Monique Lambert

VIP activation of rat anterior pituitary adenylate cyclase

The brain gut octacosapeptide VIP (vasoactive intestinal peptide) is found in discrete areas in the cerebral cortex, the hypothalamus and the posterior pituitary gland. Specific VIP binding sites are coupled to an adenylate cyclase system in synaptic membranes from guinea pig brain. Besides, the concentration of VIP in the hypothalamo-hypophysial portal vessels is much higher than in the systemic blood. The peptide has however no established function in the hypophysis. These data document the presence in the rat pituitary of functional VIP receptors existing in the form of a VIP-stimulated adenylate cyclase system and suggest that VIP might be a major peptidic activator of rat adenopituitary membrane adenylate cyclase.

Functional and molecular characterization of CCK receptors in the rat pancreatic acinar cell line AR 4-2J.

Competitive inhibition binding studies on membranes from the rat pancreatic AR 4-2J cell line revealed the predominance (80%) of low selectivity CCK receptors (KD of 1 nM and 4 nM for, respectively, CCK-8 and gastrin-17I (G-17I] over selective receptors (20% with a KD of 1 nM and 1 microM for, respectively, CCK-8 and G-17I). Amylase secretion was stimulated by low concentrations of CCK-8, G-17I and CCK-4. G-17I-induced amylase secretion was unaffected by 100 nM of the selective peripheral CCK-A receptor antagonist L-364,718, suggesting that amylase hypersecretion followed non-selective CCK receptor activation, a function normally assumed by selective CCK-A receptors in rat pancreatic acini. Direct ultraviolet irradiation of AR 4-2J cell membranes preloaded with 125I-BH-CCK-33 or 125I(Leu)G(2-17)I resulted in covalent cross-linking with, respectively, a 90 kDa protein and a 106 kDa protein, both distinct from the 81 kDa CCK binding species revealed in normal rat pancreatic membranes. Gpp[NH]p increased the dissociation rate of CCK-8 and G-17I from AR 4-2J cell membranes, indicating a coupling of receptors with guanyl nucleotide regulatory protein(s) G. [32P]ADP-ribosylation of AR 4-2J cell membranes allowed to detect the presence of two Gs alpha (the 50 kDa form predominating over the 45 kDa form) and one Gi alpha (41 kDa). However, Gi and Gs may not be involved in gastrin stimulation of amylase secretion, as Bordetella pertussis toxin and cholera toxin pretreatment of cells did not suppress G-17I-dependent amylase secretion.

Hormone-stimulated GTPase activity in rat pancreatic plasma membranes.

Pancreatic plasma membranes have an adenylate cyclase system responsive to pancreozymin and secretin [ 1-3 ] . Both hormones activate a common catalytic subunit via distinct receptors [ 1,2,4] which are not permanently coupled with the catalytic subunit [5]. Guanyl nucleotides act synergistically with these hormones [6]. In numerous eukaryotic systems, Gpp(NH)p and GTPyS are better activators than GTP and allow a quasi-irreversible activation of adenylate cyclase following binding to regulatory guanyl nucleotide site(s) 16-91; a finding which raises the question of the role of GTP hydrolysis in the adenylate cyclase system. The catecholamine-stimulated adenylate cyclase of turkey erythrocyte membranes has been shown to be associated with a catecholamine-stimulated GTPase (EC 3.6.1.-) [ 10,111. This GTPase is inhibited when adenylate cyclase is activated by isoproterenol and GTP-yS [ 121 or by cholera toxin [ 13 ,I 41. Based upon these observations a model has been proposed [ 15,161 in which adenylate cyclase oscillates between the active and inactive state and is regulated by the hydrolys~ of GTP. GTP binds to the inactive adenylate cyclase system, this complex is

Phosphorylation of protein components of isolated zymogen granule membranes from the rat pancreas

Some properties were examined of the isolated membranes of zymogen granules from the rat pancreas. It was possible to detect an endogenous protein kinase activity capable of phosphorylating 8 to 10 membrane protein constituents as well as added histones. Cyclic adenosine monophosphate (AMP), and cyclic guanosine monophosphate (GMP), to a lesser extent, exerted moderate stimulatory effects. These effects of cyclic nucleotides on membrane phosphorylation became more apparent upon addition of a partially purified soluble protein kinase also extracted from the rat pancreas.

Effector mechanisms of peptides of the VIP family

The present review is focused on the exocrine pancreas and liver where the only known effector mechanism of VIP is the activation of adenylate cyclase in plasma membranes. A two-state model of activation-deactivation of the enzyme visualizes the participation of VIP receptors and Ns, the guanyl nucleotide stimulatory protein of adenylate cyclase. In the rat pancreas, VIP and GRF receptors are indistinguishable and disulfide bridges influence their functional integrity. The antagonism of VIP and somatostatin perhaps requires, at the adenylate cyclase level, the contribution of Ni, the guanyl nucleotide inhibitory protein. The potentiation of VIP by various stimulants acting on Ca2+ movements may rely on later events, e.g., on a concerted activation of protein kinases. When comparing quantitatively peptide binding to receptors with adenylate cyclase activation, cyclic AMP levels and amylase secretion, a tool is at hand to tailor synthetic agonists and antagonists of VIP, with appropriate changes in the N-terminal moiety of the peptide (a good agonist allows efficient coupling of receptors to the adenylate cyclase system). Apart from stimulus-secretion coupling, VIP may influence protein synthesis in the rat pancreas, through the phosphorylation of ribosomal protein S6, and may alter the activity of the endoplasmic reticulum via the phosphorylation of Mr = 21 kDa and Mr = 25 kDa proteins. In rat liver membranes, high affinity VIP receptors are specifically labelled with 125I-helodermin and are coupled to adenylate cyclase (at variance with low affinity VIP receptors). These receptors are highly responsive to divalent cations and to guanyl nucleotides.

Distinct effects of the C-terminal octapeptide of cholecystokinin and of a cholera toxin pretreatment on the kinetics of rat pancreatic adenylate cyclase activity

(1) The kinetic parameters of rat pancreatic adenylate cyclase were evaluated, using GTP, p[NH]ppG or GTP gamma S as nucleotide activator, cholecystokinin as peptide hormone, and GDP beta S and dibutyryl cyclic GMP as inhibitors of guanosine triphosphate and CCK-8, respectively. The time courses of activation and the degree of activation at steady state (EA/ETOT) were compatible with a simple two-state model of activation-deactivation based on a pseudo-monomolecular activation process (rate constant kappa+1), and a deactivation process (rate constant kappa off) that included, depending on the activating nucleotide, the hydrolysis of GTP (rate constant kappa 2) and/or the dissociation of the intact nucleotide (rate constant kappa-1), so that EA/ETOT = kappa+1/(kappa+1 + kappa 2 + kappa-1). (2) The hormone CCK-8 increased the value of kappa+1 with GTP dose-dependently, from 0.2 to 10.9 min-1. The value of kappa-1 increased 0.01 to 0.3 min-1 but the value of kappa 2 was unaltered at 7 min-1, so that EA/ETOT increased 15-fold, from 4% to 61%. (3) A cholera toxin pretreatment at 30 micrograms/ml allowed also a large increase in EA/ETOT with GTP (up to 51%) but the underlying mechanism was different. It consisted of a 14-fold decrease in the kappa off value of the GTP-activated enzyme (from 7 min-1 to 0.5 min-1) that corresponded to a reduction in GTPase activity. When testing the system with p[NH]ppG, two added effects of the cholera toxin pretreatment were observed: a 4-fold increase in the value of kappa+1 (from 0.2 to 0.8 min-1) and the occurrence of a significant 0.3 min-1 value for kappa-1.

Phosphorylation in vitro of proteins in the pancreas and parotids of rats: effects of hormonal secretagogues and cyclic nucleotides.

Abstract (1) Caerulein 10 −9 M and carbamylcholine 5·10 −6 M provoked a 3-fold increase in amylase secretion and a 50% stimulation of protein phosphorylation in rat pancreas fragments. Secretin exerted similar effects, though more moderate, at 5·10 −7 M concentration, but was inefficient at low concentrations. Dibutyryl-cyclic AMP 2 mM stimulated total protein phosphorylation (+28%) but the secretory effect was subdued (+50%). The effects observed with cyclic GMP and its dibutyrylated derivative were insignificant. (2) In rat parotids, 10 −5 M isoproterenol provoked a 10-fold increase in amylase output and exerted a slight (+15%) but significant effect on protein phosphorylation. These effects were reproduced by 0·5–2·0 mM dibutyryl-cyclic AMP. Dibutyryl-cyclic GMP at a high (5 mM) concentration allowed a slight increase (+86%) in enzyme secretion with no apparent stimulation of protein phosphorylation. (3) These results indicate a clear stimulation of protein phosphorylation when hormonal stimulus-secretion coupling was involved in the pancreas acinar cells and parotids. The derivatives of cyclic AMP partially mimicked these effects, and more clearly so in the parotids than in the pancreas.

Novel GTP‐binding proteins in plasma membranes and zymogen granule membranes from rat pancreas and in pancreatic AR 4‐2J cell membranes

Photoaffinity labelling with [α‐32P]GTP allowed to detect a 54 kDa GTP‐binding protein in rat pancreatic plasma membranes and in pancreatic AR 4‐2J cell membranes. Like the 42 and 48 kDa Gsα subunits and the 41 kDa Giα subunit, this protein was absent from zymogen granule membranes. Contrastingly, a new 28 kDa GTP‐binding protein (detected by [α‐32P]GTP binding on immobilized proteins) and a 25 kDa protein (ADP‐ribosylated by botulinum toxin D) were found in all three membrane preparations. This is to our knowledge the first report on GTP‐binding proteins in zymogen granule membranes.

One-step isocratic high-performance liquid chromatographic purification of radioiodinated and radioiodinated-photoactivable derivatives of cholecystokinin

N-Hydroxysuccinimidyl-3-(4-hydroxy-3-[125I]iodophenyl)propionate (the Bolton-Hunter reagent) was conjugated with (Thr 34, NLeu 37) cholecystokinin (CCK) 31-39 in anhydrous dimethylformamide-pyridine in 20% yield. The radiolabelled peptide was purified from the reaction mixture in one step, by isocratic elution from a C18 high-performance liquid chromatographic (HPLC) column with 35% aqueous acetonitrile-0.13% heptafluorobutyric acid as eluent. The concentration of the radiolabelled peptide was estimated by UV monitoring. The acylating photoactivable radioiodinated reagent N-hydroxysuccinimidyl-N-(4-azido-2-nitrophenyl)-3-[125I]iodotyrosi ne was synthesized, purified on a C18 HPLC column by isocratic elution with 65% acetonitrile-1 mM hydrochloric acid, then conjugated with (Thr 34, NLeu 37) CCK31-39. The resulting photoactivable radioiodinated CCK analogue was purified by isocratic elution on a C18 HPLC column with 39% aqueous acetonitrile-0.1 M triethylamine phosphate (pH 3.5). The binding ability of both tracers and their non-radioactive analogues to CCK receptors was tested on rat pancreatic plasma membranes. As compared to a KD of 4.5 nM for unmodified (Thr 34, NLeu 37) CCK31-39, the KD of the radioiodinated Bolton-Hunter derivative was 3 nM, and that of the photoactivable radioiodinated derivative was 19 nM.

Internalization-sequestration and degradation of cholecystokinin (CCK) in tumoral rat pancreatic AR 4-2 J cells

Cholecystokinin (CCK) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas, after preincubation with 20 nM dexamethasone. At steady state binding at 37 degrees C (i.e., after a 5 min incubation), less than 10% of the radioactivity of [125I]BH-CCK-9 (3-(4-hydroxy-[125I]iodophenyl)propionyl (Thr34, Nle37) CCK(31-39)) could be washed away from intact cells with an ice-cold acidic medium, suggesting high and rapid internalization-sequestration of tracer. By contrast, more than 85% of the tracer dissociated rapidly after a similar acid wash from cell membranes prelabelled at steady state. In intact AR 4-2 J cells, internalization required neither energy nor the cytoskeleton framework. Tracer internalization was reversed partly but rapidly at 37 degrees C but slowly at 4 degrees C. In addition, two degradation pathways of the tracer were demonstrated, one intracellular and one extracellular. Intracellular degradation occurred at 37 degrees C but not at 20 degrees C and resulted in progressive intracellular accumulation of [125I]BH-Arg that corresponded, after 1 h at 37 degrees C, to 35% of the radioactivity specifically bound. This phenomenon was not inhibited by serine proteinase inhibitors and modestly only by monensin and chloroquine. Besides, tracer degradation at the external cell surface was still observable at 20 degrees C and yielded a peptide (probably [125I]BH-Arg-Asp-Tyr(SO3H)-Thr-Gly). This degradation pathway was partly inhibited by bacitracin and phosphoramidon while thiorphan, an inhibitor of endopeptidase EC 3.4.24.11, was without effect.