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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.
In rabbit portal vein smooth muscle cells, noradrenaline activates a Ca2+-permeable non-selective cation current (Icat) which is mediated by α1-adrenoceptors (Byrne & Large, 1988; Wang & Large, 1991). This conductance is not stimulated by a rise in intracellular Ca2+ concentration ([Ca2+]i) or by depletion of internal Ca2+ stores and therefore it is a classical receptor-operated channel (Wang & Large, 1991). The proposed physiological role of Icat is to mediate noradrenaline-evoked membrane depolarisation and open voltage-dependent Ca2+ channels (VDCCs) and to produce direct influx of Ca2+ ions to evoke vasoconstriction (Wang & Large, 1991; Inoue et al. 2001). Recently it has been shown that a member of the transient receptor potential family of proteins (TRPC6) is a central component of Icat in rabbit portal vein myocytes (Inoue et al. 2001).
Previously we investigated the transduction mechanism of Icat and showed that noradrenaline activates Icat by G-protein stimulation of phospholipase C (PLC) and hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). With regard to the two products of this reaction, intracellular dialysis of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) did not evoke Icat but the diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulated Icat (Helliwell & Large, 1997). Therefore the transduction mechanism involved the classical phosphoinositide pathway of G-protein coupled to PLC and DAG stimulation of Icat, but a significant observation was that DAG evoked Icat by a protein kinase C (PKC)-independent mechanism (Helliwell & Large, 1997). However a difficulty in the proposed model was that the amplitude and rate of activation of Icat evoked by OAG were considerably reduced compared to noradrenaline-stimulated responses (Helliwell & Large, 1997). In the present work we have investigated the effect of inositol phosphates (InsPs) on the response to OAG and the results show that Ins(1,4,5)P3 and Ins(1,4)P2 markedly increase the amplitude and rate of activation of OAG-induced Icat. These results indicate that noradrenaline utilises DAG, Ins(1,4,5)P3 and Ins(1,4)P2 for activation of Icat.
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The present work shows that intracellular Ins(1,4,5)P3 and Ins(1,4)P2 markedly potentiate the amplitude and rate of activation of DAG-evoked Icat in rabbit portal vein myocytes. Intracellular application of InsPs did not themselves stimulate channel opening but had a profound synergistic interaction with OAG which has weak agonist activity when applied on its own (Helliwell & Large, 1997; Albert & Large, 2001; present study). Therefore it appears that the InsPs prime the channel to allow more efficacious activation by OAG. It has been noted previously that the amplitude and rate of activation of OAG-induced currents were smaller than noradrenaline-evoked responses (Helliwell & Large, 1997). Quantitative comparisons show that the presence of Ins(1,4,5)P3 in the intracellular solution brings the characteristics of the OAG-induced Icat closer to those of the currents stimulated by noradrenaline (see Fig. 1). Therefore it appears that noradrenaline utilises both DAG and Ins(1,4,5)P3 for full and rapid activation of Icat. The observation that Ins(1,4)P2, a major metabolite of Ins(1,4,5)P3, markedly potentiated Icat increases the potential physiological relevance of the mechanism described in this report.
The present study does not reveal the mechanism by which Ins(1,4,5)P3 potentiates the action of OAG but it is unlikely to be due to Ca2+ release from the sarcoplasmic reticulium (SR). The high concentration of BAPTA (10 mm) would be expected to buffer any Ca2+ released from internal stores and, anyway, an increase of [Ca2+]i does not increase the amplitude of Icat (Helliwell & Large, 1996). Also any effect mediated by typical Ins(1,4,5)P3 receptors on the SR would be expected to be blocked by heparin, which did not occur.
In addition to potentiating OAG-evoked Icat, Ins(1,4,5)P3 also increased the decay rate of the OAG-induced whole-cell currents. Interestingly, Ins(1,4,5)P3 also accelerated the decay rate of the noradrenaline-induced Icat, which was not commented on previously (see Fig. 5 in Helliwell & Large, 1997). At present we have no further insight on this inhibitory action of Ins(1,4,5)P3 but it warrants further investigation.
Previously it has been shown that Ins(1,4,5)P3 activates Ca2+-permeable cation channels in human T-lymphocytes (Kuno & Gardner, 1987) and human carcinoma A431 cells (Kaznacheyera et al. 2000). More pertinent to the present work, since TRPC6 has been shown to be an essential component of Icat in portal vein myocytes, it has been demonstrated that Ins(1,4,5)P3 activates expressed human TRPC3 channels in HEK 293 cells (Kiselyov et al. 1998), although these results have been contradicted in a recent study (Trebak et al. 2003). It has also been proposed that the cation current activated by brain-derived nerve growth factor in neonatal rat pontine neurones is a TRPC3 channel activated by Ins(1,4,5)P3 (Li et al. 1999). In our work, Ins(1,4,5)P3 did not activate channels and moreover the potentiating effect of Ins(1,4,5)P3 on OAG-induced currents was not blocked by heparin whereas the Ins(1,4,5)P3-induced currents were inhibited by heparin in both HEK 293 cells (Kiselyov et al. 1998) and rat pontine neurones (Li et al. 1999). The work of Kiselyov et al. (1998) and others has been used to support the conformational-coupling model of store-operated Ca2+ entry, but Icat in rabbit portal vein myocytes is not a store-operated conductance (Byrne & Large, 1988; Wang & Large, 1991; Large, 2002). Therefore the effect of Ins(1,4,5)P3 reported in the present work represents a novel heparin-insensitive action of this intracellular mediator.
Finally it is relevant to comment on the transduction mechanism linking the α1-adrenoceptor to Icat in rabbit portal vein myocytes. In some respects the classical G-protein-PI-PLC pathway is involved, but there are two notable differences. First, DAG activates Icat via a PKC-independent mechanism (Helliwell & Large, 1997), and second, it appears that both DAG and Ins(1,4,5)P3 converge on the same target protein in the sarcolemma (present study). In many tissues Ins(1,4,5)P3 and DAG follow divergent routes with different targets (DAG activates PKC and Ins(1,4,5)P3 acts on the sarco/endoplasmic reticulum). It will be interesting to see if this synergism between Ins(1,4,5)P3 and DAG is involved in the activation of other TRP channels in other tissues.