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Biomedical subjects

S J Ashcroft

Publications and source records attributed to S J Ashcroft.

At least 37 records · Page 2Linked to original sources
PubMed1997

Superoxide dismutase activity in lymphocytes and polymorphonuclear cells of diabetic patients.

Copper-zinc superoxide dismutase (Cu,Zn-superoxide dismutase) activity was evaluated in lymphocytes and polymorphonuclear cells of insulin-dependent (n = 33) and non-insulin-dependent (n = 34) diabetic patients. A commercial method for the measurement of superoxide dismutase activity was adapted for use on a discrete analyser and evaluated for interference by other antioxidants with superoxide anion-scavenging properties. In comparison to healthy control subjects (n = 32), a significantly lower Cu,Zn-superoxide dismutase activity was found in both lymphocytes and polymorphonuclear cells of insulin-dependent (2.08 +/- 0.58 vs. 1.70 +/- 0.46 U/mg protein, p < 0.05, and 1.06 +/- 0.46 vs. 0.64 +/- 0.40 U/mg protein, p < 0.001, respectively) and non-insulin-dependent diabetic patients (2.08 +/- 0.58 vs. 1.61 +/- 0.48 U/mg protein, p < 0.01, and 1.06 +/- 0.46 vs. 0.53 +/- 0.24 U/mg protein, p < 0.001, respectively). There was a week, but significant negative correlation between age and Cu,Zn-superoxide dismutase activity in lymphocytes and polymorphonuclear cells (r = -0.22 and r = -0.28, p < 0.05, respectively), whereas no influence of gender, diabetes duration and glycaemic control was observed. The results indicate that diabetes mellitus could elicit a significant disturbance in superoxide anion-scavenging potential of lymphocytes and polymorphonuclear cells.

Adult↗
PubMed1996

Isoforms of endoplasmic reticulum Ca(2+)-ATPase are differentially expressed in normal and diabetic islets of Langerhans.

Glucose-dependent sequestration of Ca2+ into endoplasmic reticulum and its subsequent release play an important role in the control of intracellular Ca2+ concentration, which regulates insulin secretion in pancreatic beta-cells. The active uptake of cytosolic Ca2+ into endoplasmic reticulum is mediated by sarco-(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs). We found, using RT-PCR with isoform-specific primers, that SERCA 2 and SERCA 3 mRNAs are co-expressed in human and rat islets of Langerhans and in the RINm5F beta-cell line. Immunochemical analysis also revealed the existence of two SERCA proteins with molecular masses of 110 and 115 kDa in beta-cell membranes. The 115 kDa protein was identified as SERCA 2b by its reaction with an isoform-specific antibody and the 110 kDa protein most probably corresponds to SERCA 3. The presence of two functionally different SERCA isoforms raises the possibility that they are located in distinct Ca2+ stores. There is evidence that altered Ca2+ handling in the beta-cell may contribute to the decreased insulin secretion seen in non-insulin dependent diabetes mellitus (NIDDM). We therefore investigated SERCA 2 and SERCA 3 mRNA expression by quantitative RT-PCR in islets prepared from Goto-Kakizaki (GK) rats, a non-obese spontaneous model of NIDDM. We found a significant reduction (about 68%) in SERCA 3 isoform expression. Since SERCA 2 expression was not significantly reduced, these genes are independently regulated and probably play distinct roles in islets of Langerhans. The marked decrease of SERCA 3 expression may constitute a defect in Ca2+ signalling in GK rat islets which could be a component of NIDDM.

Animals↗
PubMed1996

Protein kinase C in beta-cells: expression of multiple isoforms and involvement in cholinergic stimulation of insulin secretion.

The mammalian protein kinase C (PKC) family consists of at least 11 distinct isotypes with marked differences in tissue distribution, localization, cofactor dependence and substrate specificity. Evidence exists for the expression of some of the PKC isoforms in pancreatic beta-cells but no comprehensive analysis of all the known PKC types has been accomplished. To assess the functional relevance of phosphorylation by PKC in the mechanism of insulin secretion we firstly investigated the expression of PKC isoforms in pancreatic beta-cells. The combination of reverse transcription-polymerase chain reaction (RT-PCR), Northern analysis and immunoblotting demonstrated the expression of PKC-alpha, beta II, epsilon, zeta, lambda and mu in MIN6 beta-cells. PKC-mu has not previously been detected in beta-cells. Expression of PKC-delta was also observed at the mRNA level; however, the protein could not be detected by Western blotting in MIN6 cells but was readily observed in RINm5F beta-cells. In short-term incubations, insulin release from MIN6 cells was augmented by 12-0-tetradecanoyl-phorbol-13-acetate (TPA), by carbachol, and by 40 mM K+. Culture of MIN6 cells overnight with TPA resulted in down-regulation of PKC-alpha (totally) and epsilon (partially), without significant change in the other isoforms. In such TPA-treated cells, the secretory response to TPA and to carbachol was abolished but not that elicited by high K+. It is suggested that PKC-alpha and/or epsilon may play a role in cholinergic potentiation of insulin secretion.

Animals↗
PubMed1996

A unique combination of plasma membrane Ca2+-ATPase isoforms is expressed in islets of Langerhans and pancreatic beta-cell lines.

Changes in free intracellular Ca2+ concentration regulate insulin secretion from pancreatic beta-cells. The existence of steep Ca2+ gradients within the beta-cell requires the presence of specialized Ca2+ exclusion systems. In this study we have characterized the plasma membrane Ca2+-ATPases (PMCAs) which extrude Ca2+ from the cytoplasm. PMCA isoform- and subtype-specific mRNA expression was investigated in rodent pancreatic alpha- and beta-cell lines, and in human and rat islets of Langerhans using reverse-transcription PCR with primers flanking the calmodulin-binding region of rat PMCA. The expression pattern of PMCA 1 and 2 was conserved in different species and islet-cell types since both rat and human islets of Langerhans and all cell lines tested contained the 1b and 2b forms. PMCA 4 isoform subtypes, however, were expressed in a cell-type-specific manner since beta-cells expressed PMCA 4b only, whereas in islets of Langerhans, which contain alpha, beta, delta and polypeptide-secreting cells, PMCA 4a and 4b were simultaneously present. No evidence was obtained for the expression of PMCA 3. Characterization of the beta-cell Ca2+-pump protein showed that it shared several similarities with the erythrocyte PMCA. It is a P-type ATPase; its phosphorylated intermediate was stabilized by La3+; it reacted with a PMCA-specific antibody; and it was not N-glycosylate. However, the beta-cell PMCA had a higher molecular mass than that of the erythrocyte; this difference could be explained by either predominant translation of the PMCA2 form, which has a molecular mass 3-8 kDa higher than the erythrocyte PMCA 1 and 4 proteins, or by a possible sequence insertion. Thus a unique combination of functionally distinct PMCA isoforms (1b, 2b, 4b) participates in Ca2+ homoeostasis in the beta-cell.

Alternative Splicing↗
PubMed1996

Promiscuous coupling between the sulphonylurea receptor and inwardly rectifying potassium channels.

Sulphonylureas are a class of drugs widely used to treat non-insulin-dependent diabetes mellitus. These drugs act by binding to a sulphonylurea receptor (SUR) in the pancreatic beta-cell membrane which inhibits an ATP-sensitive potassium (K-ATP) channel and thereby stimulates insulin secretion. There has been much debate as to whether SUR and the K-ATP channel are the same or separate proteins, whether SUR confers ATP-sensitivity on an ATP-insensitive pore-forming subunit, and whether sulphonylureas can also modulate other types of K-channel. We show here that SUR itself does not possess intrinsic channel activity but that it endows sulphonylurea sensitivity on several types of inwardly-rectifying K-channels. It does not necessarily confer ATP-sensitivity on these channels.

ATP-Binding Cassette Transporters↗
PubMed1995

Identification of functional ionotropic glutamate receptor proteins in pancreatic beta-cells and in islets of Langerhans.

The presence of ionotropic glutamate receptor proteins in islets of Langerhans and pancreatic beta-cell lines (MIN6, HIT T15, RINm5F) was investigated. For this purpose immunoblot analysis of beta-cell membranes was performed with subunit-specific antibodies. We identified NMDAR1 subunits of the NMDA and KA-2 subunits of the kainate receptors, but did not detect GluR1 subunits of the AMPA receptor. The receptor subunits present were shown to be glycosylated. beta-cell membranes contained specific binding sites for glutamate receptor ligands, and NMDA increased insulin secretion. These results demonstrate that ionotropic glutamate receptor proteins, similar to those in the central nervous system, are expressed in rat pancreatic beta-cells.

Amino Acid Sequence↗
PubMed1995

Characterisation of endoplasmic reticulum and plasma membrane Ca(2+)-ATPases in pancreatic beta-cells and in islets of Langerhans.

We have investigated the plasma membrane (PMCA) and endoplasmic reticulum (SERCA) Ca(2+)-ATPases involved in active transport of Ca2+ in pancreatic beta-cell lines (MIN6, HIT T15, RINm5F) and in islets of Langerhans. Under selective membrane phosphorylation conditions (at low ATP concentration, in the presence of Ca2+ and La3+ and in the absence of Mg2+ at 4 degrees C) the only labelled proteins are the phosphoenzyme intermediates of the Ca(2+)-ATPases. Under these conditions, beta-cell membranes incorporated 32P from [gamma-32P]ATP into two proteins with molecular mass on acidic SDS-polyacrylamide gels of around 115 and 150 kDa. The 150 kDa band was identified as PMCA (i) by reaction with a monoclonal anti-human erythrocyte plasma membrane Ca(2+)-ATPase antibody; (ii) by its typical tryptic cleavage pattern which generated an 80 kDa band; (iii) by lack of inhibition of its autophosphorylation by SERCA-specific inhibitors. The 115 kDa band was identified as SERCA (i) by reaction with a polyclonal anti-rat fast skeletal muscle Ca(2+)-ATPase antibody; (ii) by the concentration-dependent inhibition of its autophosphorylation by thapsigargin and 2,5-di(t-butyl)-1,4-benzohydroquinone (tBHQ), which are specific inhibitors of SERCA. The 115 kDa band was further characterised as the SERCA-2b isoform by reaction with a polyclonal rabbit antibody against the 12 C-terminal amino acids of SERCA-2b.

Animals↗
PubMed1995

A novel pancreatic beta-cell isoform of calcium/calmodulin-dependent protein kinase II (beta 3 isoform) contains a proline-rich tandem repeat in the association domain.

There is evidence for a role for calcium/calmodulin-dependent protein phosphorylation in regulation of insulin secretion but the molecular nature of the kinase(s) responsible is unknown. In this study, the screening of a neonatal rat islet cDNA library resulted in the isolation of a 2 kb clone that was 99% homologous to the beta' isoform of calcium/calmodulin-dependent protein kinase II. The predicted 589 amino acid sequence with a calculated mass of 64,976 Da contained a 24 amino acid deletion in addition to the 15 amino acid deletion that differentiates the beta' from the beta isoform, and included an 86 amino acid novel domain consisting of a tandem repeat of proline-rich residues. The expression of this new isoform of calcium/calmodulin-dependent protein kinase II (beta 3) was confirmed in beta-cell lines and testis by DNA amplification of the sequence encoding the inserted domain by reverse transcriptase-polymerase chain reaction, followed by Southern analysis.

Alternative Splicing↗
PubMed1994

Response to glucose of transcription factor binding to the rat insulin-1 gene promoter.

The mechanism by which glucose stimulates insulin gene expression has been investigated by studying the binding of nuclear proteins to a putative glucose-sensitive element (GSE) in the rat insulin-I gene promoter. Gel retardation assays showed that a specific binding activity was present in four different beta-cell lines. The binding activity was increased by glucose only in those beta-cell lines which were shown to retain glucose-regulated insulin gene transcription. However, a similar binding activity was also shown to be present in an alpha-cell line. The protein factor binding to the GSE was estimated to have a molecular weight of 27kD. This protein may play a pivotal role in glucose-regulated transcription of the insulin gene.

Animals↗
PubMed1994

Determination of the molecular mass of the native beta-cell sulfonylurea receptor.

In the present study we have determined the molecular mass of the beta-cell sulfonylurea receptor in its native form by two different experimental approaches; gel filtration chromatography and radiation inactivation analysis. We first confirmed that the denatured photolabelled MIN6 beta-cell receptor had a molecular size of 141 +/- 2 kDa (mean +/- S.E., n = 8). Under non-denaturing conditions, using gel filtration chromatography, apparent molecular masses of 166 +/- 1 kDa (mean +/- S.E., n = 3) and 182 +/- 5 kDa (mean +/- S.E., n = 4) were determined for the photoaffinity-labelled and unlabelled sulfonylurea receptor, respectively. We conclude that in the solubilized state the receptor exists as a monomer. Radiation inactivation analysis indicated that the receptor has a target size of 250 +/- 30 kDa (mean +/- S.E., n = 7). This value for the molecular mass is larger than that obtained from SDS-PAGE following photolabelling of the receptor (141 kDa) suggesting that the beta-cell sulfonylurea receptor is composed of more than one subunit in the native membrane.

ATP-Binding Cassette Transporters↗
PubMed1994

Protein phosphorylation and beta-cell function.

The central role of reversible protein phosphorylation in regulation of beta-cell function is reviewed and the properties of the protein kinases so far defined in beta cells are summarised. The key effect of Ca2+ to initiate insulin secretion involves activation of a Ca2+/calmodulin-dependent protein kinase. Potentiation of secretion by agents activating protein kinase A or C appears to involve an increase in the sensitivity of the secretory system to intracellular Ca2+. The effects of MgATP on the binding of [3H]-glibenclamide to the beta-cell sulphonylurea receptor suggest that the properties of this receptor, which controls the activity of ATP-sensitive K-channels, are modulated by phosphorylation. The identity of the kinases and phosphatases responsible is not known but the presence in beta-cell membranes of various kinases not dependent on Ca2+ or cyclic AMP, and including tyrosine kinase, is documented, together with the presence of both Ca(2+)-dependent and Ca(2+)-independent protein phosphatases. Protein phosphorylation is also involved in regulation of beta-cell Ca2+ fluxes and evidence is presented that protein kinase C activation inhibits Ca2+ signalling by reducing influx of Ca2+ into the beta cell. The identity of the Ca2+/calmodulin-dependent protein kinase activity in beta cells is discussed. Comparison of its properties towards substrates and inhibitors with those of brain Ca2+/calmodulin-dependent protein kinase II suggests that the beta-cell enzyme may be similar or identical to the brain enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-Binding Cassette Transporters↗
PubMed1994

Differential expression of the inwardly-rectifying K-channel ROMK1 in rat brain.

We have investigated the distribution of the inwardly-rectifying K-channel ROMK1 in rat brain using Northern blotting and in situ hybridization. High levels of expression were found in cortex and hippocampus but not in substantia nigra, ventromedial hypothalamus, cerebellum or striatum. PCR primers generated against ROMK1 amplified a 300 bp cDNA fragment from cortex and hippocampus identical in sequence to ROMK1.

Animals↗
PubMed1993

Characterization of Ca2+/calmodulin-dependent protein kinase in rat pancreatic islets.

We have attempted to identify islet Ca2+/calmodulin-dependent protein kinase (CaM kinase) by comparing its activity with purified brain CaM kinase II. Islet CaM kinase, in the presence of calmodulin and Ca2+, phosphorylated major endogenous substrates of 102, 57 and 53 kDa and also exogenous glycogen synthase; brain CaM kinase II phosphorylated glycogen synthase and peptides of 57 and 53 kDa. Alloxan (1 mM) inhibited the phosphorylation of glycogen synthase and the 102, 57 and 53 kDa islet peptides by islet CaM kinase; the phosphorylation of glycogen synthase and the 57 and 53 kDa substrates by brain CaM kinase II was also inhibited by alloxan. The Ca2+ and calmodulin-dependencies of phosphorylation of the endogenous islet substrates differed. In the presence of 400 nM calmodulin, half-maximal phosphorylation was attained at Ca2+ concentrations of 80 +/- 9, 401 +/- 61 and 459 +/- 59 nM for the 102, 57 and 53 kDa substrates respectively. In the presence of 10 microM Ca2+, half-maximal phosphorylation was attained at calmodulin concentrations of 9 +/- 2, 38 +/- 2.5 and 37 +/- 2 nM for the 102, 57 and 53 kDa substrates respectively. Differential centrifugation located the 102 kDa substrate in the post-100,000 g supernatant and the 57 and 53 kDa substrates in the particulate fraction. These data suggest that islet CaM kinase is similar to, if not identical with, brain CaM kinase II, but that phosphorylation of the endogenous 102 kDa substrate occurs by a distinct kinase which shows different sensitivities to Ca2+ and calmodulin. This kinase probably corresponds to CaM kinase III and the 102 kDa peptide to elongation factor 2 (EF-2), since the 102 kDa peptide was shown to undergo ADP-ribosylation in the presence of diphtheria toxin and NAD+.

Adenosine Diphosphate↗