Introduction

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Neuropeptide-Y (NPY) is a neurotransmitter that can mediate numerous acute effects in the brain and many peripheral tissues, including stimulation of food intake, inhibition of anxiety and of neurotransmitter and insulin release, regulation of cardiovascular and renal function, gut motility, and gastrointestinal and renal epithelial secretion (Wahlestedt and Reis, 1993). All of these effects occur via a family of specific G protein-coupled receptors that includes at least five members (Michel et al., 1998). All known subtypes act via pertussis toxin-sensitive G proteins to inhibit adenylyl cyclase. In some cell types, additional proximal signaling mechanisms include activation of phospholipase C, mobilization of Ca²⁺ from intracellular stores, and modulation of Ca²⁺ and K⁺ channels, but this has not been linked to a specific receptor subtype.

In addition to these acute effects, NPY can also stimulate cellular growth and cause, for example, hypertrophy of cardiomyocytes (Millar et al., 1994) or proliferation of vascular smooth muscle cells (Shigeri and Fujimoto, 1993; Zukowska-Grojec et al., 1993; Erlinge et al., 1994), renal tubular cells (Voisin et al., 1996), or colonic lamina propria lymphocytes (Elitsur et al., 1994). However, the underlying mechanisms of growth-promoting effects of NPY have not been investigated in detail. Based on analogy to other receptor systems, potential candidates for the mediation of such growth effects include signaling pathways such as phospholipase D (Boarder, 1994), protein kinase C (PKC) (Whitman and Cantley, 1988), and the mitogen-activated protein kinases (MAPK) (Post and Brown, 1996). The MAPK are a family of protein kinases that includes the extracellular signal-regulated kinases (ERK), the jun N-terminal kinases (JNK), and the p38 MAPK; they are believed to be important in the regulation of cellular growth (Post and Brown, 1996; van Biesen et al., 1996; Neary, 1997). Whereas it was originally thought that MAPK are primarily activated in response to growth factors, it is now well established that they can also be activated by G protein-coupled receptors. Depending on the cell type under investigation, MAPK activation has been reported by receptors coupling via G proteins of the G_q/11, the G_i/o, and the G_s family, with each family using a distinct pathway of MAPK activation (van Biesen et al., 1996). MAPK can also be activated in response to PKC stimulation, and this may contribute to their activation by G_q/11-coupled receptors (Post and Brown, 1996; van Biesen et al., 1996). Whether PKC is also involved in the MAPK activation by receptors coupling to the pertussis toxin-sensitive G_i/o is controversial (van Biesen et al., 1996). However, it should be noted that most of this information has been generated based on cloned and heterologously expressed receptors, which may not fully represent the physiological setting with endogenously expressed receptors. Thus, much less is known mechanistically about the regulation of MAPK by endogenously expressed receptors in general and NPY receptors in particular.

Three studies have reported ERK activation by cloned Y₁ receptors transfected into Chinese hamster ovary (CHO) cells (Nakamura et al., 1995; Mannon and Raymond, 1998; Nie and Selbie, 1998), and one study suggests that this may involve intermediary PKC activation (Mannon and Raymond, 1998). ERK activation by NPY has also been reported for primary cultures of rat coronary endothelial cells, but the underlying mechanisms have not been described (Zukowska-Grojec et al., 1998). However, to the best of our knowledge, a possible activation of phospholipase D, PKC, JNK, and p38 MAPK and mechanisms of ERK stimulation by endogenously expressed NPY receptors have not been investigated. Therefore, we have studied whether endogenously expressed NPY receptors can activate these signaling pathways and how this might occur, specifically whether intermediate PKC activation is involved in ERK stimulation. For this purpose, we have chosen human erythroleukemia (HEL) cells as a model system, because these cells are one of the best-investigated cell lines for studies of NPY receptor signaling (Motulsky and Michel, 1988; Feth et al., 1992; Michel, 1994, 1998; Michel et al., 1996).

	Experimental Procedures

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Cell Culture. HEL cells were originally obtained from Dr. T. Papayannopoulou (Department of Medicine, University of Washington, Seattle, WA). They were grown in RPMI 1640 medium supplemented with 1% L-glutamine, 1% penicillin/streptomycin, and 10% fetal calf serum. The cells were maintained in an atmosphere of 95% air and 5% CO₂ at 37°C. Before the experiments, the cells were cultured in medium without serum for 20 to 24 h. In some experiments, pertussis toxin (100 ng ml¹) was added to the serum-free medium.

Phospholipase D Assay. Phospholipase D activity was determined as phosphatidylethanol formation in the presence of ethanol, as previously described (Schmidt et al., 1998). Cellular phospholipids were labeled by incubation of cells for 20 to 24 h with [³H]oleic acid (2-2.5 µCi ml¹) in medium. After the labeling medium was replaced, the cells were equilibrated for 10 min at 37°C in Hanks' balanced salt solution (118 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 5 mM D-glucose, and 15 mM HEPES at pH 7.4). Thereafter, medium with or without agonist was added in the presence or absence of ethanol (400 mM) for 60 min. The reactions were stopped by addition of ice-cold methanol. Lipid extracts of the cells were separated on silica gel 60 plates (LK6D, Whatman, Clifton, NJ) using a mixture of ethyl acetate/2,2,4-trimethylpentane/acetic acid/water (13:2:3:10 volume ratio) as the mobile phase. Lipids were localized by iodine staining and identified by migration standards. The areas corresponding to the phosphatidylethanol standard were scraped into scintillation vials and counted with an efficacy of approximately 40%. All experiments were performed in triplicate. The formation of phosphatidylethanol was expressed relative to the total radioactivity in the phospholipid fraction.

PKC Translocation. HEL cells were collected by centrifugation (10 min, 400g) and washed into fresh RPMI 1640 medium. The cells (2-5 × 10⁶ cells) were stimulated in a total volume of 0.5 ml for the indicated times with NPY or phorbol-12-myristate-13-acetate (PMA). The reactions were stopped by adding 1.5 ml of ice-cold RPMI 1640. The samples were centrifuged for 5 min at 400g, and the supernatant was removed. The cells were lysed in 1 ml of buffer A (20 mM Tris, 2 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 50 µg ml¹ soybean trypsin inhibitor, 10 µM pepstatin A, 10 µM leupeptin, and 2 µg ml¹ aprotinin at pH 7.4) by repeated freeze thawing (three times) and thereafter centrifuged for 10 min at 12,000g. The supernatant was collected and represented the cytosolic fraction. The pellets were resuspended in 250 µl of buffer A supplemented with 1% Triton X-100, and membrane extracts were prepared by sonification (2 × 15 s). The samples were centrifuged for 10 min at 12,000g, and the supernatant represented the membrane fraction.

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MAPK Activity. Because activation of MAPK requires its tyrosine phosphorylation (Neary, 1997), we have assessed it by immunoblot detection of tyrosine-phosphorylated MAPK isoforms unless otherwise indicated. HEL cells were centrifuged for 10 min at 400g and resuspended in buffer (120 mM NaCl, 20 mM HEPES, 5 mM KH₂PO₄, 1 mM MgCl₂, 1 mM CaCl₂, 5.55 mM glucose, pH 7.4). The cells were incubated for 1 h at 37°C. Following incubation of aliquots of the cell suspension (10⁶ cells) for another 15 min in microfuge tubes, the indicated concentrations of agonist and/or inhibitor were added to yield a total volume of 250 µl. The incubations were stopped after 3 min, unless otherwise indicated, by centrifugation for 5 min at 14,000g.

Materials. [-³²P]ATP (3000 Ci mmol¹) and [³H]oleic acid (10 Ci mmol¹) were obtained from New England Nuclear (Bad Homburg, Germany). [S²⁵]PKC_19-31 was obtained from Serva (Heidelberg, Germany), NPY was obtained from Bachem (Bubendorf, Switzerland), PD 98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] was obtained from New England Biolabs (Beverly, MA), and pertussis toxin was obtained from List Biological Laboratories (Vendell, CA). Aprotinin, -glycero-phosphate, diolein, dithiothreitol, leupeptin, myelin basic protein, PMA, phenylmethylsulfonylfluoride, phosphatidylserine, soybean trypsin inhibitor, thrombin, Triton X-100, and wortmannin were obtained from Sigma (Deisenhofen, Germany). The PKC inhibitors bisindolylmaleimide I (also known as GF 109203X or Gö 6850), calphostin C, Gö 6976 (12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]-carbazole), staurosporine, and the inactive control bisindolylmaleimide V were obtained from Calbiochem (Bad Soden, Germany). BIBP 3226 ((R)-N²-diphenylacetyl-N-[(4-hydroxyphenyl)methyl]argininamide) was a kind gift of Dr. Karl Thomae AG (Biberach, Germany). All other chemicals were purchased from Merck (Darmstadt, Germany).

	Results

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Phospholipase D experiments. Incubation of HEL cells with up to 1 µM NPY for 60 min did not cause detectable activation of phospholipase D, whereas a positive control, the PKC-activating phorbol ester PMA (100 nM), enhanced phospholipase D activity 3 fold (Fig. 1).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Phospholipase D activation. HEL cells were incubated for 60 min in the absence (control) or presence of 1 µM NPY or 100 nM the PKC-activating phorbol ester PMA. Phospholipase D activity was assessed as phosphatidyl ethanol (PtdEtOH) formation in the presence of ethanol. Data are mean ± S.E. from a triplicate determination in a representative experiment; similar data were obtained in three other experiments. Data are expressed as PtdEtOH formation relative to the total radioactivity in the phospholipid fraction. **P < .01 versus control in a one-way ANOVA.

PKC experiments. Stimulation of HEL cells with 1 µM NPY for 1 to 10 min did not cause significant alterations of PKC activity in the membrane or cytosol fractions, whereas incubation with 100 nM PMA for 10 min induced a strong translocation of PKC activity from the cytosol to the membrane fraction (Fig. 2).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   PKC activation. HEL cells were incubated for the indicated times in the absence (control) and presence of 1 µM NPY or 100 nM the PKC-activating phorbol ester PMA. Thereafter, PKC activity was determined in the membrane and cytosolic fraction of these cells. Data are mean ± S.E. of four experiments and are expressed as units of PKC activity per mg of protein. *P < .05 versus control in a one-way ANOVA. NPY did not cause statistically significant alterations of PKC activity in either fraction at any time point.

MAPK Experiments. Addition of 100 nM NPY to HEL cells caused a rapid tyrosine-phosphorylation of the 42- and 44-kDa forms of ERK, which was detectable as early as 30 s after addition of NPY and was maintained for at least 20 min (Fig. 3). Therefore, all further experiments were performed with an incubation time of 3 min. The phosphorylation of the 42- and 44-kDa isoforms of ERK by NPY was concentration dependent (Fig. 4). The maximum activation was 150 to 200% over basal and occurred at NPY concentrations of 10 to 100 nM. Therefore, 100 nM NPY was used in all further experiments, unless otherwise noted. The ERK phosphorylation by 100 nM NPY was abolished by the Y₁-selective NPY receptor antagonist BIBP 3226 (1 µM, Fig. 4). When MAPK activation was determined enzymatically, incubation of HEL cells with 100 nM NPY for 3 min increased MAPK activity by 26 ± 13% relative to paired vehicle values (n = 7, P < .05, in a paired, two-tailed t test).

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Time course of NPY-stimulated ERK activation in HEL cells. Cells were incubated with 100 nM NPY for indicated times. A representative immunoblot of HEL cell lysates incubated with anti-phospho-ERK antibody (A) or anti-ERK antibody (B). C, mean ERK activation, expressed as a percentage of basal values, which was calculated from quantitative, two-dimensional densitometry of the immunoblots (see Experimental Procedures). Data are mean ± S.E. of four experiments. C, protein standard of phosphorylated ERK. B, basal value. *P and **P < .05 and < 0.01, respectively, versus control in a repeated measures ANOVA, followed by Dunnett's multiple comparison tests.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of ERK activation by NPY in HEL cells. Cells were stimulated for 3 min with indicated concentrations of NPY. Experiments with 100 nM NPY were also performed in the presence of 1 µM the Y1-selective NPY receptor antagonist BIBP 3226. Data are mean ± S.E. of six and four experiments in the absence and presence of BIBP 3226, respectively. **P < 0.01 versus control in a repeated measures one way ANOVA followed by Dunnett's multiple comparison tests. +++P < .001 in a paired two-tailed t test versus data in the absence of BIBP 3226.

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View larger version (27K): [in this window] [in a new window]   Fig. 5.   Inhibition of NPY-induced ERK activation. HEL cells were treated with or without 100 nM NPY for 3 min. Experiments were performed after pretreatment of HEL cells with pertussis toxin (100 ng ml1 PTX for 20 h) and in the presence of PD 98059 (100 µM) or wortmannin (100 nM) or without concomitant treatment (control). Data are mean ± S.E. of three to five experiments and are expressed as a percentage of basal values, i.e., those in the absence of NPY or inhibitors. NPY did not cause statistically significant stimulation of ERK in the presence of any of the inhibitors. *P < .05 versus data in the absence of inhibitor in a one-way ANOVA.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Effects of PKC inhibitors on ERK activation by NPY. Cells were treated for 3 min in the presence of 100 nM NPY and the additional presence of 3 µM staurosporine (stauro), 3 µM calphostin C (calpho), 3 µM Gö 6976, 3 µM bisindolylmaleimide I (Bis I), its negative control bisindolylmaleimide V (Bis V), or the respective vehicle. Data are mean ± S.E. of 6 to 17 experiments. **P < .01 relative to vehicle in a one-way ANOVA. Except for staurosporine, none of the PKC inhibitors caused statistically significant inhibition of ERK activation by NPY.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Time course of NPY effects on the activation of the 46- and 54-kDa forms of JNK (A) and of p38 MAPK (B) in HEL cells. Cells were incubated with 100 nM NPY for indicated times. Data are mean ± S.E. of five and three to four experiments for JNK and p38 MAPK, respectively.

	Discussion

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Signal transduction studies can be performed with tissues or cell lines endogenously expressing the receptor of interest or with cloned receptors transfected into suitable host cell lines. Both approaches have distinct advantages and disadvantages and complement each other. Thus, endogenously expressed receptors are "the real thing" and exist in a cellular environment suitable for their biological function; however, they have the disadvantages that receptor-intrinsic properties cannot easily be discriminated from those of the cellular environment and that weak signaling responses can be overlooked at physiological receptor expression levels. Cloned and transfected receptors can be expressed at greater than physiological densities, which may facilitate the detection of certain responses; on the other hand, artificially high expression levels may reveal promiscuous effector coupling that does not occur physiologically, specifically when the receptors are expressed in a cell type that does not physiologically contain these receptors and thus may quantitatively and/or qualitatively lack the appropriate signaling machinery for this receptor. In this context, it should be noted that previous studies on the coupling of cloned NPY receptors to ERK activation have used clones of CHO cells that express ¹²⁵I-labeled NPY or ¹²⁵I-labeled peptide YY binding sites at densities of 300 fmol/mg protein (Nie and Selbie, 1998), >200 fmol/10⁶ cells (Nakamura et al., 1995), or 73,000 sites/cell (Mannon and Raymond, 1998). On the other hand, the density of NPY receptors in HEL cells is 73 fmol/10⁶ cells, corresponding to 44,000 sites/cell (Feth et al., 1992).

HEL cells are a well-established model system to study the signaling properties of NPY receptors. In these cells, NPY acts on Y₁ NPY receptors to stimulate pertussis toxin-sensitive G proteins, i.e., G_i2 and G_i3, to inhibit adenylyl cyclase and mobilize Ca²⁺ from intracellular stores (Motulsky and Michel, 1988; Feth et al., 1992; Michel, 1994, 1998). Although some investigators (Daniels et al., 1989) have additionally observed a small stimulatory effect on inositol phosphate formation, we did not detect this in our HEL cells (Motulsky and Michel, 1988). Because these proximal signaling events may not fully explain the reported growth-promoting effects of NPY (see the introduction), the present study has investigated a number of signaling events that have been implicated in the regulation of cellular growth processes such as phospholipase D (Boarder, 1994); PKC (Whitman and Cantley, 1988); and the ERK, JNK, and p38 forms of MAPK (Post and Brown, 1996).

According to our experiments, a receptor-saturating concentration of NPY (Feth et al., 1992), which is maximally effective for other NPY signaling responses in HEL cells (Motulsky and Michel, 1988; Michel et al., 1990, 1992; Feth et al., 1992), fails to activate phospholipase D in HEL cells. Cloned Y₁ NPY receptors expressed in CHO cells were reported not to activate phospholipase A₂ (Selbie et al., 1995). Similarly, NPY causes only little if any phospholipase C activation in HEL cells (Motulsky and Michel, 1988; Daniels et al., 1989), and cloned NPY receptors also do not mediate phospholipase C stimulation at early time points (Selbie et al., 1995). In the present study, the same NPY concentration did not activate PKC, and similar findings have been reported for cloned NPY receptors based on different methods (Selbie et al., 1995). Taken together, these data indicate that phospholipases A₂, C, and D and PKC do not constitute a major signaling pathway for NPY receptors. Therefore, most of our study focused on activation of MAPK by NPY.

In the time frame from 30 s to 20 min, NPY did not activate the 46- or 54-kDa form of JNK or p38 MAPK, but it significantly stimulated ERK. Although most of our ERK data rely on quantitative immunoblotting, its activation by NPY was confirmed qualitatively by an enzymatic assay (Kribben et al., 1997). The quantitative differences between the two methods may involve some ERK-independent basal phosphorylation of the enzyme substrate in vitro and/or the unknown balance between protein kinases and phosphatases inside the cell, which determines the phosphorylation state measured by immunoblotting.

In the immunoblotting assay, the ERK activation occurred rapidly, i.e., within 30 s, and was maintained for at least 20 min. Although cloned Y₁ receptors expressed in CHO cells were also reported to cause maximal ERK activation at the earliest measured time point (i.e., after 1 min), that activation abated with time, and less than 20% of the original activation was detectable after 10 to 20 min (Mannon and Raymond, 1998). Although our present concentration-response data do not allow a formal calculation of EC₅₀ values, they are consistent with the previously established concentration-response relationships for NPY-induced inhibition of cAMP accumulation and for Ca²⁺ elevation in HEL cells (Motulsky and Michel, 1988, 1994, 1998; Feth et al., 1992).

The ERK activation by NPY occurred via a Y₁ receptor and, as described for cloned Y₁ NPY receptors expressed in CHO cells (Nakamura et al., 1995; Mannon and Raymond, 1998; Nie and Selbie, 1998), via a pertussis toxin-sensitive G protein. This pattern is similar to that for adenylyl cyclase inhibition and Ca²⁺ mobilization in HEL cells (Motulsky and Michel, 1988; Michel et al., 1990; Feth et al., 1992; Michel, 1998). Nevertheless, these proximal signaling responses are unlikely to account for ERK activation. Previous data from our (Kribben et al., 1997) and other laboratories (van Biesen et al., 1996) show that ERK activation by receptors coupling to pertussis toxin-sensitive G proteins does not involve cAMP lowering. Because thrombin produced much less if any ERK activation in the present study, although it causes similar Ca²⁺ elevations in HEL cells as NPY (Motulsky and Michel, 1988; Michel et al., 1996; Michel, 1998), ERK activation by NPY is also unlikely to occur secondary to Ca²⁺ increases. We rather speculate that a direct effect of G protein -subunits may be involved, as has been demonstrated for other G_i/o-coupled receptors (van Biesen et al., 1996).

MAPK activation is typically mediated by specific MAPK kinases, i.e., mitogen-activated protein/extracellular signal-related kinase kinase (MEK) in the case of ERK (Neary, 1997). Accordingly, the MEK inhibitor PD 98059 (Alessi et al., 1995) completely blocked NPY-induced ERK activation in HEL cells. ERK activation by several receptors coupling to pertussis toxin-sensitive G proteins appears to involve a phosphatidylinositol-3-kinase upstream of MEK (Post and Brown, 1996; van Biesen et al., 1996). In our study, wortmannin, which inhibits phosphatidylinositol-3-kinase (Ui et al., 1995), also blocked the ERK activation by NPY. Inhibition of NPY-stimulated ERK activation by PD 98059 and wortmannin has also been reported for cloned Y₁ NPY receptors expressed in CHO cells (Nakamura et al., 1995; Mannon and Raymond, 1998; Nie and Selbie, 1998). Interestingly, inhibition of NPY-induced ERK activation by pertussis toxin, PD 98059, and wortmannin has also been reported for cloned Y₂ NPY receptors expressed in CHO cells at a density of 800 fmol/mg protein (Nie and Selbie, 1998), indicating that this signaling mechanism may apply to multiple NPY receptor subtypes.

Whether ERK activation by receptors coupling to pertussis toxin-sensitive G proteins involves an intermediary PKC activation is controversial (van Biesen et al., 1996). Previous work with cloned NPY receptors expressed in CHO cells had suggested that PKC mediates ERK activation by NPY based on inhibitory effects of pretreatment with PMA and on the PKC inhibitors, Ro-31-8220 and sphingosine (Mannon and Raymond, 1998). In our study, the ERK activation by NPY was quantitatively similar to that achieved by the PKC-activating phorbol ester PMA, and the PKC inhibitor staurosporine reduced basal and NPY-stimulated ERK activation. However, many PKC inhibitors also have effects unrelated to PKC activation (Rüegg and Burgess, 1989). Accordingly, three other chemically distinct PKC inhibitors and a negative control, bisindolylmaleimide V, did not affect NPY-stimulated ERK activation. This finding is in good agreement with the lack of PKC activation by NPY receptors in HEL cells. Taken together, these data suggest that NPY-induced ERK activation in HEL cells does not involve intermediary PKC stimulation. Three factors should be considered to understand the discrepancy between our findings with HEL cells and those with cloned NPY receptors in CHO cells (Mannon and Raymond, 1998). First, it remains unclear how PKC can mediate ERK activation by NPY in CHO cells, since NPY receptor activation fails to activate PKC in these cells (Selbie et al., 1995). Second, it should be considered that CHO cells may lack the correct machinery and/or stochiometric ratio of its components to yield a cellular response similar to that with endogeneously expressed receptors. Third, the study proposing a role for PKC in NPY-induced ERK activation used an expression density of cloned receptors that was almost twice as high as that in HEL cells (Feth et al., 1992; Mannon and Raymond, 1998) and that may facilitate promiscuous coupling to pathways not activated by endogenous receptors.

In this context it is interesting to look at studies on other receptors coupling to ERK activation via G_i proteins. Some studies reporting a role for PKC in this coupling have relied on staurosporine (Kranenburg et al., 1997). On the other hand, studies using PKC down-regulation by extended pretreatment with phorbol ester (Cowen et al., 1996; Kribben et al., 1997) or using PKC inhibitors other than staurosporine (Bouloumie et al., 1994) have often failed to detect a role for PKC in ERK activation. When ERK activation occurs concomitantly via both pertussis toxin-sensitive and -insensitive pathways, e.g., with endothelin receptors in rat astrocytes (Kasuya et al., 1994) or bovine tracheal smooth muscle cells (Malarkey et al., 1995), the pertussis toxin-insensitive part appears to be PKC mediated, whereas the pertussis toxin-sensitive part is not. On the basis of these data, we suggest that the activation of ERK by G_i-coupled receptors does not routinely involve a PKC. The strong inhibitory effect of staurosporine on ERK activation in HEL cells may be related to ancillary properties of this compound (Kubbies et al., 1989; Rüegg and Burgess, 1989; Jalava et al., 1993; Kleinschroth et al., 1993) rather than its inhibitory effects on PKC, and staurosporine may not be a useful tool to study PKC involvement in ERK activation.

Because ERK activation frequently results in enhanced cellular growth (Post and Brown, 1996), it would have been interesting to study the effects of ERK activation by NPY on cellular growth. However, this is difficult to study in HEL cells because these cells continue to grow at a considerable rate even in the absence of serum and known growth factors. Although we were unable to detect NPY-stimulated enhancements of [³H]thymidine incorporation in HEL cells (data not shown), these data are difficult to interpret due to the high basal proliferation rate of our cells. Other cell types may be more appropriate for the study of functional consequences of ERK stimulation by NPY receptors.

In summary, our study demonstrates that NPY activates the ERK but not the JNK or p38 subfamily of MAPK in HEL cells. This activation occurs via Y₁ receptors and a G_i protein and involves a phosphatidylinositol-3-kinase and the MEK form of MAPK kinase but not PKC. The exact place of phosphatidylinositol-3-kinase in the cascade between the G_i protein and MEK remains to be determined. Similar to other G_i-coupled receptors, the -subunits of the G protein may be a proximal part of this cascade (van Biesen et al., 1996).