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Biomedical subjects

L Best

Publications and source records attributed to L Best.

At least 91 records · Page 5Linked to original sources
PubMed1984

Localization of the galactoside binding site in the lactose carrier of Escherichia coli.

The location of flurophores specifically bound to the lactose/H+ carrier of Escherichia coli was ascertained by the use of various collisional quenchers. The reporter groups were (1) the pyrenyl residue of N-(1-pyrenyl)maleimide attached to the essential cysteine residue 148, which is presumably at or near the galactoside binding site, and (2) the dansyl moieties of a series of fluorescent substrate molecules. The accessibility of these fluorophores from the lipid phase was assessed by nitroxyl-labelled fatty acids and phospholipids. By using a series of nitroxyl-labelled fatty acids carrying the quencher at different positions in the acyl chain, the position of a quenchable fluorophore with respect to the membrane normal can be determined. The accessibility of fluophores from the aqueous phase was assessed by using a water-soluble quencher, the N-methylpicolinium ion. The results of quenching studies suggest that the galactoside binding site is located within the carrier and that this binding site communicates with the aqueous phase through a pore.

Affinity Labels↗
PubMed1984

Inhibition by mepacrine and p-bromophenacylbromide of phosphoinositide hydrolysis, glucose oxidation, calcium uptake and insulin release in rat pancreatic islets.

Mepacrine and p-bromophenacylbromide were both found to impair 3H-inositol phosphate production in response to both nutrient and hormone-neurotransmitter stimuli in islets prelabelled with 3H-inositol. Both drugs also inhibited net 45Ca uptake in response to glucose or glibenclamide and considerably modified the patterns of 45Ca and 86Rb efflux from perifused islets under both basal and glucose-stimulated conditions. In addition, the oxidation of [U-14C] glucose in islets was impaired by either mepacrine or p-bromophenacylbromide. These inhibitory effects were found to be concentration-related for both mepacrine (0.01-1.0 mM) and p-bromophenacylbromide (0.03-0.3 mM) and were accompanied, in general, by a similar degree of inhibition of insulin secretion. These results suggest that both mepacrine and p-bromophenacylbromide can inhibit phospholipase C activity in intact islets, but also impair 45Ca and 86Rb fluxes and oxidation of nutrients. The diversity of these drugs' inhibitory actions makes them unsuitable tools for examining the role of specific cellular processes in the regulation of islet function.

Acetophenones↗
PubMed1984

Impairment of insulin release by methylation inhibitors.

The possible participation of enzymatic methylation reactions in the process of insulin release was investigated in rat pancreatic islets. The combination of 3-deazaadenosine and DL-homocysteine impaired the incorporation of 3H-methyl from L-[methyl-3H]methionine into endogenous islet proteins and phospholipids, but failed to affect turnover in the phosphatidylinositol cycle. The inhibitors of methylation decreased insulin release evoked by D-glucose or the combinations of D-glucose and gliclazide, L-leucine and L-glutamine, or Ba2+ and theophylline. The inhibitors of methylation did not impair either the oxidation of D-glucose or affect its capacity to decrease K+ conductance, stimulate Ca2+ inflow and provoke 45Ca accumulation in pancreatic islets. It is proposed that, in the process of insulin secretion, a methyl acceptor protein and/or phospholipid play(s) a limited modulatory role in the coupling of cytosolic Ca2+ accumulation to exocytosis.

Animals↗
PubMed1984

Activation of protein kinase C by a tumor-promoting phorbol ester in pancreatic islets.

Rat pancreatic islet homogenates display protein kinase C activity. This phospholipid-dependent and calcium-sensitive enzyme is activated by diacylglycerol or the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In the presence of TPA, the Ka for Ca2+ is close to 5 microM. TPA does not affect phosphoinositide turnover but stimulates [32P]- and [3H]choline-labelling of phosphatidylcholine in intact islets. Exogenous phospholipase C stimulates insulin release, in a sustained and glucose-independent fashion. The secretory response to phospholipase C persists in media deprived of CaCl2. It is proposed that protein kinase C participates in the coupling of stimulus recognition to insulin release evoked by TPA, phospholipase C and, possibly, those secretatogues causing phosphoinositide breakdown in pancreatic islets.

Animals↗
PubMed1984

The stimulus-secretion coupling of amino acid-induced insulin release: metabolism of L-asparagine in pancreatic islets.

The metabolism of L-asparagine in pancreatic islets was investigated. The deamidation of L-asparagine and the conversion of aspartate to oxalacetate, by transamination, may occur in both the cytosol and mitochondria. Oxalacetate is then converted to pyruvate in part via phosphoenolpyruvate and in part via malate. The latter modality, by consuming NADH and generating NADPH, may lead to changes in the redox state of the cytosolic NADH/NAD+ and NADPH/NADP+ couples. Such changes may in turn account, in part at least, for the capacity of L-asparagine to augment insulin release induced by certain nutrient secretagogues.

Acetyl Coenzyme A↗
PubMed1984

Enhanced de novo synthesis of phosphatidic acid and phosphatidylinositol in rat pancreatic islets exposed to nutrient or neurotransmitter stimuli.

A stimulation of [3H]glycerol incorporation into phosphatidic acid and phosphatidylinositol was observed upon exposure of rat pancreatic islets to the nutrient secretagogues alpha-ketoisocaproate and glucose, or to the neurotransmitter stimuli carbamylcholine and cholecystokinin. These effects were associated with reduced labeling of phosphatidylcholine and, in some cases, phosphatidylethanolamine. The modified patterns of [3H]glycerol incorporation into islet phospholipids persisted in the absence of added Ca2+, but were abolished by excess EDTA. Nutrient, but not neurotransmitter, secretagogues also stimulated the incorporation of [3H]glycerol into triacylglycerols. The results suggest that the stimulation of islets with the above classes of secretagogues is accompanied by enhanced de novo synthesis of acidic phospholipids.

Animals↗
PubMed1984

Effects of ionophore A23187 and Ba++ ions on labelling of phospholipids in rat pancreatic islets.

Ionophore A23187, either in the presence or absence of added Ca2+ or Mg2+, caused a marked accumulation of [32P]-phosphatidic acid in pancreatic islets pre-labelled with 32 Pi. A similar effect was observed following the addition of 4 mM Ba2+ ions in the absence of added Ca2+. Neither agent caused a significant modification of labelling in other lipid fractions, although there was a persistent trend towards reduced labelling of phosphatidylcholine and phosphatidylethanolamine. Ionophore A23187 also potentiated the incorporation of 3H-glycerol into phosphatidic acid and reduced the incorporation of this precursor into phosphatidylcholine. In islets pre-labelled with 3H-glycerol and subsequently exposed to A23187 or Ba2+, no significant changes were observed in label associated with either phospholipids or neutral glycerolipids. These results suggest that ionophore A23187 and Ba2+ ions can divert the synthesis of phospholipids resulting in increased formation of phosphatidic acid at the expense of non-acidic phospholipids, principally phosphatidylcholine. We tentatively suggest that this effect may be the result of inhibition by Ca2+ of the breakdown of phosphatidic acid to diglyceride, an enzymic step which may regulate the relative amounts of acidic and neutral phospholipids.

Animals↗
PubMed1984

Nutrient and hormone-neurotransmitter stimuli induce hydrolysis of polyphosphoinositides in rat pancreatic islets.

Preincubation of rat pancreatic islets with 3H-inositol, and subsequent exposure, in the presence of LiCl, to either glucose or carbamylcholine resulted in a rapid stimulation of 3H-inositol 1,4,5-triphosphate and 3H-myo-inositol 1,4-bisphosphate formation, the level of which reached a plateau after about 5 min of stimulation. Both stimuli also caused an approximately linear accumulation of 3H-myo-inositol 1-phosphate. The amounts of 3H-inositol phosphates formed were dependent on the concentration of LiCl. Studies of 32P-labeling of islet ATP, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and phosphatidylinositol 4-phosphate revealed that these approached isotopic equilibrium after about 240-min incubation, whereas 32P-labeling of phosphatidylinositol, phosphatidic acid, phosphatidylcholine, and phosphatidylethanolamine proceeded at a lower rate. Carbamylcholine provoked an immediate fall in 32P-PtdIns(4,5)P2 and, to a lesser extent, 32P-phosphatidylinositol 4-phosphate. Glucose caused a similar response although, in this case, the most marked decline was in a more polar 32P-labeled lipid. Cholecystokinin-pancreozymin was also found to induce 32P-PtdIns(4,5)P2 hydrolysis, although the ionophore A23187 was without effect. Both carbamylcholine and glucose induced an increase in 32P-phosphatidic acid. The results provide two independent pieces of evidence suggesting that phospholipase C-mediated hydrolysis of polyphosphoinositides occurs as an early response in rat islets to either nutrient or neurotransmitter secretagogues.

Adenosine Triphosphate↗
PubMed1984

Does glucose affect phospholipase A2 activity in pancreatic islets?

Rat pancreatic islet homogenates catalyzed the production of labelled oleate and lysophosphatidylcholine from 1-palmitoyl-2-[1-14C] oleoylphosphatidylcholine and di[1-14C] stearoylphosphatidylcholine, respectively. The phospholipase A2 was activated by Ca2+ (Ka approximately 0.5 mM) and inhibited by mepacrine. No difference in enzymic activity was found in glucose-deprived and glucose-stimulated islets, respectively.

Animals↗
PubMed1983

Stimulation of phosphoinositide breakdown in rat pancreatic islets by glucose and carbamylcholine.

In the presence of Li+, glucose, 2-ketoisocaproate and carbamylcholine induced the rapid formation of 3H-inositol phosphates in rat pancreatic islets prelabelled with 3H-inositol. The production of labelled inositol phosphates continued up to 20 min of incubation. Glibenclamide and ionophore A23187 had no significant effect on labelled inositol phosphate production. The effects of carbamylcholine and to a lesser extent, glucose were found to persist in the absence of added Ca2+, but both were strongly inhibited by excess EGTA. In general, the rise in 3H-inositol phosphate production was associated with a fall in lipid bound radioactivity, although the latter was found to occur more slowly, and was of a smaller magnitude than labelled inositol phosphate formation. The results suggest that nutrient secretagogues and cholinergic agonists stimulate hydrolysis of phosphoinositides in pancreatic islets by a phospholipase C mechanism. This effect is Ca2+-dependent, but probably not triggered by increased Ca2+ uptake into the islet.

Animals↗
PubMed1983

The stimulus-secretion coupling of glucose-induced insulin release: fuel metabolism in islets deprived of exogenous nutrient.

The fuel hypothesis for insulin release postulates that the secretory response to nutrient secretagogues reflects their capacity to augment catabolic fluxes in pancreatic islet cells. Hence, both the oxidation of exogenous nutrients and their effect upon the metabolism of endogenous nutrients should be taken into consideration to account for the stimulation of insulin release. In the present work, an attempt was made to quantify the respective contribution of carbohydrates, fatty acids, and amino acids in the respiration of islets deprived of exogenous nutrient. The metabolism of glycerol, fatty acids, and amino acids was found to account for the major part of the basal respiratory rate. Glucose modestly decreased the oxidation of endogenous fatty acids, lowered the production of NH4+, but did not impair the oxidative catabolism of 2-keto acids derived from endogenous amino acids. These findings suggest that the catabolism of noncarbohydrate nutrients largely contributes to the respiration of the islets, even when the latter are exposed to circulating glucose in its physiological concentration.

Amino Acids↗
PubMed1983

Phosphatidylinositol and phosphatidic acid metabolism in rat pancreatic islets in response to neurotransmitter and hormonal stimuli.

Carbamylcholine produced a concentration-dependent stimulation of labelling of phosphatidylinositol and phosphatidic acid in rat islets of Langerhans following preincubation with 32PO43(-). The time course of these effects suggested that the initial action of carbamylcholine was to stimulate phosphatidic acid production, presumably by causing hydrolysis of phosphatidylinositol. This conclusion was substantiated by experiments in which islet phospholipids were pre-labelled with [3H]arachidonic acid. Under these conditions, carbamylcholine caused a loss of radioactivity from phosphatidylinositol, together with an increase in labelling of phosphatidic acid. The effects of carbamylcholine on islet phospholipid labelling were not dependent upon the presence of added Ca2+, but were abolished by EDTA and by atropine. An apparent stimulation of phosphatidylinositol and phosphatidic acid metabolism was also induced by cholecystokinin-pancreozymin, whereas glucagon, arginine, glibenclamide and thyrotropin had no significant effect. The data suggest that enhanced activity of the so-called phosphatidylinositol cycle may be an important event in regulating secretory activity of islets in response to certain neurotransmitter and hormonal stimuli. Furthermore, the results are compatible with the hypothesis that increased phospholipid metabolism may play a role in the modulation of ionic fluxes during stimulation by such agents.

Animals↗
PubMed1983

Effects of nutrient secretagogues upon phospholipid metabolism in rat pancreatic islets.

Rat pancreatic islets labelled with [32P]Pi were used to investigate the effects of nutrient secretagogues upon phospholipid metabolism. Stimulatory concentrations of glucose, 4-methyl-2-oxopentanoate, 3-phenylpyruvate and 2-endo-aminonorbornane-2-carboxylic acid caused a marked and specific stimulation of labelling with [32P]Pi of phosphatidylinositol, phosphatidic acid and the polyphosphoinositides. The effects of glucose were concentration-related and were inhibited by mannoheptulose and menadione. Enhanced phospholipid labelling persisted in the absence of added calcium ions but was abolished by excess EDTA. The time-course of labelling of these phospholipids in response to glucose contrasted with that previously observed using carbamylcholine in that the accumulation of radioactivity in phosphatidic acid and in phosphatidylinositol occurred in parallel. We conclude that glucose, in common with other nutrient secretagogues or those which promote the metabolism of endogenous nutrients, causes a marked stimulation of turnover in the phosphatidylinositol cycle. This effect requires the integrity of nutrient catabolism and is probably not dependent upon the stimulation of Ca2+ uptake from the extracellular medium.

Adenosine Triphosphate↗
PubMed1982

Unifying description of the effect of membrane proteins on lipid order. Verification for the melittin/dimyristoylphosphatidylcholine system.

The effect of melittin on lipid order in dimyristoylphosphatidylcholine bilayers is investigated by means of Raman spectroscopy and fluorescence anisotropy using diphenylhexatriene as fluorescence probe. In the fluid lipid phase, the Raman results indicate a slight increase in the conformational order of the lipid chains, and the fluorescence anisotropy results indicate a considerable increase in the rigid-body orientational order of the lipid chains. These results are contrasted with the reported decrease of the deuterium magnetic resonance order parameter. A consistent interpretation of the complete set of experimental data is presented according to which proteins induce a tilt of the preferred axes of lipid orientation and increase the orientational order with respect to these axes. The values of the tilt angle and the orientational order parameter at the surface of proteins are determined from the experimental data within a continuum model of lipid-protein interaction. The same values are obtained for melittin, Ca/Mg-ATPase, and cytochrome c oxidase, suggesting that different membrane proteins affect the lipid order in the same way.

Bee Venoms↗
PubMed1982

Phosphoinositide metabolism in human blood platelets: effects of two types of divalent cation chelators.

Phospholipid metabolism was investigated in platelets in relation to secretion of 5-hydroxytryptamine. Both thrombin and the ionophore, A23187, induced secretion of 5-hydroxytryptamine and platelet aggregation accompanied by the specific breakdown of phosphatidyl inositol. The products detected corresponded to phosphatidic acid and a substance which co-chromatographed with polyphosphoinositides and lysophosphatidyl inositol. Thrombin-induced aggregation, secretion and phosphatidyl inositol metabolism were all prevented by EDTA, but only the secretory response was inhibited by the intracellular calcium chelator chlortetracycline (CTC). In contrast, A23187 induced turnover of phosphatidyl inositol in the presence of EDTA even though aggregation and secretion were prevented. All three responses to A23187 were inhibited by chlortetracycline. Thus, platelet aggregation, secretion and phospholipid metabolism can be dissociated by the use of two types of divalent cation chelators: EDTA and CTC. Furthermore, these findings suggest that thrombin and A23187 induce phosphoinositide metabolism by two distinct mechanisms.

Blood Platelets↗