John MacDermot

Pituitary adenylate cyclase-activating polypeptide: a novel, long-lasting, endothelium-independent vasorelaxant.

The vasoactivity of the 27- and 38-amino acid forms of the novel peptide pituitary adenylate cyclase-activating polypeptide (PACAP) was tested in vitro. Both forms of PACAP caused endothelium-independent vasodilation (assayed by their vasodilator action on rabbit aorta). When superfused for 1 min the relaxation EC50 of PACAP27 was 23 +/- 8 nM and of PACAP38 was 152 +/- 66 nM. PACAP was 100-fold more potent than vasoactive intestinal polypeptide (VIP) (PACAP27 shows 68% amino acid sequence homology with VIP), and had a prolonged duration of action, a 1 min exposure to 1 microM PACAP27 lasting 135 +/- 7 min and to 1 microM PACAP38 108 +/- 3 min. Adenylate cyclase activity in homogenates of rabbit aortic smooth muscle cells was increased by PACAP27 and PACAP38 with EC50s of 4.4 and 0.73 nM, respectively. PACAP27 and PACAP38 are potent, long-lasting, endothelium-independent vasodilators.

Human calcitonin gene-related peptide activates adenylate cyclase and releases prostacyclin from human umbilical vein endothelial cells.

1 Endothelial cells of human umbilical vein were isolated and cultured in vitro. 2 In these cells there was a concentration‐dependent release of prostacyclin and activation of adenylate cyclase by human calcitonin gene‐related peptide (hCGRP). The concentration of hCGRP for half‐maximum activation of adenylate cyclase (Kact) by hCGRP was 190 nm. 3 Bradykinin induced a ten fold greater release of prostacyclin than CGRP, but did not activate adenylate cyclase. 4 hCGRP may exert its potent vasodilator properties by stimulating release of vasorelaxant substances, including prostacyclin from endothelial cells.

Neuropathic pain in Anderson-Fabry disease: pathology and therapeutic options.

An inherited deficiency of the enzyme alpha-galactosidase A is manifest clinically as Anderson-Fabry disease. Most affected patients present with severe peripheral pain in childhood or early adult life, and frequently progress to multi-organ failure by the 4th or 5th decades. The present review examines the probable mechanisms that contribute to pain in these patients, and outlines some of the treatment options that are currently used. The successful outcome of the first two trials of enzyme replacement therapy suggest that this disease might be amenable in the future to gene therapy.

Imidazolines stimulate release of insulin from RIN-5AH cells independently from imidazoline I1 and I2 receptors

The effect on insulin release of efaroxan, an alpha 2-adrenoceptor antagonist and a highly potent drug at imidazoline I1 receptors, and the effects of seven other imidazoline compounds selective for the imidazoline I1 or I2 receptors, were studied in the rat insulinoma cell line RIN-5AH. The cells released insulin in response to glucose (0.3-10 mM), and efaroxan (100 microM) potentiated glucose-induced insulin release. (-)-Adrenaline completely displaced the binding of [125I]p-iodoclonidine to membranes of RIN-5AH cells, indicating that these cells do not express imidazoline I1 receptors. Cirazoline and idazoxan (100 microM), both highly potent drugs at imidazoline I2 receptors, and the guanidines guanoxan and amiloride (200 microM), also promoted insulin release from RIN-5AH cells. Irreversible blockade of imidazoline I2 receptors with 10 microM clorgyline did not prevent the stimulatory effects of cirazoline or idazoxan; however, these compounds completely reversed the inhibition by diazoxide (250 microM), an opener of ATP-dependent K+ channels (K+ATP channels), of glucose-induced insulin release. These data indicate that the imidazoline/guanidine compounds promote insulin release from RIN-5AH cells, by interacting with a novel binding site related to K+ATP channels that does not represent any of the known imidazoline I1 or I2 receptors.

Prostacyclin analogues reduce ADP‐ribosylation of the α‐subunit of the regulatory Gs‐protein and diminish adenosine (A2) responsiveness of platelets

1 Prostacyclin and adenosine activate adenylate cyclase in human platelet membranes and inhibit platelet aggregation. Results are presented which show that prolonged incubation of platelets with iloprost (a stable prostacyclin analogue) results in a reduction in the capacity for adenylate cyclase activation by the adenosine analogue 5′‐(N‐ethyl)‐carboxamidoadenosine (NECA), NaF, guanyl‐5′‐yl imidodiphosphate or GTP. However, iloprost pretreatment resulted in no change in the binding of [3H]‐NECA to platelet membranes. 2 These results contrast with those obtained after pretreatment with 2‐chloroadenosine which revealed no change in NaF or guanyl‐5′‐yl imidodiphosphate sensitivity of adenylate cyclase. Pretreatment with 2‐chloroadenosine resulted in reduced NECA‐dependent adenylate cyclase activation, and loss of [3H]‐NECA binding sites. 3 The heterologous desensitization of adenosine A2‐receptors by iloprost is accompanied by a loss (greater than 80%) of a 45 kDa protein from the plasma membrane, as revealed by [32P]‐ADP‐ribosylation in the presence of cholera toxin. 4 It is proposed that this example of heterologous desensitization is mediated by elimination of Gsα, a subunit of the stimulatory guanyl nucleotide‐binding regulatory protein.

Infection by HIV‐1 blocked by binding of dextrin 2‐sulphate to the cell surface of activated human peripheral blood mononuclear cells and cultured T‐cells

1 Structural analogues of a sulphated polysaccharide, dextrin sulphate, were synthesized and tested for their ability to block infection by HIV‐1. Using the T‐cell lines, C8166 and HPB‐ALL, and the laboratory adapted strains of HIV‐1.MN, HIV‐1.IIIb and HIV‐1.RF, dextrin 2‐sulphate (D2S) combined the best combination of high anti‐HIV‐1 activity (95% inhibitory concentration (IC95) = 230 nM) and low anticoagulant activity. It also blocked infection of activated peripheral blood mononuclear (PBMN) cells by five primary viral isolates at an IC95 of 230–3700 nM depending upon the primary viral isolate tested. 2 In saturation binding studies, [3H]‐D2S bound to a cell surface protein on HPB‐ALL cells in a specific and saturable manner with a Kd of 82 ± 14 nM and a Bmax of 4.8 ± 0.3 pmol/106 cells. It bound to other human T‐cell lines in a similar manner. 3 There was very little binding of [3H]‐D2S to freshly isolated PBMN cells (Bmax 0.18 ± 0.03 pmol/106 cells) and these cells could not be infected by HIV‐1. Culture of PBMN cells in lymphocyte growth medium (LGM) containing IL‐2 did not significantly change the Bmax of [3H]‐D2S. In contrast, PBMN cells which had been cultured with phytohaemagglutinin (PHA; 5 μg ml−1) for 72 h had a Bmax of [3H]‐D2S binding of 7.2 ± 0.1 pmol/106 cells and these cells could be infected by HIV‐1. Removal of the PHA and further culture of the PBMN cells in LGM containing IL‐2 resulted in a fall in the Bmax to 2.0 ± 0.1 pmol/106 cells. The Kd of binding did not change significantly during the course of these experiments. 4 [3H]‐D2S did not bind to freshly isolated erythrocytes or to erythrocytes which had been cultured in PHA for 72 h. 5 These results suggest that there is a relationship between the expression of the [3H]‐D2S binding protein on the plasma membrane of PBMN cells and the susceptibility of these cells to infection by HIV‐1.

Differential sensitivities of the prostacyclin and nitric oxide biosynthetic pathways to cytosolic calcium in bovine aortic endothelial cells

1 Bovine aortic endothelial cells were cultured in vitro, and shown to release both prostacyclin (PGI2; Kact = 24.1 nm) and endothelium‐derived relaxing factor (EDRF, NO; Kact = 0.7 nm) in a concentration‐dependent manner when exposed to bradykinin. 2 The bradykinin‐dependent release of PGI2 (but not EDRF) was inhibited by 1 μm isoprenaline or 5 μm forskolin, and the inhibitory effect of isoprenaline could be reversed by the β2‐adrenoceptor antagonist, ICI 118551. In contrast, isoprenaline had no capacity to inhibit PGI2 release stimulated by exogenous arachidonic acid. 3 Exposure of cells to bradykinin increased the cytosolic concentration of Ca2+ ions ([Ca2+]i; Kact = 4.8 nm), and the effect was inhibited by both 1 μm isoprenaline and 5 μm forskolin. 4 In similar experiments, exposure of cells to ionomycin also increased [Ca2+]i and the values of [Ca2+]i were calibrated in terms of the ionomycin concentration. In subsequent experiments involving exposure of endothelial cells to selected concentrations of ionomycin, it was possible to show that the biosynthesis of NO was triggered at ionomycin concentrations about one tenth of that required for PGI2 biosynthesis and that these corresponded to a [Ca2+]i threshold of 350 nm for PGI2 release while that for EDRF release was less than 200 nm. 5 These differences in Ca2+ ion sensitivity explain the selective inhibition of bradykinin‐stimulated PGI2 biosynthesis (to the exclusion of NO biosynthesis) by isoprenaline or forskolin, both of which attenuate bradykinin‐dependent increases in [Ca2+]i.

Characterization of the thromboxane receptor mediating prostacyclin release from cultured endothelial cells

The thromboxane A2 (TXA2) mimetic, 9,11-dideoxy-11,9-epoxymethano-prostaglandin F 2 alpha (U46619), mobilized calcium in the bovine aortic endothelial cell line AG4762 and stimulated release of prostacyclin from these cells. The U46619-stimulated release of prostacyclin could be inhibited by TXA2 antagonists with the order of potency [Is-[1 less than a, 2 less than b(5z), 3 less than b, 4 less than a]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo- [2.2.1]hept-2-yl]-5- heptenoic acid (SQ29548) greater than 4-[2-(4-chlorobenzene-sulphonamido) ethyl]phenylacetic acid (BM13505) greater than 4-[2-(phenylsulphonamido)-ethyl]phenoxyacetic acid (BM13177), which was consistent with release being mediated by a TXA2 (TP) receptor. The TP receptor ligands, [3H]SQ29548 and 9,11-dimethylmethano-16(3-[125I]iodo-4-hydroxyphenyl)-13,14-dih ydr o-13-aza- 15-omega-o-tetranor-thromboxane ([125I]-PTA-OH), both appeared to bind to a homogenous population of sites in AG4762 cell membranes. The affinities of [3H]SQ29548 and [125I]PTA-OH were approximately 10 nM and approximately 0.3 nM, respectively, and the density of sites labelled by either ligand was approximately 25 fmol/mg protein. Under conditions where equilibrium was approached, the specific binding of [3H] SQ29548 or [125I]PTA-OH was displaced by SQ29548, BM13505 and BM13177 with the same order of potency and similar apparent affinities as in the functional assay, suggesting that these binding sites represent bona fide TP receptors.

PROSTACYCLIN‐DEPENDENT ACTIVATION OF ADENYLATE CYCLASE IN A NEURONAL SOMATIC CELL HYBRID: PROSTANOID STRUCTURE‐ACTIVITY RELATIONSHIPS

1 Prostacyclin activates adenylate cyclase of the NCB‐20 neuronal hybrid cell line. 2 There is a guanosine 5′‐triphosphate requirement for the activation of adenylate cyclase by 5,6β‐dihydroprostacyclin (a stable analogue of prostacyclin). 3 Steady‐state kinetic analysis of the activation of adenylate cyclase by 5,6β‐dihydroprostacyclin suggests a simple non‐cooperative bimolecular interaction between the ligand and a single receptor population. 4 Structure‐activity relationships of selected prostanoids elucidated certain functional requirements for activation of adenylate cyclase.

Inactivation of platelet‐derived growth factor‐BB following modification by ADP‐ribosyltransferase

Arginine‐specific ADP‐ribosyltransferase (ART1) is expressed on the surface of a number of cell types, and catalyses the transfer of ADP‐ribose from NAD+ to target proteins. We investigated whether extracellular proteins such as growth factors may serve as substrates for this enzyme, with subsequent alteration in their biological activity. Experiments were performed with rat skeletal muscle membranes and V79 Chinese hamster lung fibroblasts with doxycycline‐inducible expression of human ART. From a panel of growth factors, platelet‐derived growth factor‐BB (PDGF‐BB) was found to be the best substrate for ART1, whereas the structural homologue PDGF‐AA was not a substrate. Under conditions of maximum labelling 5 mol ADP‐ribose was incorporated per mol of PDGF‐BB. Purified (ADP‐ribosyl)‐PDGF‐BB did not stimulate a mitogenic or chemotactic response in human pulmonary smooth muscle cells, and showed a reduced capacity to bind to PDGF receptors in competition binding experiments, when compared to unmodified PDGF‐BB. PDGF‐dependent [3H‐methyl]‐thymidine incorporation was measured in the ART1‐transfected fibroblast cell line at physiological concentrations of PDGF‐BB, and without addition of extracellular NAD+. Fibroblasts expressing human ART1 at the cell surface showed reduced mitogenic responses to PDGF‐BB, but not to PDGF‐AA. This loss of mitogenic response in cells expressing ART1 activity was reversed by the addition of agmatine (an ART1 substrate). In conclusion, we propose that PDGF‐BB‐dependent signalling may be regulated by post‐translational modification of the growth factor by ART1 at the cell surface. This has been demonstrated in membranes of rat skeletal muscle, and the reaction confirmed in ART1‐transfected fibroblasts.