Neuronal Ion Channel Signalling Pathways

ISSN 0898-6568/98 $19.00 1 .00 PII S0898-6568(97)00133-2

Cell. Signal. Vol. 10, No. 5, pp. 303–311, 1998 Copyright  1998 Elsevier Science Inc.

TOPICAL REVIEW

Neuronal Ion Channel Signalling Pathways: Modulation by Angiotensin II Colin Sumners* and Craig H. Gelband Department of Physiology, College of Medicine, University of Florida, Gainesville, FL 32610, USA

ABSTRACT. The brain contains both angiotensin II (Ang II) type 1 (AT1) and Ang II type 2 (AT2) receptors. Neuronal AT1 receptors mediate the stimulatory actions of Ang II on blood pressure, water and salt intake, and secretion of vasopressin. In contrast, neuronal AT2 receptors have been implicated in the stimulation of apoptosis and as being antagonistic to AT1 receptors. The physiological actions of Ang II in the brain, whether mediated by AT1 or AT2 receptors, involve changes in neuronal activity that are initiated by changes in the activity of membrane ionic currents and channels. This review focusses on the intracellular signalling pathways that couple neuronal AT1 and AT2 receptors to changes in the activity of membrane K1 and Ca21 currents and channels. As will become clear from our discussion, the signalling pathways that are modulated by neuronal AT1 and AT2 receptors are quite distinct. cell signal 10;5:303–311, 1998.  1998 Elsevier Science Inc. KEY WORDS. Potassium current, Calcium current, AT1 receptor, AT2 receptor, Protein kinase C, Arachidonic acid, Phospholipase A2

INTRODUCTION The peptide angiotensin II (Ang II) has well-known peripheral actions on cardiovascular regulation, hormone secretion, and growth of vascular smooth muscle cells [1]. Aside from these functions, it is now widely recognised that mammalian brain contains all of the components of the renin-angiotensin system, including Ang II–specific receptors [2, 3]. Furthermore, it has been demonstrated that Ang II elicits important receptor-mediated actions in the brain [4, 5]. High densities of Ang II receptors are located in the hypothalamus and brainstem [2, 3], and Ang II acts at specific neuronal receptors in these areas to modulate the activity of neuronal pathways. These actions ultimately lead to physiological changes such as increased blood pressure, altered baroreflex modulation, increased water and sodium intake, and increased secretion of vasopressin [6–15]. More recently, it has been shown that mammals contain two major sub-types of Ang II receptors, the Ang II type 1 (AT1) and Ang II type 2 (AT2) receptors, both of which exist in the brain [16]. These sub-types were initially identified on the basis of differential pharmacological properties [17, 18]. Briefly, AT1 receptors have a high affinity for losartan, whereas AT2 receptors have a high affinity of PD123,177, PD123,319, and CGP42112A [17, 18]. Cloning studies have since revealed that the AT1 and AT2 receptors are *Address all correspondence to Colin Sumners, P. O. Box 100274, Gainesville, FL 32610. Tel: (352) 392-4485; Fax: (352) 846-0270; E-mail: csumners @phys.med.ufl.edu Received 3 July 1997; and accepted 18 August 1997.

quite different molecules. Both are similar in size (359 amino acids for the AT1 vs. 363 amino acids for the AT2), and hydropathy analyses of the sequences revealed that both have a seven transmembrane-spanning domain structure, consistent with G-protein–coupled receptors [19–22]. However, AT1 and AT2 receptors are only 32–34% identical, based upon amino acid sequence [19–22]. In consideration of these differences, it is perhaps not surprising that AT1 and AT2 receptors have very different functions. AT1 receptors, which are widespread in peripheral tissues such as blood vessels, kidney, adrenal cortex, and liver [16], are also localised in the hypothalamus and brain stem in areas such as the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the median eminence, the subfornical organ (SFO), the organum vasculosum of the lamina terminalis, the solitary tract nucleus, and the dorsal vagal nucleus [23– 26]. The AT1 receptors within these brain areas are responsible for mediating the afore-listed stimulatory effects of Ang II on cardiovascular regulation, fluid balance, and hormone secretion [27–29]. In addition, it is known that these AT1 receptor–mediated actions of Ang II include modulation of brain noradrenergic neurons [29–31]. In contrast, AT2 receptors are mostly localised to discrete brain areas such as the mediodorsal thalamic nuclei, ventral septum, inferior olive (IO), locus coeruleus (LC), and lateral septum [23–26, 32] and do not appear to have a major involvement in the stimulatory actions of Ang II on blood pressure, drinking, and vasopressin secretion [27–29]. In fact, the physiological role(s) of AT2 receptors is not well understood. The fact that neonate animals express far higher levels of AT2 than

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AT1 receptors in brain or other tissues led to the suggestion that these sites somehow take part in development and differentiation [24, 25, 33]. This idea is somewhat supported by studies that demonstrated that Ang II, through AT2 receptors, induces neurite outgrowth of NG108-15 neuroblastoma x glioma hybrid cells [34]. In addition, mutant mice that lack the gene encoding the AT2 receptor demonstrated deficits in brain-controlled behaviour such as exploration and spontaneous movements [35, 36], suggesting that the AT2 receptor has a role in normal brain development. Another study has also linked AT2 receptors to programmed cell death (apoptosis) [37]. In that study, activation of AT2 receptors on PC12W pheochromocytoma cells (an in vitro model of rat sympathetic neurons) led to apoptosis [37]. In agreement with this, we have determined that Ang II, through AT2 receptors, causes apoptosis of neurons cultured from neonate rat hypothalamus and brain stem [38]. It has also been suggested that AT2 receptors mediate effects that are opposite or antagonistic to effects mediated by AT1 receptors. For example, the afore-described stimulation of neurite outgrowth in NG108-15 cells was counteracted by stimulation of AT1 receptors [34]. Other studies have shown that blockade of periventricular brain AT2 receptors potentiates the Ang II-induced (AT1 receptor-mediated) stimulation of vasopressin release and drinking [39]. The idea that brain AT2 and AT1 receptors have opposite or antagonistic effects is also supported by the demonstration that these receptor sub-types are co-localised in certain areas (e.g., anteroventral third ventricle, superior colliculus) of adult rats [23–26, 32]. In summary, although the functions of central AT1 receptors are quite well defined, the roles of AT2 receptors in the brain (and other tissues) are less well established. Effects of Angiotensin II on Neuronal Membrane Ionic Currents and Channels Despite the well-documented presence and functional roles of Ang II on neurons, relatively few studies have addressed the modulatory actions of Ang II on neuronal membrane ionic currents and channels. These currents (and the underlying channels) are the basis of neuronal action potentials (APs). Thus, an understanding of how neuronal membrane ionic currents are modulated by Ang II is extremely important because the frequency and firing pattern of APs is the basic regulator of all physiological and behavioural events mediated by a given neuron. Of equal importance is an understanding of the intracellular signalling events that are responsible for the modulatory actions of Ang II on membrane ionic currents. This is critical because perturbations of these signalling mechanisms would lead to altered influences of Ang II on neuronal activity and on the physiological events mediated by a given neuron. In the next paragraphs, we will briefly consider the effects of Ang II, through AT1 and AT2 receptors, on neuronal membrane ionic currents/channels and, where available, neuronal activity. For both Ang II receptor sub-types, much of the discussion will centre on our studies using neurons co-cultured

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from newborn rat hypothalamus and brain stem [40]. For the sake of simplicity, these will be referred to as “cultured neurons” in subsequent sections. AT1 RECEPTORS. Studies using neurons in situ or brain slice preparations have shown that selective activation of AT1 receptors by Ang II elicits an increase in neuronal firing rate in specific regions such as the PVN, the SFO, the rostral ventrolateral medulla, and the SON [41–44]. Similar increases in neuronal excitation elicited by Ang II through AT1 receptors have been observed in cultured neurons [45]. Whole-cell and single-channel voltage-clamp procedures have been used to investigate the changes in membrane ionic currents that account for AT1 receptor–mediated increases in neuronal excitation. In cultured neurons, Ang II, through AT1 receptors, elicits a decrease in neuronal net outward ionic current (Ino) [46]. In these cells, Ino consists mainly of Na1, K1, and Ca21 current. Therefore, for Ang II to decrease Ino, it must either attenuate K1 current or potentiate Na1 or Ca21 current. Few data are available on the actions of Ang II on Na1 current in cultured neurons. However, Ang II (in the presence of 1 mM PD123,319 to block AT2 receptors) caused significant decreases in a voltage-dependent delayed rectifier K1 current (IK(v), formerly referred to as IK) and in transient A-type K1 current (IA), effects mediated by AT1 receptors [47, 48]. Consistent with the latter effect is the finding that Ang II caused an AT1 receptor– mediated decrease in single-channel-open probability (NPo) of an A-type K1 channel in the same cultured neurons [48]. Studies in cultured neurons have also shown that Ang II elicits an AT1 receptor–mediated stimulation of voltage-dependent Ca21 current (ICa) [47]. The preceding observations on IA agree with studies that determined that Ang II (through AT1 receptors) decreased IA in neurons from the SFO, SON, and PVN magnocellular areas contained in brain slices [49–51]. In summary, the decreases in neuronal IK(v) and IA and the increase in ICa elicited by Ang II are consistent with the observed increases in neuronal excitation discussed earlier [41–44]. Furthermore, recent studies demonstrate that AT1 receptors are localised on cultured catecholaminergic neurons [52] and that stimulation of AT1 receptors on cultured neurons causes release of norepinephrine (NE; Sumners et al., unpublished observations). Thus, the observed changes in IK(v), IA, and ICa may underlie the increases in neuronal excitation that ultimately lead to NE release. AT2 RECEPTORS. Studies using neurons in situ or in a brain slice preparation suggest that the AT2 receptor–mediated effects of Ang II on neuronal firing rate are site specific. For example, iontophoretic application of Ang II onto IO neurons in situ elicited an excitatory effect mediated by AT2 receptors [53]. In contrast, stimulation of AT2 receptors in the LC contained in slices caused a depression of excitatory post-synaptic potentials and of glutamate-induced depolarisations [54]. Whole-cell voltage-clamp procedures have been used to investigate the changes in membrane ionic currents that account for the AT2 receptor–mediated

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changes in neuronal excitation. In cultured neurons, selective activation of AT2 receptors by Ang II (in the presence of losartan to block AT1 receptors) caused a significant potentiation of both IK(v) and IA [55]. These effects are clearly opposite to the decreases in IK(v) and IA produced by Ang II through AT1 receptors [47, 48]. Furthermore, CGP42112A, which acts as an AT2 receptor agonist at low concentrations, also elicited stimulatory effects on IK(v) and IA [56]. Cell-attached patch-clamp experiments have shown that stimulation of AT2 receptors by either Ang II (plus losartan) or CGP42112A elicits a stimulatory effect on a delayed rectifier K1 [K(v)] channel that is consistent with the stimulatory effects on IK(v) [57]. Stimulation of AT2 receptors by Ang II does not seem to influence voltage- dependent ICa in cultured neurons [55] but does inhibit T-type Ca21 current in undifferentiated NG108-15 neuroblastoma x glioma cells [58]. In summary, the stimulatory effects of Ang II on IK(v) and IA in cultured neurons suggest that activation of AT2 receptors leads to a hyperpolarisation or decrease in neuronal excitability. This is consistent with one of the afore-mentioned studies from brain slices [54]. However, it appears that voltage-clamp studies must be performed in more specific brain areas (in slices or in cultured neurons) that are rich in AT2 receptors (e.g., IO or LC). In this way, discrepancies between observed changes in neuronal currents and excitability may be resolved. Furthermore, identification of the type of neuron on which AT2 receptors are found will allow the determination of whether AT2 receptor–mediated changes in ionic currents and neuronal activity modify release of a particular transmitter. One study has shown that AT2 receptors are present on cultured catecholaminergic neurons [52], but there is no evidence that these receptors control catecholamine release. Role of Intracellular Signalling Pathways in Angiotensin II Receptor–Modulated Neuronal K1 and Ca21 Currents and Channels Activation/inhibition of membrane ionic currents and their underlying channels by G-protein–coupled receptors can occur by direct (membrane-delimited) coupling of the G protein to the channel or by indirect modulation by protein kinases or phosphatases [59]. As stated earlier, both AT1 and AT2 receptors fall into the G-protein–coupled category of plasma membrane receptors [19–22]. In the following sections, we will consider the signal transduction pathways (both direct and indirect) that couple neuronal AT1 and AT2 receptors to K1 and Ca21 currents and channels. The focus of our discussion will be on studies performed in cultured neurons, with reference to experiments from other cells, if available. Included within this discussion will be a review of the information that is available on the various signalling pathways that are known to be modulated by AT1 and AT2 receptors. AT1 RECEPTOR–MEDIATED SIGNALLING PATHWAYS. Most of the information on AT1 receptor–mediated signal-transduction pathways has been obtained from peripheral tissues

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and cells. Many studies have revealed that AT1 receptors are coupled to either activation of phospholipase C (PLC) and stimulation of phosphoinositide (PI) hydrolysis or to inhibition of adenylyl cyclase, depending on the cell or tissue type [60–65]. These pathways are modulated presumably by Gq (or a Gq family member such as G11 or G13) and Gi, respectively. More recent studies have indicated that AT1 receptor signalling pathways are not so straightforward and have shown that activation of AT1 receptors can lead to the stimulation of phospholipase D and phospholipase A2 activities in certain cell types [66–68]. In addition, Ang II causes an AT1 receptor–mediated stimulation of the Ras/Raf/mitogen activated protein (MAP) kinase and JAK [Janus kinase/ STAT (signal transducers and activators of transcription)] pathways [69, 70]. These pathways may be activated by Gbg sub-units. In the brain, activation of AT1 receptors in the median eminence leads to a stimulation of PI hydrolysis [71]. Similarly, our studies in cultured neurons have revealed that Ang II stimulates, through AT1 receptors, PI hydrolysis with generation of inositol 1,4,5-triphosphate (IP3) and subsequent increases in intracellular Ca21 concentration—[Ca21]int—and activation of protein kinase C (PKC) [40, 47, 72]. AT1 receptor–mediated stimulation of PI hydrolysis has also been observed in NG108-15 neuroblastoma x glioma cells [73, 74]. Recently, Ang II has been shown to elicit an AT1 receptor–mediated stimulation of the Ras/Raf pathway, with subsequent increases in MAP kinase activity, in cultured neurons [75, 76]. Thus, as in peripheral tissues and cells, activation of neuronal AT1 receptors leads to modulation of a number of different signalling pathways. SIGNALLING PATHWAYS OF AT1 RECEPTOR–MODULATED

Considering that PKC, Ca21, and IP3 are known modulators of neuronal ion channels [77–83], studies were performed to determine their possible role in the modulation of neuronal IK(v), IA, and ICa after AT1 receptor activation. These studies are summarised as follows. With respect to IK(v), the AT1 receptor–mediated inhibition of this current was partly reduced by intracellular application of anti-Gq/11a antibodies [47] and was totally abolished by the non-selective PLC inhibitor U73122 (10 mM; unpublished observations). U73343 (10 mM), an inactive analogue of U73122, did not modify the inhibition of IK(v) elicited by Ang II. Taken together, these data suggest that the AT1 receptor–mediated reduction of IK(v) involves a Gq/11a protein and activation of PLC, which is not surprising. However, this situation may not be so straightforward, and the role of Gq/11a needs to be more firmly established. The anti-Gq/11a antibodies may be working by preventing the dissociation of Gbg from Gq/11a9 and so it could be argued that it is the Gbg that are responsible for the signal transduction. Further complications arise from the fact that recent reports have shown that Ang II (through AT1 receptors) stimulates PI hydrolysis in vascular smooth muscle cells through a mechanism that activates a soluble tyrosine kinase (pp60c-src), which then phosphorylates (and activates) PLCg [84]. This proposed mechanism does not require a Gqa protein and is IONIC CURRENTS/CHANNELS.

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unusual because G-protein–coupled receptors normally activate PLCb through Gq/11a and PLCg is normally activated by tyrosine kinase receptors. Such a mechanism may be partly responsible for the negative modulation of IK(v) by AT1 receptors in cultured neurons. For example, the reduction in neuronal IK(v) elicited by Ang II is abolished by intracellular application of polyclonal anti-PLCg antibodies [47] and is partly reversed by the tyrosine kinase inhibitor genistein (10 mM) but not by its inactive analogue daidzein (10 mM; unpublished observations). The AT1 receptor–mediated reduction in neuronal IK(v) elicited by Ang II was mimicked either by intracellular injection of IP3 or superfusion of the PKC agonist phorbol-12myristate-13-acetate (PMA) [47]. Further, this effect of Ang II was partly reduced by treatment of cultures with either of the PKC antagonists calphostin C or PKC inhibitory peptide 19-31 (PKCIP) or by chelation of [Ca21]int with 1,2-bis(2-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid (BAPTA) [47]. These data argue for an involvement of both PKC and [Ca21]int in the negative modulatory effects of Ang II on IK(v) in cultured neurons, through AT1 receptors. These data also suggest that another Ca21-dependent pathway, aside from PKC, is important in this response. Support for roles of both PKC and [Ca21]int in the negative modulation of IK(v) by Ang II also comes from experiments with a synthetic 25 amino acid peptide that corresponds to cytoplasmic loop 3 of the AT1a receptor (AT1a/i3) [85]. Intracellular application of AT1a/i3 elicited a decrease in neuronal IK(v) similar to that obtained with Ang II. This effect was partly blocked by PKCIP, by BAPTA, or by IP3 receptor antibodies. Combined intracellular application of both PKCIP and BAPTA totally blocked the reduction in IK(v) elicited by AT1a/i3, indicating that both PKC and [Ca21]int are critical for this response. The fact that all recordings of IK(v) were performed in the presence of the Ca21 channel blocker CdCl2 and that the AT1 receptor–mediated decrease in IK(v) was partly reduced by anti-IP3 receptor antibodies indicates that IP3-sensitive intracellular Ca21 stores are important in this response. Because [Ca21]int also has a role in the negative modulation of neuronal IK(v) after AT1 receptor activation, the possibility that another Ca21-dependent mechanism is required for this response was investigated. Calcium/ calmodulin-dependent protein kinase II (CAM kinase II) is a known modulator of ion channels and is activated by Ang II through AT1 receptors in vascular smooth muscle cells [86]. Thus, we tested the idea that calmodulin and CAM kinase II take part in the AT1 receptor modulation of IK(v) in cultured neurons. Preliminary studies have shown that the AT1 receptor–mediated reduction in neuronal IK(v) is partly blocked by either the calmodulin antagonist W-7 (10 mM) or the specific CAM kinase II inhibitor KN-93 (10 mM) (unpublished observations). In summary, these data indicate that the AT1 receptor– mediated inhibitory effect of Ang II on neuronal IK(v) occurs by an indirect pathway involving the activation of PLC and subsequent increases in the activity of PKC and CAM kinase II. These findings are summarised as a flow diagram in

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Figure 1. Although the data so far provide a basic framework for understanding how AT1 receptor activation leads to inhibition of IK(v), many questions remain. For example, the mechanism of PLC activation and the nature of the PLC involved are not clear. Other questions concern the mechanisms by which PKC and CAM kinase II modulate IK(v). For example, are they direct effects on the channel proteins? Furthermore, which biophysical properties of the K1 channel do these enzymes modulate? These questions, and many others, remain subjects of intense investigation. The intracellular signalling pathways that underlie the AT1 receptor–mediated inhibition of IA are less well defined. However, in preliminary studies, we have determined that the PKC activator PMA decreases IA in cultured neurons and causes a dramatic decrease in the open probability (NPo) of A-type K1 channels in these cells (unpublished observations). Thus, the effects of PMA are similar to those of Ang II (through AT1 receptors) on IA and A-type K1 channels [48]. This may indicate that the inhibitory effects of Ang II on neuronal IA involve PKC, similar to its effect on IK(v) (Fig. 1). The stimulatory effects of Ang II on neuronal ICa by AT1 receptors also appear to occur through an indirect signalling pathway. In many respects, this pathway is similar to that which mediates the inhibitory effects of Ang II on IK(v). For example, the stimulation of neuronal ICa by Ang II and AT1a/ i3 requires both Gq and PLC [47, 85]. A major difference, however, is that Ang II– and AT1a/i3-stimulated ICa is completely abolished by the PKC antagonists calphostin C and PKCIP [47, 85]. Thus, the AT1 receptor–mediated stimulation of ICa involves PKC alone, rather than a dual PKC/ CAM kinase II as is the case for modulation of IK(v). These proposed pathways are summarised as a diagram in Figure 1, and (as with the modulation of IK(v)) many questions remain concerning the exact mechanisms. AT2 RECEPTOR–MEDIATED SIGNALLING PATHWAYS.

Compared with the AT1 receptors, the signal transduction pathways that are modulated after AT2 receptor activation are less clear. Most of the information on AT2 receptor– mediated signalling pathways has been obtained from tumour cell lines, as well as from our own studies on cultured neurons from newborn rat hypothalamus and brain stem. As stated previously, AT2 receptors belong to the G-protein– coupled class of plasma membrane receptors, based on hydropathy analysis of the deduced amino acid sequence [21, 22]. However, expression cloning studies and earlier experiments in PC12W pheochromocytoma cells failed to demonstrate any G-protein coupling for the AT2 receptor; that is, no modulation of signalling pathways such as PI hydrolysis, [Ca21]int, PKC, adenylyl cyclase, and phospholipase A2 (PLA2) after activation of the AT2 receptor [21, 22, 89]. Subsequent studies in cultured neurons indicated that AT2 receptors couple through Gi proteins [56, 85], and this was confirmed by studies that demonstrated that AT2 receptors from rat foetus co-precipitate with Gia2 and Gia3 proteins [90]. As previously stated, earlier experiments failed to ob-

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FIGURE 1. Summary of the in-

tracellular signalling pathways in the AT1 receptor–mediated inhibition of neuronal IK(v) and IA, and stimulation of ICa. PLC, phospholipase C; PiP2, phosphotidylinositol 4,5-bisphosphate; DAG, diacylglycerol; IP3, inositol 1,4,5-triphosphate; IP3-R, IP3 receptor; PKC, protein kinase C; CAM kinase II, calcium/calmodulin-dependent protein kinase II. Solid arrows represent stimulatory pathways for which there is support from published (see refs. [69, 70, 77–83, 87, 88]) or preliminary (see text) studies. Dashed arrows represent inhibitory pathways for which there is support from published (see refs. [77–83]) or preliminary (see text) studies.

serve any intracellular changes in classical signal transduction pathways [21, 22, 89]. However, many of these conclusions were reached on the basis of studies in transfected cells. In more recent experiments, stimulation of AT2 receptors in neonatal rat cardiac myocytes resulted in a stimulation of PLA2 activity and consequent release of arachidonic acid (AA) [91]. In addition, Ang II was shown to cause activation of a Ca21-independent isoform of PLA2 in primary cultures of rabbit proximal tubule epithelia [92]. Our studies in cultured neurons revealed that selective activation of AT2 receptors leads to stimulation of PLA2 activity and AA release [93]. It should also be noted that many studies have demonstrated that stimulation of AT2 receptors can lead to modulation of intracellular signalling pathways that take part in cell growth processes. For example, results from PC12W cells and murine neuroblastoma (N1E-115) cells indicate that activation of AT2 receptors leads to stimulation of phosphotyrosine phosphatase [37, 94, 95]. More specifically,

one study in PC12W cells has shown that Ang II, through AT2 receptors, stimulates MAP kinase phosphatase 1 (MKP-1), which is a tyrosine phosphatase [37]. Similarly, we have determined that activation of AT2 receptors in cultured neurons leads to a weak stimulation of MKP-1 gene expression (unpublished observations). However, our data show that AT2 receptor activation has much more dramatic effects on the activity of serine/threonine phosphatase type 2A (PP-2A) in cultured neurons [96]. Ang II, through AT2 receptors, stimulates neuronal PP-2A through a pertussis toxin–sensitive G protein [96]. MAP kinases are potential targets for, and can be inhibited by, PP-2A and MKP-1 [97, 98], and, in further studies, we and others have shown that MAP kinase activities are inhibited by activation of AT2 receptors in both cultured neurons and PC12W cells [37, 75]. SIGNALLING PATHWAYS OF AT2 RECEPTOR–MODU-

Gi proteins, PP-2A, AA, and AA metabolites such as leukotrienes, hydroxyeiLATED IONIC CURRENTS/CHANNELS.

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cosatetranoic acid (HETE), and prostaglandins are known modulators of K1 channels and currents in neurons and other cells [59, 99–107]. Therefore, studies were performed to determine their possible roles in the stimulation of neuronal IK(v) after AT2 receptor activation. These studies are summarised as follows. The stimulatory actions of Ang II and CGP42112A on IK(v) in cultured neurons were abolished by pre-treatment of cells with pertussis toxin [56], suggesting the involvement of a Gi or Go protein in the response. Further, the stimulatory effect of Ang II on neuronal IK(v) was completely inhibited by intracellular application of Gia antibodies but not by similar application of either anti-Goa or anti-Gq/11a antibodies [56]. This suggests that AT2 receptor–mediated stimulation of IK(v) requires a Gia protein. However, it has also been established that Gbg sub-units are important regulators of neuronal ion channels [102]. Thus, another interpretation of the data is that the anti-Gia antibody prevents dissociation of the bg complex from the Gia protein, thus preventing interaction of the bg subunits with the K1 channel or intracellular pathways that modulate the K1 channel. Further studies have determined that the stimulatory effects of Ang II on IK(v) and IA in cultured neurons do not require cAMP, cGMP, PKC, IP3-sensitive Ca21 stores, phosphodiesterase enzymes, or phosphotyrosine phosphatase enzymes [56]. However, the Ang II–stimulated increase in IK(v) was abolished by the selective PP-2A inhibitors okadaic acid (OA; [56]) and nodularin (unpublished observations) and by intracellular application of anti-PP-2A antibodies [56] but not by the inactive OA analogue norokadaone [56]. At the single-channel level, OA (3 nM) abolished the stimulatory actions of Ang II on a neuronal K(v)-like channel (unpublished observations), providing further evidence for the importance of PP-2A in this response. Support for a role of Gi and of PP-2A in the AT2 receptor–mediated stimulation of neuronal IK(v) has come from experiments with a synthetic 22 amino acid peptide that corresponds to the third cytoplasmic loop of the AT2 receptor (PEP-22; AT2/i3) [108]. Similar to the effects of Ang II, intracellular injection of AT2/i3 caused a stimulation of neuronal IK(v) that was abolished by intracellular application of anti-Gia antibodies or by pre-treatment of neurons with OA [108]. Taken together, these data suggest that the AT2 receptor–mediated stimulation of neuronal IK(v) requires a Gi protein and PP-2A. Furthermore, the third cytoplasmic loop of this receptor appears to be critical for transducing the effects of Ang II on neuronal IK(v). As stated earlier, Ang II elicits an AT2 receptor–mediated stimulation of PLA2 activity in cultured neurons and other cells [91–93]. AA and AA metabolites, which are formed as a result of PLA2 activation, are known modulators of K1 channels in neurons and other cells [101–107]. In addition, a recent study has suggested that lipoxygenase (LO) metabolites of AA can link pertussis toxin–sensitive G proteins to serine/threonine phosphatases that regulate K1 channel activity in pituitary tumour cells [109]. A similar scenario exists for the calcium-dependent modulation of K1

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current in bullfrog sympathetic neurons [100]. In cultured neurons, our studies have shown that the stimulation of neuronal IK(v) by Ang II through AT2 receptors is partly (z 75%) blocked by the PLA2 inhibitors 4-BPB (10 mM) or anti-flammin-2 (20 mM) or by intracellular application of anti-PLA2 antibodies (1:1000) [93]. Furthermore, the effects of Ang II on neuronal IK(v) are mimicked by AA (10– 100 mM) [93]. The effects of both Ang II and AA on neuronal IK(v) are inhibited by inhibition either of LO enzymes or of PP-2A but not by inhibition of cyclooxygenase (CO) enzymes, which yield prostaglandins from AA (unpublished observations). These data lend support to the idea that stimulation of IK(v) by Ang II requires AA and LO metabolites of AA (such as leukotrienes or 12-HETE), and the proposed pathways are summarised as a diagram in Fig. 2. The fact that inhibition of PP-2A completely blocks the stimulation of IK(v) by AA suggests that PP-2A may be a final event in the pathway that causes stimulation of IK(v) and K(v) channel activity. In general terms, the preceding findings demonstrate that the stimulation of neuronal IK(v) by Ang II through AT2 receptors involves an indirect pathway (i.e., through intracellular messengers). At this time, the involvement of a direct membrane-delimited pathway (with PP-2A as a final event; Fig. 2) cannot be excluded, because inhibition of PLA2 causes only z 75% inhibition of Ang II’s effects. In addition, the involvement of other metabolites of AA, such as epoxygenase metabolites, cannot be excluded. The preceding discussion has centred on IK(v), because the signalling pathways involved in AT2 receptor– modulated IA have not been defined. However, IA was measured in many of the afore-cited experiments on IK(v) (the same recording conditions can be used for each current) and exhibited many of the same changes in response to the various treatments. Thus, it is likely that Ang II–stimulated IA also involves PLA2 and AA (Fig. 1). In cultured neurons, stimulation of AT2 receptors does not modulate Ca21 current (ICa). However, in non-differentiated NG108-15 neuroblastoma x glioma cells, AT2 receptor stimulation caused a decrease in T-type Ca21 current [58]. Consistent with the proposed signalling pathways in this cell type, the AT2 receptor modulation of T-type Ca21 current required activation of a phosphotyrosine phosphatase rather than PP-2A [58]. Thus, with the information available at present, it appears that the ionic currents that are modulated by AT2 receptor activation, as well as the signalling pathways taking part in this modulation, are unique to the cell type. SUMMARY In the preceding sections, we have considered the intracellular signalling pathways that couple AT1 and AT2 receptors to their ion channel effectors in neurons. It is apparent that each sub-type of the Ang II receptor utilises a complex intracellular signalling cascade for ultimate modulation of K1 and Ca21 currents. The signalling pathways are very different. For example, the AT1 receptor–mediated decreases

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FIGURE 2. Summary of the in-

tracellular signalling pathways in the AT2 receptor–mediated stimulation of neuronal IK(v) and IA. PLA2, phospholipase A2; PP-2A, serine/threonine phosphatase type 2A; LO, lipoxygenase; HPETE, hydroperoxyeicosatetranoic acid; HETE, hydroxyeicosatetranoic acid. Solid arrows represent stimulatory pathways for which there is support from published (see refs. [99–107]) or preliminary (see text) studies. Dotted arrows are putative pathways.

in neuronal IK(v) involve Ca21-dependent pathways such as PKC and CAM kinase II, whereas AT2 receptor–mediated increases in IK(v) involve AA, AA metabolites, and PP-2A. The fact that these pathways are quite distinct is probably not surprising, because, in most cases, AT1 and AT2 receptors exist on different populations of neurons and mediate different functions. In a few cases, AT1 and AT2 receptors do exist within the same brain nucleus, and recent studies have shown that AT1 and AT2 receptors co-exist on a small population of cultured neurons [52]. In fact, stimulation of these AT1 and AT2 receptors on the same neuron leads to opposing actions on IK(v) [52]. However, there is no evidence that an interaction between the intracellular signalling pathways has a role in these opposite actions of Ang II. On the basis of our present understanding, we would speculate that the point of interaction may be at the level of the K1 channel. It is also apparent from the preceding discussion that each of the Ang II receptor sub-types modulates multiple signalling pathways in neurons. For example, aside from the pathways taking part in the modulation of K1 and Ca21 cur-

rents, AT1 receptors can stimulate messengers involved in neuromodulation and cell growth [69, 70, 75, 76], and AT2 receptors can modulate pathways involved in apoptosis and cell differentiation [37, 75]. Whether the short-term (ion current/channel) and “longer term” changes induced by Ang II are part of the same continuum of events has yet to be established. The intricacies of the signalling pathways involved in AT1 and AT2 receptor–modulated neuronal K1 and Ca21 currents also require further definition. The authors thank Jennifer Brock and Pia Jacobs for typing the manuscript. This work was supported by grants from the National Institutes of Health, NS-19441 and HL-49130 (CS), and HL-52189 (CHG), and from the Council for Tobacco Research (CHG).

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