Specific interaction between the hop1 intracellular loop 3 domain of the human PAC(1) receptor and ARF.

The PAC(1), VPAC(1) and VPAC(2) receptors are members of the secretin (Group II) family of G protein-coupled receptors. All members of this family activate adenylate cyclase and several have also been shown to activate phospholipase C. We have recently reported that the rat VPAC(1), VPAC(2) and PAC(1) receptors activate phospholipase D and that distinct pathways are utilised by two intracellular loop 3 splice variants of PAC(1), one of which is ARF-dependent. Phospholipase D activation by the hop1, but not the null (short), form of the PAC(1) receptor is sensitive to brefeldin A, an inhibitor of GTP exchange at ARF. We have expressed the null and hop1 intracellular loop 3 domains of the human PAC(1) receptor in bacteria as GST-fusion proteins and used them as peptide affinity matrices to determine whether a functional interaction exists between these domains and ARF. Using this GST pull-down assay, we have shown binding of the small G protein ARF6 to the hop1 but not the null domain of this receptor.

Additional signals from VPAC/PAC family receptors.

The receptors for the neuropeptides vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptide are strong activators of adenylate cyclase, but recent evidence suggests that they can elicit a number of additional intracellular signals. Some of these are likely to be downstream of the conventional adenylate cyclase pathway, but it is now clear that others reflect novel primary coupling events of the receptors.

ADP-ribosylation factor-dependent phospholipase D activation by VPAC receptors and a PAC(1) receptor splice variant.

The VPAC(1) and VPAC(2) receptors for vasoactive intestinal polypeptide and the PAC(1) receptor for pituitary adenylate cyclase-activating polypeptide are members of a subfamily of G protein-coupled receptors (GPCRs). We recently reported that phospholipase D (PLD) activation by members of the rhodopsin group of GPCRs occurs by at least two routes, one of which seems to involve the small G protein ADP-ribosylation factor (ARF) and its physical association with GPCRs. Here we report that rat VPAC and PAC(1) receptors can also stimulate PLD (albeit less potently than adenylate cyclase) in transfected cells and also in cells where they are natively expressed. PLD responses of the VPAC receptors and the hop1 spice variant of the PAC(1) receptor but not its null form are sensitive to brefeldin A (BFA), an inhibitor of GTP exchange at ARF. The presence of the hop1 cassette in the rat PAC(1) receptor facilitates PLD activation in the absence of marked changes in ligand binding, receptor internalization, and adenylate cyclase activation, with some reduction in phospholipase C activation. Both VPAC(2) and PAC(1-hop1) (but not PAC(1-null)) receptors were shown to associate with immunoprecipitates directed against native or epitope-tagged ARF. A chimeric construct of the VPAC(2) receptor body with intracellular loop 3 (i3) of the PAC(1-null) receptor mediated BFA-insensitive activation of PLD, whereas the response of the corresponding PAC(1-hop1) construct was BFA-sensitive. Motifs in i3 of the PAC(1-hop1) receptor may act as critical determinants of coupling to ARF-dependent PLD activation by contributing to the GPCR:ARF interface.

Mechanisms of phospholipase C activation by the vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide type 2 receptor.

The vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide type 2 (VPAC(2)) receptor was shown to induce both [(3)H]inositol phosphate ([(3)H]InsP)and cAMP production in transfected COS7 cells and in GH(3) cells where it is natively expressed. Neither cholera toxin nor forskolin could elicit an equivalent [(3)H]InsP response, suggesting independent coupling of the two pathways. The VPAC(2) receptor-mediated [(3)H]InsP response was partially inhibited by pertussis toxin (Ptx) and by the G beta gamma-sequestering C-terminal fragment of GRK2 (GRK2-ct) in COS7 and GH(3) cells, whereas responses of control receptors were unaffected. Blockers of receptor-activated Ca(2+) influx pathways (Co(2+) and SKF 96365) also partially inhibited VPAC(2) receptor-mediated [(3)H]InsP responses. This inhibition was not present in the component of the response remaining after Ptx treatment. A range of blockers of voltage-sensitive Ca(2+) channels were ineffective, consistent with the reported lack of these channels in COS7 cells. The data suggest that the VPAC(2) receptor may couple to phospholipase C through both Ptx-insensitive and Ptx-sensitive G proteins (G(q/11) and G(i/o), respectively) to generate [(3)H]InsP. In addition to G beta gamma, G(i/o) activation appears to require receptor-activated Ca(2+) entry. This is consistent with the possibility that not only G alpha(q/11)-responsive and G beta gamma-responsive isoforms of phospholipase C but also Ca(2+)-responsive forms may contribute to the overall [(3)H]InsP response.

Gonadotropin-releasing hormone receptor activation of extracellular signal-regulated kinase and tyrosine kinases in transfected GH3 cells and in alphaT3-1 cells.

GH3 cells were stably transfected with the wild-type murine GnRH receptor and a clonal cell line selected on the basis of inositol phosphate production and PRL/GH release in response to GnRH. This cell line (wt28) was characterized by [125I]GnRH analog binding, [3H]inositol phosphate response to GnRH, and hormone secretion. We examined the activation of the mitogen-activated protein kinase isoforms, extracellular signal-regulated kinase 1/2 (ERK1/2) and tyrosine kinases in wt28 cells and alphaT3-1 cells (which express a native GnRH) using specific phospho-ERK1/2 and phosphotyrosine antibodies. Concentration-response and time-course data revealed that a sustained ERK1/2 response was seen only in aT3-1 cells. Furthermore, GnRH-induced tyrosine phosphorylation was detectable in alphaT3-1 cells, but not in wt28 cells. Activators for several different signaling pathways revealed distinct differences between the cell types. Protein kinase C activation by phorbol 12,13-dibutyrate was very effective in alphaT3-1 cells at phosphorylation of both ERK1/2 and tyrosine, whereas raising cAMP levels using forskolin also strongly increased wt28 cell ERK1/2 phosphorylation. Only the tyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation in wt28 cells. The lack of sustained ERK1/2 phosphorylation in wt28 cells could be the result of minimal tyrosine kinase activation by GnRH compounded by a different pathway profile for ERK1/2 activation. When pervanadate and GnRH were combined, ERK1/2 phosphorylation was synergistic and sustained in wt28 cells, whereas the response was additive in alphaT3-1 cells. In sum, the intracellular pathways leading to ERK1/2 and tyrosine phosphorylation in alphaT3-1 and wt28 cells are distinct; thus, activating GnRH receptors in each of the two cell types leads to different sequelae of events regarding ERK1/2 activation.

The prevalence and correlates of binge eating in a British community sample of women with a history of obesity.

OBJECTIVE: To estimate the frequency of binge eating and binge eating disorder and their correlates in a sample of women with a history of obesity. METHOD: A group of women who had been found in a previous community study to have a body mass index > or = 30 were studied using self-report measures (n = 74) and interview (n = 62). RESULTS: One subject met criteria for binge eating disorder, while 24% reported binging. Subjects who reported binging were significantly more likely to have a past history of depressive illness. DISCUSSION: Prevalence of binge eating, binge eating disorder, and psychopathology was broadly in keeping with that found in North America. In addition, there were nonsignificant trends towards a positive family history of obesity, of childhood obesity, of early onset of dieting, of excessive concern about weight and shape, and a recent history of weight reduction. Further study is required to elucidate the causes of binge eating in the obese.

The effect of Paf antagonists on bronchial hyperresponsiveness induced by Paf, propranolol or indomethacin.

1. Intravenous administration of platelet activating factor, Paf (600 ng kg-1 h-1) to ventilated anaesthetised guinea-pigs induced bronchial hyperresponsiveness to i.v. acetylcholine. 2. Pretreatment of ventilated, anaesthetised guinea-pigs with the beta-adrenoceptor antagonist, propranolol, or the non-steroidal anti-inflammatory drug, indomethacin, induced bronchial hyperresponsiveness to i.v. histamine. 3. Paf-induced bronchial hyperresponsiveness was significantly attenuated by pretreatment with three different Paf antagonists, CV-3988, BN 52021 and WEB 2086. 4. Pretreatment of guinea-pigs with CV-3988, BN 52021 or WEB 2086 at doses inhibiting Paf-induced bronchial hyperresponsiveness, had no significant effect on propranolol or indomethacin-induced bronchial hyperresponsiveness. 5. It is suggested that bronchial hyperresponsiveness induced by propranolol or indomethacin is not secondary to Paf release in the guinea-pig.

The effect of platelet-activating factor on histamine and muscarinic receptor function in guinea pig airways.

Platelet-activating factor (PAF) administered i.v. or by aerosol to guinea pigs elicited an increase in airway responsiveness to both acetylcholine and histamine when compared with guinea pigs exposed to the vehicle bovine serum albumin (BSA). After i.v. PAF, the ED100 for acetylcholine and histamine was 5.4 and 3.4 micrograms/kg, respectively, in comparison with 17 and 6 micrograms/kg, respectively, before PAF, representing approximately a 3- and 2-fold increase in responsiveness, respectively. After aerosol PAF (500 micrograms), the ED100 for acetylcholine and histamine was 13 and 7 micrograms/kg, respectively, whereas after aerosolized BSA, the ED100 for acetylcholine and histamine was 23 and 12 micrograms/kg, respectively, representing approximately a 2-fold increase in responsiveness. However, airway smooth muscle obtained from these PAF- or BSA-treated animals did not exhibit any differences in contractile response to histamine or acetylcholine in vitro. Likewise, there were not significant differences in the binding affinity or receptor density between PAF- and BSA-treated tissues with [3H]quinuclidinylbenzilate or [3H]pyrilamine binding, which were used to identify muscarinic and H1-histamine receptors, respectively. Furthermore, histamine and carbachol-induced phosphoinositide hydrolysis was similar in PAF- and BSA-treated tracheal smooth muscle preparations. Thus, PAF induces airway hyperresponsiveness in the guinea pig that is not related to changes in airway smooth muscle or to changes in muscarinic and histamine (H1) receptor density or function.

Effect of platelet agonists on airway reactivity and intrathoracic platelet accumulation.

1 Intravenous infusion of platelet activating factor (Paf), adenosine diphosphate (ADP), collagen and the thromboxane-mimetic U46619 induced a dose-related accumulation of 111indium-labelled platelets into the thoracic region of anaesthetized guinea-pigs. 2 Intravenous infusion of Paf increased the reactivity of the airways to the spasmogen histamine. Such changes were not observed following treatment with ADP, collagen or U46619. 3 Paf-induced bronchial hyperreactivity is not secondary to pulmonary platelet recruitment, changes in basal lung function or cardiovascular changes. 4 Paf-induced bronchial hyperreactivity is not dependent upon the endogenous generation of ADP or thromboxane.

The effect of platelet activating factor on pulmonary beta-adrenoceptors.

An intravenous infusion of platelet activating factor (Paf) in the guinea-pig elicits an increase in bronchial responsiveness to the spasmogens, histamine and bombesin. Airways obstruction induced by bombesin in Paf-treated animals is poorly reversed by isoprenaline compared to comparable airways obstruction induced by bombesin in vehicle-treated animals. Isoprenaline induced a comparable dose-related relaxation in vitro of tracheal smooth muscle isolated from Paf- and vehicle-treated animals. No change in beta-adrenoceptor numbers or binding affinity was observed in lungs removed from Paf-treated animals in comparison with those from vehicle-treated animals, or after direct incubation with Paf in vitro. The reduced bronchodilator responsiveness to isoprenaline in Paf-treated animals is not related to changes in pulmonary beta-adrenoceptor function. These results suggest that non-spasmogenic elements may contribute to airways obstruction induced in hyper-responsive animals.

Sustained intrauterine release of levonorgestrel over five years.

Two models of levonorgestrel-releasing intrauterine devices were studied. Model A was designed to deliver 20 micrograms/day, and model B was designed to deliver 30 micrograms/day. Plasma concentrations of levonorgestrel were determined in blood samples taken from women who used the levonorgestrel-releasing intrauterine devices (IUDs) and who participated in a clinical trial for more than 5 years. During clinical studies IUDs have been removed for various reasons between 8 and 40 months after insertion. The rate of release of levonorgestrel from the IUDs was calculated by determining the remaining amount of levonorgestrel. Plasma concentrations of levonorgestrel ranged between a mean +/- standard deviation (SD) concentration of 166 +/- 75 pg/ml and 131 +/- 32 pg/ml for the first 18 months of use and between 101 +/- 37 pg/ml and 74 +/- 15 pg/ml at 24 and 60 months after insertion of the IUD. The plasma concentrations from 24 months through 60 months were significantly lower than concentrations measured during initial months of IUD use, but not between the two devices. There was a strong correlation between the time of use and the amount of levonorgestrel lost from the IUDs. The calculated mean daily release of levonorgestrel was 17.6 micrograms for model A and 22.2 micrograms for model B. This gives a calculated lifetime of more than 6 years for a levonorgestrel-releasing IUD.

PIP: 2 models of levonorgestrel-releasing IUDs were studied. Model A was designed to deliver 20 mcg/day and model B 30 mcg/day. Plasma concentrations of levonorgestrel were determined in blood samples taken from who had used the levonorgestrel-releasing IUDs and who had participated in a clinical trial for more than 5 years. During clinical studies, IUDs have been removed for various reasons between 8-40 months after insertion. The rate of release of levonorgestrel from the IUDs was calculated by determining the remaining amount of levonorgestrel. Plasma concentrations of levonorgestrel ranged between a mean +or- standard deviation concentration of 166 +or- 75 pg/ml and 131 +or- 32 pg/ml for the 1st 18 months of use and between 101 +or- 37 pg/ml and 74 +or- 15 pg/ml at 24 and 60 months after IUD insertion. Plasma concentrations from 24 to 60 months were significantly lower than concentrations measured during the initial months of IUD use, but not between the 2 devices. There was a strong correlation between the time of use and the amount of levonorgestrel lost from the IUDs. The calculated mean daily release of levonorgestrel was 17.6 mcg for model A and 22.2 mcg for model B. This gives a calculated lifetime of more than 6 years for a levonorgestrel-releasing IUD.

CV3988 inhibits in vivo platelet aggregation induced by PAF-acether and collagen.

Platelet activating factor (PAF-acether) was shown to cause a fall in the circulating platelet count and blood pressure (BP) in anaesthetised rabbits. CV3988 caused a dose-dependent inhibition of these responses to a low dose of PAF-acether (150 ng X kg-1). CV3988 itself was found to cause a fall in both BP and platelet count in vivo. Collagen (40 micrograms X kg-1) i.v. caused sudden death in rabbits and pretreatment with CV3988 (5 mg X kg-1) caused a 62% inhibition of the platelet count response to collagen without significantly increasing the survival rate. In the rat, collagen (40 micrograms X kg-1) was not lethal and CV3988 caused a dose-dependent inhibition of the fall in platelet count due to collagen, but this action was short-lived. CV3988 also caused agonist actions in the rat. These results show that CV3988 inhibits the effects of PAF-acether in vivo and suggests that PAF-acether may play a role in mediating collagen-induced platelet aggregation in vivo.

Cholesterol and HDL-cholesterol values in women during use of subdermal implants releasing levonorgestrel.

Plasma concentrations of cholesterol, HDL-cholesterol, and levonorgestrel were determined in two groups of women using levonorgestrel-releasing subdermal implants. One group used six capsules (NORPLANT)*; the other six covered rods. Plasma concentrations of levonorgestrel among NORPLANT users averaged 700 pg/ml in the first two weeks of use, decreased to 300 pg/ml at 8 weeks, and to about 230 pg/ml by 50 weeks. Concentrations among covered rod users were 1.4 to 1.7 times higher at comparable time periods. Total serum cholesterol and HDL-cholesterol were decreased as compared with controls at all sampling intervals during the 114 weeks of the trial, although the differences did not meet tests of significance at all time periods. Decreases during the test period were of the order of 10 percent, except for total cholesterol among covered rod users where the decrease was less. Cholesterol to HDL-cholesterol ratios did not differ significantly from control values at any sampling period.

Contraception with long-acting subdermal implants. A five-year clinical trial with Silastic covered rod implants containing levonorgestrel. The International Committee for Contraception Research (ICCR) of the Population Council.

A total of 189 women volunteered to accept subdermal implants for contraception. The implants were "covered rods", consisting of a core rod containing equal parts by weight of levonorgestrel and polydimethylsiloxane and sealed inside a thin-walled tube of Silastic tubing with medical adhesive. In one study 78 women used 4 3cm rods (study 07) and in the other 111 women used 6 3cm rods. In 5 years of use there were no pregnancies in either group. Terminations because of menstrual problems were twice as frequent among the 4-rod users than among users of the 6 rods. Menstrual pattern analysis is presented for the two rod regimens and compared with the previously reported patterns for the 6-capsule regimen (NORPLANT). Long--term in vivo release rates are also presented.

Long-term reversible contraception with levonorgestrel-releasing Silastic rods.

Subcutaneously placed Silastic capsules containing levonorgestrel are effective for 5 years and have a higher continuation rate than other methods of reversible contraception. Six 3 cm capsules are required to achieve satisfactory circulating levels of levonorgestrel. Two 4 cm covered Silastic rods containing levonorgestrel, which are easier to manufacture, insert, and remove than the capsules, produce similar in vitro release rates. This study compared clinical and metabolic effects as well as bleeding patterns in 23 women using either six capsules (n = 11) or two covered rods (n = 12). Serum levels of levonorgestrel, lipids, and lipoproteins as well as frequency of elevated progesterone levels were compared in serum samples obtained before treatment and 1, 6, 12, 18, and 24 months after insertion with the two systems. While bleeding patterns were similar for users of the two systems, rod users had slightly higher serum levels of levonorgestrel and a lower incidence of cycles with elevated progesterone levels. Therefore, rods could replace capsules as a long-term, reversible contraceptive method.