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Biomedical subjects

J G McCormack

Publications and source records attributed to J G McCormack.

At least 91 records · Page 5Linked to original sources
PubMed1991

The cholinergic regulation of intracellular calcium in the human neuroblastoma, SH-SY5Y.

The regulation of intracellular calcium by cholinergic agonists was investigated in the human neuroblastoma SH-SY5Y, loaded with fura-2. The resting free Ca2+ concentration in this cell line was 199 +/- 14 nM (mean +/- SEM, n = 19). At 1 mM extracellular Ca2+, high concentrations of carbachol and acetylcholine evoked a biphasic change in intracellular Ca2+ concentration, consisting of a transient initial peak followed by a decline to a plateau that was significantly higher than the basal level. Carbachol (0.5 mM) and acetylcholine (10 microM) caused a maximal increase in the intracellular Ca2+ concentration, reaching a peak of 465 +/- 52 (mean +/- SEM, n = 12) and 422 +/- 48 nM (mean +/- SEM, n = 7), respectively, in less than 4 s. This initial calcium transient declined to a plateau of 268 +/- 36 and 240 +/- 27 nM for carbachol and acetylcholine, respectively, in approximately 40 s. The plateau persisted until the agonist was displaced by the addition of antagonist. Atropine, hexahydrosiladifenidol (HHSD), pirenzepine, and methoctramine inhibited the carbachol-evoked initial calcium transient with Ki values of 0.85 +/- 0.05, 8.3 +/- 1.6, 411 +/- 36, and 240 +/- 46 nM (mean +/- SEM, n = 3), respectively, and the acetylcholine-induced initial calcium transient with Ki values of 0.48 +/- 0.18, 13.5 +/- 8.5, 192 +/- 32, and 414 +/- 25 nM (mean +/- SEM of two experiments), respectively, results suggesting that an M3 muscarinic receptor was predominantly mediating these effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1990

Measurement of matrix free Mg2+ concentration in rat heart mitochondria by using entrapped fluorescent probes.

1. The concentration of free Mg2+ ([Mg2+]m) within the matrix of isolated rat heart mitochondria was measured after loading of the mitochondria with the fluorescent Mg2+ indicators mag-indo-1 and mag-fura-2. No detectable change in total mitochondrial magnesium content occurred during loading with the indicators. Apparent Kd values for Mg2+ of 3.7 mM and 2.3 mM were obtained for mag-indo-1 and mag-fura-2 respectively within mitochondria permeabilized to bivalent cations with ionomycin and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These values are 2.7- and 1.8-fold greater respectively than those obtained for the free acid forms of the dyes in incubation medium. 2. Based on the above Kd values, mitochondrial matrix Mg2+ concentrations were found to lie in the range 0.8-1.5 mM in the absence, or immediately after the addition, of a respiratory substrate. 3. Incubation of mitochondria in the presence of respiratory substrate, but in the absence of external Mg2+, led to a time-dependent decline in [Mg2+]m to about half the initial values after 5 min. This was accompanied by a fall in the total mitochondrial magnesium content from 12.7 to 7.0 nmol/mg of protein. 4. ADP (0.5 mM), ATP (0.5 mM) or 10 mM-NaCl had no significant effect on the fall in [Mg2+], whereas 1 microM-nigericin blocked, and 0.3 microM-valinomycin accelerated, the fall. 5. External Mg2+ concentrations above 1 mM progressively inhibited and reversed the decline in free and total mitochondrial Mg2+.

Animals↗
PubMed1990

The use of the Ca2(+)-sensitive intramitochondrial dehydrogenases and entrapped fura-2 to study Sr2+ and Ba2+ transport across the inner membrane of mammalian mitochondria.

In extracts of rat heart mitochondria, Sr2+ mimicked the activatory effects of Ca2+ on the Ca2(+)-sensitive intramitochondrial enzymes, pyruvate dehydrogenase phosphate phosphatase, isocitrate dehydrogenase (NAD+), and 2-oxoglutarate dehydrogenase, but at about tenfold higher concentrations (effective range approximately 1-100 muM) in each case. Ba2+ had no effect on extracted phosphatase, but did mimic the effect of Ca2+ on the other two enzymes with effective concentration ranges similar to those of Sr2+; as with Ca2+ and Sr2+, effective Ba2+ ranges were slightly (2-3-fold) raised by increases in ATP/ADP. In intact uncoupled rat heart mitochondria, the effects of Sr2+ and Ba2+ on the pyruvate and 2-oxoglutarate dehydrogenases were essentially similar to their effects in extracts. In fully coupled rat heart or liver mitochondria, the effective concentration ranges of extramitochondrial Sr2+, leading to activation of the matrix enzymes, were always approximately tenfold higher than those for Ca2+ under all conditions. Ba2+ did not affect pyruvate dehydrogenase in coupled mitochondria, but was shown to activate 2-oxoglutarate dehydrogenase in heart or liver mitochondria, and also isocitrate dehydrogenase (NAD+) in the latter; effective concentration ranges for extramitochondrial Ba2+ were approximately 100-fold greater than those for Ca2+, and like those for Ca2+ and Sr2+, were affected markedly by Mg2+ and spermine (which inhibit and promote mitochondrial Ca2+ uptake, respectively) but, in contrast to Ca2+ and Sr2+, they were hardly affected at all by Na+ (which promotes mitochondrial Ca2+ egress). Ba2+ effects were also blocked by ruthenium red (an inhibitor of mitochondrial Ca2+ uptake), but not so effectively as its blockage of the effects of Sr2+ and Ca2+. Ba2+ and Sr2+ both mimicked the inhibitory effects of extramitochondrial Ca2+ on the Na+/Ca2+ exchanger, but only Sr2+ could mimic Ca2+ in exchanging for internal Ca2+ by this mechanism. Both Sr2+ and Ba2+ changed the fluorescent properties of fura-2 or indo-1 in a similar manner to Ca2+, but with higher kd values. In fura-2-loaded rat heart mitochondria, increases in matrix Sr2+ and Ba2+ and the effects of the transport effectors could be readily demonstrated.

Animals↗
PubMed1990

The role of mitochondrial Ca2+ transport and matrix Ca2+ in signal transduction in mammalian tissues.

The pyruvate, NAD(+)-isocitrate and 2-oxoglutarate dehydrogenases are key regulatory enzymes in intramitochondrial oxidative metabolism in mammalian tissues, and can all be activated by increases in Ca2+ in the micromolar range. There is now mounting evidence that hormones and other stimuli which act by increasing cytosolic Ca2+ also, as a result, cause increases in mitochondrial matrix Ca2+ and hence activation of these enzymes, suggesting that the primary physiological function of mitochondrial Ca2(+)-transport is to be involved in this relay mechanism. This may also explain how in such circumstances rates of ATP production may be increased to meet the greater demand, but without any decreases in ATP/ADP occurring.

Animals↗
PubMed1990

Glucose-induced activation of pyruvate dehydrogenase in isolated rat pancreatic islets.

1. Rat pancreatic islets were isolated and then maintained in culture for 2-4 days before being incubated in groups of 100 in the presence of different glucose (0-20 mM) or CaCl2 (1.2-4.2 mM) concentrations, or with uncoupler. 2. Increases in extracellular glucose concentration resulted in increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase in the islets, with half-maximal effects around 5-6 mM-glucose. Increasing extracellular glucose from 3 to 20 mM resulted in a 4-6-fold activation of pyruvate dehydrogenase within 2 min. 3. The total enzyme activity was unchanged, and averaged 0.4 m-unit/100 islets at 37 degrees C. 4. These changes in active pyruvate dehydrogenase were broadly similar to changes in insulin secretion by the islets. 5. Increasing extracellular Ca2+ or adding uncoupler also activated pyruvate dehydrogenase to a similar degree, but only the former was associated with increased insulin secretion.

Animals↗
PubMed1990

Effects of non-esterified fatty acid availability on insulin stimulated glucose utilisation and tissue pyruvate dehydrogenase activity in the rat.

Fatty acids in cardiac muscle compete with glucose for oxidation, thereby inhibiting glucose utilisation. It is not clear whether a similar mechanism is important in resting skeletal muscle. We used the hyperinsulinaemic euglycaemic clamp technique in conscious rats fasted for 20 h to examine the effects of increased plasma non-esterified fatty acid levels (approximately 1 mmol/l) on glucose metabolism. Insulin was infused at 75 mU/h (plasma insulin, 2.27 +/- 0.21 micrograms/l) or 300 mU/h (16.41 +/- 0.47 micrograms/l). An increase in non-esterified fatty acid levels decreased clamp glucose requirement and 3-3H-glucose turnover by 35% (p less than 0.001) when the higher insulin dose was used but there was no change at the lower dose. At both insulin infusion rates, clamp blood lactate and pyruvate responses suggested inhibition of muscle glycolysis by elevated plasma non-esterified fatty acid concentrations. Quadriceps muscle glycogen deposition during the clamps was enhanced by increased non-esterified fatty acid availability at the lower insulin dose (p less than 0.001) but not at the higher insulin concentration. Activation of pyruvate dehydrogenase during the clamps was partially inhibited by increased plasma non-esterified fatty acid in the heart, adipose tissue and quadriceps muscle. This was evident at both insulin levels in heart but only at the higher insulin concentration in muscle (p less than 0.002). The findings are consistent with an inhibition of glycolysis in skeletal muscle of mixed fibre type as a result of increased fatty acid availability. At low rates of glucose flux glycogen synthesis may compensate for decreased glycolysis so that glucose turnover is not decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1990

Suppression of neutrophil and lymphoproliferative responses in vitro by itraconazole but not fluconazole.

Itraconazole and fluconazole are triazole compounds recently licensed for the therapy of systemic fungal infections. At 10 micrograms/ml concentrations, itraconazole was found to suppress neutrophil chemotaxis, random movement, deoxyglucose uptake and hexose-monophosphate shunt activity to the same extent as ketoconazole, an older generation azole antifungal. Itraconazole was also found to suppress mitogen-induced lymphocyte transformation to the same extent as ketoconazole at concentrations as low as 1 microgram/ml. By contrast, significant inhibition of both neutrophil and lymphocyte functions was not observed with fluconazole at concentrations as high as 50 micrograms/ml. These results suggest that fluconazole may be less immunotoxic than itraconazole, and may be more suitable for use in immunocompromised patients.

Antifungal Agents↗
PubMed1990

Mucor cerebral abscess associated with intravenous drug abuse.

We report on a case of a 26-year-old intravenous narcotic abuser with a primary cerebral mucormycotic abscess caused by Rhizopus oryzae. He was treated with a combination of intravenous and intraventricular amphotericin B and surgical drainage with a successful outcome. There was no evidence that his infection was acquired by the rhinocerebral route, it seems likely that he injected himself with a contaminated batch of narcotic or amphetamine. Mucormycosis presenting in this way has been described previously but this is only the second such case to survive. Early diagnosis and treatment is essential for a favourable outcome in this condition.

Adult↗
PubMed1990

In vitro immunosuppressive and anti-phagocytic properties of the exopolysaccharide of mucoid strains of Pseudomonas aeruginosa.

The exopolysaccharide (EPS) of Pseudomonas aeruginosa was found to significantly inhibit neutrophil random movement, chemotaxis and degranulation at concentrations as low as 0.3 microgram/ml. Neutrophil adherence, respiratory burst and bactericidal capacity were inhibited by EPS concentrations of greater than or equal to 3 micrograms/ml. Similarly, mitogen-induced lymphocyte transformation was more sensitive to the inhibitory effects of EPS than natural-killer cell cytotoxicity. These results cannot be explained by simple mechanical blockade, as additions of EPS as late as 48 h after the initiation of lymphocyte cultures still resulted in a significant inhibition of lymphocyte transformation. However, the inhibitory effects of EPS can be reversed by extensive washing of treated lymphocytes. These results suggest that the propensity of mucoid P. aeruginosa to persist in cystic fibrosis may be explained in part by the ability of EPS to interfere with host immunity.

Blood Bactericidal Activity↗
PubMed1989

Effects of spermine on mitochondrial Ca2+ transport and the ranges of extramitochondrial Ca2+ to which the matrix Ca2+-sensitive dehydrogenases respond.

1. Spermine has previously been reported to be an activator of mitochondrial Ca2+ uptake [Nicchitta & Williamson (1984) J. Biol. Chem. 259, 12978-12983]. This is confirmed in the present studies on rat heart, liver and kidney mitochondria by using the activities of the Ca2+-sensitive intramitochondrial dehydrogenases (pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases) as probes for matrix Ca2+, and also, for the heart mitochondria, by using entrapped fura-2. 2. As also found previously [Damuni, Humphreys & Reed (1984) Biochem. Biophys. Res. Commun. 124, 95-99], spermine activated extracted pyruvate dehydrogenase phosphate phosphatase. However, it was found to have no effects at all on the extracted NAD+-isocitrate or 2-oxoglutarate dehydrogenases. It also had no effects on activities of the enzymes in mitochondria incubated in the absence of Ca2+, or on the Ca2+-sensitivity of the enzymes in uncoupled mitochondria. 3. Spermine clearly activated 45Ca uptake by coupled mitochondria, but had no effect on Ca2+ egress from mitochondria previously loaded with 45Ca. 4. Spermine (with effective Km values of around 0.2-0.4 mM) caused an approx. 2-3-fold decrease in the effective ranges of extramitochondrial Ca2+ in the activation of the Ca2+-sensitive matrix enzymes in coupled mitochondria from all of the tissues. The effects of spermine appeared to be largely independent of the other effectors of mitochondrial Ca2+ transport, such as Mg2+ (inhibitor of uptake) and Na+ (promoter of egrees). 5. In the most physiological circumstance, coupled mitochondria incubated with Na+ and Mg2+, the presence of saturating spermine (2 mM) resulted in an effective extramitochondrial Ca2+ range for matrix enzyme activation of from about 30-50 nM up to about 800-1200 nM, with half-maximal effects around 250-400 nM-Ca2+. The implications of these findings for the regulation of matrix and extramitochondrial Ca2+ are discussed.

Animals↗
PubMed1989

The role of Ca2+ ions in the regulation of intramitochondrial metabolism and energy production in rat heart.

In the heart and other mammalian tissues, there are three exclusively intramitochondrial dehydrogenases that occupy key regulatory sites in oxidative metabolism which can be activated by increases in Ca2+ in the approximate range 0.05-5 microM; they are the pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases. Activation of these enzymes can be demonstrated within intact mitochondria, incubated under expected physiological conditions, when the extramitochondrial concentration of Ca+ is raised within the expected physiological range. Recent studies with fura-2-loaded mitochondria have established that matrix Ca2+ is indeed in the 0.02-2 microM range as the enzymes are activated. There is now good evidence that in the rat heart, increases in cytoplasmic [Ca2+] caused by various inotropic agents result in increases in intramitochondrial Ca2+ and activation of these dehydrogenases. It is argued therefore that matrix Ca2+ may thus be a key regulator of oxidative phosphorylation under such circumstances. The major advantage of such a mechanism of dehydrogenase-based control of this process would be to the energy homeostasis of the cell by allowing stimulated ATP production without the need to decrease the ATP/ADP ratio. Therefore it is also proposed that the major function of the mitochondrial Ca2+-transport system is to regulate matrix Ca2+, and that the ability of mitochondria to buffer the extramitochondrial concentration of Ca2+ may thus only be reserved for pathophysiological conditions of abnormal sarcolemmal Ca2+ influx as perhaps may occur in ischaemia-reperfusion.

Animals↗