Multivesicular exocytosis in rat pancreatic beta cells.

AIMS/HYPOTHESIS: To establish the occurrence, modulation and functional significance of compound exocytosis in insulin-secreting beta cells. METHODS: Exocytosis was monitored in rat beta cells by electrophysiological, biochemical and optical methods. The functional assays were complemented by three-dimensional reconstruction of confocal imaging, transmission and block face scanning electron microscopy to obtain ultrastructural evidence of compound exocytosis. RESULTS: Compound exocytosis contributed marginally (<5% of events) to exocytosis elicited by glucose/membrane depolarisation alone. However, in beta cells stimulated by a combination of glucose and the muscarinic agonist carbachol, 15-20% of the release events were due to multivesicular exocytosis, but the frequency of exocytosis was not affected. The optical measurements suggest that carbachol should stimulate insulin secretion by ∼40%, similar to the observed enhancement of glucose-induced insulin secretion. The effects of carbachol were mimicked by elevating [Ca(2+)](i) from 0.2 to 2 µmol/l Ca(2+). Two-photon sulforhodamine imaging revealed exocytotic events about fivefold larger than single vesicles and that these structures, once formed, could persist for tens of seconds. Cells exposed to carbachol for 30 s contained long (1-2 µm) serpentine-like membrane structures adjacent to the plasma membrane. Three-dimensional electron microscopy confirmed the existence of fused multigranular aggregates within the beta cell, the frequency of which increased about fourfold in response to stimulation with carbachol. CONCLUSIONS/INTERPRETATION: Although contributing marginally to glucose-induced insulin secretion, compound exocytosis becomes quantitatively significant under conditions associated with global elevation of cytoplasmic calcium. These findings suggest that compound exocytosis is a major contributor to the augmentation of glucose-induced insulin secretion by muscarinic receptor activation.

Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells.

The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.

Voltage-dependent formation of gramicidin channels in lipid bilayers.

The formation kinetics of gramicidin A channels in lipid bilayer membranes has been characterized as a function of voltage for different solution conditions and membrane composition. The frequency of channel events was measured during the application of voltage ramps and counted in given intervals, a procedure that eliminated the effects of drift in gramicidin concentration. The formation rate was found to increase strongly with voltages up to approximately 50 mV and then to level off slightly. The shape of the voltage dependence was independent of lipid solvent and ramp speed but differed for different ions and different solution concentrations. This suggested an ion occupancy effect on the formation rate that was further supported by the fact that the minimum of the formation rate was shifted toward the equilibrium potential in asymmetric solution concentrations. The effects are explained in terms of a model that contains two contributions to the voltage dependence, a voltage-dependent ion binding to the monomers and a polarization of monomers by the applied electric field and by the occupied ions. The theory is found to give a good fit to experimental data.

Tight coupling between electrical activity and exocytosis in mouse glucagon-secreting alpha-cells.

alpha-Cells were identified in preparations of dispersed mouse islets by immunofluorescence microscopy. A high fraction of alpha-cells correlated with a small cell size measured as the average cell diameter (10 microm) and whole-cell capacitance (<4 pF). The alpha-cells generated action potentials at a low frequency (1 Hz) in the absence of glucose. These action potentials were reversibly inhibited by elevation of the glucose concentration to 20 mmol/l. The action potentials originated from a membrane potential more negative than -50 mV, had a maximal upstroke velocity of 5 V/s, and peaked at +1 mV. Voltage-clamp experiments revealed the ionic conductances underlying the generation of action potentials. alpha-Cells are equipped with a delayed tetraethyl-ammonium-blockable outward current (activating at voltages above -20 mV), a large tetrodotoxin-sensitive Na+ current (above -30 mV; peak current 200 pA at +10 mV), and a small Ca2+ current (above -50 mV; peak current 30 pA at +10 mV). The latter flowed through omega-conotoxin GVIA (25%)- and nifedipine-sensitive (50%) Ca(2+)-channels. Mouse alpha-cells contained, on average, 7,300 granules, which undergo Ca(2+)-induced exocytosis when the alpha-cell is depolarized. Three functional subsets of granules were identified, and the size of the immediately releasable pool was estimated as 80 granules, or 1% of the total granule number. The maximal rate of exocytosis (1.5 pF/s) was observed 21 ms after the onset of the voltage-clamp depolarization, which is precisely the duration of Ca(2+)-influx during an action potential. Our results suggest that the secretory machinery of the alpha-cell is optimized for maximal efficiency in the use of Ca2+ for exocytosis.

Voltage-gated and resting membrane currents recorded from B-cells in intact mouse pancreatic islets.

1. The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial cells in intact pancreatic islets. Immunostaining in combination with confocal microscopy revealed that the superficial cells consisted of 35 % insulin-secreting B-cells and 65 % non-B-cells (A- and D-cells). 2. Two types of cell, with distinct electrophysiological properties, could be functionally identified. One of these generated oscillatory electrical activity when the islet was exposed to 10 mM glucose and had the electrophysiological characteristics of isolated B-cells maintained in tissue culture. 3. The Ca2+ current recorded from B-cells in situ was 80 % larger than that of isolated B-cells. It exhibited significant (70 %) inactivation during 100 ms depolarisations. The inactivation was voltage dependent and particularly prominent during depolarisations evoking the largest Ca2+ currents. 4. Voltage-dependent K+ currents were observed during depolarisations to membrane potentials above -20 mV. These currents inactivated little during a 200 ms depolarisation and were unaffected by varying the holding potential between -90 and -30 mV. 5. The maximum resting conductance in the absence of glucose, which reflects the conductance of ATP-regulated K+ (KATP) channels, amounted to approximately 4 nS. Glucose produced a concentration-dependent reduction of KATP channel conductance with half-maximal inhibition observed with 5 mM glucose. 6. Combining voltage- and current-clamp recording allowed the estimation of the gap junction conductance between different B-cells. These experiments indicated that the input conductance of the B-cell at stimulatory glucose concentrations ( approximately 1 nS) is almost entirely accounted for by coupling to neighbouring B-cells.

Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells.

We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.

Cytoplasmic Ca2+ oscillations in human leukemia T-cells are reduced by 50 Hz magnetic fields.

The effect of 50 Hz magnetic fields on the cytosolic calcium oscillator in Jurkat E6.1 cells was investigated for field strengths within the range from 0 to 0.40 mT root mean square. The intracellular Ca2+ concentration data were collected for single Jurkat cells that exhibited a sustained spiking for at least 1 h while repeatedly exposing them to an alternating magnetic field in 10-min intervals interposed with nonexposure intervals of the same length. The obtained data were analysed by computing spectral densities of the Ca2+ oscillating patterns for each of these 10-min intervals. For every single-cell experiment the spectra of all exposure as well as nonexposure periods were then averaged separately. A comparison between the resulting averages showed that the total spectral power of the cytosolic Ca2+ oscillator was reduced by exposure of the cells to an alternating magnetic field and that the effect increased in an explicit dose-response manner. The same relationship was observed within the 0-10 mHz (10 x 10(-3) Hz) subinterval of the Ca2+ oscillation spectrum. For subintervals at higher frequencies, the change caused by the exposure to the magnetic field was not significant.

Amplification of electromagnetic signals by ion channels.

Cells may respond to the exposure of low-frequency electromagnetic fields with changes in cell division, ion influx, chemical reaction rates, etc. The chain of events leading to such responses is difficult to study, mainly because of extremely small energies associated with low-frequency fields, usually much smaller than the thermal noise level. However, the presence of stochastic systems (for instance, ion channels) provides a basis for signal amplification, and could therefore, despite the low signal-to-noise ratio of the primary response, lead to the transmission of weak signals along the signaling pathways of cells. We have explored this possibility for an ion channel model, and we present a theory, based on the formalism of stochastically driven processes, that relates the time averages of the ion channel currents to the amplitude and frequency of the applied signal. It is concluded from this theory that the signal-to-noise ratio increases with the number of channels, the magnitude of the rate constants, and the frequency response of the intracellular sensing system (for instance, a calcium oscillator). The amplification properties of the stochastic system are further deduced from numerical simulations carried out on the model, which consists of multiple identical two-state channels, and the behavior for different parameters is examined. Numerical estimates of the parameters show that under optimum conditions, even very weak low-frequency electromagnetic signals (<100 Hz and down to 100 microT) may be detected in a cellular system with a large number of ion channels.

The influence of 50-Hz magnetic fields on cytoplasmic Ca2+ oscillations in human leukemia T-cells.

We have studied the effects of 50-Hz 100-microT rms magnetic fields on intracellular Ca2+ concentration in the Jurkat T lymphocyte variant E6.1 using fluorescent probes Indo-1 and Fura-2. We found, however, that the pattern of intracellular Ca2+ fluctuations also depended on the agent used for cell attachment, in our case the polypeptide poly-L-lysine. In order to isolate possible effects of magnetic field exposure from those of poly-L-lysine, the action of polypeptide on cytosolic Ca2+ was studied as well. It was found that a 10(-7)% concentration of polypeptide triggered prolonged Ca2+ spiking. Higher (10(-4)%) concentrations induced rapid increases in intracellular Ca2+ followed by high, unstable Ca2+ levels. The response of these cells to the monoclonal antibody anti-CD3 was also inhomogeneous, similar to one caused by poly-L-lysine. The effect of magnetic field exposure was studied on cells initially exhibiting (1) non-oscillating, low Ca2+ concentration and (2) prolonged Ca2+ concentration oscillations. In case (1) the result was negative. In case (2), statistically significant changes were found: the oscillation amplitude was reduced on average by 30%, and the frequency composition was shifted towards higher frequencies.

The frequency response of ion channel currents in a combined diffusion and barrier type model.

The dynamics of ion transport through channels has been calculated using a small signal analysis method. This is applied to the equations describing the rates of ion transport through channels represented by a pair of entrance barriers in series with an interior diffusion regime. The responses to combinations of applied DC and AC electric fields, assumed to be constant in space, have been calculated and are represented as complex admittances. On examining the admittance properties, the channel is found to behave electrically as a double reactance network, which can give rise to resonance effects. A further interesting property is the limiting slope of the current amplitude at high frequencies, which is found to exhibit an f-2/3 dependence for small entrance barriers and an f-1 dependence for large entrance barriers. This points to the possibility of using noise measurements for drawing conclusions about ion transport properties through channels.