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Neuropeptide Y (NPY) and somatostatin (SRIF) interact with receptors that are members of the seven transmembrane domain-, Gi/Go-coupled receptor family to affect the activity of a wide variety of cells. In neuronal cells, both NPY and SRIF receptors couple to a similar range of cellular effectors including inhibition of voltage-dependent Ca2+ currents (Lewis et al., 1986; Walker et al., 1988), inhibition of adenylyl cyclase activity (Heisler et al., 1982; Westlind-Danielsson et al., 1987), stimulation of Ca2+ entry (Miyoshi et al., 1989; Lynch et al., 1994) and mobilization of intracellular Ca2+ (Perney & Miller, 1989; Okajima & Kondo, 1992). SRIF receptor activation also modulates a variety of K+ currents (Mihara et al., 1987; Wang et al., 1989). Where examined, virtually all the effects of NPY and SRIF in neuronal cells have been shown to be mediated by pertussis toxin-sensitive G-proteins.
A variety of subtypes of NPY receptor have been proposed, with the classification primarily based on the rank order of potencies of agonist peptide analogues of NPY (reviewed in Wahlestedt & Reis, 1993). In general, the classification for NPY receptors have been based on the in vitro pharmacology of the native receptors, and the recent cloning of 3 different NPY receptors, Y1 (Krause et al., 1992; Larhammer et al., 1992), Y2 (Gerald et al., 1995; Rose et al., 1995) and Y4 (Bard et al., 1995), that exhibit pharmacological profiles similar to previously characterized native receptors, confirms the utility of such an approach.
In contrast, data from radioligand binding studies suggested the existence of two major classes of SRIF receptor (e.g. SS1/ SS2, SRIF1/SRIF2, SOMA/SOMB) before the cloning of 5 different SRIF receptor genes (sst1-sst5, reviewed in Hoyer et al., 1995a). Extensive pharmacological profiles of the recombinant receptors have been determined (e.g. Raynor et al., 1993a,b; Patel & Srikant, 1994; Hoyer et al., 1995b; Schoeffter et al., 1995; Castro et al., 1996) and these data, when combined with structural information about the receptors, have led to the proposal of 2 classes of SRIF receptor, SRIF1 and SRIF2 (Hoyer et al., 1995a). The SRIF1 class comprises sst2, sst3 and sst5 receptors, and appears to correspond to the SS1 binding site (Hoyer et al., 1995a,b), while the SRIF2 class comprises ssti and sst4 receptor and corresponds to the SS2 binding site (Hoyer et al., 1995a; Schoeffter et al., 1995). Recently, attempts have been made to correlate the pharmacological profiles determined for the recombinant receptors with those of native receptors in isolated preparations, for example to determine the SRIF receptor involved in SRIF inhibition of ion transport in rat colon (sst2-like McKeen et al., 1995), SRIF inhibition of firing rates in rat locus coeruleus neurones (sst2-like, Chessell et al., 1996) and SRIF contraction of human saphenous veins (also sst2-like, Dimech et al., 1995).
SH-SY5Y cells are a human neuroblastoma cell line that has been used as a system for the investigation of the signal transduction mechanism of many human neurotransmitter receptors (reviewed in Vaughan et al., 1995). Both SRIF (Frie-derich et al., 1993) and NPY (McDonald et al., 1995) have been shown to inhibit the voltage-dependent Ca2+ currents in SH-SY5Y cells via pertussis toxin-sensitive mechanisms. It was proposed that NPY acted via NPY Y2 receptors to inhibit the Ca2+ currents (McDonald et al., 1995). The type(s) of SRIF receptor present on SH-SY5Y cells is not known. It is also not known whether either NPY or SRIF receptors couple to cellular effectors other than Ca2+ currents in SH-SY5Y cells. Both δ and μ opioid receptors (Seward et al., 1990; 1991), as well as receptors for the neuropeptide nociceptin (Connor et al., 1996b), have been shown to inhibit voltage-dependent Ca2+ channels in SH-SY5Y cells. We have recently shown that δ and μ opioid receptors and receptors for nociceptin also couple to the mobilization of intracellular Ca2+ in SH-SY5Y cells, via a novel pathway that requires the simultaneous activation of Gq-coupled receptors such as muscarinic receptors (Connor & Henderson, 1996; Connor et al., 1996b). In this study we have sought to determine whether the native receptors for NPY and SRIF can couple to effectors in addition to voltage-dependent Ca2+ channels in SH-SY5Y cells. We find that both NPY and SRIF, when applied in the presence of the cholinoceptor agonist carbachol, can couple to the mobilization of intracellular calcium in SH-SY5Y cells; and further, that this Ca2+ mobilization is mediated via NPY Y2-like receptors and sst2-like receptors, respectively. A preliminary account of this work has been presented to the British Pharmacological Society (Connor et al., 1996a; Yeo & Henderson, 1996).
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The principle findings of this study are that SRIF, via an sst2-like receptor, and NPY, via an NPY Y2-like receptor, can couple to the mobilization of intracellular Ca2+ in SH-SY5Y cells. Both SRIF (Freiderich et al., 1993) and NPY (McDonald et al., 1995) have been previously shown to inhibit a voltage-dependent, N-type, calcium current in SH-SY5Y cells through pertussis toxin (PTX) sensitive G-proteins. Similarly, the mobilization of [Ca2+]i by sst2-like and NPY Y2-like receptors seen in this study was also dependent on the presence of functional pertussis toxin-sensitive G-proteins.
The identification of the NPY receptor subtype involved in the mobilization of intracellular Ca2+ was based on the rank order of potency of a series of NPY analogues. PYY(3–36), an N-terminally truncated PYY analogue that is at least 200 fold more selective for the rat (Dumont et al., 1994) and human (Gerald et al., 1995) Y2 receptor than Y1 receptor, was the most potent compound tested. In contrast, the C-terminally modified NPY analogue [Leu31,Pro34] NPY, which has at least 1000 fold greater affinity for the cloned human Y1 receptor than the cloned human Y2 receptor (Gerald et al., 1995), was much less potent than PYY(3–36) or NPY, and the expense of the material precluded construction of a full concentration-response curve. This rank order of potency: PYY(3 36) ≥ NPY ≥[Leu31,Pro34] NPY, is consistent with an NPY Y2 receptor-mediated effect. If a Y1 receptor were involved the rank order of potency of the compounds should have been reversed (Wahlestedt & Reis, 1993). If an NPY Y3-like receptor were involved, PYY(3–36) would have been expected to be inactive (Wahlestedt & Reis, 1993); if the response had been mediated by an NPY Y4-like receptor, [Leu31,Pro34] NPY would have been expected to be approximately as potent as PYY(3–36) or NPY itself (Bard et al., 1995). Recently, an NPY Y1-receptor antagonist has been developed (BIBP3226, Rudolf et al., 1994), unfortunately we were unable to obtain a sample of the compound for this study. The EC50 for NPY mobilization of intracellular Ca2+ in SH-SY5Y cells was about 15 nM, which is similar to the EC50 obtained for inhibition by NPY of voltage-dependent Ca2+ currents in these cells (EC50 approximately 50 nM; McDonald et al., 1995), a response also attributed to Y2 receptor activation.
In the absence of potent SRIF receptor antagonists (see Hoyer et al., 1994), the identification of the SRIF receptor type involved in the mobilization of intracellular Ca2+ was also based on the rank order of potency of SRIF analogues. This rank order: BIM-23027 ≥ SRIF > > L-362855 > > > BIM-23056, is consistent with activation of an sst2-like receptor. In mouse fibroblasts expressing recombinant hsst2, the rank order of potency of the above compounds for inhibition of [125I]-[Tyr11]-SRIF binding and stimulation of extracellular acidification was identical to that obtained here (Castro et al., 1996). In contrast, BIM-23027 was inactive in binding assays performed on fibroblasts expressing the hssti, while L-362855 and BIM-23056 displayed similar potencies to displace [125I]-[Tyr11]-SRIF binding (Castro et al., 1996). In a previous study of hsst1 expressed in Chinese hamster ovary (CHO) cells, BIM-23027, BIM-23056 and L-362855 had IC50s of greater than 1 μM against [125I]-CGP 23996 binding (Raynor et al., 1993a). Similarly, in CHO cells expressing hsst4 the compounds used in this study displace [125I]-CGP 23996 with a rank order SRIF > L-362855 > BIM-23056 ≥ BIM-23027 (Raynor et al., 1993b), quite different from that obtained here for mobilization of Ca2+. In the present study, BIM-23056 was inactive as either an agonist or antagonist at concentrations up to 1 μM, given that BIM-23056 has affinities of 11 nM and 6 nM, respectively, for hsst3 and hsst5 receptors expressed in CHO-K1 cells (Patel & Srikant, 1994), its lack of effect in SH-SY5Y cells suggests that neither sst3 nor sst5 receptors were involved in the mobilization of Ca2+. There is at present no information regarding the SRIF binding sites present on SH-SY5Y cells, nor has the expression of SRIF receptors in these cells been examined by use of molecular biological techniques. It is possible that SH-SY5Y cells express more than one kind of SRIF receptor, and in the absence of potent and selective agonists and antagonists for sst1 and sst4 receptors it is impossible to rule out completely a contribution of these receptor types to the mobilization of [Ca2+]i observed in this study. Nevertheless, the rank order of agonist potency seen in this study indicates a predominant involvement of sst2-like receptors in the mobilization of Ca2+ in SH-SY5Y cells.
The mobilization of intracellular Ca2+ by NPY and SRIF applied in the presence of carbachol is indistinguishable from that mediated by δ and μ-opioid receptor agonists in SH-SY5Y cells (Connor & Henderson, 1996). We never observed an elevation of [Ca2+]i in the presence of any of the agonists alone, and the elevations of [Ca2+]i were blocked when carbachol and either SRIF or NPY were applied in the continued presence of atropine. As with the δ-opioid induced elevations of [Ca2+]i, the extent to which muscarinic receptors were activated did not seem to be critical for the agonist-induced Ca2+ mobilizations, because NPY elevated [Ca2+]i by the same amount and with identical potency when applied in the presence of either 1 μM or 100 μM carbachol. Finally, when either NPY or SRIF was applied together with a maximally effective concentration of the δ-opioid agonist DPDPE, the elevations of [Ca2+]i were not additive, which suggests that the 3 receptors were acting via a common signal transduction pathway to mobilize intracellular Ca2+.
The precise mechanism by which NPY and SRIF mobilize Ca2+ in SH-SY5Y cells is not clear. Direct coupling of NPY or SRIF receptors to phospholipose C (PLC) is unlikely. There is no evidence that NPY or SRIF receptors can couple to G proteins of the Gq family, whose α subunits directly activate PLC (Exton, 1996). There are isoforms of PLC that can be directly activated by the βγ subunits of Gi/Go proteins (Exton, 1996), and SH-SY5Y cells contain PLC-β3, one of the βγ responsive isoforms (Yeo, Kelly and Henderson, unpublished observations), but neither NPY nor SRIF elevated [Ca2+]i by themselves. Concomitant muscarinic receptor activation is clearly necessary for the Gi/Go-coupled receptor mobilization of Ca2+, as we have previously shown that simultaneous application of a muscarinic antagonist with a δ opioid agonist was sufficient to block the opioid-induced rise in [Ca2+]i, regardless of whether the carbachol concentration was 1 μM or 100 μM (Connor & Henderson, 1996). The precise nature of the link between muscarinic receptor occupancy and the NPY or SRIF receptor mobilization of [Ca2+]i is not clear. However, it is possible that for some types of PLC, βγ stimulation of the enzyme requires prior activation by αq; in a manner analogous with the βγ stimulation of some forms of adenylyl cyclase, which requires prior priming with αs (Tang & Gilman, 1991).
A similar signal transduction pathway to that described here (i.e. Gi/Go coupling to elevation of intracellular Ca2+ in the presence of a permissive Gq-coupled receptor activation) has been observed in the neuroblastoma × glioma hybrid cell line NG108-15 (Okajima & Kondo, 1992; Okajima et al., 1993). In these cells δ-opioids, SRIF and noradrenaline mobilized intracellular Ca2+ only when applied in the presence of bradykinin or ATP as the ‘permissive’ Gq-coupled receptor agonist. The mobilization of Ca2+ by δ-opioids, SRIF and noradrenaline was mediated via pertussis toxin-sensitive G-proteins and appeared to be accompanied by an increase in the amount of IP3 produced following bradykinin receptor activation (Okajima et al., 1993). The identification of this novel signal transduction pathway in two cell types suggests that it may be a mechanism common to many cell types for the coupling of Gi/Go-coupled receptors to intracellular Ca2+ stores.
NPY receptors have not previously been shown to be coupled to the mobilization of intracellular Ca2+ via a pathway that requires the concomitant activation of another receptor. NPY receptor activation has been shown to mobilize intracellular Ca2+ in cultured dorsal root ganglion cells (Perney & Miller, 1989), human erythroleukaemia cells (Motulsky & Michel, 1988), SK-N-MC neuroblastoma cells (Aakerlund et al., 1990) and cultured vascular smooth muscle cells (Mihara et al., 1989). These mobilizations of Ca2+ by NPY were also mediated by pertussis toxin-sensitive G-proteins. However, where examined, the receptors responsible were of the Y1 type (Aakerlund et al., 1990; Shigeri et al., 1991; Feth et al., 1992). In the dorsal root ganglion, human erythroleukaemia and vascular smooth muscle cells an NPY-stimulated increase in IP3 production has been shown to accompany the elevations of intracellular Ca2+, suggesting that NPY receptors can couple directly to PLC (Daniels et al., 1989; Perney & Miller, 1989; Shigeri et al., 1995), which is clearly not the case in the present study. It is possible that in cell types other than SH-SY5Y, Y1 receptors are activating isoforms of PLC that can be stimulated by the βγ subunits of pertussis toxin-sensitive G-protein heterotrimers (Exton, 1996). Alternatively, it is possible that under the conditions in which some of the previous studies were performed (i.e. cells in suspension, Motulsky & Michel, 1988; Aakerlund et al., 1990), ‘priming’ agents such as ATP could have been released from damaged cells in the cuvette and interacted with the Y1 agonists added subsequently. It was in these conditions that δ-opioid mobilization of Ca2+ in NG108-15 cells was first noted (Tomura et al., 1992). Nevertheless, this study, taken together with those described above, indicates that there are several possible pathways for NPY to mediate mobilization of intracellular Ca2+.
In contrast to NPY receptor activation, native SRIF receptors have previously been shown to mobilize Ca2+ only in NG-108 cells (Okajima & Kondo, 1992), although there is some evidence that SRIF can stimulate phosphoinositide hydrolysis in various brain regions (Lachowicz et al., 1994), and all 5 subtypes of cloned human sstr activate PLC when het-erologously expressed in COS-7 cells (Akbar et al., 1994). The receptor type(s) responsible for the mobilization of intracellular Ca2+ in neuronal cell lines have not been identified, and as outlined above, the precise mechanism by which SRIF mobilizes Ca2+ in neuronal cells is not known.
This study demonstrates that in SH-SY5Y cells NPY Y2 receptors and native sst2-like receptors can interact with muscarinic cholinergic systems to promote the mobilization of intracellular Ca2+. This interaction is the same as between μ and δ opioid receptor agonists and carbachol in SH-SY5Y cells, and may represent a common signal transduction pathway for Gi/Go-coupled receptors. As previously shown for μ and δ opioid receptors and receptors for nociceptin (Seward et al., 1990; 1991; Connor & Henderson, 1996; Connor et al., 1996b); this study shows that NPY Y2 receptors and SRIF receptors can couple to more than one effector in SH-SY5Y cells (Freiderich et al., 1993; McDonald et al., 1995). Careful investigation of the interactions between NPY and SRIF receptors and other plasma membrane receptors may lead to new insights into the cellular consequences of SRIF and NPY receptor activation, such interactions may be common in an in vivo situation, where cells are exposed to a many neuro-transmitters and neuromodulator substances.