. Numerical simulations regarding the geometric model of the electrode were performed primarily for the electric field analysis. The dielectric responses of membrane for apoptotic HL-60 cells induced by bufalin were detected. The membrane capacitance of the cells was found to fall from an initial value of 15.6 ± 0.9 mF/cm² to 6.4 ± 0.6 mF/cm² after a 48 h treatment with 10 nM bufalin. However, the membrane conductance remained almost constant at (2.25 ± 1.1) × 10³ S/m² during the first 12 h of bufalin treatment and then increased distinctly to (4.2 ± 1.3) × 10³ S/m² thereafter. Scan electron microscopy (SEM) studies of the cells revealed a decreased complexity in cell membrane morphology following bufalin treatments, suggesting that the observed changes in the membrane capacitance was dominated by the alterations of cell surface structures. The results demonstrate that the ROT technique gives a quantitative analysis of the toxic damage by chemicals to cells and can be exploited in the testing and development of new pharmaceuticals and active cell agents.     

The role of exocytosis in the activation of the chloride conductance in Chinese hamster ovary cells (CHO) stably expressing CFTR

 The aim of this study was to examine the question of whether activation of wt-CFTR (wild-type cystic fibrosis transmembrane conductance regulator) by cAMP and the opening of a Cl^–conductance is paralleled by exocytosis and corresponding increases in membrane capacitance. To this end three types of Chinese hamster ovary (CHO) cells were examined: a control group of CHO cells; a group of CHO cells stably expressing wt-CFTR at high levels (also called BQ2-CHO); and a group of CHO cells stably expressing the frequent mutation ΔF508-CFTR. Whole-cell patch-clamp studies were performed to measure the membrane voltage (V _m), the membrane conductance (G _m) and the membrane capacitance (C _m). C _m was assessed by a two-frequency lock-in amplifier method. Forskolin (Fsk, 0.1 μmol/l) and isobutylmethylxanthine (IBMX, 0.1 mmol/l) were used to increase cytosolic cAMP. It is shown that Fsk and IBMX had no effect on V _m and G _m in control CHO and ΔF508-CFTR-CHO cells. Fsk and IBMX depolarized wt-CFTR-expressing CHO cells significantly (from –40 ± 1.5 to –32 ± 1.6 mV, n = 41) and enhanced G _m strongly from 5.0 ± 0.9 to 36 ± 3.9 nS (n = 65). The conductance increase was mostly for Cl^–, because under stimulated conditions a reduction in bath Cl^–concentration depolarized these cells further and significantly from –26 ± 1.8 to –10 ± 1.2 mV (n = 16). This conductance had the characteristic wt-CFTR selectivity of Br^– > Cl^– > I^–(n = 16). Despite this large increase in the Fsk- and IBMX-induced conductance C _m was not altered significantly (15.5 versus 15.7 pF, n = 50). These data indicate that stable overexpression of wt-CFTR but not of ΔF508-CFTR in CHO cells induces a cAMP-activated Cl^–conductance. The activation of this large conductance obviously proceeds with little if any exocytosis. Received: 13 May 1997 / Received after revision: 1 July 1997 / Accepted: 7 July 1997     

Membrane capacitance increases induced by histamine and cyclic AMP in single gastric acid-secreting cells of the guinea pig

Summary During acid secretion, gastric parietal cells undergo profound morphological changes including formation of the apical secretory membrane. To examine the mechanism of histamine-induced increases in the apical membrane area at the single cell level, we monitored the membrane capacitance by applying a time-resolved phase-sensitive detection method to singly isolated parietal cells of guinea pig. A real-time increase in the membrane capacitance was detected within several min after stimulation with histamine. An H2-blocker (cimetidine), but not an H1-blocker (pyrilamine), inhibited the histamine response. Dibutyryl cyclic AMP mimicked the histamine effect. The capacitance response to histamine was sensitive to cytosolic Ca²⁺, temperature and N-ethylmaleimide. The histamine response was inhibited by intracellular application of a non-hydrolyzable ATP analog (AMP-PNP) and an isoquinolinesulfonamide derivative that works as an inhibitor of protein kinase A (H-8). These results indicate that in parietal cells, elevation of intracellular cyclic AMP induces exocytotic insertion of intracellular membranes into the plasma membrane, presumably by activating protein kinase A.     

Ca2+-and voltage activated K+ channel in apical cell membrane of gallbladder epithelium fromTriturus

The presence of Ca²⁺- and voltage-activated K⁺ channels was directly demonstrated in the apical cell membrane of gallbladder epithelium by patch-clamp single-channel current recording. In K⁺-depolarized epithelial cells, negative pipette potentials induced outward current steps when the patch-pipette was filled with Na⁺-rich solution and these current steps were not affected by the presence or absence of Cl^–. When K⁺-rich solution was in the pipette and K⁺-depolarized cells were examined, the current-voltage relations were linear with a single-channel conductance of 140 pS and polarity was reversed at 0 mV. In excised inside-out membrane patches, raising the free Ca²⁺ concentration of the medium facing the inner side of the membrane from 10^–7 to 10^–6 M evoked a marked increase in open state probability of the channels without affecting the elementary current steps. This suggests that intracellular Ca²⁺ as a second messenger plays a crucial role in the regulatory mechanism of the membrane potential by modulating the high-conductance apical K⁺ channels.     

Kiss-and-run exocytosis and fusion pores of secretory vesicles in human β-cells

Exocytosis of secretory vesicles results in the release of insulin from pancreatic β-cells, although little is known about this process in humans. We examined the exocytosis of single secretory vesicles and their associated fusion pores in human β-cells by cell-attached capacitance and conductance measurement. Unitary capacitance steps were observed, consistent with the exocytosis of single secretory vesicles. These were often coincident with increases in patch conductance representing the presence of a stable fusion pore. In some events, the fusion pore closed, mediating kiss-and-run, which contributed 20% of the exocytotic events. The cAMP-raising agent forskolin (5 μM) doubled the relative contribution of kiss-and-run. This effect was confirmed visually in MIN6 cells expressing a fluorescent granule probe. Thus, we demonstrate the unitary capacitance steps and fusion pores during single vesicle exocytosis in human β-cells. Furthermore, these secretory vesicles can undergo rapid recycling by kiss-and-run, and this process is up-regulated by cAMP.     

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: Biochemical analysis of eight mutants

Purine biosynthesis was studied in 8 mutants of Chinese hamster cells which require purines for growth and in wild-type cells which do not show this nutritional requirement. Of these, 6 mutants, ade ^–B, ade^–D, ade^–E, ade^–F, GAT^–, and AT^–, were shown to accumulate metabolic intermediates not accumulated by wild-type cells. These intermediates were shown to be compounds unique to the adenylic acid biosynthetic pathway by the following criteria: (a) their radioisotopic labeling properties, (b) their response to agents which specifically inhibit known enzymatic steps in the pathway, (c) their chromatographic properties, and (d) spectrophotometric analysis. Two mutants, ade^–A and ade^–C, accumulate no detectable compounds not accumulated by the wild type. These 2 mutants are believed to be defective in steps very early in the purine biosynthetic pathway. The sites of the defects in the other mutants are proposed, and the usefulness of these mutants is discussed.