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Biomedical subjects

C J Kirk

Publications and source records attributed to C J Kirk.

At least 91 records · Page 5Linked to original sources
PubMed1986

The labelling of polyphosphoinositides with [32P]Pi and the accumulation of inositol phosphates in vasopressin-stimulated hepatocytes.

When hepatocytes were incubated with [32P]Pi, the kinetics for the labelling of the monoester phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were similar to each other and slightly slower than that for the labelling of the gamma-phosphate of ATP. Analysis of the water-soluble 3H-labelled materials derived from [3H]inositol-labelled hepatocytes revealed that, in addition to inositol and its mono-, bis- and tris-phosphates (Ins, InsP, InsP2 and InsP3), these cells contained two unidentified radioactive compounds which co-eluted with InsP on anion-exchange chromatography. When [3H]inositol-labelled hepatocytes were stimulated with 0.23 microM-vasopressin in the presence of 10 mM-Li+, there was an accumulation of radioactivity in InsP, InsP2 and InsP3 but not in Ins or the two unidentified compounds. Further analysis of these inositol phosphates by h.p.l.c. revealed that vasopressin also stimulates the accumulation of inositol tetrakisphosphate (InsP4) in these cells. Vasopressin-stimulated InsP and InsP2 accumulations were maximal in the presence of 1-10 mM-Li+ but InsP3 accumulation continued to increase up to 50 mM-Li+. Accumulated inositol phosphates were retained within the cell. Li+ from 1 to 50 mM did not influence the extent of vasopressin-stimulated inositol lipid degradation in hepatocytes. In the absence of Li+, radioactivity in vasopressin-stimulated hepatocytes accumulated almost entirely in free inositol. The vasopressin-stimulated accumulation of inositol phosphates in the presence of 10 mM-Li+ was abolished by a V1-vasopressin antagonist. Inositol phosphate accumulation was not influenced by ionophore A23187, dimethyl sulphoxide or indomethacin.

Adenosine Triphosphate↗
PubMed1986

Changes in free cytosolic calcium and accumulation of inositol phosphates in isolated hepatocytes by [Leu]enkephalin.

Isolated hepatocytes from fed rats were used to study the effects of the opioid peptide [Leu]enkephalin on intracellular free cytosolic Ca2+ ([Ca2+]i) and inositol phosphate production. By measuring the fluorescence of the intracellular Ca2+-selective indicator quin-2, [Leu]enkephalin was found to increase [Ca2+]i rapidly from a resting value of 0.219 microM to 0.55 microM. The magnitude of this response was comparable with that produced by maximally stimulating concentrations of either vasopressin (100 nM) or phenylephrine (10 microM). The opioid-peptide-mediated increase in [Ca2+]i showed a dose-dependency comparable with the activation of phosphorylase, but it preceded the increase in phosphorylase alpha activity. Addition of [Leu]enkephalin to hepatocytes prelabelled with myo-[2-3H(n)]inositol resulted in a significant stimulation of inositol phosphate production. At 10 min after hormone addition, there were increases in the concentrations of inositol mono-, bis- and tris-phosphate fractions of 12-, 9- and 14-fold respectively. No effect was apparent on the glycerophosphoinositol fraction. The effect of 10 microM-[Leu]enkephalin on inositol phosphate production was significantly greater than that obtained with 10 microM-phenylephrine, but marginally smaller than that induced by 100 nM-vasopressin. However, at these concentrations all three agonists gave a comparable increase in [Ca2+]i and activation of phosphorylase a. These data provide evidence for [Leu]enkephalin acting via a mechanism involving a mobilization of Ca2+ as a result of increased phosphatidylinositol turnover.

Animals↗
PubMed1986

Changes in biliary lipid concentrations in bile duct obstruction: an experimental study.

Changes in biliary concentrations of bile acids, phospholipids and cholesterol and biliary pressures were measured in dogs. These parameters were studied during 7-day periods of partial biliary obstruction, of varying degrees, and after 24-hour and 48-hour periods of complete obstruction. The sample were obtained via an exteriorized but intact enterohepatic circulation allowing the introduction of varying degrees of obstruction and bile sampling. Biliary obstruction reduced the concentration of all biliary lipids especially when the obstruction produced pressures in excess of 75% of the maximum biliary secretion pressure. Only immediately after the release of a 48-hour period of complete obstruction did the risk of cholesterol supersaturation of bile occur. However, at that time there was a greatly reduced concentration of lipids in the bile and the amount of cholesterol that could potentially have precipitated was very small. It is suggested that this supersaturation would not play a significant role in the formation of gallstones.

Animals↗
PubMed1985

Ca2+ transport and Ca2+-dependent ATP hydrolysis by Golgi vesicles from lactating rat mammary glands.

Ca2+ transport across mammary-gland Golgi membranes was measured after centrifugation of the membrane vesicles through silicone oil. In the presence of 2.3 microM free Ca2+ the vesicles accumulated 5.8 nmol of Ca2+/mg of protein without added ATP, and this uptake was complete within 0.5 min. In the presence of 1 mM-ATP, Ca2+ was accumulated at a linear rate for 10 min after the precipitation of intravesicular Ca2+ with 10 mM-potassium oxalate. ATP-dependent Ca2+ uptake exhibited a Km of 0.14 microM for Ca2+ and a Vmax. of 3.1 nmol of Ca2+/min per mg of protein. Ca2+-dependent ATP hydrolysis exhibited a Km of 0.16 microM for Ca2+ and a Vmax. of 10.1 nmol of Pi/min per mg of protein. The stoichiometry between ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase varied between 0.3 and 0.7 over the range 0.03-8.6 microM-Ca2+. Both Ca2+ uptake and Ca2+-stimulated ATPase were strongly inhibited by orthovanadate, which suggests that the major mechanism by which Golgi vesicles accumulate Ca2+ is through the action of the Ca2+-stimulated ATPase. However, Ca2+ uptake was also decreased by the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone), indicating that it may occur by other mechanisms too. The effect of CCCP may be related to the existence of transmembrane pH gradients (delta pH) in these vesicles: the addition of 30 microM-CCCP reduced delta pH from a control value of 1.06 to 0.73 pH unit. Golgi vesicles also possess a Ca2+-efflux pathway which operated at an initial rate of 0.5-0.57 nmol/min per mg of protein.

Adenosine Triphosphate↗
PubMed1985

Hormonal regulation of fat esterification & oxidation in hepatocytes from fed rats: modulation by 3-mercaptopicolinate.

3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), decreased esterification of [1-14C] oleate and [1-14C] myristate in hepatocytes from fed rats. In the absence of 3-mercaptopicolinate, adrenaline, noradrenaline, vasopressin or angiotensin II increased esterification to triacylglycerol of [1-14C] oleate but not [1-14C] myristate. Cyclic AMP decreased esterification of both oleate and myristate. In the presence of 3-mercaptopicolinate, stimulation of oleate esterification by the catecholamines, vasopressin or angiotensin II was increased, and stimulatory effects of these hormones on myristate esterification were observed. Adrenaline, noradrenaline, vasopressin or angiotensin II increased 14CO2 production from both [1-14C] oleate and [1-14C] myristate but the degree of stimulation was similar in the absence or presence of 3-mercaptopicolinate. The results indicate a role for the catecholamines and angiotensin II in the regulation of liver fat metabolism and emphasize the potential importance of changes in activity of PEPCK as determinants of hepatic carbon flux.

Animals↗
PubMed1985

Two automated methods for measuring plasma thiocyanate compared.

In an interlaboratory comparison of two continuous-flow analytical procedures for measuring thiocyanate, we used ferric nitrate (y) and p-phenylenediamine/pyridine (x) as colorimetric reagents to measure its concentrations in plasma of 100 consecutive patients attending a peripheral vascular disease clinic. The results correlated well (r = 0.987, p less than 0.001; y = 0.938x + 1.2 mumol/L). However, there were small, systematic, positive differences between the phenylenediamine values and the corresponding ferric nitrate values (paired t = 5.4, p less than 0.001). These differences were linearly related to the means of the pairs of results (r = 0.42, p less than 0.001; y = 0.0739x - 2). Nevertheless, when we used previously determined cutoff points the two sets of SCN concentrations concurred completely in classifying the 100 patients as smokers or nonsmokers. On the basis of self-classification by 71 of these subjects, the measurement techniques had a sensitivity of 91% and a specificity of 75%; when five patients claiming to be nonsmokers but found to have abnormally high values for carboxyhemoglobin (2.7 - 6.9%) were reclassified as smokers, specificity increased to 89%.

Autoanalysis↗
PubMed1984

Rapid accumulation of inositol phosphates in isolated rat superior cervical sympathetic ganglia exposed to V1-vasopressin and muscarinic cholinergic stimuli.

An accumulation of 3H-labelled inositol phosphates is observed when prelabelled rat superior cervical sympathetic ganglia are exposed to [8-arginine]vasopressin or to muscarinic cholinergic stimuli. The response to vasopressin is much greater than the response to cholinergic stimuli. The response to vasopressin is blocked by a V1-vasopressin antagonist, and oxytocin is a much less potent agonist than vasopressin. Vasopressin causes no increase in the cyclic AMP content of ganglia. These ganglia therefore appear to have functional V1-vasopressin receptors that are capable of activating inositol lipid breakdown, but no V2-receptors coupled to adenylate cyclase. The first [3H]inositol-labelled products to accumulate in stimulated ganglia are inositol trisphosphate and inositol bisphosphate, suggesting that the initiating reaction in stimulated inositol lipid metabolism is a phosphodiesterase-catalysed hydrolysis of phosphatidylinositol 4,5-bisphosphate (and possibly also phosphatidylinositol 4-phosphate). This response to exogenous vasopressin occurs in ganglia incubated in media of reduced Ca2+ concentration. The physiological functions of the V1-vasopressin receptors of these ganglia remain unknown.

Animals↗
PubMed1984

Determination of mitochondrial calcium content in hepatocytes by a rapid cellular fractionation technique. Vasopressin stimulates mitochondrial Ca2+ uptake.

Stimulation of hepatocytes with vasopressin (10 nM) in the presence of 1.25 mM extracellular Ca2+ increased glycogen phosphorylase activity 4-fold within 15s and provoked a rapid efflux of cell-associated Ca2+. Vasopressin also caused a transient increase in the Ca content of a mitochondria-rich fraction separated within seconds of hormone stimulation by a rapid fractionation technique [Shears & Kirk (1984) Biochem. J. 219, 375-382]. The Ca content of this fraction was restored to the control value within 2 min of hormone addition. These results indicate that mitochondria are not the source of the cell-associated Ca which is mobilized in the cytosol of vasopressin-stimulated hepatocytes. Rather, these organelles buffer the increase in cytosol [Ca2+] attributable to Ca mobilization from non-mitochondrial sources.

Animals↗
PubMed1984

Characterization of a rapid cellular-fractionation technique for hepatocytes. Application in the measurement of mitochondrial membrane potential in situ.

A rapid cellular-fractionation technique [ Hoek , Nicholls & Williamson (1980) J. Biol. Chem. 255, 1458-1464] was further characterized by using hepatocytes. Of the mitochondrial marker-enzyme activity, 80% was routinely separated from 71-98% of the total cell activities of marker enzymes for plasma membranes, Golgi-membranes, endoplasmic reticulum, lysosomes and cytosol. The mitochondria were contaminated with 53% of cell nuclei. [3H]Triphenylmethylphosphonium ion (TPMP+) was added to hepatocytes in an attempt to measure cellular transmembrane electrical potentials. After rapid cell fractionation the electrical potential between mitochondria in situ and the incubation medium was found to be 202 mV. This value was slightly increased when hepatocytes were treated with oligomycin, but substantially decreased by oligomycin plus an uncoupler of oxidative phosphorylation. Although estimates of TPMP+ binding were obtained, substantial difficulties prevented the accurate measurement of the electrical potential across the plasma membrane. It is concluded that TPMP+ may be employed to demonstrate the integrity of mitochondria during the fractionation procedures. However, the cation is inadequate for the determination of the separate components of the electrical potential between the mitochondrial matrix and the incubation medium.

Animals↗
PubMed1984

Determination of mitochondrial calcium content in hepatocytes by a rapid cellular-fractionation technique. Alpha-adrenergic agonists do not mobilize mitochondrial Ca2+.

A rapid cellular fractionation technique [the preceding paper, Shears & Kirk (1984) Biochem. J., 219, 375-382] was employed to separate a mitochondria-rich fraction from hepatocytes within seconds. Mitochondrial Ca was estimated to be no more than 41% of total cell Ca. At least half of the mitochondrial Ca was present in an energy-dependent pool; 20% of total cell Ca was accessible to EGTA within 10s. The alpha-adrenergic agonist phenylephrine stimulated glycogen phosphorylase activity by 100% within 0.5 min and induced a loss of 20% of total cell Ca after 10 min from the EGTA-inaccessible pool. However, between 0.5 and 10 min after the addition of phenylephrine to hepatocytes there was no significant change in the Ca content of the mitochondria-rich fraction. Hepatocytes that were preloaded with Ca2+ during 90 min incubation at 0-4 degrees C expelled this cation during 20 min incubation at 37 degrees C. After this time, phenylephrine failed to alter the Ca content of a mitochondria-rich fraction. It is concluded that alpha-adrenergic agonists do not mobilize Ca2+ from hepatocyte mitochondria.

Animals↗
PubMed1984

Analysis of the metabolic turnover of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Validation of novel analytical techniques by using 32P-labelled lipids from erythrocytes.

We have developed methods that yield estimates of the 32P content of each of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, thus extending the information available from studies of the labelling of these lipids in intact cells or membrane preparations. The analyses are undertaken with the deacylated lipids. Assay of the 5-phosphate of phosphatidylinositol 4,5-bisphosphate is achieved by the use, under conditions of first-order kinetics, of a 5-phosphate-specific phosphomonoesterase present in isolated erythrocyte membranes [Downes, Mussat & Michell (1982) Biochem. J. 203, 169-177]. Assay of the 4-phosphate of phosphatidylinositol 4-phosphate and of the total monoester phosphate content (4-phosphate plus 5-phosphate) of phosphatidylinositol 4,5-bisphosphate employs alkaline phosphatase from bovine intestine. The radioactivity of the 1-phosphate is that remaining as organic phosphate after exhaustive alkaline phosphatase treatment. The methodology has been validated by using lipids from human erythrocytes: these contain no 32P in their 1-phosphate. These methods should be of substantial value in studies of the many cells that show rapid hormonal perturbations of phosphatidylinositol 4,5-bisphosphate metabolism.

Alkaline Phosphatase↗
PubMed1984

The role of phosphatidylinositol 4,5 bisphosphate breakdown in cell-surface receptor activation.

The activation of Ca2+-mobilising receptors on hepatocytes and many other cells leads to a prompt reduction in the cellular content of inositol phospholipids. The primary event which underlies these changes is, most probably, a phospholipase C-catalysed attack upon phosphatidylinositol 4,5 bisphosphate. The receptor-mediated breakdown of this lipid in stimulated cells is: (i) not mediated by an increase in cytosol [Ca2+] and (ii) closely coupled to receptor occupation. Phosphatidylinositol 4,5 bisphosphate degradation may be studied by measuring the appearance of the water-soluble product, inositol trisphosphate (and its metabolites: inositol bisphosphate and inositol monophosphate), in stimulated cells. Recent evidence indicates that inositol trisphosphate and the lipid soluble product of phosphatidylinositol 4,5 bisphosphate breakdown, 1,2 diacylglycerol, may act as 'second messengers' which mediate the effects of many extracellular signals in stimulated cells.

Animals↗
PubMed1984

Hormonal regulation of ketogenesis in hepatocytes from fed rats before and after glycogen depletion.

Vasopressin, angiotensin II and the catecholamines decreased ketogenesis from oleate but increased ketogenesis from butyrate in hepatocytes from fed rats. The hormones increase CO2 production from both oleate and butyrate. It is suggested that whereas the mitochondrial uptake of butyrate is linked to its rate of oxidation, that of oleate is independent of its intramitochondrial metabolism, and consequently the oxidation of oleate to CO2 occurs at the expense of ketogenesis. Effects of the hormones on ketogenesis from oleate or butyrate were not observed after pre-treatment of the hepatocytes with dibutyryl cyclic AMP for 1 hour. The insensitivity of ketogenesis to the hormones after this treatment (which mimics the effects of acute carbohydrate deprivation in vivo) questions the physiological significance of hormonal regulation of ketogenesis other than at the onset of starvation.

Angiotensin II↗
PubMed1983

Rapid breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+-mobilizing hormones.

Rat hepatocytes rapidly incorporate [32P]Pi into phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; their monoester phosphate groups approach isotopic equilibrium with the cellular precursor pools within 1 h. Upon stimulation of these prelabelled cells with Ca2+-mobilizing stimuli (V1-vasopressin, angiotensin, alpha 1-adrenergic, ATP) there is a rapid fall in the labelling of PtdIns4P and PtdIns(4,5)P2. Pharmacological studies suggest that each of the four stimuli acts at a different population of receptors. Insulin, glucagon and prolactin do not provoke disappearance of labelled PtdIns4P and PtdIns(4,5)P2. The labelling of PtdIns4P and PtdIns(4,5)P2 in cells stimulated with vasopressin or angiotensin initially declines at a rate of 0.5-1.0% per s, reaches a minimum after 1-2 min and then returns towards the initial value. The dose-response curves for the vasopressin- and angiotensin-stimulated responses lie close to the respective receptor occupation curves, rather than at the lower hormone concentrations needed to evoke activation of glycogen phosphorylase. Disappearance of labelled PtdIns4P and PtdIns(4,5)P2 is not observed when cells are incubated with the ionophore A23187. The hormone-stimulated polyphosphoinositide disappearance is reduced, but not abolished, in Ca2+-depleted cells. These hormonal effects are not modified by 8-bromo cyclic GMP, cycloheximide or delta-hexachlorocyclohexane. The absolute rate of polyphosphoinositide breakdown in stimulated cells is similar to the rate previously reported for the disappearance of phosphatidylinositol [Kirk, Michell & Hems (1981) Biochem. J. 194, 155-165]. It seems likely that these changes in polyphosphoinositide labelling are caused by hormonal activation of the breakdown of PtdIns(4,5)P2 (and may be also PtdIns4P) by the action of a polyphosphoinositide phosphodiesterase. We therefore suggest that the initial response to hormones is breakdown of PtdIns(4,5)P2 (and PtdIns4P?), and that the simultaneous disappearance of phosphatidylinositol might be a result of its consumption for the continuing synthesis of polyphosphoinositides.

Angiotensin II↗
PubMed1983

A simple assay method for determination of the specific radioactivity of the gamma-phosphate group of 32P-labelled ATP.

A simple and rapid assay method is described for determining the specific radioactivity of the gamma-phosphate group of 32P-labelled ATP. Labelled ATP is incubated with cyclic AMP, cyclic AMP-dependent protein kinase and histone H2A under conditions leading to maximum phosphorylation of the histone. The specific radioactivity of the gamma-phosphate group of the 32P-labelled ATP is calculated from the total amount of [32P]Pi incorporated into a standard amount of histone. This assay method uses inexpensive commercially available materials, and it yields an accurate specific radioactivity with as little as 0.25 nmol of 32P-labelled ATP.

Adenosine Triphosphate↗
PubMed1982

Molecular events following activation of muscarinic receptors: the role of inositol phospholipids.

Muscarinic cholinergic agonists can provoke a wide variety of physiological responses in target tissues. This can be achieved by mobilisation of calcium from intracellular and extracellular stores. The members of the family of Ca2+-mobilizing receptors to which the muscarinic receptor belongs share the ability to provoke a calcium-independent decrease in the concentration of inositol phospholipids. This response is closely coupled to agonist binding and is thus implicated in the mechanism whereby muscarinic and related receptors bring about elevated levels of Ca2+ in cells. Another widespread response to activation of muscarinic receptors is inhibition of adenylate cyclase possibly through direct interaction with a nucleotide regulatory protein. Circumstantial evidence and analogy with alpha 2-adrenergic receptors suggests that adenylate cyclase inhibition may be mediated by a different form of the muscarinic receptor to that involved in calcium mobilisation.

Animals↗