SUMMARY1. Two glass micro-electrodes were inserted into neighbouring cells from rat or mouse pancreatic segments, superfused in vitro. The tip of a third glass micro-electrode, filled with 2 M-AChCl, was placed just outside the acinus under investigation. Membrane potential and resistance, and changes in these parameters in response to short pulses of ACh stimulation, were recorded.2. The resting current-voltage relationship, obtained by injecting 100 msec depolarizing or hyperpolarizing current pulses through one of the intracellular micro-electrodes and recording the membrane potential with the other intracellular electrode, was linear within the range -5 to -60 mV.3. Injecting depolarizing or hyperpolarizing current (d.c.) through one of the intracellular micro-electrodes, the membrane potential (as measured with the other intracellular micro-electrode) could be set at various levels. The effect of ACh at different membrane potentials was investigated. When the acinar cell membrane was hyperpolarized, the amplitude of ACh-evoked depolarization was increased, while ACh-evoked depolarization was reduced when the membrane potential was reduced by depolarizing current, and finally changed into a hyperpolarization at very low membrane potentials. In each acinus investigated (rat and mouse), there was a linear relationship between amplitude of ACh-evoked potential change (AV) (+ value or -
The electrical communication network in the mouse pancreatic acinar tissue has been investigated using simultaneous intracellular recording with two separate microelectrodes and direct microscopical control of the localizations of the microelectrode tips.All cells within one acinus were electrically coupled, and the coupling coefficient (the electrotonic potential change in a cell neighboring to the cell into which current is injected [Vz] divided by the electrotonic potential change in the cell of current injection [VI]) between two cells near each other (<50/zm) was always close to 1. Cells farther apart (50-100/xm) were, in some cases, coupled; in other cases, there was no coupling at all. Coupling coefficients varied between 0 and 1. There was rarely electrical coupling over distances of more than 110/zm.Using microiontophoretic acetylcholine (ACh) application, it was possible to evoke almost complete electrical uncoupling of two previously coupled pancreatic or lacrimal acinar cells from different acini or within one acinus. The effects were fully and quickly reversible. While the ACh-evoked uncoupling in the pancreas was associated with membrane depolarization, ACh caused hyperpolarization in the lacrimal acinar cells. The uncoupling was associated with a very marked reduction in electrical time constant, indicating a reduction in input capacitance (effective surface cell membrane area).The concentrations of stimulants needed to evoke reduction in pancreatic cellto-cell coupling were 1 /zM for ACh, 0.14 nM for caerulein, and 3 nM for bombesin. These concentrations are smaller than those required to evoke maximal enzyme secretion.
SUMMARY1. Segments of mouse pancreatic or exorbital lacrimal gland were superfused with saline solutions. Under visual control two micro-electrodes were inserted into neighbouring cells within the same acinus or into neighbouring acini. Cell to cell electrical coupling was assessed by injecting rectangular current pulses through one electrode and measuring the electrotonic potential change in the same cell (V1) and in the neighbouring cell (V2). Acetylcholine (ACh) was added locally to impaled acini by micro-ionophoresis from an extracellular micropipette.2. Exposure of the tissues to a Krebs solution equilibrated with 100 % C02 caused a rapid increase in the size of electrotonic potential changes in the current injection cell and disappearance of the electrotonic potential changes in a neighbouring acinus or cell. This electrical uncoupling of previously coupled cells was rapidly reversible upon return to a solution equilibrated with 95 % 02 and 5 % CO2.3. Reduction of electrical intercellular coupling was also obtained using smaller C02 concentrations (50, 20 or 10 %). In these cases the effects developed more slowly and were less dramatic. Reducing the extracellular HCO3 concentration enhanced the uncoupling effect of 10 or 20 % C02. However, weak uncoupling effects were still observed using 10 or 20 % C02 in combination with a high bicarbonate concentration maintaining a constant extracellular pH (7.4).4. Reductions in extracellular pH (down to 5.5) achieved by varying combinations of Tris base and Tris HC1 had no effect on electrical coupling. Brief periods of anoxia (100 % N2) also had no effect.5. Exposure to 20 % C02 markedly enhanced the uncoupling effect of a brief ionophoretic pulse of ACh.6. Exposure of the tissue to 10 mM-NH4Cl, a procedure expected to increase the intracellular pH, counteracted the uncoupling effect of ACh. During sustained uncoupling caused by a sustained ACh stimulation a brief period of exposure to NH4C1 caused an immediate and fully reversible recoupling.7. It is concluded that variations in intracellular pH have marked effects on the electrical coupling between neighbouring cells in the pancreatic and lacrimal acinar tissue.
Segments of mouse parotid were placed in a superfusion chamber. Surface acini were impaled by one or two micro-electrodes for measurement of membrane potential and resistance. The acinus under investigation was stimulated by micro-iontophoretic application of acetylcholine (ACh) or adrenaline. Neighbouring acinar cells were electrically coupled. Electrical coupling between acinar cells only occurred within restricted domains probably corresponding to an acinus or a group of acini. Passing direct current through one intracellular electrode, the resting potential of an acinus could be set at desired levels and the dependency of the ACh-evoked potential change on the resting potential investigated. The ACh null potential (initial effect) was about--60 mV. A delayed hyperpolarizing effect of ACh could not be reversed. The initial ACh-evoked potential change was sensitive to alterations in extracellular Na, K and Cl concentration. The delayed ACh-evoked hyperpolarization was blocked by ouabain, exposure to Na-free or K-free solutions. It is concluded that ACh increases mainly K and Na membrane conductance causing K efflux and Na influx with a subsequent Na activation of an electrogenic Na pump.
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