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Biomedical subjects

N Fleming

Publications and source records attributed to N Fleming.

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

Regulation of the cAMP signal transduction pathway by protein kinase C in rat submandibular cells.

Treatment of rat submandibular acinar cell extracts with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused the dose-dependent activation of protein kinase C (PKC), assessed by the phosphorylation of a novel and highly specific substrate. This effect was duplicated by a diacylglycerol, but not by the 4 alpha-phorbol ester 4 alpha-phorbol 12,13-didecanoate. The TPA elevation of PKC was blocked by the PKC inhibitors H-7 and sangivamycin. In intact cells, TPA caused the translocation of PKC from cytosol to membrane, consistent with its known mode of activation. The beta-adrenergic agonist, isoproterenol, stimulated cAMP levels which were significantly reduced by preactivation of PKC. This inhibitory PKC effect was reversed by H-7. When cAMP was stimulated at the post-receptor level, however, by forskolin, NaF or GTP[gamma S], PKC did not inhibit, but rather enhanced the cyclic nucleotide response. Since PKC phosphorylated an endogenous protein of 55 kDa, the size of the beta 1 receptor, these findings indicate that, as in other cell types, PKC can desensitize adenylate cyclase by direct phosphorylation of the beta receptor, but potentiate the cAMP response by a post-receptor mechanism. In mucin secretion studies in the model, TPA alone caused the cAMP-independent release of up to 44% total mucin, which was much less than additive with the isoproterenol response. When the cAMP-mucosecretory response was stimulated at the adenylate cyclase level by forskolin, however, the TPA + forskolin effects were additive. These findings on the modulation of cAMP by PKC indicate cross-talk regulation in the phosphoinositide-cAMP signal transduction pathways in submandibular acinar cells.

Animals↗
PubMed1992

Arachidonic acid regulates the phosphoinositide signal transduction pathway in submandibular acinar cells.

Modulation of the phosphoinositide signal transduction pathway by arachidonic acid (AA) in collagenase-dispersed rat submandibular acinar cells was investigated. The muscarinic agonist, carbachol, stimulated PIP2 hydrolysis and the generation of IP3 to five-fold the control levels. This response was inhibited by 75% on pre-treatment of cells with AA. The AA inhibitory effect was not duplicated by a range of prostaglandins and leukotrienes and was not reversed by blockers of the cyclo-oxygenase and lipoxygenase synthetic pathways, indicating that AA action was not mediated by eicosanoid metabolites. Additional experiments confirmed that the enzyme, protein kinase C, was also not a mediator of the AA effect. Arachidonic acid did not affect the uptake of radioactive inositol into acinar cells, but it did inhibit the incorporation of inositol into inositol phospholipids of the phosphoinositide cycle. In studies on inositol phospholipid turnover, AA alone reduced the level of PIP2 but not of PIP or PI. Under conditions of PI cycle stimulation with carbachol, AA significantly lowered PIP2 and PIP but not PI. These findings suggest that arachidonic acid may regulate the phosphoinositide response by inhibiting the synthetic phase of the cycle at a locus distal to PI generation.

Animals↗
PubMed1990

Muscarinic M3 receptors are coupled to two signal transduction pathways in rat submandibular cells.

The receptor subtypes involved in muscarinic-induced phosphoinositide hydrolysis and adenylate cyclase inhibition in rat submandibular acinar cells were characterized by comparing the inhibitory potencies of four muscarinic antagonists on the two signal transduction responses. Carbachol-induced phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis was inhibited by all antagonists with a potency rank order of 4-diphenylacetoxy-N-methyl piperidine methobromide (4-DAMP) = atropine much greater than pirenzepine much greater than AF-DX 116 (P less than 0.01). The same rank order was observed in antagonist-reversal of the reduction of cAMP caused by carbachol in the model. These findings suggest that muscarinic effects are mediated by M3 receptors in both the phosphoinositide and adenylate cyclase pathways in the submandibular gland.

Animals↗
PubMed1989

G regulatory proteins and muscarinic receptor signal transduction in mucous acini of rat submandibular gland.

The involvement of G regulatory proteins in muscarinic receptor signal transduction was examined in electrically permeabilized rat submandibular acinar cells. The guanine nucleotide analog, GTP gamma S, caused the dose dependent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to release IP3. This response was insensitive to pertussis toxin treatment and was duplicated by NaF but not by GDP beta S. Enhanced IP3 synthesis was observed with a combination of GTP gamma S and carbachol. Exogenous IP3, as well as carbachol and GTP gamma S, provoked the release of sequestered 45Ca2+ from non-mitochondrial stores. In intact cells, carbachol significantly reduced the level of cyclic AMP induced by the beta-adrenergic agonist, isoproterenol, to 69% of its normal value. Pertussis toxin abolished this inhibitory action of carbachol on cyclic nucleotide levels. These results suggest that muscarinic receptors are coupled to two separate G regulatory proteins in submandibular mucous acini-the pertussis toxin-insensitive Gp of the phosphoinositide transduction pathway associated with elevated cytosolic calcium levels, and the pertussis toxin-sensitive Gi inhibitory protein of the adenylate cyclase complex.

Adenylate Cyclase Toxin↗
PubMed1989

Training induced physiological and metabolic changes associated with improvements in running performance.

The purpose of the present study was to examine the relationship between improvements in running performance and some of the prominent physiological and metabolic adaptations to endurance exercise training. Twelve male undergraduates agreed to participate in this study (trial group), aged matched physical education students provided a control group. Running performance, assessed as a five km time trial, improved from 19.69 +/- 2.24 to 19.22 +/- 2.03 min in the trial group (P less than 0.01) after training. Maximal oxygen uptake values increased from 56.0 +/- 6.1 to 60.7 +/- 5.4 ml.kg-1.min-1, the running speed equivalent to a blood lactate reference concentration of 4 mmol.l-1 (V-4 mM) increased from 3.79 +/- 0.77 to 4.04 +/- 0.71 m.s-1, and the rate of oxygen consumption at 3.58 m.s-1 (running economy) increased from 43.3 +/- 3.2 to 45.0 +/- 3.4 ml.kg-1.min-1 (P less than 0.01). The control group did not show any significant changes. The improved five km times in the trial group were significantly correlated (r = -0.71; P less than 0.01) with changes in the running economy rather than changes in the VO2 max (r = -0.07; ns), or V-4 mM (r = -0.13; ns) suggesting the increased rate of oxygen utilization reflected a greater oxidative degradation of metabolic substrates together with a slower rate of lactate production.

Humans↗
PubMed1989

Intubation with low-dose atracurium in children.

The objective of this study was to compare intubating conditions and neuromuscular effects using smaller doses of atracurium (0.25 mg/kg and 0.3 mg/kg) with the recommended dose of 0.4 mg/kg for intubation in children anesthetized with halothane, N2O and oxygen undergoing strabismus repair. All patients (10 in each group) had good or excellent intubating conditions at 80% depression of twitch height [T1 of train-of-four (TOF) stimulation]. Mean times to intubation were 2.6 +/- 0.2 minutes following 0.25 mg/kg and 2.2 +/- 0.2 minutes following 0.3 mg/kg. These times were significantly longer (P less than 0.05) than the mean intubation time of 1.5 +/- 0.2 minutes following 0.4 mg/kg. Mean times to recovery, defined as times from injection of atracurium to return of T1 of TOF to 10%, 25%, and 95% of control measurements, were significantly shorter with the smaller doses. Atracurium at these low doses may provide an alternative to succinylcholine for intubating children during halothane anesthesia for surgical procedures lasting 20-30 min.

Anesthesia↗
PubMed1988

Different tachykinin receptor subtypes are coupled to the phosphoinositide or cyclic AMP signal transduction pathways in rat submandibular cells.

Tachykinins of different classes (NK1, NK2, NK3) caused the concentration-dependent synthesis of IP3 in rat submandibular acinar cells with the potency rank order of NK1 greater than NK2 greater than NK3. Enhancement of IP3 was not affected by pertussis toxin treatment. The reverse rank order was found in the tachykinin inhibition of isoproterenol-induced cAMP synthesis and this inhibition was abolished by pertussis toxin, an inactivator of the adenylate cyclase Gi regulatory protein. It is suggested that different tachykinin receptor subtypes are preferentially coupled to phospholipase C or adenylate cyclase by separate G regulatory proteins in rat submandibular acinar cells.

Adenylate Cyclase Toxin↗
PubMed1987

Muscarinic, alpha 1-adrenergic and peptidergic agonists stimulate phosphoinositide hydrolysis and regulate mucin secretion in rat submandibular gland cells.

Three classes of agonists associated with Ca2+-mobilization--alpha 1-adrenergic (methoxamine), muscarinic (carbachol) and peptidergic (substance P, SP)--significantly stimulated the secretion of mucin from enzymatically-dispersed rat submandibular gland acinar cells. The same three secretagogues also caused the hydrolysis of membrane inositol phospholipids, resulting in elevated cellular levels of inositol phosphates, particularly inositol 1,4,5-trisphosphate (IP3). Exogenous IP3 elicited the dose-dependent release of mucin in dispersed cells suggesting that agonist-generated endogenous IP3 may provoke a secretory response. IP3 and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in combination, stimulated an additive secretion of mucin in the model. The potential use of these two agents as specific probes of the IP3- and diacylglycerol-associated legs of the polyphosphoinositide (PPI) breakdown pathway is indicated. Although all three agonists shared a common action in stimulating PPI hydrolysis, their effects on the beta-adrenergic mucosecretory response were inconsistent. A brief preincubation of cells with carbachol or SP significantly reduced the subsequent isoproterenol (IPR)-provoked secretion of mucin, whereas methoxamine plus IPR stimulated an additive response. The mechanisms underlying these opposite effects are not known. Failure of IP3 or TPA to modify IPR responses suggests that modulation of the beta response may operate at a locus before the generation of diacylglycerol and IP3, possibly at the level of signal transduction. The study indicates a role for Ca2+-mobilizing agonists in controlling submandibular mucin secretion and provides evidence that receptor-linked phosphoinositide hydrolysis is an early stage in their stimulus-secretion coupling mechanism.

Animals↗
PubMed1987

Bacteria and the mucus blanket in experimental small bowel bacterial overgrowth.

Self-filling blind loops were created experimentally in jejunal segments of specific pathogen-free male Wistar rats, and the loop contents and mucosa were examined over an 8-week period for evaluation of the interaction between mucus and luminal bacteria. Corresponding jejunal segments from rats that did not undergo surgery were used as controls. Proliferation of anaerobic bacteria developed in the test animals by the first week after surgery. Despite anaerobic bacterial proliferation, no adherence by bacteria to the intestinal microvillus surface was observed by scanning or transmission electron microscopy. Rather, bacteria were present within the mucus layer overlying the intestinal mucosal surface. Immunoassay of goblet cell mucin demonstrated an increase in the proportion of mucin present in the intestinal lumen and a decrease in mucin levels in the jejunal mucosa. These results suggest that the interaction of bacteria with mucus is an important mechanism of protection of the mucosal surface in experimental small bowel bacterial overgrowth.

Animals↗
PubMed1986

A versatile transition metal salt reaction for a wide range of common biochemical reagents: an instantaneous and quantifiable color test.

A rapid and sensitive spot test amenable to visual or spectrophotometric quantitation has been developed for a wide variety of biochemical reagents by utilizing the transition metal salt cupric chloride and its large number of related colored compounds. This assay is potentially a widely applicable multipurpose test for rapidly detecting the presence of unknown substances. Combination of the test sample with the working reagent results in the immediate formation of a distinctive colored product that may be precipitable. Some compounds require the further addition of sodium hydroxide in order to generate the distinctively colored product. Distinctive reactions occur with the following reagents, and their limit of visual detection is indicated in parentheses: ammonium bicarbonate (12.5 mM), ammonium acetate (25 mM), ammonium hydroxide (0.1%), ammonium sulfate (2%), ammonium persulfate (0.02 mM), L-(+)-cysteine (0.07 mM), dithiothreitol (DTT) (1.25 mM), EDTA (0.6 mM), ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (5 mM), D-glucose (6 mM), glycerol (0.3%), imidazol (12.5 mM), DL-methionine (100 mM), mercaptoethanol (0.05%), sodium azide (19 mM, 0.1%), sodium dithionite (0.25%), sodium metabisulfite (25 mM), sodium nitrite (6.2 mM), sodium periodate (3.1 mM), sodium sulfite (12.5 mM), sodium thiosulfite (12.5 mM), sucrose (6 mM), and N,N,N',N'-tetramethylethylenediamine (0.05%). A distinctive exothermic reaction occurs with hydrogen peroxide, but without color change. Compounds reacting insignificantly include 50 mM Tris buffer, urea, N,N'-methylene bisacrylamide, sodium dodecyl sulfate, isopropyl alcohol, sodium fluoride, trichloroacetic acid, phenol, mannose, K2HPO4, guanidine HCl, chloramine-T, magnesium chloride, and boric acid, where the solids were tested at approximately 10 mg/ml. Spectrophotometric standard curves were developed for DTT and sodium azide utilizing the clear supernatants resulting from these reactions. Combinations of at least four reagents could be discriminated, as demonstrated with mixtures of glucose, sodium azide, EDTA, and DTT. In addition ammonium sulfate could be detected to a limit of 4% in the presence of protein, DTT, and EDTA in a 50 mM Tris buffer. Spot tests were developed which utilized reagent-impregnated filter paper and gave distinctive colored products on addition of 5 microliter of test sample.

Centrifugation↗
PubMed1986

Effects of a phorbol ester and diacylglycerols on secretion of mucin and arginine esterase by rat submandibular gland cells.

The effects of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and a diacylglyceride, 1-oleoyl-2-acetyl-glycerol (OAG) on the secretion of two major exocrine products by dispersed rat submandibular cells were investigated. TPA stimulated the release of acinar cell mucin and ductal cell protease (arginine esterase) in a dose- and time-dependent manner. Mucin secretion was also provoked by OAG, which, however, had no effect on arginine esterase release. The unsaturated diacylglycerol, 1,2-diolein, elicited a greater mucosecretory response than did OAG at the same concentration, while the saturated 1,2-distearin produced a smaller response. Mucin and enzyme secretion caused by TPA or OAG in the rat submandibular model was not inhibited by either of two putative antagonists, the antipsychotic drug, fluphenazine, and the antibiotic, polymyxin B. The involvement of extracellular Ca2+ in TPA-induced secretion was examined by comparing responses of cells maintained in normal or Ca2+-free medium, or in medium containing the ionophore A23187. Although extracellular Ca2+ was not an absolute requirement for a secretory response, the results indicate a synergistic relationship between TPA and Ca2+ in stimulating the release of both mucin and arginine esterase. These results suggest a role for the Ca2+-, phospholipid-dependent enzyme, protein kinase C in the secretory mechanism of mucous and serous cells in the submandibular gland. This is consistent with the proposal that receptor-mediated hydrolysis of membrane phosphoinositides is an initial event in stimulus-response coupling in exocrine cells.

Animals↗
PubMed1986

Chronic hypoglycemia increases brain glucose transport.

Glucose transport into the brain is depressed in chronically hyperglycemic (diabetic) rats. To determine whether hypoglycemia has the opposite effect, brain transport of hexoses and other substrates was examined in chronically and acutely hypoglycemic rats. We produced chronic hypoglycemia by implanting insulin-secreting tumors or insulin-releasing osmotic mini-pumps or by repeated injection of protamine zinc insulin (PZI) and acute hypoglycemia by intravascular injection of regular insulin. Blood-brain barrier (BBB) transport was measured using the brain uptake index (BUI) method. In the three models of chronic hypoglycemia, brain glucose extraction was increased compared with controls. The extraction of deoxyglucose and several other hexoses was also increased by chronic hypoglycemia. Acute hypoglycemia had no effect on brain transport. The transport of other substrates was either not affected or depressed, suggesting increased brain hexose transport is specific. Studies of freeze-blown brain in insulinoma-engrafted rats showed that brain glucose levels were depressed while creatine phosphate, ATP, and glucose 6-phosphate were maintained. Tumor removal led to a reversion of brain glucose transport to control rates but only after 5-25 days. These findings support the view that glucose transport across the BBB is modulated by chronic alterations in the ambient glucose concentration. They also may explain why some patients with chronic hypoglycemia tolerate low blood glucose concentrations.

Animals↗
PubMed1984

Cholera-induced mucin secretion from rat intestine: lack of effect of cAMP, cycloheximide, VIP, and colchicine.

Purified cholera enterotoxin (20-50 micrograms) and dialyzed cholera filtrate (50-125 mg) increased net glycoprotein synthetic and secretory rates in rat intestinal epithelium. Specific goblet cell mucin secretion was increased 5- to 10-fold. However, other agents that increase intestinal cAMP and accelerate glycoprotein synthesis did not enhance mucin secretion. This was true for dibutyryl cAMP (10(-3) and 10(-2) M) with or without theophylline (10(-3) M) and isoproterenol (10(-4) M) with or without dibutyryl cAMP (10(-3) M). Hyperosmotic mannitol (450 mosmol/l), which increases fluid secretion but does not affect cAMP, and vasoactive intestinal peptide (2 X 10(-7) M), which increases both fluid secretion and cAMP, both failed to increase mucin secretion, implying that fluid "washout" of mucin adherent to the mucosal surface is not responsible for cholera-induced mucin secretion. Cycloheximide, an inhibitor of cholera diarrhea in vivo (20 mg/kg) or in vitro (1 mM), effectively abolished [3H]leucine incorporation into protein but did not affect cholera-induced mucin secretion. Colchicine (10-50 mg/kg) given to block microtubule assembly was similarly without effect on mucin secretion. These findings suggest that there is a dissociation of electrolyte/fluid and mucin secretory processes and cast doubt on the widely accepted notion that all cholera effects are mediated via the well-known adenylate cyclase-cAMP mechanism.

Animals↗
PubMed1984

Effect of substance P on exocrine secretion by rat submandibular gland cells.

The effects of the undecapeptide, substance P(SP), on the secretion of mucin and proteolytic enzymes from dispersed cells of the rat submandibular gland were studied. The peptide, at a concentration of 1 X 10(-7) M, stimulated the release of 31.9 +/- 3.0% (mean +/- SEM) of intracellular mucin over 40 min, compared with 12.5 +/- 1.5% in untreated controls (p less than 0.01). This effect was duplicated by the homologous peptides, physalaemin, and eledoisin-related peptide. Substance P action was not affected by pre-incubation of cells with phentolamine or propranolol and was therefore independent of adrenergic stimulation. Furthermore, SP did not enhance the intracellular concentrations of cyclic AMP or cyclic GMP, confirming that cyclic nucleotides were not involved in its stimulus-secretion coupling mechanism. The isoproterenol-stimulated secretion of mucin from dispersed cells was reduced to 75.7% of the normal response (p less than 0.01) after a brief exposure to SP. This inhibitory effect was probably mediated by intracellular events rather than by direct effects on cell surface receptors. However, mucin release after treatment with SP followed by norepinephrine (NE) was 161% of that caused by NE alone (p less than 0.01) and may reflect an additive response to the independent stimulation of SP and NE receptors. Substance P and related peptides had no effect on arginine esterase secretion in the experimental model, although a response was elicited by alpha- and beta-adrenergic agonists. It is, therefore, proposed that serous cells of the granular convoluted tubule in the rat submandibular gland lack substance P receptors.

Animals↗