Introduction

Top Abstract Introduction Methods Results Discussion References

NPY, a 36-residue peptide abundant especially in the forebrain, is known to participate especially in regulation of vascular tone (Malmstrom, 1997), of feeding (e.g., Stanley et al., 1992) and of neuropeptide and anterior pituitary hormone secretion (Kalra and Crowley, 1992). At least six subtypes of NPY receptor have been described to date, showing a large degree of sequence variation (Larhammar, 1997). All of these peptides possess the seven hydrophobic folds characteristic of the rhodopsin family of G protein-linked receptors.

Among the known mammalian NPY receptors, the Y₁ species (including possible splicing variantssee Larhammar, 1997) apparently represents a large fraction of physiologically expressed NPY binding molecules, especially in the neural matrix. Activation of this receptor is known to stimulate the in vivo release of oxytocin (Parker and Crowley, 1993), and the release of LHRH from hypothalamic tissue (Kalra et al., 1992). Stimulation of the peripheral vascular Y₁ complement is vasotonic (Malmstrom, 1997) and diuretic (Bischoff et al., 1997).

Most of these activities could be coordinated through a multiple regulation of the Y₁ agonist signal by ions, transducers and effectors, which appears to have evolved both at the level of the Y₁ receptor structure and in the features of the agonistic peptides. Thus, agonist binding requires interaction with several extracellular and transmembrane domains of the Y₁ receptor molecule (for a review, see Du et al., 1997), resulting in a high sensitivity of the attachment to chaotropic influences, including those related to the ionic environment (Parker et al., 1996a). This is distinct from the binding to, for example, the Y₂ site, which shows little ion or chaotrope sensitivity (possibly due to accommodation of a long carboxyl-terminal primary binding epitope; Parker et al., 1996a, 1996b). The Y₁ receptor also appears to be easily down-regulated by agonistic peptides (Parker et al., 1996b), possibly in connection to a large susceptibility to secondary interactions with neighboring proteins. Regulatory interactions could also be facilitated by the noncontinuous localization of the binding epitopes on both termini of Y₁-active NPY analogue peptides (see Daniels et al., 1995).

Binding of agonists to many receptors coupled to G proteins is attenuated by high levels of guanosine polyphosphates, which force a change in the conformation of the nucleotide-binding site of the transducer molecule that in turn modifies the conformation of the attached receptor to decrease the affinity of ligand association (Mixon et al., 1995). Based on the existing reports using subtype-nonselective unmodified NPY analogs (Unden and Bartfai, 1984; Walker and Miller, 1988), association of agonist peptides with the brain Y₁ receptor should also be sensitive to G protein activators and inhibitors. The Y₁ receptor is also linked to activity of phosphoinositide-specific phospholipase C (Selbie et al., 1995) and could be involved in the activity of ion channel systems (Hastings et al., 1997). Although these classes of physiological regulators are generally presumed to act as effectors located apart from a receptor molecule proper, there is evidence of direct association of rhodopsin family receptors with phospholipase C (e.g., Aiyar et al., 1989; Biddlecome et al., 1996). Such interactions could result in perturbations sufficient to affect the highly sensitive agonist binding to the Y₁ receptor. Physical engagement of metabolic effectors could facilitate the regulation of activity, especially for metabotropic peptide receptors triggering cascade events, including the Y₁ receptor. Indeed, it is known that at least G_q-associating receptors would extensively interact with the phospholipase C effectors that serve as physiological amplifiers of the GTPase activity of G-protein (Biddlecome et al., 1996). However, the influence of such association on ligand-binding parameters would be expected to vary considerably among G protein-linked receptors, depending on organization of binding sites and properties of the agonists involved.

Phospholipase inhibitors, especially those of phospholipase A₂, are known to influence ligand-binding activity of both G protein-associating receptors and ion channel receptors. Thus, quinacrine is known to alter the affinity of cholinergic receptors (e.g., O'Donnell and Howlett, 1991). Ion channel-active drugs are also known to cross-react in the binding of ligands to several classes of receptors, as shown for nicotinic receptors with Ca⁺⁺ channel blockers (Siegel and Lukas, 1986). It is therefore of interest to also examine the possible interactions of these antagonists with NPY-binding sites.

In this report, we show that ligand association with the Y₁ receptor in four areas of the rat brain is strongly and uniformly sensitive to guanosine polyphosphates or to inhibitors of phospholipase C, much less sensitive to inhibitors of other phospholipase groups and insensitive to antagonists of several ion channels.

	Methods

Top Abstract Introduction Methods Results Discussion References

Chemicals. LP-PYY, hPYY(3-36) and hPP were purchased from Bachem California (Los Angeles, CA). pPYY and rPP were purchased from Peninsula Laboratories (Los Angeles, CA). Other chemicals were purchased from either Sigma Chemical (St. Louis, MO) or Calbiochem (La Jolla, CA). BIBP-3226, a selective Y₁ receptor antagonist, was a gift from Dr. Karl Thomae GmbH (Biberach, Germany).

Phospholipase assays. These assays were performed similar to the procedure of Claro et al. (1989). Briefly, the particles were resuspended in Tris-maleate buffer (20 mM; pH 7.0) containing 10 mM LiCl, 2 mM sodium cholate, 6 mM MgCl₂, 3 mM EGTA (neutralized to pH 7.0 with NaOH) and 1 mM CaCl₂ (corresponding to ~100 nM free Ca⁺⁺ as estimated by the procedure of Raaflaub, 1960), at 0.5 mg of particle protein/ml and 10 µM [³H]phosphatidylinositol-4,5-bisphosphate [inositol-2,-3³H(N); DuPont-NEN, Cambridge, MA] and after the addition of 100 µM of inhibitors incubated for 20 min at 24°C, in a total volume of 0.2 ml. The reaction was stopped by adding 0.8 ml of 2:1:0.4 (v/v) mixture of chloroform, isopropanol and 1 N HCl and 0.25 ml of 1 N HCl/4 mM EGTA and shaking. After centrifugation for 5 min at 6000 × g_max, 0.5 ml of the upper phase was mixed with 20 volumes of a liquid scintillation medium and counted in a Beckman (Palo Alto, CA) model LS 3801 liquid scintillation counter.

Iodinated peptides. [¹²⁵I](Leu³¹,Pro³⁴)hPYY, hPYY(3-36) and hPP were either purchased from NEN (Cambridge, MA), or iodinated as described (Parker et al., 1996a). The commercial peptides, monoiodinated by the chloramine-T procedure, had specific activities close to the theoretical (2170 Ci/mmol) and were labeled mainly in the carboxyl-terminal tyrosine residue (75-90%, as ascertained by exhaustive tryptic digestion followed by Bio-Gel P-4 chromatography; Parker et al., 1996a) or by high-performance liquid chromatography using the procedures of Walker and Miller (1988). Full-length PYY analogs iodinated in our laboratory had specific activities in excess of 1000 Ci/mmol and contained both carboxyl-terminally and amino-terminally labeled peptides. No important affinity differences were observed in the binding of these ligands relative to carboxyl-terminally monoiodinated peptides. All of the assay paradigms were tested at least once with commercial iodinated peptides. No significant differences were noted for either Y₁ or Y₂ site-selective tracers. The iodinated peptides were stored at 60°C. Preservation of NPY/PYY analogs over assay incubations was evaluated by Bio-Gel P-4 chromatography (Parker et al., 1996a). Routinely, <3% of the input of [¹²⁵I]-labeled NPY or PYY derivatives was fragmented over the assay incubation, and no degraded peptides could be detected in particle-bound tracers by either gel filtration or high-performance liquid chromatography.

Tissue preparation. Rat brains were rapidly excised and frozen in dry ice before slicing with a cryomicrotome to obtain 0.3-mm-thick coronal sections. All stereotaxic coordinates listed below refer to the atlas of Paxinos and Watson (1986). Tissue was excised from the following areas: PAR1, the parietal cortex area 1, ~1 mm deep and 4 mm long at 0.6 to 3 mm behind bregma; PIR, the piriform cortex area, ~1 mm deep and 3 mm long at 0.6 to 3 mm behind bregma; AHA, the anterior hypothalamic area, trapezoidal cuts 1 to 1.5 mm deep on either side of the third ventricle, 0.8 to 2.0 mm behind bregma; and HIPP, the anterior hippocampal area (mainly the CA1-CA3 zones), triangular cuts (side length ~1.5 mm) taken 1.8 to 3 mm behind bregma.

Particle isolation. This was accomplished as described (Parker et al., 1996a). The particles were stored at 60°C.

Ligand-binding assays. The assay buffer and most of the conditions were similar to those described previously (Parker et al., 1996a). The assay buffer contained 10% sucrose, 20 mM HEPES-NaOH (pH 7.4), 0.25 mg/ml bacitracin, 10 µg/ml each of leupeptin, pepstatin, aprotinin, chymostatin and antipain, 0.5 mM each of phenylmethylsulfonyl fluoride, benzamidine and diisopropylfluorophosphate and 2 mg/ml of proteinase-free bovine serum albumin. Before all assays, the particulates were resedimented once (for 10 min at 6000 × g_max; Eppendorf 5413 centrifuge; Brinkmann, Westbury, NJ, operated at 5°C) from the assay buffer. The assay volume was 0.4 ml. The assays were incubated for 100 min at 24°C, using 25 µg/ml of particulate protein (as measured by the Coomassie Brilliant Blue procedure). The [¹²⁵I]-labeled peptides were input at 50 pM. Competition assays utilized the nonlabeled NPY or PYY analogs at 0.01 to 300 nM, using up to 16 different concentrations. Unlabeled LP-PYY at 100 nM was used to define the nonspecific binding. Saturation assays were done at 10 different inputs in the range of 5 to 500 pM of [¹²⁵I]LP-PYY, using 100 nM unlabeled LP-PYY at each concentration of the labeled ligand to define the nonspecific binding. The assay incubations were terminated by centrifugation at 5°C, as specified above. All pretreatments were for 20 min at 24°C in the receptor assay buffer containing the appropriate concentration of drug or drugs examined, followed by sedimentation as above, resuspension in the assay buffer without the drug and resedimentation. Steady-state assays consisted of ligand association (at 50 pM of [¹²⁵I]LP-PYY) over 100 min in the standard assay buffer in the absence of drugs (however, in the presence of 100 nM unlabeled LP-PYY for the nonspecific binding control samples), followed by the addition of volume of the drug tested, dissolved at a concentration of 1 mM in the assay buffer (or volume of the assay buffer only, for the control samples), and an additional incubation of 20 min at 24°C, before recovery of particulates by sedimentation at 5°C. The final pellets were surface-washed with cold assay buffer, and radioactivity was measured in excised tube bottoms using a -scintillation counter (Micromedic; Rohm and Haas, Philadelphia, PA) at an efficiency of 51%. Particulate protein and absorbance at 400 nm (used as an index of preservation of membrane structure) were not significantly changed by any of the agents tested at concentrations of up to 100 µM.

Data analysis. Receptor binding parameters were calculated in the LIGAND program (Munson and Rodbard, 1980). Constants for modulation of NPY analog binding by nonpeptide ligands were obtained from biexponential or logistic curve fitting. Mean values of the binding data recorded at discrete molar inputs (usually 100 µM) of various drugs were compared by Tukey's tests after an analysis of variance and in some cases by Student's t tests.

	Results

Top Abstract Introduction Methods Results Discussion References

Displacement of the Y₁ ligand by NPY receptor agonists and antagonists. As expected from previous studies, proportions of the binding of subtype-selective ligands varied greatly among the brain areas examined, whereas the apparent affinity was generally much higher with the Y₂ ligand (table 1). At 50 pM of [¹²⁵I]-labeled ligands, the Y₁ binding represented >85% of the specifically bound radioactivity in the parietal cortex but <35% in circumventricular hypothalamic particulates (table 1). The affinity ranges found for PYY/NPY analogs at the Y₁ receptor were generally similar for the respective compound across the brain areas tested (fig. 1).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   A, Competition of [125I](Leu31,Pro34)hPYY binding to particulates from four areas of rat brain by peptide YY-derived ligands. All binding data were corrected for the binding at 100 nM nonlabeled LP-PYY. The Kdiss values for nonlabeled (Leu31,Pro34)hPYY (LP-PYY) ranged from 74 to 106 pM (n = 6-12; see table 1). For porcine/rat peptide YY (pPYY(1-36)), the corresponding values, in pM, were 234 ± 33 with PAR1, 290 ± 79 with PIR, 300 ± 45 with AHA and 293 ± 50 with HIPP (n = 3 for all). For human peptide YY(3-36) (hPYY(3-36)) at 100 nM, the displacement was between 50% and 60% of that obtained at 100 nM unlabeled LP-PYY, and half of this displacement was reached between 8 and 18 nM of hPYY(3-36) (n = 2 for all). For abbreviations and other details, see the text. B, Competition binding of [125I](Leu31,Pro34)hPYY to particulates from four areas of rat brain by the Y1-site selective antagonist BIBP-3226 and by NPY/PYY ancestral peptide, rPP. The displacement of the Y2-selective ligand [125I]hPYY(3-36) by BIBP-3226 from anterior hypothalamic particulates (AHA) is included for comparison. The Ki values of the Y1 ligand with BIBP-3226, in nM, were 6.5 ± 0.8 with PAR1, 11 ± 2 with PIR, 6.8 ± 1.3 with AHA and 8.5 ± 2.1 with HIPP particulates (n = 3 for all). For rPP, the corresponding values for the Y1 ligand with the above tissues (in nM) were 12.6 ± 0.9, 12.8 ± 7.4, 23.3 ± 9.9 and 13.4 ± 6.1 (n = 2 for all). The displacement of total [125I]LP-PYY binding by 1 µM rPP was <45%, and that of the specific binding (as defined at 100 nM LP-PYY) was 55% to 60%; there was <5% displacement at inputs up to 1 nM. As detailed in the text, similar profiles were obtained with hPP. For abbreviations and other details, see the text.

Nucleotide sensitivity of the Y₁ binding. The binding of LP-PYY to particles from all brain areas studied was highly sensitive to guanosine polyphosphates but not to GMP (fig. 2). The binding was not sensitive to the purinergic ligands ADPS and ATPS and other adenosine or pyrimidine nucleoside polyphosphates (data not shown). However, the active guanine nucleotides did not inhibit more than 75% of the specific LP-PYY binding even at inputs of 1 mM. As seen in figure 2, no significant differences in the inhibition of the Y₁ binding by the various guanosine polyphosphates were noted among the tissues studied at a saturating concentration of 100 µM. The four-tissue means for Y₁ binding at 100 µM of any of the guanosine polyphosphates were in no case significantly different in post hoc Tukey testing, whereas the differences between any of the guanosine polyphosphates and GMP or any of the adenine or pyrimidine nucleoside polyphosphates tested were highly significant (P < .01 in all cases; these significances are not indicated in figure 2).

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Nucleotide sensitivity of steady-state Y1 receptor binding in four rat brain areas. The data are expressed as percentage of the binding for a nucleotide relative to the binding in the absence of nucleotides. The results represent averages (plus S.E.M.) of at least two separate assays in the presence of 100 µM of the respective nucleotide, with the nonspecific binding defined at 100 nM of unlabeled LP-PYY. Any adenine nucleotides (including the purinergic ligands ATPS and ADPS), UTP and CTP were not active at up to 100 µM (also see Results). For abbreviations and other details, see the text.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Profiles of inhibition of the binding of Y1-receptor selective ligand [125I](Leu31,Pro34)hPYY to particulates from rat parietal cortex by GTP, GTPS, GDP and GDPS in absence (open symbols) or in the presence (filled symbols) of ATP. The nucleotides were used at 1 to 100,000 nM and ATP at 1 mM. The IC50 values (in µM) obtained without and with ATP were 14.9 ± 1.9 and 0.096 ± 0.014 for GTP, 0.0597 ± 0.0079 and 0.0307 ± 0.00493 for GTPS, 10.0 ± 1.9 and 0.14 ± 0.02 for GDP and 0.953 ± 0.19 and 0.593 ± 0.088 for GDPS (n = 2 in all cases). With GTPS as competitor, at 1 mM ATP the IC50 values for AHA and HIPP were 0.06 ± 0.009 and 0.04 ± 0.007 µM, respectively (n = 2 for both). For other details, see the text.

Sensitivity of Y₁ binding to a receptor mimic peptide. Modulation of the Y₁ binding by MAS-7, a receptor mimic in the docking region and also at the nucleotide site of the G protein alpha subunit, was quite similar with particulates from the four brain areas examined, with an average IC₅₀ value of 13 ± 2 µM, and the half-inhibition range was 11 to 17 µM (fig. 4). Essentially the same profiles for the response of [¹²⁵I]LP-PYY binding to MAS-7 were observed without ATP or in the presence of 0.1 to 1 mM ATP. The response of the PAR1 Y₁ binding to MAS-7 showed little change over a large range of particle protein input (legend of fig. 4).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Inhibition of the binding of Y1-receptor selective ligand [125I](Leu31,Pro34)hPYY to particulates from four rat brain areas by mastoparan analogue MAS-7. The labeled ligand was input at 50 pM. The IC50 values (from single-component logistic fits), in µM, were 11 ± 1.4 at PAR1 (n = 2), 11.5 ± 0.25 at PIR (n = 4), 13.3 ± 2.7 at AHA (n = 2) and 17.1 ± 5.4 at HIPP (n = 2) particulates. At 100 µM of MAS-7, inhibition of the specific binding (as defined at 100 nM of unlabeled LP-PYY) ranged from 93.6% to 100%. Assays for PAR1 and HIPP particulates were done without ATP in the assay buffer, while 1 mM ATP was present in assays using PIR and AHA particulates. A similar inhibition profile (IC50 = 15 µM) was observed for PAR1 particulates assayed at 5-fold larger particle protein input of 125 µg.

Lack of sensitivity of Y₁ binding to ion-channel blockers, a protein kinase C inhibitor and adrenergic blockers. Steady-state Y₁ binding was not sensitive to any of the ion channel blockers tested; the maximum decrease at 100 µM was <20% of the control binding (data not shown). The channel blockers inactive toward the steady-state brain particulate Y₁ receptor binding included dihydropyridine antagonists of the L-type Ca²⁺ channel, nimodipine and nitrendipine, the phenylalkylamine L-channel antagonist verapamil, the inhibitors of ATP-sensitive K⁺ channels glyburide and tolazamide and the Na⁺ channel/transport inhibitors, amiloride and ethylisopropylamiloride (EIPA). Pretreatment with any of these agents at 100 µM did not induce a significant reduction in the subsequent Y₁ binding. The protein kinase C inhibitor H-7 also was not active at 100 µM either in inhibition of the steady-state Y₁ binding or by way of pretreatment.

Sensitivity of Y₁ binding to phospholipase inhibitors. The phospholipase C inhibitors U-73122, ET-18-O-CH₃ and D609 at 100 µM produced different degrees of reduction of the steady-state Y₁ binding to particulates from the brain areas studied (fig. 5). For the above agents, the degree of dissociation of the steady-state binding was quite similar to the extent of irreversible Y₁ binding inhibition produced by pretreatment at 100 µM. Data for PAR1 Y₁ binding after preincubation with U-73122 over the dose range of 0.3 to 100 µM are shown in figure 6. U-73122 was consistently the most active inhibitor, with IC₅₀ values ranging from 2 to 6 µM in direct competition (fig. 6). Pretreatment of PAR1 particulates with U-73122 at 0.3 to 100 µM reduced the subsequent [¹²⁵I]LP-PYY binding with an inflexion at 10 µM (fig. 6), parallel and quantitatively similar to the profile of direct inhibition by U-73122 at PAR1 shown in the same graph. The control aminosteroid U-73343 over the same molarity range reduced the Y₁ binding insignificantly by <15% (figs. 5 and 6). The phospholipid ether ET-18-O-CH₃ induced a strong inhibition of the Y₁ binding, but its potency was much lower than that of U-73122 (fig. 5). The xanthate drug D609 was also active in all assay conditions, although its activity at 100 µM was significantly lower than that of U-73122 with particulates from all areas and also somewhat lower than that of ET-18-O-CH₃ (fig. 5).

View larger version (50K): [in this window] [in a new window]   Fig. 5.   Sensitivity of the steady-state binding of [125I](Leu31,Pro34)hPYY in four brain areas to phospholipase inhibitors. Data are averages of at least two separate determinations for each agent tested. The results are expressed as percent of binding relative to control particulates (treated after 100 min of ligand association with volume of the assay buffer for 20 min at 24°C). The results are shown with 1 S.E.M. For U-73122 means, significant differences in Tukey tests vs. the activity of ET-18-OCH3 are indicated by asterisks (*, >95%; **, >99% confidence); the differences between U-73122 and D609 means were significant above 99% confidence for any tissue. For all PLC inhibitors, the least common differences significant at the level of 95% and 99% confidence relative to the corresponding controls are indicated by & and &&, respectively. There was no significant sensitivity to 100 µM Tween 80, CHAPS, PIP2, and phosphatidylethanolamine, and several steroids tested (see Results). For further details, see the text.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Profiles of inhibition of the binding of [125I](Leu31,Pro34)hPYY to particulates from rat brain areas by phospholipase inhibitor U-73122. A profile of LP-PYY binding after pre-treatment of PAR1 particulates by 0.3 to 100 µM of U-73122 (see the text) is also shown. The IC50 values, in µM, were 2.1 ± 0.36 for PAR1 (n = 5), 2.2 ± 0.23 for PIR (n = 8), 4.4 ± 0.85 for AHA (n = 3) and 5.3 ± 1.1 for HIPP particulates (n = 5). After pretreatment of PAR1 particulates with U-73122, the half-maximal decrease of [125I]LP-PYY binding was found at 10.7 ± 1.1 µM (n = 2).

Lack of sensitivity of Y₁ binding to phospholipase substrates, detergents and steroids. In view of the pronounced sensitivity of the brain area particulate Y₁ binding to phospholipase C inhibitors, it was of interest to check for possible sensitivity of the binding to phospholipase substrates or activators. Phospholipase C substrates and activators PIP2 and phosphatidylethanolamine at 100 µM did not significantly decrease the specific binding of [¹²⁵I]LP-PYY. (It should be noted that both compounds at 100 µM, but not at 10 µM, strongly increased both the apparent total and nonspecific binding of the Y₁ agonist.) The nonionic detergent Tween 80 and the zwitterionic detergent CHAPS, tested as controls of the possible role of surfactant comicellation in the observed inhibition of Y₁ binding by phospholipase C blockers (see James et al., 1995), did not inhibit the steady-state Y₁ binding at 100 µM (i.e., at a molar excess over total particulate phospholipid of 2) over 20 min at 24°C. It should be noted, however, that treatment at 0° to 4°C with 1 to 10 mM of CHAPS (but not of Tween 80) resulted in a progressive loss of the Y₁ binding, linked to solubilization of the particulate lipid and to an inactivating extraction of the Y₁ receptor. Cholesterol, estradiol, progesterone and pregnenolone 4-sulfate (used as steroid controls against PLC-active aminosteroid U-73122, and also to test for possible sex steroid cross-reactivity in the Y₁ binding) were not inhibitory at 100 µM (data not shown).

Inhibition of phospholipase C activity by the antagonists tested. U-73122, ET-18-OCH₃ and, to a lesser extent, D609 significantly inhibited the activity of phosphatidylinositol-hydrolyzing phospholipase C as assayed in AHA or PAR1 particulates (fig. 7). U-73343 was not significantly active. Under the assay conditions used (free Ca²⁺ of ~100 nM, at pH 7.0), ET-18-O-CH₃ appeared to be the most active phospholipase C blocker.

View larger version (57K): [in this window] [in a new window]   Fig. 7.   Comparison of the inhibition of [3H]phosphatidylinositol-4,5-bisphosphate hydrolysis in particulates from rat brain parietal cortex (PAR1) and anterior hypothalamic area (AHA) by phospholipase inhibitors used in this study. The data represent binding at 1 mM CaCl2 input (or ~100 nM of Ca++; see the text), in a medium also containing 6 mM MgCl2 and 3 mM EGTA, over 20 min at 24°C. The results were corrected for the binding in the absence of CaCl2. Asterisks indicate differences significant vs. the control means at the level of 95% (*) and 99% (**) confidence in Student's t tests. For other details, see the text.

Sensitivity of the Y₁ binding to cotreatment with G protein and phospholipase-active drugs. As seen in figure 8, U-73122 inhibited, with particulates from three brain areas, most of the Y₁ binding not sensitive to guanine nucleotides. At 20 to 100 µM GTPS, this binding represented about one third of the specific Y₁ binding displaceable by 100 nM of LP-PYY (see also figs. 2 and 3). Scatchard estimates from saturation assays (fig. 8, insets and legend) showed, with particulates from all areas, a somewhat higher affinity for this component relative to the total specific Y₁ binding. Similar affinity trends for the guanine nucleotide-insensitive Y₁ binding were noted in competition of [¹²⁵I]LP-PYY by unlabeled LP-PYY (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Displacement of Y1 binding to particulates from three brain areas by U-73122 without (A) and with (B) 20 µM of GTPS in the assay. The data (from three assays for each area) are shown with 1 S.E.M. The estimated IC50 values (in µM of U-73122) without and with 20 µM GTPS were 2.2 ± 0.4 vs. 1.23 ± 0.1 at PIR, 3.9 ± 0.2 vs. 2.9 ± 0.6 at AHA and 6 ± 0.2 vs. 4.6 ± 0.6 at HIPP particulates, respectively. The insets show Scatchard plots of the saturation binding of [125I]LP-PYY at 5 to 500 pM; the values of the bound variable for both axes are expressed in fmol/mg particle protein. The estimated Kdiss values (in pM [125I]LP-PYY) without and with 20 µM GTPS were 79 ± 6 vs. 74 ± 9 at PIR, 98 ± 19 vs. 52 ± 9 at AHA and 98 ± 9 vs. 80 ± 7 at HIPP particulates, respectively (n = 3).

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Inhibition of the Y1 binding to particles from rat piriform cortex by G protein and PLC active agents. The same set of molar inputs was used for all agents. A, Inhibition by GTPS and U-73122 alone or paired. The data are averages of three assays using nine molar inputs. For GTPS alone and U-73122 alone, the data could be best represented by single-component logistic fits (with IC50 values of 45 ± 4.4 nM and 1.92 ± 0.37 µM, respectively.) For the joint treatment, the data best fitted the biexponential model, with half-inhibitions at 29.4 ± 11 nM and 1.49 ± 0.43 µM. B, Inhibition by MAS-7 and U-73122 alone or paired. The data are averages of two assays. All inhibition profiles were best fitted to a single-component model. The IC50 values were 11.7 ± 3.4 µM for MAS-7 alone (also included in the averages for PIR area shown in fig. 4) and 2.19 ± 0.2 µM for U-73122 alone (also included in the averages for PIR of fig. 6). The half-displacement corresponded to an input of 1.21 ± 0.19 µM of each chemical for the joint treatment. Asterisks indicate significant differences (at P < .05 in Student's t test) between inhibition by U-73122 alone and that by U-73122 plus MAS-7.

	Discussion

Top Abstract Introduction Methods Results Discussion References

Sensitivity to guanine nucleotides of the brain binding of [¹²⁵I]NPY and [¹²⁵I]PYY, agonists that have no clear preference for NPY receptor subtypes, was shown previously by Unden and Bartfai (1984) and by Walker and Miller (1988), before discovery of the subtypes. The present study provides, to our knowledge, the first direct characterization of the sensitivity to guanosine polyphosphates for brain sites labeled by a ligand selective for the Y₁ subgroup of the NPY receptor. The Y₁-selective ligand used can also label, with high affinity, the structurally related Y₄ and Y₅ sites (Gehlert et al., 1996, 1997). However, the low affinity and displacement activity against [¹²⁵I]LP-PYY observed with pancreatic polypeptides for rat forebrain sites in this and previous work (Parker et al., 1996a) indicate that most of the binding is to the Y₁ subtype. We also present the first evidence for sensitivity of Y₁ ligand binding to inhibitors of phosphatidylinositol-specific PLC. With rat forebrain Y₁ sites, there could exist a direct link between regulation of a peptidergic signal through an interaction of the classic G protein nucleotide exchange stimulated by the receptor (Mixon et al., 1995) and of GTP hydrolysis stimulated by a phospholipase effector known to potently act on G alpha subunits (Biddlecome et al., 1996).

The affinity of the Y₁ receptor in homologous competition and saturation assays found in this work is essentially in agreement with previous reports on selective Y₁ binding in areas of rodent brain (Dumont et al., 1995; Parker et al., 1996a). Unlabeled LP-PYY was significantly more potent than pPYY(1-36) in displacement of [¹²⁵I]LP-PYY, probably due to a closely similar carboxyl-terminal epitope. The affinity profiles for LP-PYY were quite similar with particulates from the four brain areas studied, indicating an absence of major differences in structure or environment of the binding sites. The binding parameters observed in this work for the Y₁-selective antagonist BIBP-3226 are close to values reported for displacement of [¹²⁵I]NPY from rat brain areas by this chemical (Wieland et al., 1995), as is the low activity of BIBP-3226 at the Y₂ site. The low affinity that we observed with particulates from four brain areas in competition of the Y₁ binding by rPP confirms a generally low number of Y₄/rPP sites in rat forebrain (Parker et al., 1996a). This affinity is in a good agreement with displacement of the binding of PYY or LP-PYY by pancreatic polypeptides in cell lines expressing only the Y₁ subtype (Mannon et al., 1994; Gehlert et al., 1997).

The uniform sensitivity of a considerable portion of the Y₁ binding to guanosine polyphosphates across the areas studied indicates the importance of a direct coupling of the Y₁ receptor with G proteins in its attachment of agonist peptides. Optimization of the apparent activity of GTP and GDP by ATP should be due to saturation of nonselective phosphatase activities (e.g., Salomon and Rodbell, 1975) and highlights participation of the guanine nucleotide-site "switch" in ligand association with the Y₁ receptor.

Our findings do not support an important participation of common neuronal ion channels in the process of Y₁ ligand binding. Among the compounds tested, the phenylalkylamine L-channel blocker verapamil is known to influence binding of radioligands to many classes of both peptide and non-peptide receptors (e.g., Siegel and Lukas, 1986). While NPY and Y₁ site agonists are known to stimulate the mobilization of Ca⁺⁺ (Daniels et al., 1989) and to assist the activity of K⁺ channels in neuronal systems (Hastings et al., 1997), this might be effected by Y₁ receptor interactions that do not involve the agonist binding site or via processes located downstream to the receptor. The lack of activity with the adrenergic antagonists tested also corroborates the selectivity of the binding profiles found with the Y₁ agonist.

Sensitivity of the Y₁ binding to MAS-7 (a peptide mimetic at the receptor-docking site of G proteins, (e.g., Higashijima et al., 1990) was highly similar across the areas tested. The observed IC₅₀ values for MAS-7 inhibition of the brain particulate Y₁ site were quite similar to ED₅₀ values for activation of GDP/GTP exchange by this mastoparan analog with G proteins reconstituted into liposomes (Higashijima et al., 1990). This would indicate that the inhibition of Y₁ binding is connected to a competitive activation of the nucleotide "switch" by MAS-7.

Among phospholipase inhibitors tested, the potent PtdIns-selective PLC blocker U-73122 (Bleasdale et al., 1990) was by far the most active (and also largely irreversible) inhibitor of the Y₁ ligand binding. The control aminosteroid U-73343 (Bleasdale et al., 1990) and four other steroids were inactive, indicating lack of selective effects of the steroid nuclei tested. The PLC substrates/activators PIP2 and phosphatidylethanolamine were also not inhibitory. Two PLC inhibitors not chemically related to U-73122 were also active against the Y₁ binding. The PtdIns-PLC-selective phospholipid ether ET-18-O-CH₃ (Powis et al., 1992) was more active than a xanthate selective for phosphatidylcholine-specific PLC, D-609 (e.g., Muller-Decker, 1989). Detergents Tween 80 and CHAPS also did not inhibit the Y₁ binding at up to 100 µM, indicating that surfactant-like amphiphilic structure or lipid comicellating activity present in most PLC blockers (James et al., 1995) are not primary determinants of the observed inhibition. The activity of two other PLC-selective blockers, ET-18-OCH₃ and D609, was lower than that of U-73122. However, ET-18-OCH₃ was the most active PtdIns-PLC inhibitor under the assay conditions used. This could be related to preferences of individual antagonists for PLC subclasses or isoenzymes and to analytical factors such as micellar size of the substrate and the extent of membrane fluidization needed for activity of a PLC isoenzyme (see, e.g., James et al., 1995). Quinacrine, a phospholipase A₂ inhibitor that affects the muscarinic ligand binding (O'Donnell and Howlett, 1991), only weakly dissociated the steady-state Y₁ binding and did not block the Y₁ site. The low activity of wortmannin, a blocker of receptor-coupled PLD, which also inhibits phosphatidylinositol 3-kinase (Mollinedo et al., 1994), and inactivity of propranolol, a phosphatidate phosphohydrolase inhibitor of the PLD cascade (see Mollinedo et al., 1994), lend further support for a specific role of PtdIns-specific PLCs in brain area Y₁ binding.

The guanine nucleotide-insensitive brain Y₁ binding could be competed or dissociated by either U-73122 or MAS-7, possibly pointing to a connection of PLC and G protein-responsive elements of the Y₁ binding site. Mastoparan and derivatives, beside mimicking receptors at docking sites of G proteins, may stimulate PLC activity independent of interactions with G proteins (Schnabel et al., 1997). As in the case of coupling with the G protein nucleotide site, an interaction of the mastoparan derivative with PLC would competitively weaken the coupling of the Y₁ receptor with the enzyme and thus interfere with the attachment of the Y₁ ligand. A different status for this component was also indicated by a consistently higher affinity than found for the total Y₁ binding. Pairing of U-73122 and GTP--S did not result in a significant synergism in the inhibition of Y₁ binding, indicating largely independent sites of action. An interaction sensitive to a PLC inhibitor is more likely to be centered on a PLC molecule. However, elements of carboxyl-terminal portions of G protein alpha subunit, known to participate in neuropeptide receptor docking and interaction with PLC (Conklin et al., 1996), could also be involved. Further mechanistic evidence for an interaction involving both G protein and PLC sequences is provided by the synergic inhibition of Y₁ binding by U-73122 and MAS-7, a peptide known to interact with membrane-associated or liposome-reconstituted G protein alpha subunits (Higashijima et al., 1990), as well as with PLC (Schnabel et al., 1997). It is not clear whether participation of a G protein would be obligatory for attachment of the Y₁ receptor to a PLC molecule. Elucidation of a more precise mechanism for this interaction would require reconstitution of the corresponding complex in a form capable of high-affinity ligand binding, as already done for the m1 muscarinic receptor (Biddlecome et al., 1996).

The multiple interaction of PLC, G protein and receptor triggered by the binding of agonistic peptides could be shared by other peptide receptors known to interact with phospholipase systems, including the vasopressin Y₁ and oxytocin receptors (see also Conklin et al., 1996). Thus, evidence was presented that the liver vasopressin V1 receptor could directly associate with a PLC isoenzyme (Aiyar et al., 1989). Precoupling of the epidermal growth factor receptor with PLC- in the absence of ligand attachment was also documented (Langgut and Ogilvie, 1995). A physical association of PLC and the Y₁ receptor is strongly supported by our finding of sensitivity to U-73122 for a sizable component of the Y₁ binding that is insensitive to guanine nucleotides. Sensitivity of all Y₁ binding to MAS-7, on the other hand, could indicate that the guanine nucleotide-refractory component interacts with aspects of PLC attached to the docking region of the G protein involved. The PLC/mastoparan sensitive region of G alpha subunits can have important epitopes located close to its ultimate carboxyl terminus (Conklin et al., 1996).

The high sensitivity to guanosine polyphosphates and phospholipase inhibitors shown in this study augments the number of mechanisms known to be involved in the regulation of Y₁ receptor activity. The Y₁ receptor is to some degree unique among neuropeptide receptors in possessing a highly segmented agonist-binding domain (see Du et al., 1997) and also in requiring noncontinuous and widely separated binding epitopes in agonist peptides (Daniels et al., 1995). This delicate binding assembly can be easily perturbed by an array of chaotropic influences, including temperature (Parker MS, Crowley WR and Parker SL, in preparation), common ions (Parker et al., 1996a) and alkylating agents and nonionic chaotropes (Parker et al., 1996b), thus weakening or even terminating the association of the Y₁ receptor with G protein transducers, and perhaps also with PLC effectors. However, as already shown for another neuropeptide receptor (Aiyar et al., 1989), association of the Y₁ receptor with PLC enzymes may not be easy to dismantle and may require proteolysis (observed with PLC- in a number of systems; see, e.g., Blank et al., 1993). This could point to a novel signal transduction mechanism connected to phosphoinositide signaling. On the other hand, attachment to a large PLC molecule could also serve to vectorially promote receptor sequestration and internalization, known to be readily induced by agonist peptides in the case of the brain Y₁ receptor (Parker et al., 1996b).