Expression of functional Y1 receptors for neuropeptide Y in human Ewing's sarcoma cell lines

Summary In the human Ewing's sarcoma cell line WE-68, saturation analysis using³H-labelled neuropeptide Y ([³H]NPY) as the radioligand disclosed a homogeneous population of binding sites with a dissociation constant (K _d ) of 4.5 nM and maximal binding capacity (B _max) of 712 fmol/mg cell protein. Besides the WE-68 cell line, ten other human Ewing's sarcoma cell lines (FM-62, HS-80, HT-78, HT-M1-78, NT-68, RM-82, RS-63, VH-64, WE-M1-68, WE-M2-68) were also found to display NPY receptors withK _d varying from 3.5 nM to 10.7 nM andB _max=247–3744 fmol/mg cell protein. NPY, its natural analogues and the Y₁-receptor-specific peptide ligand [Leu³¹, Pro³⁴]NPY inhibited [³H]NPY binding in the potency order: [Leu³¹,Pro³⁴]NPYhuman NPYpeptide YY (PYY)>> salmon pancreatic polypeptide (PP) > human PP>porcine NPY_13–36NPY_22–36. In the Ewing's sarcoma cell lines NPY provoked inhibition of forskolin-stimulated cyclic AMP formation by up to 98%. Pertussis toxin alleviated the cyclic-AMP-inhibitory response to NPY. In isolated Ewing's sarcoma plasma membranes pertussis toxin [³²P]ADP-ribosylated a 41-kDa protein. The ability of NPY and analogues to inhibit cyclic AMP accumulation paralleled their potencies in displacing radioligand binding. By contrast, a cell line derived from an atypical form of Ewing's sarcoma did not express specific and functional NPY receptors. These results demonstrate that conventional Ewing's sarcoma cells possess G_i-potein-coupled NPY receptors of the Y₁ type, which upon interaction with NPY, PYY, and PP mediate inhibition of cyclic AMP generation.Abbreviations NPY neuropeptide Y - PP pancreatic polypeptide - PYY peptide YY - VIP vasoactive intestinal peptide     

Characterization of Binding of an RGD Mimetic, [3H]-SC-52012, to Platelet GPIIb/IIIa

In order to compare binding of small peptide mimetics on activated vs resting platelets and with fibrinogen (fgn) on activated platelets, the binding of [³H]-SC-52012, a low molecular weight (483) mimetic of the RGDF sequence present in fgn, was evaluated. This compound is a potent inhibitor of fgn binding to activated platelets, IC, 9.0 ± 0.6 nM (mean ± SEM), and inhibits ADP induced human platelet aggregation (IC, 44 ± 5 nM). The dissociation constant (Kd) of [³H]-SC-52012 was 21.6 ± 4.7 nM (n = 13) in ADP-induced human washed platelets while the Kd for resting platelets was 156 ± 8.3 nM (n = 3). The maximum number of binding sites on ADP-activated and resting platelets were 60846 ± 7158 and 59464 ± 5898 molecules/platelet, respectively. By comparison, results with [¹²⁵I]-fgn binding to activated platelets gave values of 363 ± 73 nM and 58046 ± 6386 molecules/platelet (n = 8) for the Kd and receptor number, respectively. These data suggest that the small molecule binds regardless of activation state of the platelet with only a change in affinity. [³H]-SC-52012 could be displaced by unlabelled SC-52012 with an IC₅₀ of 135 ± 20 nM.     

Inhibition of glucose stimulated insulin secretion by neuropeptide Y is mediated via the Y1 receptor and inhibition of adenylyl cyclase in RIN 5AH rat insulinoma cells

Summary Neuropeptide Y (NPY) has been shown to inhibit insulin secretion from the islets of Langerhans. We show that insulin secretion in the insulinoma cell line RIN 5AH is inhibited by NPY. ¹²⁵I-Peptide YY (PYY) saturation and competition-binding studies using NPY fragments and analogues on membranes prepared from this cell line show the presence of a single class of NPY receptor with a Y1 receptor subtype-like profile. Inhibition of insulin secretion in this cell line by NPY fragments and analogues also shows a Y1 receptor-like profile. Both receptor binding and inhibition of insulin secretion showed the same orders of potency with NPY > [Pro³⁴]-NPY > NPY 3–36 > > NPY 13–36. The Y1 receptor antagonist, BIBP 3226, blocks NPY inhibition of insulin secretion from, and inhibits ¹²⁵I-PYY binding to, RIN 5AH cells. Northern blot analysis using a Y1-receptor specific probe shows that NPY Y1 receptors are expressed by RIN 5AH cells. Y5 receptors are not expressed in this cell line. Neuropeptide Y inhibition of insulin secretion is blocked by incubation with pertussis toxin, implying that the effect is via a G-protein (G_i or G_o) coupled receptor. Neuropeptide Y inhibits the activation of adenylyl cyclase by isoprenaline in RIN 5AH cell lysates, and the stimulation of cAMP by glucagon-like peptide-1 (7–36) amide (GLP-1). It also blocks insulin secretion stimulated by GLP-1, but not by dibutyryl cyclic AMP. Hence, we suggest that NPY inhibits insulin secretion from RIN 5AH cells via a Y1 receptor linked through G_i to the inhibition of adenylyl cyclase. [Diabetologia (1998) 41: 1482–1491] Received: 10 November 1997 and in final revised form: 16 June 1998     

DPP-IV inhibition enhances the antilipolytic action of NPY in human adipose tissue

Context: Dipeptidyl peptidase IV (DPP‐IV) inactivates the incretin hormone glucagon‐like peptide. It can also affect the orexigenic hormone neuropeptide Y (NPY_1–36) which is truncated by DPP‐IV to NPY_3–36, as a consequence NPY’s affinity changes from receptor Y1, which mediates the antilipolytic function of NPY, to other NPY receptors. Little is known whether DPP‐IV inhibitors for the treatment of type 2 diabetic (T2DM) patients could influence these pathways. Aims: To investigate the in vitro effects of NPY with DPP‐IV inhibition in isolated abdominal subcutaneous (AbdSc) adipocytes on fat metabolism, and assessment of NPY receptor and DPP‐IV expression in adipose tissue (AT). Methods: Ex vivo human AT was taken from women undergoing elective surgery (body mass index: 27.5 (mean ± s.d.) ± 5 kg/m², age: 43.7 ± 10 years, n = 36). Isolated AbdSc adipocytes were treated with human recombinant (rh)NPY (1–100 nM) with and without DPP‐IV inhibitor (1 M); glycerol release and tissue distribution of DPP‐IV, Y1 and Y5 messenger RNA (mRNA) were measured and compared between lean and obese subjects. Results and conclusion: rhNPY reduced glycerol release, an effect that was further enhanced by co‐incubation with a DPP‐IV inhibitor [control: 224 (mean ± s.e.) ± 37 μmol/l; NPY, 100 nM: 161 ± 27 μmol/l**; NPY 100 nM/DPP‐IV inhibitor, 1 M: 127 ± 14 μmol/l**; **p < 0.01, n = 14]. DPP‐IV was expressed in AbdSc AT and omental AT with relative DPP‐IV mRNA expression lower in AbdSc AT taken from obese [77 ± 6 signal units (SU)] vs. lean subjects (186 ± 29 SU*, n = 10). Y1 was predominantly expressed in fat and present in all fat depots but higher in obese subjects, particularly the AbdSc AT‐depot (obese: 1944 ± 111 SU vs. lean: 711 ± 112 SU**, n = 10). NPY appears to be regulated by AT‐derived DPP‐IV. DPP‐IV inhibitors augment the antilipolytic effect of NPY in AT. Further studies are required to show whether this explains the lack of weight loss in T2DM patients treated with DPP‐IV inhibitors.     

Characterization of a [3H]glutamate binding site in rat pineal gland: Enhanced affinity following superior cervical ganglionectomy

Abstract: Glutamate, an excitatory neurotransmitter/neuromodulator involved in cell-to-cell communication within the central nervous system, is now believed to play a role in neuroendocrine function. In this study we describe a single, saturable, stereospecific, and temperature-, time-, and pH-dependent binding site for glutamate in the pineal gland of the rat (K_d= 612 ± 23 nM, B_max= 3.17 ± 0.33 pmol/mg protein). After removal of the sympathetic innervation to the pineal gland, [³H]glutamate binding displayed a higher apparent affinity (K_d= 412 ± 28 nM) (P < 0.05) without a change in binding site number (B_max= 3.60 ± 0.24 pmol/mg protein). No difference in [³H]glutamate binding site number was observed in pineal glands obtained from animals sacrificed during the middle of the light and dark periods. These data suggest a possible modulatory role for a glutamate binding site in pineal gland function.     

Identification and functional studies of a specific peptide YY-preferring receptor in dog adipocytes.

Specific binding sites for peptide YY (PYY) and neuropeptide Y (NPY) as well as functional responses were identified in dog adipocytes. Studies were carried out using the radioligand [125I-Tyr1]monoiodo-PYY on crude adipocyte membranes. [125I]PYY bound to dog adipocyte membranes with a high affinity (156 +/- 24 pM) and binding capacity of 314 +/- 48 fmol/mg protein. Competition studies revealed a higher affinity of the binding sites for PYY than NPY (inhibition constants were 118 +/- 17 pM and 300 +/- 53 pM, respectively, P < or = 0.001). NPY analogs displaced [125I]PYY specific binding with the following order of potency: NPY-(13-36) > NPY-(18-36) > NPY-(22-36) > [Leu31-Pro34]NPY. Neither adrenergic nor adenosine agents (activating or inhibiting other antilipolytic systems) interacted with [125I]PYY binding sites. So [125I]PYY binding was specific, saturable, and reversible. Lipolysis experiments performed with PYY, NPY, and NPY analogs confirm the relative order of potency found in competition experiments. The data agree with the definition of PYY-preferring receptor which resembles a Y2 receptor subtype since NPY-(13-36), a specific Y2 receptor agonist, inhibited binding and lipolysis in a similar way to PYY, whereas [Leu31-Pro34]NPY did not. No difference was observed in the antilipolytic response between IC50 values measured on omental, perirenal, and subcutaneous fat deposits. Moreover, PYY and NPY (10(-6) M) significantly attenuated forskolin-stimulated cAMP levels, involving inhibition of adenylyl cyclase as a transmembrane signaling mechanism. Cross-linking of bound [125I]PYY to membranes indicated that the mol wt of the receptor was 62K. The relative importance of such a receptor on fat cells alongside another powerful antilipolytic receptor--the alpha 2-adrenoceptor--is discussed.     

Agonist-independent modulation of L-type Ca currents by basal Gs protein activities in single guinea pig ventricular myocytes

The modulation of L-type Ca²⁺ currents (I _Ca,L) by the basal activities of G proteins was studied in adult guinea pig ventricular myocytes by whole-cell patch-clamp techniques. With intrapipette guanosine triphosphate (GTP) (100 μM), a specific inhibition of G_i proteins by pertussis toxin (PTX) produced an increase in the basal density of I _Ca,L (from 11.0 ± 0.8, n = 13, to 25.0 ± 2.0 pA/pF, n = 11, at 0 mV test potential). In addition, PTX shifted the forskolin (Fsk) concentration–I _Ca,L response relation significantly leftward (EC₅₀ = 63.7 ± 12.5 vs 625 ± 75 nM). With intrapipette guanosine diphosphate (GDP)βS (1 mM), the Fsk–I _Ca,L relation was also shifted leftward (EC₅₀ = 197 ± 18.3 vs 781 ± 82.5 nM). However, chronic GDPβS dialysis accelerated the rundown of I _Ca,L significantly, suggesting a potential contribution of G_s proteins in maintaining basal I _Ca,L. In contrast, intra-pipette GTPγS (100 μM) produced a transient rise in I_Ca,L from 11.0 ± 3.0 to 22.8 ± 7.0 pA/pF (in 3.4 min after whole-cell formation at 0 mV, n = 9), presumably through the activation of G_s proteins. It was followed by a gradual decline in I _Ca,L (to 15.5 ± 3.5 pA/pF), which was still enhanced by Fsk (EC₅₀ = 1450 ± 98 nM), indicating that the current decay was not solely due to rundown but to activation of G_i proteins. G_s, in addition to G_i proteins, show sufficient basal activity to modulate I _Ca,L in an agonist-independent manner. Received: October 19, 2000 / Accepted: February 24, 2001     

A novel binding site for the native hepatic acute‐phase protein α1‐antitrypsin expressed on the human hepatoma cell line HepG 2 and intestinal cell line Caco 2

Abstract: Aims/Background:α1‐antitrypsin (α1‐AT) is a hepatic acute phase protein which predominantly inhibits neutrophil elastase. Besides this major function, we have also previously shown that α1‐AT markedly increased H‐ferritin mRNA expression and ferritin synthesis in the human hepatoma cell line HepG 2. These actions suggest that α1‐AT might interact with HepG 2 cells via a specific cell surface binding site. Methods and Results: Using radio‐labelled native α1‐AT, we observed saturable binding to HepG 2 cells with a dissociation constant (Kd) of 63.3±6.9 nM and a maximal density of binding sites (Bmax) of 0.34 ±0.05 pmol/10⁶ cells equivalent to 195800±29200 sites/cell. The binding of [¹²⁵I]α1‐AT was time dependent with a calculated association rate constant of 9.22±1.84×10⁴×M^?1×min^?1. Binding was highly specific since other acute phase proteins or protease inhibitors failed to block binding. Although α1‐AT‐trypsin, α1‐AT‐elastase and the pentapeptide FVYLI, the minimal binding sequence for the SEC receptor, increased [¹²⁵I]α1‐AT binding, in long term experiments these complexes failed to influence the number of α1‐AT binding sites. Specific, saturable binding of [¹²⁵I]α1‐AT was also found on the human intestinal epithelial Caco 2 cells, but not on fibroblast or leukaemic cell lines. Conclusion: These experiments demonstrate a specific, high affinity binding site for native α1‐AT on HepG 2 and Caco 2 cells, cell lines derived from tissues involved in the acute phase response.     

Characterization of the μ and δ Opioid Receptors in the Brain of the C57BL/6 and DBA/2 Mice, Selected for Their Differences in Voluntary Ethanol Consumption

Genetically determined differences in the activity of the hypothalamic β-endorphin system have been demonstrated between the C57BL/6 (alcohol-preferring) and DBA/2 (alcohol-aversive) inbred strains of mice. The present studies examined the distribution and density of the μ and δ receptors in specific brain regions that may mediate the rewarding and reinforcing effects of ethanol, using quantitative auto-radiography and the specific μ agonist FK 33–824 and 6 agonist DPDPE, in their iodinated form. ¹²⁵I-FK 33–824 recognizes a high affinity binding site in brain membrane preparations from both the C57BL/6 (K_d= 1.37 ± 0.22 nM; B_max= 80 ± 12.3 fmol/mg protein) and DBA/2 mice (K_d= 1.02 ± 0.16 nM; B_max= 39.5 ± 9.6 fmol/mg protein), whereas ¹²⁵I-DPDPE binds to a high affinity binding site in brain membranes from both the C57BL/6 (K_d= 1.08 ± 0.34 nM; B_max= 24.4 ± 4.5 fmol/mg protein) and DBA/2 mice (K_d= 0.68 ± 0.24 nM; B_max= 15.3 ± 3.7 fmol/mg protein). The auto-radiographic studies demonstrated differences in the density of the μ opioid receptors between the two strains of mice in brain nuclei that are not directly related to the brain reward system. However, strain-related differences in the density of δ opioid receptors were observed in regions of the limbic system known to mediate the positive reinforcing effects of many drugs of abuse. The density of δ receptors was significantly higher in the ventral tegmental area and nucleus accumbens of the C57BL/6 mice. The results of the present study support the hypothesis that genetically determined differences exist in the density of opioid receptors in distinct regions of the brain between the C57BL/6 and DBA/2 inbred strains of mice, which may play a role in controlling their voluntary ethanol consumption.     

Type I and type II insulin-like growth factor receptors and their function in human Ewing's sarcoma cells

Summary Binding studies using recombinant human¹²⁵I-labelled insulin-like growth factor I ([¹²⁵I]IGF-I) revealed IGF-I receptors in three Ewing's sarcoma cell lines withK _d ranging from 74×10^–12 M to 100×10^–12 M andB _max=36–63 fmol/mg cell protein. [¹²⁵I]IGF-I binding was displaced by IGF-I, IGF-II and insulin with IC₅₀ values of 1.5 nM, 6.3 nM and 0.7 M respectively. Recombinant human [¹²⁵I]IGF-II radioligand-binding assays in the cell lines disclosed specific binding sites for IGF-II withK _d=(110–175)×10^–12 M andB _max varying from 21 fmol/mg to 72 fmol/mg cell protein. Neither IGF-I nor insulin displaced [¹²⁵I]IGF-II binding. IGF-I was found to increase basal glucose transport by maximally 1.5 times with EC₅₀=0.9 nM IGF-I. The efficacy and potency of IGF-II on glucose uptake were comparable to those of IGF-I whereas insulin was ineffective. IGF-I and IGF-II also provoked stimulation of glycogen synthesis in Ewing's sarcoma cells. The maximal glycogenic response was reached at 0.01 M IGF-I and 0.1 M IGF-II, the EC₅₀ value being approximately 1 nM IGF-I and 2 nM IGF-II. Insulin did not significantly influence glycogen formation. IGF-I and IGF-II but not insulin increased DNA synthesis in Ewing's sarcoma cells. The maximal mitogenic response was obtained with 10 nM IGF-I or IGF-II with an EC₅₀ value of about 0.7 nM for both peptides. -IR-3, a monoclonal antibody specific for the IGF type I receptor, effectively blocked IGF-I- and IGF-II-mediated metabolic responses. In conclusion, the data show that IGF-I and IGF-II induce rapid and longterm biological responses in Ewing's sarcoma cells exclusively through interaction with IGF type I receptors.     

In vitro studies on the relative potency of bronchodilator agents

This study describes a rapid in vitro assay for the order of potency of bronchodilator drugs using specific binding of (−)-[³H] dihydroalprenolol ([³H]DHA) to rat lung membranes. Under linear conditions with respect to tissue, specific binding of [³H]DHA showed saturability, rapid kinetics of association and dissociation of radioligand, and sterospecificity. Nanomolar (nM) concentrations for 50% inhibition (IC₅₀±SE) for the bronchodilator drugs examined were as follows: albuterol, 1485±170; isoproterenol, 136±53; procaterol, 162±28; terbutaline, 3310±934; and zinterol, 51±8.3. A comparison of binding studies using rat lung tissue membranes and similar preparations of rat heart and skeletal muscle demonstrated that lung tissue had 7 to 8 times more receptor sites (B_max) for [³H]DHA than heart or skeletal muscle. Adenyl cyclase activit of the rat lung membrane preparation almost doubled in the presence of (−)-isoproterenol. Displacement of specific (³H)DHA binding in membrane preparations may provide useful data for evaluating bronchodilator compounds.     

Gliclazide produces high-affinity block of KATP channels in mouse isolated pancreatic beta cells but not rat heart or arterial smooth muscle cells

Aims/hypothesis: Sulphonylureas stimulate insulin secretion by closing ATP-sensitive potassium (K_ATP) channels in the pancreatic beta-cell membrane. K_ATP channels are also found in other tissues, including heart and smooth muscle, where they link cellular metabolism to electrical activity. The sulphonylurea gliclazide blocks recombinant beta-cell K_ATP channels (Kir6.2/SUR1) but not heart (Kir6.2/SUR2A) or smooth muscle (Kir6.2/SUR2B) K_ATP channels with high potency. In this study, we examined the specificity of gliclazide for the native (as opposed to recombinant) K_ATP channels in beta cells, heart and smooth muscle. Methods: The action of the drug was studied by whole-cell current recordings of native K_ATP channels in isolated pancreatic beta-cells and myocytes from heart and smooth muscle. Results: Gliclazide blocked whole-cell beta-cell K_ATP currents with an IC ₅₀ of 184 ± 30 nmol/l (n = 6–10) but was much less effective in cardiac and smooth muscle (IC ₅₀s of 19.5 ± 5.4 μmol/l (n = 6–12) and 37.9 ± 1.0 μmol/l (n = 5–10), respectively). In all three tissues, the action of the drug on whole-cell K_ATP currents was rapidly reversible. In inside-out patches on beta-cells, gliclazide (1 μmol/l) produced a maximum of 66 ± 13 % inhibition (n = 5), compared with more than 98 % block in the whole-cell configuration. Conclusion/interpretation: Gliclazide is a high-potency sulphonylurea which shows specificity for the pancreatic beta-cell K_ATP channel over heart and smooth muscle. In this respect, it differs from glibenclamide. The difference in the maximal block observed in the excised patch and whole-cell recordings from beta-cells, may be due to the absence of intracellular Mg-nucleotides in the excised patch experiments. [Diabetologia (2001) 44: 1019–1025] Received: 21 March 2001 and in revised form: 30 April 2001     

Peptide-YY and neuropeptide-Y inhibit vasoactive intestinal peptide-stimulated adenosine 3',5'-monophosphate production in rat small intestine: structural requirements of peptides for interacting with peptide-YY-preferring receptors

Previous binding studies indicated that peptide-YY (PYY) and neuropeptide-Y (NPY) shared a common PYY-preferring receptor site in rat small intestinal epithelium. We showed here that PYY and NPY inhibited vasoactive intestinal peptide (VIP)-stimulated cAMP production in epithelial cells isolated from rat small intestine and examined their structure-activity relationship. Inhibition of VIP-stimulated cAMP by PYY or NPY is time and dose dependent; half-maximal effects were observed for 10 and 107 nM, respectively. In contrast, the structurally related peptide, pancreatic polypeptide, was only active at 1 microM. PYY or NPY reduced the efficacy of VIP by about 50% without altering its potency. Both peptides also suppressed prostaglandin E1-, prostaglandin E2-, and forskolin-stimulated cAMP production and reduced basal cAMP levels. Their inhibitory effects were observed throughout the small intestine, including duodenum, jejunum, and ileum, but not in large intestine. PYY or NPY and epinephrine (through alpha 2-adrenergic receptors) did not exert additive inhibitory effects on intestinal cAMP production. Several fragments of PYY and NPY were used to characterize their structural requirement for inhibiting VIP-stimulated cAMP production and competing with [125I]PYY for binding to intestinal membranes. A highly significant correlation was observed between IC50 values measured in the two assays. No partial sequence of PYY retained the full activity of intact PYY, but the C-terminal portion of PYY was shown to be much more important than the N-terminal portion. Deletion of 21 amino acids from the N-terminus [PYY-(22-36)] only resulted in a 4- to 5-fold decrease in potency compared to that of PYY-(1-36). In contrast, PYY-(27-36) exhibited a drastic loss of potency. The N-terminal fragments PYY-(1-22) and PYY-(1-28) also had very low potencies. Similar results were obtained with NPY fragments. These results provide the first insight on the negative coupling of PYY-preferring receptors with the cAMP production system in small intestine and evidence of the crucial role of the C-terminal portion of PYY in interaction with these receptors.     

Characterization of the receptors for peptide-YY and avian pancreatic polypeptide in chicken and pig brains

We have previously identified the peptide-YY (PYY) receptor on porcine brain membranes as a 50-kDa protein after chemical cross-linking. PYY receptors are discretely distributed in the brain of various mammals, to which neuropeptide-Y (NPY), but not pancreatic polypeptide (PP), bind with great specificity. The present study was carried out in order 1) to identify and characterize the PYY receptor in the avian brain, 2) to compare it with the APP receptor that had been demonstrated in the cerebellum, and 3) to examine [125I]APP-binding activity in the porcine brain. [125I]PYY was bound to chicken brain membranes via high affinity (Kd = 2.19 x 10(-10) M) and low affinity (Kd = 1.93 x 10(-7) M) components. The binding sites were highly specific for PYY and APP as well as for NPY and PPP, coupled to a guanine nucleotide regulatory protein, and distributed in various brain areas, including the cerebellum. The C-terminal fragments of PYY, PYY-(17-36) and PYY-(24-36), exhibited low potency in inhibiting binding, but behaved like full agonists. Porcine brain membranes, on the other hand, possessed two orders of the APP-binding sites, a high affinity component (Kd = 4.24 x 10(-9) M) and a low affinity component (Kd = 3.08 x 10(-7) M). APP binding showed a high specificity for APP, but not for PPP, NPY, or PYY. The binding activity was highest in the pituitary gland, followed by the hippocampus, amygdala, cerebral cortex, hypothalamus, and cerebellum. Guanosine 5'-O-thiotriphosphate, a nonhydrolyzable GTP analog, did not inhibit the binding of [125I]APP to porcine or chicken brain membranes, which ran counter to the results of PYY receptors in both species. Cross-linking studies have demonstrated that receptor-bound [125I]APP is cross-linked to a protein of 67 kDa without disulfide-linked subunits in both porcine and chicken brain membranes. In the latter species, [125I]PYY and [125I]NPY were also cross-linked to the same 67-kDa proteins, which were different from the receptor proteins (50 kDa) in mammalian species. These results indicate that chicken brain has receptors specific for PYY and NPY, as was found in mammalian brains, and that PYY, NPY, and PP act in the brain through interaction at multiple receptor sites, which are similar to and shared by other members of the PP family. Furthermore, the finding that APP-binding sites in porcine brain are more specific than those in avian brain suggests that an endogenous peptide similar to APP may exist in porcine brain.     

Thrombin Binding to Platelets Defines Functional Receptors: Inhibition of Thrombin-Induced Platelet Activation by Catalytically-Inactivated Thrombin

It has been widely questioned as to whether the observed binding of a-thrombin to intact platelets defines receptors coupled to signal transduction or merely thrombin binding sites. We have now shown that at α-thrombin concentrations sufficient to induce a full shape change response without aggregation (0.1 nM), PPACK-thrombin (that is, α-thrombin treated with the irreversible active site inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethylketone) dose-dependently inhibits platelet shape change (IC₅₀～70 nM), the concomitant increases in [Ca²⁺Ii (IC₅₀～75 nM) and ATP secretion (IC₅₀～50 nM). Since PPACK-thrombin competes fully in the binding of a-thrombin to high, moderate and low affinity sites on intact platelets, these results show that this binding defines functional receptors coupled to platelet activation.     

Evidence for further heterogeneity of the receptors for neuropeptide-Y and peptide-YY in tumor cell lines derived from neural crest.

The expression and structure of the receptors for neuropeptide-Y (NPY) and peptide-YY (PYY) were studied in 16 human and rodent tumor cell lines derived from the neural crest by ligand binding and cross-linking techniques using [125I]Bolton-Hunter-NPY, [125I]PYY, and various forms of monoiodinated NPY and PYY. Although NPY-binding sites were observed in most of the tumor cells, PYY-binding sites were found only on the human neuroblastoma cell lines SMS-MSN, SMS-KAN, SK-N-MC, and MC-IXC and the human Ewing's sarcoma cell line SK-ES. The differential labeling of the NPY/PYY receptors on these cell lines suggests that the NPY/PYY receptors are more heterogeneous than previously described as the Y1, Y2, and Y3 receptor subtypes. Cross-linking studies demonstrate that the Y1 and Y2 receptors for NPY/PYY are structurally different (mol wt, 70 and 50 kilodaltons, respectively) and that the 70- and 50-kilodalton receptor proteins are coexpressed in certain tumor cell lines. This could explain at least in part why cell lines show a relative specificity for Y1/Y2 classification, observed as the inhibition by both C-terminal fragments and Y1-specific analogs on the NPY/PYY binding to membrane receptors. Collectively, the present study suggests further heterogeneity of the NPY/PYY receptors and the existence of multiple receptor proteins in the tumor cell lines derived from the neural crest.     

Neuropeptide Y reduces acetylcholine release and vagal bradycardia via a Y2 receptor-mediated, protein kinase C-dependent pathway

The co-transmitter neuropeptide Y (NPY), released during prolonged cardiac sympathetic nerve stimulation, can attenuate vagal-induced bradycardia. We tested the hypothesis that NPY reduces acetylcholine release, at similar concentrations to which it attenuates vagal bradycardia, via pre-synaptic Y2 receptors modulating a pathway that is dependent on protein kinase A (PKA) or protein kinase C (PKC). The Y2 receptor was immunofluorescently colocalized with choline acetyl-transferase containing neurons at the guinea pig sinoatrial node. The effect of NPY in the presence of various enzyme inhibitors was then tested on the heart rate response to vagal nerve stimulation in isolated guinea pig sinoatrial node/right vagal nerve preparations and also on ³H-acetylcholine release from right atria during field stimulation. NPY reduced the heart rate response to vagal stimulation at 1, 3 and 5 Hz (significant at 100 nM and reaching a plateau at 250 nM NPY, p < 0.05, n = 6) but not to the stable analogue of acetylcholine, carbamylcholine (30, 60 or 90 nM, n = 6) which produced similar degrees of bradycardia. The reduced vagal response was abolished by the Y2 receptor antagonist BIIE 0246 (1 μM, n = 4). NPY also significantly attenuated the release of ³H-acetylcholine during field stimulation (250 nM, n = 6). The effect of NPY (250 nM) on vagal bradycardia was abolished by the PKC inhibitors calphostin C (0.1 μM, n = 5) and chelerythrine chloride (25 μM, n = 6) but not the PKA inhibitor H89 (0.5 μM, n = 6). Conversely, the PKC activator Phorbol-12-myristate-13-acetate (0.5 μM, n = 7) mimicked the effect of NPY and significantly reduced ³H-acetylcholine release during field stimulation. These results show that NPY attenuates vagal bradycardia via a pre-synaptic decrease in acetylcholine release that appears to be mediated by a Y2 receptor pathway involving modulation of PKC.     

Evidence for direct action of alloxan to induce insulin resistance at the cellular level

Summary To determine whether long-term insulin deficiency alters insulin movement across the endothelium, plasma and lymph dynamics were assessed in dogs after alloxan (50 mg/kg; n = 8) or saline injection (n = 6). Glucose tolerance (K_G) and acute insulin response were assessed by glucose injection before and 18 days after treatment. Two days later, hyperglycaemic (16.7 mmol/l) hyperinsulinaemic (60 pmol · min⁻¹· kg⁻¹) glucose clamps were carried out in a subset of dogs (n = 5 for each group), with simultaneous sampling of arterial blood and hindlimb lymph. Alloxan induced fasting hyperglycaemia (12.9 ± 2.3 vs 5.7 ± 0.2 mmol/l; p = 0.018 vs pre-treatment) and variable insulinopenia (62 ± 14 vs 107 ± 19 pmol/l; p = 0.079). The acute insulin response, however, was suppressed by alloxan (integrated insulin from 0–10 min: 155 ± 113 vs 2745 ± 541 pmol · l⁻¹· 10 min⁻¹; p = 0.0027), resulting in pronounced glucose intolerance (K_G: 0.99 ± 0.19 vs 3.14 ± 0.38 min⁻¹; p = 0.0002 vs dogs treated with saline). During clamps, steady state arterial insulin was higher in dogs treated with alloxan (688 ± 60 vs 502 ± 38 pmol/l; p = 0.023) due to a 25 % reduction in insulin clearance (p = 0.045). Lymph insulin concentrations were also raised (361 ± 15 vs 266 ± 27 pmol/l; p = 0.023), such that the lymph to arterial ratio was unchanged by alloxan (0.539 ± 0.022 vs 0.533 ± 0.033; p = 0.87). Despite higher lymph insulin, glucose uptake (R_d) was significantly diminished after injection of alloxan (45.4 ± 2.5 vs 64.3 ± 6.5 μmol · min⁻¹· kg⁻¹; p = 0.042). This was reflected in resistance of target tissues to the lymph insulin signal (ΔR_d/Δlymph insulin: 3.389 ± 1.093 vs 11.635 ± 2.057 · 10⁻⁶· l · min⁻¹· kg^–1· pmol⁻¹· l⁻¹; p = 0.012) which correlated strongly with the K_G (r = 0.86; p = 0.0001). In conclusion, alloxan induces insulinopenic diabetes, with glucose intolerance and insulin resistance at the target tissue level. Alloxan treatment, however, does not alter lymph insulin kinetics, indicating that insulin resistance of Type 1 (insulin-dependent) diabetes mellitus reflects direct impairment at the cellular level. [Diabetologia (1998) 41: 1327–1336] Received: 3 November 1997 and in final revised form: 2 June 1998     

Endothelin-1 receptors in the human myometrium: evidence for different binding properties in post-menopausal as compared to premenopausal and pregnant women

OBJECTIVE We investigated the binding properties of the endothelin receptors in the human myometrium in clinical situations associated with different ovarian steroid levels. SUBJECTS AND METHODS Binding properties of the endothelin receptors were studied in myometrial membranes from post-menopausal women (n= 12), myomatous premenopausal women (n= 14) and pregnant women (n= 14), using ¹²⁵I-labelled endothelin-1. RESULTS The mean (SD) maximal receptor density (B_max) was significantly higher in samples from premenopausal and pregnant women than from post-menopausal women (983 ± 196, 1116 ± 201 and 490 ± 145 pmol/g protein, respectively). Receptor affinity (K_d) did not differ significantly between these groups. Among the pregnant women, mean B_max and K_d values were similar in those who electively underwent Caesarean section prior to the onset of labour and those operated on during the second stage of spontaneous labour. Binding properties of myometrial membranes of either pre or post-menopausal women were unaffected by the presence of high levels of beta-oestradiol or progesterone in the medium. Among samples of premenopausal women, no significant difference was found in binding properties between those operated on either during mid-follicular phase or during mid-luteal phase. CONCLUSIONS In clinical situations associated with relatively high levels of ovarian steroids, the density of endothelin receptors in the myometrium is higher than in situations associated with low ovarian steroid level. Ovarian steroids may exert their influence via the production of other mediators. Changes in density of the endothelin receptor, induced by change in ovarian steroids activity, might play a role in the regulation of myometrial contractility.     

Antagonism of P2Y12 reduces physiological thromboxane levels

Antiplatelet therapy for the management of patients with cardiovascular risks often includes a combination therapy of aspirin and clopidogrel, acting through inhibition of thromboxane generation and blockade of G_i-coupled P2Y₁₂ receptor, respectively. We hypothesized that ADP acting through P2Y₁₂ regulates physiological thromboxane levels. The serum thromboxane levels in mice (n?=?3) dosed with clopidogrel and prasugrel were decreased by 83.1?±?5.3% and 94.26?±?1.75% respectively compared to untreated mice. Pre-treatment of human blood (n?=?3) ex vivo with active metabolites of clopidogrel or prasugrel led to a reduction in thromboxane levels to 16.3?±?3.2% and 4.9?±?0.8% respectively, compared to untreated human serum. We also evaluated serum thromboxane levels in P2Y receptor null mice (n?=?4). Whereas serum thromboxane levels in P2Y₁ null mice were similar to those in wild type littermates, those in the P2Y₁₂ null mice were inhibited by 83.15?±?3.8%. Finally, in a pilot study, serum thromboxane levels were reduced by 76.05?±?8.41% in healthy human volunteers (n?=?6) upon dosing with clopidogrel, compared to the levels before dosing. In conclusion, P2Y₁₂ antagonism alone can decrease physiological thromboxane levels. Thus, this study could pave way the for newer/modified treatment regimens for the management of patients with thrombotic complications who are allergic or non-responsive to aspirin.