Synergism between inositol phosphates and diacylglycerol on native TRPC6-like channels in rabbit portal vein myocytes

Abstract

In rabbit portal vein myocytes noradrenaline activates a non-selective cation current (Icat) which involves a transient receptor potential protein (TRPC6). Previously we have shown that the diaylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulates Icat via a protein kinase C (PKC)-independent mechanism, and in the present study we have investigated the interaction between inositol phosphates (InsPs) and OAG on Icat. With whole-cell recording of Icat from freshly isolated rabbit portal vein myocytes the amplitude and rate of activation of noradrenaline-evoked Icat were much greater than those of OAG-induced Icat. Inclusion of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in the pipette solution did not evoke Icat but greatly potentiated the amplitude and rate of activation of OAG-induced Icat. With isolated outside-out patches Ins(1,4,5)P3 markedly increased the rate of activation and the open probability of OAG-evoked channel activity, with no change in unitary conductance, channel mean open times or burst durations. The effects of Ins(1,4,5)P3 were mimicked by Ins(2,4,5)P3, 3-F-Ins(1,4,5)P3 and Ins(1,4)P2 but not by Ins(1,3,4,5)P4 and the potentiating effects of InsPs were not inhibited by heparin. Therefore it is concluded that both DAG and InsPs are necessary for full activation of Icat by noradrenaline and the effect of InsPs is via a heparin-insensitive mechanism and represents a novel action of InsPs.

Figure 1. Effect of Ins(1,4,5)P₃ on OAG-induced whole-cell currents

Ai, typical response to bath application of 100 μm noradrenaline recorded in the absence of Ins(1,4,5)P₃ in the patch pipette solution. Aii and iii show records of OAG-evoked whole-cell currents in the absence and presence respectively of 10 μm Ins(1,4,5)P₃ in the pipette solution. In all cases the holding potential was −50 mV and breaks in the traces correspond to 2 min intervals. The asterisk denotes when whole-cell configuration was obtained. B, mean data for peak amplitude (i), activation rate (ii), latency (iii) and decay rate rate (iv) for noradrenaline-evoked (n = 9) and OAG-induced whole-cell currents activated in the absence (n = 5) or presence of 1 μm (n = 5), 10 μm (n = 9) and 100 μm Ins(1,4,5)P₃ (n = 6) in the pipette solution. Activation rate (pA s⁻¹) was calculated from 10–90 % of the rise time of the currents. *P < 0.05, **P < 0.01, compared to OAG-evoked currents in the absence of Ins(1,4,5)P₃. Note that with noradrenaline Ins(1,4,5)P₃ was not included in the pipette solution.

Figure 2. Effect of Ins(1,4,5)P₃ on OAG-induced single channel currents in outside-out patches

A, effect of 40 μm OAG in the absence of Ins(1,4,5)P₃ in the pipette solution; B, effect of 40 μm OAG with 10 μm Ins(1,4,5)P₃ in the pipette solution. The continuous lines represent closed states and the dashed lines open states in the expanded traces. Holding potential was −50 mV. Note the multiple openings in B due to higher channel activity and the different time scales for the upper records in A and B. C and D show mean data from OAG-evoked channel currents in the absence (n = 8) and presence of Ins(1,4,5)P₃ (n = 5–9). *P < 0.05, **P < 0.01, compared to OAG-evoked channel currents in the absence of Ins(1,4,5)P₃.

Figure 3. Properties of single OAG-induced channel currents activated in the presence of 10 μm Ins(1,4,5)P₃

A, OAG-induced channel currents evoked in the presence of 10 μm Ins(1,4,5)P₃ recorded from the same patch at different membrane potentials. Note the higher activity and longer openings at +50 mV. B, I-V relationship from two different patches of OAG-induced channel currents activated in the presence (•) and absence (○) of 10 μm Ins(1,4,5)P₃ and both had unitary conductances of 25 pS and E_r of +6 mV and +9 mV respectively. C and D, open time and burst duration distributions of OAG-evoked channel currents induced in the presence of 10 μm Ins(1,4,5)P₃ at −50 mV. Both distributions could be fitted by the sum of two time exponentials.

Figure 4. Effect of InsPs on OAG-induced channel currents in outside-out patches

A, B and C show that inclusion of, respectively, 3-F-Ins(1,4,5)P₃, Ins(2,4,5)P₃ and Ins(1,4)P₂ in the pipette solution produced similar effects to Ins(1,4,5)P₃ on OAG-induced channel currents. D, inclusion of 1 mg ml⁻¹ heparin in the pipette solution did not prevent the effect of Ins(1,4,5)P₃ of potentiating OAG-induced single channel currents. E and F show mean data of time to maximum NP_o and maximum NP_o of OAG-evoked channel activity in the presence of InsPs (n = 5–8). *P < 0.05, **P < 0.01, compared to OAG-evoked channel currents in the absence of InsPs in the pipette solution.