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Biomedical subjects

R L Dormer

Publications and source records attributed to R L Dormer.

At least 37 records · Page 2Linked to original sources
PubMed1992

Targeting aequorin to the endoplasmic reticulum of living cells.

The photoprotein aequorin has been engineered with an ER targeting sequence at the N-terminus, with and without KDEL at the C-terminus, so that it locates in the ER-secretory pathway. For the first time the free Ca2+ has been quantified inside the ER and shown to be 5-20 times that in the cytosol. In COS cells free Ca2+ in the ER ranged from 1-5mM at 37 degrees C, decreasing 2-5-fold within 1 min of exposure to the Ca2+ ionophore ionomycin in the absence of external Ca2+.

Aequorin↗
PubMed1992

An antibody against a CFTR-derived synthetic peptide, incorporated into living submandibular cells, inhibits beta-adrenergic stimulation of mucin secretion.

An antibody raised against a peptide in the first nucleotide-binding domain (NBD) of CFTR [1], incorporated into intact rat submandibular acini by hypotonic swelling, inhibited beta-adrenergic stimulated mucin secretion, without affecting cyclic AMP rise. The data are the first to show that a CFTR-antibody-containing cell results in defective stimulation of mucin secretion, as is seen in CF cells, and that this can be reversed by an excessive increase in cyclic AMP.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Engineering the CA(2+)-activated photoprotein aequorin with reduced affinity for calcium.

Two stage PCR has been used to introduce single amino acid substitutions into the EF hand structures of the Ca(2+)-activated photoprotein aequorin. Transcription of PCR products, followed by cell free translation of the mRNA, allowed characterisation of recombinant proteins in vitro. Substitution of D to A at position 119 produced an active photoprotein with a Ca2+ affinity reduced by a factor of 20 compared to the wild type recombinant aequorin. This recombinant protein will be suitable for measuring Ca2+ inside the endoplasmic reticulum, the mitochondria, endosomes and the outside of live cells.

Aequorin↗
PubMed1992

CFTR mechanism.

Explore the source record for details and available documents.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Abnormalities in intracellular regulation in cystic fibrosis.

Abnormalities in intracellular regulation in cystic fibrosis (CF) result from a given mutation in the cytoplasmic domain of CFTR, which renders it unable to respond correctly to agonists acting at the cell surface. This results in altered composition of epithelial secretions, which leads to some of the clinical manifestations of CF. Investigation of cyclic AMP- and Ca(2+)-mediated pathways controlling secretion is crucial for understanding how CFTR fails to respond to stimuli and how to reverse the defect in a CF cell. It should be feasible either to upregulate abnormal CFTR activity or to bypass the defect in the cell by stimulating a compensatory signalling pathway. Although their mechanism of action is unknown, one class of compounds, the methylxanthines, have been shown to reverse a fundamental CF abnormality in CF salivary cells and in non-epithelial cells overexpressing CFTR. This affords the exciting possibility that agents acting on intracellular signal transduction pathways will prove to be useful in devising new drug strategies for CF.

Cell Membrane↗
PubMed1991

Introduction of BAPTA into intact rat submandibular acini inhibits mucin secretion in response to cholinergic and beta-adrenergic agonists.

Incorporation of the calcium chelator BAPTA into isolated, intact rat submandibular acini by hypotonic swelling, resulted in complete inhibition of carbamylcholine, noradrenaline and isoproterenol stimulation of mucin secretion. No effects of intracellular BAPTA on cell viability or beta-adrenergic stimulation of cyclic AMP formation were observed. The data are the first to demonstrate that calcium is necessary for beta-adrenergic stimulation of secretion and suggest that Ca2+ provides a common link in the triggering of exocytosis.

Animals↗
PubMed1990

An altered phosphoprotein in cystic fibrosis.

Phosphorylation of calmodulin-dependent regulator proteins has been studied in control and CF submandibular glands. Results showed that a 61,000 molecular weight calmodulin-binding protein was less phosphorylated in CF glands than control (p less than 0.001 for difference). The altered calmodulin-binding protein cross-reacted with an antiserum against a known calmodulin-dependent protein phosphatase, calcineurin. An alteration in a protein with calcineurin-like activity in CF epithelial cells would provide a link between defective beta-adrenergic responses and altered Ca2(+)-mediated events in CF and thus might be directly related to the genetic defect.

Adolescent↗
PubMed1990

Dissociation between cyclic AMP rise and mucin secretion in response to a beta-adrenergic agonist.

A novel hypotonic swelling method has been used to introduce cyclic AMP phosphodiesterase (cAMP PDE) into intact rat submandibular acini. Acini swollen in the presence of cAMP PDE showed no cyclic AMP rise in response to the beta-agonist isoproterenol; however, stimulation of mucin secretion was not impaired. Control experiments showed that acini swollen in the absence of cAMP PDE showed normal cyclic AMP rise and mucin secretion responses. The results showing a dissociation between cyclic AMP rise and mucin secretion suggest that cyclic AMP is not the sole mediator of beta-adrenergic stimulation of epithelial cell secretion. The method of intracellular manipulation described will allow (i) investigation of key regulators of secretory events, shown to be defective in CF cells. (ii) determination of the biochemical function of proteins which are candidates for the biochemical defect in CF.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Defective phosphorylation of a calmodulin-binding protein in cystic-fibrosis submandibular glands.

Calmodulin-binding proteins in fractions purified from human submandibular glands by calmodulin-Sepharose were phosphorylated with [gamma-32P]ATP, in the absence of exogenous protein kinase. The major proteins phosphorylated had molecular masses of 45, 51 and 61 kDa. Phosphorylation was increased by activators of protein kinase C and inhibited by H-7. Phosphorylation of the 61 kDa band was markedly decreased in cystic-fibrosis submandibular glands.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Introduction of cyclic AMP phosphodiesterase into rat submandibular acini prevents isoproterenol-stimulated cyclic AMP rise without affecting mucin secretion.

Cyclic AMP phosphodiesterase has been incorporated into isolated rat submandibular acini by hypotonic swelling. This resulted in complete inhibition of the cyclic AMP rise stimulated by isoproterenol (10 microM), but had no effect on the stimulation of mucin secretion. Acini swollen in the absence of cyclic AMP phosphodiesterase showed similar cyclic AMP and mucin secretion responses to those of unswollen acini. The dissociation between cyclic AMP rise and mucin secretion was not due to stimulation of different beta-receptor subtypes since both responses to isoproterenol were inhibited by the beta 1 antagonist atenolol, but not by the beta 2 antagonist, butoxamine. The results are the first to directly demonstrate that a maximally effective concentration of isoproterenol can increase mucin secretion in the absence of a detectable increase in cyclic AMP.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Increased phosphoinositide breakdown by phospholipase C in erythrocyte membranes from patients with cystic fibrosis.

Phosphoinositide phospholipase C activity has been measured in erythrocyte membranes from age-matched control and CF subjects. Inositol phospholipids were labelled with [3H]myo-inositol and control experiments demonstrated that the [3H]-labelled products released by incubation of membranes with Ca2+ were derived specifically from erythrocytes (a) by purification of erythrocytes on cellulose columns, (b) by demonstration that the phospholipase C activity was inhibited by 10 mmol/l neomycin but not by 1 mmol/l p-methylsulphonylfluoride. The [3H]-labelled products were shown to be inositol phosphates by their elution from anion-exchange columns. Membranes from CF patients showed increased phospholipase C activity compared to controls which did not correlate with the degree of [3H]inositol labelling of the membranes, with pancreatic function as assessed by serum immunoreactive trypsin or with medications taken by the patients.

Adolescent↗
PubMed1989

Formation of inositol polyphosphates in cultured human sweat duct cells in response to cholinergic stimulation.

Inositol phosphate formation in response to cholinergic stimulation was studied in cultured human sweat duct cells, prelabelled with myo-[2-3H]inositol. Formation of inositol mono-, bis-, tris- and tetrakisphosphates was increased after 15 min stimulation by 30 microM carbachol. Formation of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate was significantly increased within 1 min at carbachol concentrations between 10 microM and 100 microM. No detectable increase in inositol 1,4,5-trisphosphate formation was observed at 15 s or 1 min, but an increase was observed after 15 min at a carbachol concentration of 30-100 microM. The data are consistent with an involvement of inositol polyphosphates in the biphasic response of ion transport, to cholinergic stimulation in these cells (see Pederson, P.S. (1986) 6th Professional Conference "Broken Arrow 1986". Genetic and Eptihelial Dysfunction in Cystic Fibrosis (Riordan, J.R. and Buchwalds, M., eds.), Alan Liss, New York and Pedersen, P.S. (1987) Med. Sci. Res. 15, 769-770) and suggest a different pattern of metabolism from exocrine acinar cells.

Calcium↗