SUMMARY1. Anion replacement experiments were performed on superfused in vitro mouse pancreatic tissue and the effects on the electrical response of acinar cells to ACh investigated.2. Electrical measurements were made with two micro-electrodes inserted into electrically coupled cells. ACh was applied by microionophoresis. Potential recordings were taken before, during and after changeover from the control superfusion fluid, containing Cl-, to one containing the substituted anion.3. From the results obtained the tested anions were classified into three groups: I, Cl--like anions: Br-, I-and NO3-, causing either no change or a negative displacement of the ACh null-potential, compared to that measured in the control C--containing solution, and only small changes in the resting and stimulated electrical properties of the acinar cell, II, ions less permeable than Cl-: isethionate, acetate, sulphate and hippurate, showing a positive displacement of the ACh null-potential and a similar or increased resting cell input resistance, and III, methylsulphate and benzenesulphonate, causing a negatively displaced ACh null-potential but showing changes in the resting electrical properties of the acinar cells characteristic of anions in group II.4. The ACh null-potential sequence, in order of decreasing negativity, was NO3-3 benzenesulphonate 3 I-3 methylsulphate > Br-3 Cl-> isethionate > acetate 3 sulphate > hippurate.5. Experiments involving the use of bicarbonate demonstrated that it does not contribute significantly to the value of the ACh null-potential.6. The sequence of the anions in group I were compared to the Eisenman series I, suggesting that the ACh-opened Cl-pathway comprises a large hydrated ion channel bearing a lining of weak positive charges. 7. A quantitative relationship was sought between the ACh null-potential and extracellular Cl-. It was found that a tenfold reduction in the extracellular concentration resulted in a 15 mV positive shift of the null-potential.

1. A two-micro-electrode voltage-clamp technique was applied to a study of the resting properties of mouse pancreatic acinar cell membranes and the action of acetylcholine (ACh). 2. The resting voltage-current relation was linear. The specific membrane resistance was calculated to be about 10 k omega cm2. This value was doubled after removal of Cl from the tissue bath superfusion solution. 3. At a holding potential equal to the spontaneous resting potential (about -35 mV) micro-ionophoretic ACh application evoked inward current. Reversal of the polarity of the ACh-evoked current occurred at about - 15 mV. 4. The voltage dependence of the ACh-evoked current displayed inward rectification. This inward rectification could not be accounted for by the constant field equation. 5. The dose-response curves for ACh-evoked inward current were compared in the same units with dose-response curves for ACh-evoked depolarization. Half-maximal depolarization was consistently obtained at a lower dose of ACh than half-maximal inward current. 6. During steady-state exposure of the pancreatic tissue segments to Cl-free sulphate solution the ACh reversal potential was about + 10 mV and the voltage-current relationship for the ACh-controlled channels showed inward rectification. Removal of external Na from the Cl-free solution virtually abolished ACh-evoked inward current. 7. The resting pancreatic acinar cell membrane is electrically inexcitable. The relative permeabilities of the major monovalent ions appears to be PC1/PNa/PK = 2/0.23/1. The ACh-evoked inward current is largely carried by Na. Dose-response curves for ACh-evoked depolarization and inward current in the same acinar units are different, in such a way that the depolarization response saturates at lower ACh concentrations than the current response.

Peptide hormones, like neurotransmitters, are traditionally thought to activate cells by interacting with receptor sites accessible only from the extracellular space. However, there is no available evidence that establishes whether intracellular injections of peptide secretagogues can or cannot initiate cell activation. In view of recent demonstration that peptide hormones can penetrate the intracellular space in some tissues and the reports that intracellular injections of the neurotransmitter, dopamine, into acinar cells of cockroach salivary gland cause stimulation it seems of fundamental importance to test directly whether introduction of peptide secretagogues inside acinar cells of mammalian exocrine tissue can induce cell activation without first interacting with the outer surface of the external cell membrane. The data presented here show that injections of the secretagogue peptides caerulein and bombesinnonapeptide (bombesin-NP) into pancreatic acinar cells fail to evoke the characteristic potential and conductance changes that are observed following extracellular applications of these peptides.

SUMMARY1. Acinar cell membrane potential and resistance were measured from superfused segments of mouse pancreas, in vitro, using intracellular glass micro-electrodes. One or two extracellular micropipettes containing caerulein, bombesin nonapeptide (Bn) or acetylcholine (ACh) were placed near to the surface of the impaled acinus. The secretagogues were ejected rapidly from the micropipettes by ionophoresis.2. Each secretagogue evoked a similar electrical response from the impaled acinar cell: membrane depolarization and a simultaneous reduction in input resistance. The duration of cell activation from caerulein ionophoresis was longer than that observed for ACh and Bn. The cell response to the peptide hormone applications could be repeated in the presence of atropine.3. The minimum interval before the onset of cell depolarization after caerulein ionophoresis was determined. Values ranged between 500 and 1000 msec. The minimum latencies after Bn ionophoresis were 500-1400 msec.4. With two electrodes inserted into electrically coupled acinar cells, direct measurements of the caerulein and Bn null potentials were made. At high negative membrane potentials an enhanced depolarization was evoked by caerulein ionophoresis. At low negative membrane potentials the caerulein stimulation produced a diminished depolarization, and at membrane potentials less than -10 mV acinar cell hyperpolarizations were observed. A similar series of responses was obtained in experiments where Bn ionophoresis was used. The caerulein and the Bn null potentials were always contained within -10 to -15 mV.5. The results describe the almost identical electrical response of acinar cells to stimulation by ACh, caerulein and bombesin. All three secretagogues have similar null potentials and latencies of activation on acinar cells. The bombesin latency responses appear as short as those measured for caerulein and provide electrophysiological evidence that Bn acts directly on acinar cells. The findings support the hypothesis that ACh, caerulein and Bn, though acting on different receptors, evoke the observed changes in electrical properties of acinar cell membranes, through a common pathway.