1. The patch-clamp technique of whole-cell current recording was applied to single, enzymatically isolated, rat pancreatic acinar cells to investigate the current responses evoked by internal perfusion of inositol polyphosphates (InsP(x)). The InsP(x) were included in the solution filling the recording pipette and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3; 10 mum) evoked transient current responses generally of less than 1 min duration, inositol 2,4,5-trisphosphate (Ins(2,4,5)P3; 10 mum) evoked smaller current transients while inositol 1,3,4,5-tetrakisphosphate (InsP4; 10 mum) evoked no detectable current response. However, in the presence (in external bathing solution) of the phospholipase A, inhibitor 4-bromophenacyl bromide (4-BPB; 8 mum) all three of the InsP(x) now evoked prolonged current responses lasting for several minutes. The current responses to all three InsP(x) were abolished by inclusion of the Ca2+ chelator EGTA (5 mm) in the internal, pipette-filling solution indicating that the responses are calcium dependent and reflect the effect of the InsP(x) in increasing intracellular Ca2+. Inositol 1,3,4,5,6-pentophosphate (InsP5) induced no current response when tested up to 20 mum in the presence or absence of 4-BPB. 2. The potentiating effect of 4-BPB on the InsP(x)-induced current responses was not mimicked by application of arachidonic acid (AA) oxidation inhibitors; indomethacin (20 mum), nordihydroguaiaretic acid (20 mum) or proadifen (SKF525A, 100 mum). The effects of 4-BPB were countered however, by the inclusion of 2 mum AA in the external solution. The results suggest that the 4-BPB potentiates the response by inhibiting the activity of phospholipase A2, thereby reducing the formation of AA. 3. In the presence of 4-BPB (8 muM) the InsP(x)-evoked responses were dose dependent with an increase in both the amplitude and speed of onset with increasing concentrations. In the presence of 4-BPB InsP4 was as efficient as Ins(1,4,5)P3 both in terms of speed of onset and amplitude of responses; the efficacy and dissociation constant (K(d)) for both of these InsP(x) were the same at 1 muM and 45 nm respectively. Ins(2,4,5)P3 was always less effective, with an efficacy and K(d) of 10 mum and 750 nm respectively. 4. If 4-BPB was applied after the current responses evoked by the InsP(x) were over, or if guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) was included in the recording pipette then the phospholipase inhibitor gave rise to an additional, prolonged, current response. This effect of 4-BPB was abolished by the inclusion of heparin in the pipette solution, indicating that heparin inhibits the receptors for InsP(x) and that the effect in the presence of GTPgammaS is mediated by stimulation of G-proteins coupled to phospholipase C activity and InsP(x) production. 5. In the presence of a lower concentration of 4-BPB (2 mum) InsP4, even at 12 mum, gave no current response and the effect of 2 mum Ins(1,4,5)P3 was small. When the 2 mum Ins(1,4,5)P3 was combined with 10 mum InsP4 in the presence of 2 mum 4-BPB there was a much enhanced response of longer duration than that induced by the Ins(1,4,5)P3 itself. This indicates a marked synergism between the Ins(1,3,5)P3 and InsP4 that is manifest in the presence of the lower concentration of the phospholipase A, inhibitor. 6. During the prolonged, steady-state component of the 4-BPB-potentiated InsP(x) responses the removal of external Ca2+ resulted in a partial, reversible inhibition of the current responses indicating that Ca2+ influx, in addition to Ca2+ release from intracellular stores, is a component in sustaining the current responses. 7. Overall the data indicate that the cellular level of AA is an important modulator of InsP(x) regulation of cytosolic free Ca2+ concentration. AA appears to regulate the InsP(x)-induced Ca2+ mobilization and/or the synergism between different InsP(x). The effect of AA in the regulation of pancreatic Ca2+ signalling is probably at the level of inhibition of the InsP(x)-receptor channels and/or through facilitation of the plasma membrane Ca2+ pump. The implications of these data are discussed.
