Introduction

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Neuropeptide Y (NPY), a 36-amino acid residue peptide isolated initially from porcine brain (Tatemoto, 1982; Tatemoto et al., 1982), is widely distributed in the central and peripheral nervous systems. It has been demonstrated that NPY exerts a variety of biological effects in relation to feeding, memory, emotion, blood pressure, cardiac contractility, and intestinal secretion through multiple specific receptors (Wahlestedt and Reis, 1993; Balasubramaniam, 1997). Five distinct NPY receptors have been cloned up to date, all of which have been shown to belong to the superfamily of receptors coupled with guanine nucleotide-binding regulatory proteins (G proteins) with putative seven transmembrane hydrophobic domains (Michel et al., 1998). Indeed, it has been shown that most, if not all, NPY receptors are coupled to inhibition of adenylyl cyclase and/or modification of free intracellular Ca²⁺ concentrations through pertussis toxin [islet-activating protein (IAP)]-sensitive G proteins (Michel, 1991; Wahlestedt and Reis, 1993)

G proteins are a family of heterotrimer proteins composed of -, -, and -subunits. Activation of G proteins by agonist-receptor complexes facilitates the dissociation of GDP from the -subunits and the subsequent binding of GTP. These activated -subunits of the G proteins (_GTP) dissociate from -subunits to modulate the activity of second messenger-generating enzymes such as adenylyl cyclase and phospholipases. The -subunits have intrinsic high-affinity GTP-hydrolyzing activity, by which GTP on the -subunit is converted into GDP and inorganic orthophosphate. The GDP-bound -subunits (_GDP) recombine with -subunits to terminate the activation cycle of G proteins. This characteristic feature of the receptor-mediated G protein activation/deactivation processes provides with several experimental techniques with which to assess functional coupling between the receptors and the G proteins. The authors revealed that agonist-induced increase in high-affinity GTPase activity is available even in crude membrane preparations from discrete brain regions for the detection of the functional interaction between several receptors and their respective G proteins, especially when associated with adenylyl cyclase inhibition (Odagaki and Fuxe, 1997). Because NPY has been shown to elicit inhibition of adenylyl cyclase or reduction in cAMP accumulation in cortex (Westlind-Danielsson et al., 1987; Widdowson and Halaris, 1991; Widdowson et al., 1991; Karelson et al., 1995), hippocampus (Petrenko et al., 1987; Widdowson and Halaris, 1991; Karelson et al., 1995), striatum (Westlind-Danielsson et al., 1988), hypothalamus (Chance et al., 1989), and medulla oblongata (Harfstrand et al., 1987; Ny and Grundemar, 1997), it is likely feasible to detect the NPY-mediated high-affinity GTPase activity of the G proteins coupled with NPY receptors in brain membranes. To our knowledge, however, there has not been such a report. Most recently, functional activation of G proteins coupled with NPY receptor subtypes was reported in rat brain slices (Primus et al., 1998) by means of the application of in vitro autoradiography of agonist-induced [³⁵S]guanosine-5'-O-(3-thio)triphosphate ([³⁵S]GTPS) binding (Sim et al., 1995) to the activation of NPY receptor subtypes.

In addition to the conventional mode of G protein activation (i.e., a receptor-mediated process described earlier), several lines of evidence have indicated that a number of compounds, such as cationic amphiphilic peptides, activate G proteins directly in a receptor-independent manner (Mousli et al., 1990; Odagaki et al., 1998a). NPY and its C-terminal fragments have been shown to be capable of releasing histamine from mast cells (Grundemar and Hakanson, 1991; Shen et al., 1991; Grundemar et al., 1994; Emadi-Khiav et al., 1995; Mousli et al., 1995) and of activating purified G_i/o (Mousli et al., 1995) through a nonspecific and receptor-independent mechanism as for other cationic amphiphilic neuropeptides such as substance P and venom peptides such as mastoparan (Mousli et al., 1990). The possible implication of such a mechanism of action of NPY in its multiple biological effects has not been fully considered thus far.

Odagaki et al. (1997) have shown that high-affinity GTPase activity in rat brain membranes can be stimulated by mastoparan in a manner that is distinct from that for the receptor-mediated G protein activation (i.e., through direct and receptor-independent activation of IAP-sensitive G proteins). In the present study, the effects of NPY on high-affinity GTPase activity in rat cerebral cortical membranes were investigated to elucidate the involvement of NPY receptor-mediated as well as receptor-independent modes of action. The GABA_B receptor-mediated action on high-affinity GTPase activity was studied in parallel as a representative of receptor-dependent G protein activation.

	Experimental Procedures

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Membrane Preparation. Male Sprague-Dawley rats (200-250 g) were sacrificed by decapitation, and their brains were quickly removed. The dissected cerebral cortex was homogenized in 5 ml of ice-cold TED buffer (5 mM Tris · HCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.4) containing 10% (w/v) sucrose with a motor-driven Teflon/glass tissue grinder (20 strokes). All of the following centrifuge procedures were carried out at 0-4°C. Subsequent to the centrifugation of the homogenate at 1000g for 10 min, the supernatant was decanted to another centrifuge tube, whereas the pellet was vortexed in 5 ml of TED/sucrose buffer followed by another centrifugation at 1000g for 10 min. The combined supernatant was washed twice by centrifugation at 9000g for 20 min and resuspended in 10 ml of TED buffer. The suspension was kept on ice for 30 min followed by the final centrifugation at 35,000g for 10 min, and the resulting pellet was resuspended in 50 mM Tris · HCl buffer (pH 7.4) to produce the homogenate with a protein concentration ranging from 1.6 to 3.2 mg/ml. The homogenate was divided into 150-µl aliquots in plastic tubes, frozen quickly on fine-grained dry ice, and stored at 80°C until use.

Measurement of GTP Hydrolysis. GTP-hydrolyzing activity was assayed by measuring the radioactivity of the ³²P_i released from [-³²P]GTP derived from an enzymatic conversion of GTP to GDP and inorganic orthophosphate by the incubated membranes. The thawed membranes were diluted with 50 mM Tris · HCl buffer (pH 7.4), and 25-µl aliquots of the membranes corresponding to 4 to 8 µg of protein were incubated at 30°C for 15 min in the reaction mixture (final volume, 100 µl), which contained 50 mM Tris · HCl (pH 7.4), 0.3 µM [-³²P]GTP, 2 mM (or indicated concentrations of) MgCl₂, 0.5 mM ATP, 0.5 mM adenylylimidodiphosphate, 5 mM phosphocreatine, 50 U/ml creatine phosphokinase, 50 µg BSA, 0.1 mM EDTA, 0.2 mM EGTA, 0.2 mM dithiothreitol, 0.5 mM cAMP, 0.5 mM 3-isobutyl-1-methylxanthine, and 100 mM NaCl. The low-affinity GTPase activity was determined as the GTP hydrolysis in the presence of 100 µM unlabeled GTP, which was subtracted from the total activity to define the high-affinity GTPase activity. The enzyme reaction was terminated by transfer of the tubes to an ice bath followed by the addition of 500 µl of 20 mM phosphoric acid containing 5% (w/v) activated charcoal. The tubes were kept chilled for about 30 min and centrifuged at 13,000g for 10 min. An aliquot (200 µl) from the supernatant fraction was pipetted onto the solid scintillator (Ready Cap; Beckman, Fullerton, CA). After being dried overnight, the radioactivity (cpm) of each sample was counted for 5 min with a liquid scintillation spectrometer. The GTP-hydrolyzing activity was expressed as pmol of released ³²P_i/mg protein/15 min.

Data Analysis. All results were presented as the mean ± S.E. The increase in high-affinity GTPase activity elicited by -aminobutyric acid (GABA) and (±)-baclofen was analyzed by computer-assisted nonlinear regression software originally designed for enzyme reactions in accordance with the Michaelis-Menten equation to determine the maximal percent increase above basal value and the concentration eliciting the half-maximal effect (EC₅₀). Statistical analysis was performed by using Student's paired two-tailed t test with a value of P < .05 considered significant.

Materials. [-³²P]GTP (30 Ci/mmol) was purchased from DuPont NEN Research Products (Boston, MA). [Leu³¹,Pro³⁴]NPY (porcine), NPY(13-36) (porcine), desamido-NPY, and pancreatic polypeptide (PP; rat) were obtained from Phoenix Pharmaceuticals Inc. (Mountain View, CA). NPY, GABA, and all reagents for the GTPase assay were obtained from Sigma Chemical Co. (St. Louis, MO). (R)-N²-(Diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]argininamide (BIBP3226) was purchased from Peninsula Laboratories Inc. (Belmont, CA).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Stimulation of High-Affinity GTPase Activity through GABA_B Receptor Activation. Our previous studies on agonist-induced high-affinity GTPase activity in rat (Odagaki and Fuxe, 1995a,b) and human (Odagaki et al., 1998b) brain membranes have shown that receptor-mediated G protein activation is strictly dependent on the presence of Mg²⁺ in the assay medium regardless of the receptor subtypes involved. This is also the case with GABA_B receptor-mediated G protein activation in rat cerebral cortical membranes assessed by the increase in high-affinity GTPase activity elicited by the GABA_B receptor agonist (±)-baclofen. As demonstrated in Fig. 1, GTP-hydrolyzing activity was significantly augmented by the addition of 1 mM (±)-baclofen in the presence of MgCl₂ but not in the absence of MgCl₂. The increase in high-affinity GTPase activity induced by 1 mM (±)-baclofen was strictly dependent on the presence of millimolar concentrations of Mg²⁺ (Fig. 1, inset).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Effects of various concentrations of MgCl2 on the GTP hydrolysis in rat cerebral cortical membranes. The amount of 32Pi released from 0.3 µM [-32P]GTP during an incubation time of 15 min was determined in the absence (, ) and presence (, ) of 1 mM (±)-baclofen. The assay was performed in the absence and presence of 0.5, 2, 5, and 20 mM MgCl2. The activity in the presence of 100 µM GTP (, ) was subtracted from the total activity (, ) to define the high-affinity GTPase activity. Values are mean ± S.E. of four experiments carried out in duplicate. ***P < .001, analyzed by Student's paired two-tailed t test. Inset, effects of various concentrations of MgCl2 on the (±)-baclofen-stimulated high-affinity GTPase activity. The increase in high-affinity GTPase activity elicited by 1 mM (±)-baclofen was determined in the absence and presence of 0.5, 2, 5, and 20 mM MgCl2.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effects of (±)-baclofen and GABA on the high-affinity GTPase activity in rat cerebral cortical membranes. The percent increase in high-affinity GTPase activity by the addition of increasing concentrations of (±)-baclofen (, ) and GABA (, ) was determined in the absence (, ) and presence (, ) of 2 mM MgCl2. Values are mean ± S.E. of three or four experiments carried out in duplicate. The basal high-affinity GTPase activities were 344.6 ± 3.5 (n = 4) and 396.7 ± 11.3 (n = 7) pmol/mg protein/15 min in the absence and presence of 2 mM MgCl2, respectively.

Stimulation of High-Affinity GTPase Activity by NPY through Two Modes of Action. The effect of MgCl₂ concentrations on GTP hydrolysis in the absence and presence of 10 µM NPY in rat cerebral cortical membranes was investigated. As shown in Fig. 3, the addition of 10 µM NPY significantly augmented the GTP-hydrolyzing activity not only in the presence of MgCl₂ but, against our expectation, also in the absence of MgCl₂. When the increase in high-affinity GTPase activity elicited by 10 µM NPY was plotted as a function of the concentrations of MgCl₂, the NPY-sensitive high-affinity GTPase activity showed a bimodal pattern (Fig. 3, inset). Thus, the maximum augmentation of high-affinity GTPase activity by the addition of 10 µM NPY was seen in the presence of millimolar concentrations of MgCl₂, as well as in the absence of MgCl₂.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effects of various concentrations of MgCl2 on the GTP hydrolysis in rat cerebral cortical membranes. The amount of 32Pi released from 0.3 µM [-32P]GTP during an incubation time of 15 min was determined in the absence (, ) and presence (, ) of 10 µM NPY. The assay was performed in the absence and presence of 0.5, 2, 5, and 20 mM MgCl2. The activity in the presence of 100 µM GTP (, ) was subtracted from the total activity (, ) to define the high-affinity GTPase activity. Values are mean ± S.E. of four experiments carried out in duplicate. *P < .05, **P < .01, analyzed by Student's paired two-tailed t test. Inset, effects of various concentrations of MgCl2 on the NPY-stimulated high-affinity GTPase activity. The increase in high-affinity GTPase activity elicited by 10 µM NPY was determined in the absence and presence of 0.5, 2, 5, and 20 mM MgCl2.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effects of NPY on the high-affinity GTPase activity in rat cerebral cortical membranes. The percent increase in high-affinity GTPase activity by the addition of increasing concentrations of NPY was determined in the absence () and presence () of 2 mM MgCl2. Values are mean ± S.E. of four experiments carried out in duplicate. The basal high-affinity GTPase activities were 340.8 ± 9.7 (n = 4) and 436.0 ± 138.8 (n = 4) pmol/mg protein/15 min in the absence and presence of 2 mM MgCl2, respectively.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Effects of various concentrations of MgCl2 on the GTP hydrolysis in rat cerebral cortical membranes. The amount of 32Pi released from 0.3 µM [-32P]GTP during an incubation time of 15 min was determined in the absence (, ) and presence (, ) of 100 nM NPY. The assay was performed in the absence and presence of 0.5, 2, 5, and 20 mM MgCl2. The activity in the presence of 100 µM GTP (, ) was subtracted from the total activity (, ) to define the high-affinity GTPase activity. Values are mean ± S.E. of four experiments carried out in duplicate. *P < .05, **P < .01, analyzed by Student's paired two-tailed t test. Inset, effects of various concentrations of MgCl2 on the NPY-stimulated high-affinity GTPase activity. The increase in high-affinity GTPase activity elicited by 100 nM NPY was determined in the absence and presence of 0.5, 2, 5, and 20 mM MgCl2.

Effects of BIBP3226, a Selective NPY Y-1 Receptor Antagonist, on NPY-Stimulated High-Affinity GTPase Activity. The antagonistic effects of BIBP3226 on the NPY-stimulated high-affinity GTPase activity were investigated under different assay conditions (i.e., in the absence and presence of 2 mM MgCl₂). In the presence of 2 mM MgCl₂ (Fig. 6), high-affinity GTPase activity was stimulated by 9.6 ± 0.7% (n = 4) and 42.8 ± 0.9% (n = 4) by means of the addition of NPY at 100 nM and 10 µM, respectively. The increase in high-affinity GTPase activity by 100 nM NPY was almost completely inhibited by the addition of BIBP3226 at 1 and 10 µM (by 83.4 ± 2.6 and 92.8 ± 7.0%, respectively). On the other hand, the increase in the activity by 10 µM NPY was inhibited by BIBP3226 in a concentration-dependent manner but partially even in the presence of the highest concentration (10 µM) of BIBP3226. BIBP3226 at 1 and 10 µM inhibited the NPY (10 µM)-stimulated high-affinity GTPase activity by 34.1 ± 1.5 and 58.6 ± 3.9%, respectively. In the absence of MgCl₂, the effects of BIBP3226 on the NPY-induced stimulation of the high-affinity GTPase activity were determined only in the presence of 10 µM NPY, because submicromolar concentrations of NPY were unable to stimulate the activity in the absence of MgCl₂ (Fig. 4). As demonstrated in Fig. 7, 10 µM NPY stimulated high-affinity GTPase activity by 23.4 ± 2.6% (n = 4) even in the absence of MgCl₂. However, this increase was not inhibited at all by the addition of BIBP3226 at 1 and 10 µM.

View larger version (76K): [in this window] [in a new window]   Fig. 6.   Effects of BIBP3226 on the NPY-elicited high-affinity GTPase activity in rat cerebral cortical membranes in the presence of 2 mM MgCl2. The high-affinity GTPase activity was determined in the absence and presence of NPY (100 nM and 10 µM) and in the absence and presence of BIBP3226 (1 and 10 µM) in the presence of 2 mM MgCl2. Values are mean ± S.E. of four experiments carried out in duplicate, and expressed as percent of the respective basal activity in the absence of NPY and BIBP3226 (555.1 ± 38.2 pmol/mg protein/15 min).

View larger version (95K): [in this window] [in a new window]   Fig. 7.   Effects of BIBP3226 on the NPY-elicited high-affinity GTPase activity in rat cerebral cortical membranes in the absence of MgCl2. The high-affinity GTPase activity was determined in the absence and presence of NPY (10 µM) and in the absence and presence of BIBP3226 (1 and 10 µM) in the absence of MgCl2. Values are mean ± S.E. of four experiments carried out in duplicate and expressed as percent of the respective basal activity in the absence of NPY and BIBP3226 (445.3 ± 10.9 pmol/mg protein/15 min).

Effects of NPY-Related Peptides on High-Affinity GTPase Activity. The effects of four NPY-related compounds [i.e., porcine [Leu³¹,Pro³⁴]NPY, porcine NPY(13-36), desamido-NPY, and rat PP] on GTP-hydrolyzing activity were investigated in the presence of 2 mM MgCl₂ (Fig. 8). Of these four peptides, only [Leu³¹,Pro³⁴]NPY (porcine) was able to stimulate the activity at submicromolar concentrations. The secondary, or further, increase in the activity was elicited by micromolar concentrations of [Leu³¹,Pro³⁴]NPY (porcine). Rat PP stimulated the high-affinity GTPase activity only at concentrations higher than 1 µM and to a slight extent. Two other compounds, NPY(13-36) porcine and desamido-NPY were unable to stimulate the activity at least up to 0.3 and 1.0 µM, respectively. The effects of higher concentrations of these two peptides on the high-affinity GTPase activity were indeterminable because the low-affinity GTPase activity in the presence of 100 µM unlabeled GTP was nonspecifically inhibited by these compounds at higher concentrations.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Effects of NPY-related peptides on the high-affinity GTPase activity in rat cerebral cortical membranes. The percent increase in high-affinity GTPase activity by the addition of increasing concentrations of porcine [Leu31,Pro34]NPY (; n = 2), porcine NPY(13-36) (; n = 3), rat PP (; n = 2), and desamido-NPY (; n = 2) was determined in the presence of 2 mM MgCl2. The effects of NPY(13-36) (porcine) and desamido-NPY at concentrations higher than 0.3 and 1.0 µM, respectively, were indeterminable because of nonspecific inhibitory effects on the low-affinity GTPase activity in the presence of 100 µM unlabeled GTP. The mean values of two or three experiments carried out in duplicate are presented without error bars for a sake of clarity. The basal high-affinity GTPase activities were 344.4 ± 7.6 (n = 9) pmol/mg protein/15 min.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

In the present study, it was shown that the Mg²⁺ dependence was quite different between NPY-stimulated high-affinity GTPase activity and GABA_B receptor-mediated activity, representative of the conventional mode of G protein activation. Thus, the high-affinity GTPase activity stimulated by 10 µM NPY was not dependent on the existence of Mg²⁺ in the assay medium but was detectable even in the absence of MgCl₂. The significant revelation of the NPY-sensitive high-affinity GTPase activity even in the absence of Mg²⁺ reminds us of the same phenomenon observed in the previous study in which the wasp venom peptide mastoparan was used as a stimulant (Odagaki et al., 1997). Mastoparan has been shown to activate directly IAP-sensitive G proteins without the existence of a specific receptor for the peptide (Higashijima et al., 1988). Because it has been reported that NPY has the same physicochemical feature as cationic amphiphilic peptides like mastoparan (Mousli et al., 1990) and that NPY is indeed capable of directly activating the G_i/o or of inducing histamine release from mast cells in a receptor-independent manner (Grundemar and Hakanson, 1991; Shen et al., 1991; Grundemar et al., 1994; Emadi-Khiav et al., 1995; Mousli et al., 1995), the increase in high-affinity GTPase activity elicited by 10 µM NPY in the absence of Mg²⁺ was likely derived from the receptor-independent process.

The increase in high-affinity GTPase activity elicited by the lower concentration (100 nM) of NPY was strictly dependent on the existence of Mg²⁺, which indicates that the first phase of the biphasic pattern evoked by NPY in the presence of 2 mM MgCl₂ is probably derived from the G proteins activated through specific cell-surface receptors for NPY, as demonstrated for other cases of receptor-mediated G protein activation (e.g., the above-mentioned GABA_B receptor-coupled high-affinity GTPase activity). This NPY receptor subtype was defined as the NPY Y-1 receptor, because the stimulatory effect of 100 nM NPY was almost completely antagonized by the highly selective NPY Y-1 receptor blocker BIBP3226 (Rudolf et al., 1994). This conclusion is in good agreement with the previous studies on the distribution of the NPY receptor subtypes in the rat brain (Dumont et al., 1995, 1996), in which the predominant existence of the NPY Y-1 receptors was shown in the cerebral cortex. On the contrary, the increase in the high-affinity GTPase activity by 10 µM NPY in the absence of Mg²⁺ was at all not inhibited by BIBP3226. This failure of inhibition by BIBP3226 is compatible with the notion that the NPY-stimulated response in the absence of Mg²⁺ is mediated through a receptor-independent mechanism of action. The NPY (10 µM)-induced increase in high-affinity GTPase activity in the presence of 2 mM MgCl₂ was partially inhibited by BIBP3226, indicating that this response was composed of two distinct components: the NPY Y-1 receptor-mediated increase and probably the receptor-independent portion undisplaceable by the NPY receptor antagonist.

Under a physiological condition in which millimolar concentrations of MgCl₂ are present, the conventional NPY receptor-dependent mechanism dominates when the concentrations of NPY are lower than 1 µM, whereas the receptor-independent, direct G protein activation may become gradually prominent as the NPY concentrations are increased. However, the latter mode of action is unlikely to play a significant role, at least under physiological conditions, when considering the extremely high level of necessary concentrations of NPY. Nevertheless, the possibility of involvement of direct G protein activation by NPY in some pathological situations could not completely excluded, provided synaptic concentrations of NPY are dramatically altered by severely pathological conditions. It has been reported that plasma NPY concentrations are altered to a great extent according to sympathetic nerve activity (Wahlestedt and Reis, 1993).

Although the implication of direct activation of G proteins by high concentrations of NPY in physiological and/or pathological situations in vivo remains to be elucidated in future studies, the effects of high concentrations of NPY on the adenylyl cyclase activity are of great interest. Petrenko et al. (1987) reported that forskolin-stimulated adenylyl cyclase activity in rat hippocampal membranes was inhibited by NPY in a concentration-dependent manner with a concentration eliciting half-maximal inhibition of 73 nM, with a statement in the text that NPY at concentrations above 5 µM produced a second inhibitory phase that did not reach saturation at concentrations up to 20 µM and was not GTP dependent. By using biophysical techniques, McLean et al. (1990) showed that micromolar concentrations of NPY altered the membrane bilayer structure, to which they ascribed the inhibition of isoproterenol-stimulated cAMP accumulation by low micromolar concentrations of NPY in smooth muscle cells. Such membrane perturbation may lead to an alteration of membrane fluidity, which underlies the receptor-independent, direct activation of G proteins by high concentrations of NPY demonstrated in the present study. Although almost all other investigators did not test the effect of such high concentrations of NPY, the concentration-response curve for the inhibitory effect of NPY on isoproterenol-stimulated adenylyl cyclase activity in rat hypothalamic membranes appears to not show saturation even at 10 µM (Chance et al., 1989).

Recently, the activation of [³⁵S]GTPS binding in the rat brain by NPY was reported through the use of in vitro autoradiography (Primus et al., 1998). In their study, the increase in [³⁵S]GTPS binding by NPY Y-1 receptor activation was reported to predominate over that elicited by the activation of NPY Y-2 receptor in the frontal cortex. This result is consistent with the data of the present study indicating that the activation of G proteins elicited by lower concentrations of NPY is mediated mainly through NPY Y-1 receptor subtype. Although they did not mention the possibility of involvement of the receptor-independent effect of NPY, the requirement of high concentrations (1-10 µM) of NPY for the detection of NPY-elicited increase in [³⁵S]GTPS binding, as well as the apparent lack of saturability even at the highest concentration (10 µM), may suggest that the direct G protein activation mediated through nonreceptor mechanism underlies this phenomenon, as demonstrated in the NPY-stimulated high-affinity GTPase activity in rat cerebral cortical membranes in the present study.

The effects of four NPY-related peptides on the high-affinity GTPase activity are consistent with the argument described previously. Desamido-NPY, which has been shown to be devoid of an agonistic activity at NPY receptors (Wahlestedt et al., 1990), does not stimulate the high-affinity GTPase activity at least up to 1 µM, which is in good contrast with the stimulatory effect of submicromolar NPY. The high-affinity GTPase activity was stimulated by submicromolar concentrations of porcine [Leu³¹,Pro³⁴]NPY (an agonist at NPY Y-1, Y-4, and Y-5 receptors; Gerald et al., 1996) but not by porcine NPY(13-36) (a Y-2 and Y-5 agonist; Gerald et al., 1996) or rat PP (a selective and potent Y-4 agonist; Gerald et al., 1996), indicating that only the NPY Y-1 receptor subtype was involved in the stimulatory effects of submicromolar concentrations of NPY. The higher concentrations of rat PP stimulated the high-affinity GTPase activity but to a lesser extent compared with the effects of NPY and [Leu³¹,Pro³⁴]NPY (porcine). This increase likely derives from the receptor-independent activation of G proteins, because PP has been shown to evoke histamine release, slightly but substantially, from mast cells (Grundemar and Hakanson, 1991). The possibility of involvement of NPY Y-4 or Y-5 receptor subtype is also excluded from the point of view of receptor distribution. The Y-4 receptor mRNA is abundantly expressed in testis and lung but faintly in the brain (Lundell et al., 1996), and [¹²⁵I]PP (human) binding sites are detectable only in hypothalamus and brainstem, not in cortex, in the rat brain (Trinh et al., 1996). The NPY Y-5 receptor is a "feeding" receptor that is involved in the regulation of food intake, which is localized mainly in hypothalamic nuclei and undetectable in cortical region except for cingulate cortex (Gerald et al., 1996). The increase in [³⁵S]GTPS binding coupled with NPY Y-5 receptor subtype was also undetectable in rat frontal cortex (Primus et al., 1998).

In conclusion, the data of the present investigation regarding the NPY-elicited increase in high-affinity GTPase activity in the rat cerebral cortical membranes indicate that there are two distinct mechanisms of action underlying the activation of G proteins by NPY according to the concentrations of NPY used. In the presence of low concentrations of NPY, the conventional receptor-mediated mechanism is mainly used to activate G proteins through NPY Y-1 receptor subtype in the rat cerebral cortex. When the concentrations of NPY are increased, the secondary unconventional mechanism for G protein activation is driven in a receptor-independent manner in addition to the receptor-mediated mode of action. Although the implication of such receptor-independent mode of action for G protein activation induced by high concentrations of NPY for the in vivo function of the central nervous system remains to be elucidated, this atypical G protein activation by NPY might account for the complicated, and occasionally paradoxical, experimental results with regard to a variety of biological effects of NPY in conjunction with the existence of multiple NPY receptor subtypes.