scholarly journals Calcium metabolism in rat hepatocytes

1978 ◽  
Vol 170 (3) ◽  
pp. 615-625 ◽  
Author(s):  
S Foden ◽  
P J Randle
Keyword(s):  
Cyclic Amp ◽  
Time Course ◽  
Rat Hepatocytes ◽  
Ionophore A23187 ◽  
Total Calcium ◽  
Free Medium ◽  
Adrenergic Agonists ◽  
Calcium Efflux ◽  
Similar Time ◽  
Carbonyl Cyanide

1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.

scholarly journals Stimulation by glucose of cyclic AMP accumulation in mouse pancreatic islets is mediated by protein kinase C

1988 ◽  
Vol 253 (1) ◽  
pp. 229-234 ◽  
Author(s):  
P Thams ◽  
K Capito ◽  
C J Hedeskov
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Pancreatic Islets ◽  
Time Course ◽  
Ionophore A23187 ◽  
Kinase C ◽  
Cyclic Amp Accumulation ◽  
Mouse Pancreatic Islets

The mechanism of glucose-stimulated cyclic AMP accumulation in mouse pancreatic islets was studied. In the presence of 3-isobutyl-1-methylxanthine, both glucose and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, enhanced cyclic AMP formation 2.5-fold during 60 min of incubation. Both TPA-stimulated and glucose-stimulated cyclic AMP accumulations were abolished by the omission of extracellular Ca2+. The Ca2+ ionophore A23187 did not affect cyclic AMP accumulation itself, but affected the time course of TPA-induced cyclic AMP accumulation, the effect of A23187 + TPA mimicking the time course for glucose-induced cyclic AMP accumulation. A 24 h exposure to TPA, which depletes islets of protein kinase C, abolished the effects of both TPA and glucose on cyclic AMP production. Both TPA-induced and glucose-induced cyclic AMP productions were inhibited by anti-glucagon antibody, and after pretreatment with this antibody glucose stimulation was dependent on addition of glucagon. Pretreatment of islets with TPA for 10 min potentiated glucagon stimulation and impaired somatostatin inhibition of adenylate cyclase activity in a particulate fraction of islets. Carbamoylcholine, which is supposed to activate protein kinase C in islets, likewise stimulated cyclic AMP accumulation in islets. These observations suggest that glucose stimulates islet adenylate cyclase by activation of protein kinase C, and thereby potentiates the effect of endogenous glucagon on adenylate cyclase.

THE STEROIDOGENIC RESPONSE OF ADULT RAT ADRENOCORTICAL CELLS IN MONOLAYER CULTURE

1973 ◽  
Vol 56 (3) ◽  
pp. 537-549 ◽  
Author(s):  
M. J. O'HARE ◽  
A. MUNRO NEVILLE
Keyword(s):  
Cyclic Amp ◽  
Time Course ◽  
Monolayer Culture ◽  
Zona Fasciculata ◽  
Adrenocortical Cells ◽  
Similar Time ◽  
Adult Rat ◽  
Monolayer Cultures ◽  
Corticosterone Secretion ◽  
Steady Rate

SUMMARY Quantitative aspects of corticosteroidogenesis were examined in monolayer cultures of cells from the zona fasciculata and zona reticularis of the adult rat adrenal cortex, maintained for up to 4 months. Corticosterone secretion continued at a steady rate in cultures maintained with corticotrophin (ACTH), the output of maximally stimulated cultures being approximately 12 μg/106 adrenocortical cells/day. In the absence of ACTH, a small amount of 20α-hydroxypregn-4-en-3-one, but no detectable corticosterone, was secreted, resulting in a fluorogenic steroid output 1/125 th of that of maximally stimulated cultures. Restimulation with ACTH of cultures maintained for up to 2 months in its absence resulted in maximum levels of corticosterone secretion after 4–5 days of continuous ACTH treatment. The levels of corticosterone secretion attained on restimulation were similar to those observed in cultures maintained with ACTH from the out set. Withdrawal of ACTH resulted in a fall in steroid output which took 10 days to reach final unstimulated levels. Trophic stimulation of corticosteroidogenesis with a similar time-course was obtained with both cyclic AMP and dibutyryl cyclic AMP, the latter being the more effective.

scholarly journals Hormonal control of fructose 2,6-bisphosphate concentration in isolated rat hepatocytes

1983 ◽  
Vol 214 (3) ◽  
pp. 829-837 ◽  
Author(s):  
R Bartrons ◽  
L Hue ◽  
E Van Schaftingen ◽  
H G Hers
Keyword(s):  
Cyclic Amp ◽  
Pyruvate Kinase ◽  
Time Course ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Hormonal Control ◽  
Isolated Rat Hepatocytes ◽  
Free System ◽  
Significant Difference ◽  
Dependent Protein

The ability of glucagon and of adrenaline to affect the concentration of fructose 2,6-bisphosphate in isolated hepatocytes was re-investigated because of important discrepancies existing in the literature. We were unable to detect a significant difference in the sensitivity of the hepatocytes with regard to the effect of glucagon to initiate the interconversion of phosphorylase, pyruvate kinase, 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase, and also to cause the disappearance of fructose 2,6-bisphosphate. In contrast, we have observed differences in the time-course of these various changes, since the interconversions of phosphorylase and of pyruvate kinase were at least twice as fast as those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. When measured in a cell-free system in the presence of MgATP, the cyclic AMP-dependent interconversion of pyruvate kinase was 5-10-fold more rapid than those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. These data indicate that 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase are relatively poor substrates for cyclic AMP-dependent protein kinase; they also support the hypothesis that the two catalytic activities belong to a single protein. Adrenaline had only a slight effect on the several parameters under investigation, except for the activation of phosphorylase. In the absence of Ca2+ ions from the incubation medium, however, adrenaline had an effect similar to that of glucagon.

CALPAIN-MEDIATED CYTOSKELETAL DEGRADATION CORRELATES WITH STIMULATION OF PLATELET PROCOAGULANT ACTIVITY

1987 ◽  
Author(s):  
P F J verhallen ◽  
E M Bevers ◽  
P Comfurius ◽  
W M A Linkskens ◽  
R F A Zwaal
Keyword(s):  
Outer Surface ◽  
Time Course ◽  
Degradation Products ◽  
Cytoskeletal Proteins ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Calpain Activity ◽  
Similar Time ◽  
Coagulant Activity ◽  
Relationship Of

We have shown earlier that the negatively charged phospholipid phosphatidylserine (PS), which becomes translocated from the inner surface to the outer surface of the plasma membrane upon platelet activation, is responsible for platelet procoagulant activity. Studies with erythrocytes have suggested a role for cytoskeletal proteins in the regulation of transmembrane asymmetry of PS. The possibility that platelet cytoskeletal proteins are involved in the loss of transmembrane asymmetry of PS, was explored by correlative investigations of both platelet prooagulant activity and activity of calpain, an endogenous Ca 2+ -dependent thiol-protease, known to hydrolyze major cytoskeletal proteins (e.g.: filamin, talin, myosin). Platelet procoagulant activity was assayed by determination of the prothrombinase activity under conditions at which the catalytic PS-surface was rate-limiting. Calpain-activity was monitored by the appearance of known degradation products of major cytoskeletal proteins. The following results were obtained: (1) The ability of thrombin, collagen, collagen & thrombin, or the Ca -ionophore A23187 to stimulate platelet procoagulant activity closely correlated with their ability to stimulate platelet calpain-activity (2). Generation of platelet procoagulant activity upon platelet stimulation by collagen & thrombin or by A23187 exhibited a time course identical to the development of calpain-activity. In addition, the local anesthetics dibucaine and tetracaine, shown to gradually stimulate calpain activity, were able to generate platelet procoagulant activity with a similar time course. (3) Using a Ca2+ buffering system and A23187 to equilibrate intracellular- and extracellular free Ca2+ , it was found that the Ca2+ -response relationship of both platelet calpain- and pro-coagulant-activity was identical. From these findings we conclude that the degradation of cytoskeletal proteins destroys their putative interactions with PS, enabling this lipid to participate in transbilayer movement, leading to the formation of a procoagulant outer surface of the platelet.

scholarly journals Inhibition by calcium ions of adenosine cyclic monophosphate formation in sealed pigeon erythrocyte ‘ghosts’. A study using the photoprotein obelin

1978 ◽  
Vol 176 (1) ◽  
pp. 53-66 ◽  
Author(s):  
A K Campbell ◽  
R L Dormer
Keyword(s):  
Cyclic Amp ◽  
Initial Rate ◽  
Ionophore A23187 ◽  
Erythrocyte Ghosts ◽  
Adrenergic Agonists ◽  
Intact Cells ◽  
Beta Adrenergic ◽  
Beta Adrenergic Agonists ◽  
The Relationship ◽  
Stimulation Of

1. Sealed pigeon erythrocyte ‘ghosts’ were prepared containing ATP and the Ca2+-activated photoprotein obelin to investigate the relationship cyclic AMP formation and internal free Ca2+. 2. The ‘ghosts’ were characterized by (a) morphology (optical and electron microscopy), (b) composition (haemoglobin, K+, Na+, Mg2+, ATP, obelin), (c) permeability to Ca2+, assessed by obelin luminescence and (d) hormone sensitivity (the effect of beta-adrenergic agonists and antagonists on cyclic AMP formation). 3. The effect of osmolarity at haemolysis and ATP at resealing on these parameters was investigated. 4. Sealed ‘ghosts’, containing approx. 2% of original haemoglobin, 150mM-K+, 0.5MM-ATP, 10(3)–10(4) obelin luminescence counts/10(6) ‘ghosts’, which were relatively impermeable to Ca2+ and in which cyclic AMP formation was stimulated by beta-adrenergic agonists over a concentration range similar to that for intact cells, could be prepared after haemolysis in 6mM-NaCl3mM-MgCl2/50mM-Tes, pH7, and resealing for 30min at 37 degrees C in the presence of ATP and 150mM-KCl. 5. The initial rate of adrenaline-stimulated cyclic AMP formation in these ‘ghosts’ was 30–50% of that in intact cells and was inhibited by the addition of extracellular Ca2+. Addition of Ca2+ to the ‘ghosts’ resulted in a stimulation of obelin luminescence, indicating an increase in internal free Ca2+ under these conditions. 6. The ionophore A23187 increased the rate of obelin luminescence in the ‘ghosts’ and also inhibited the adrenaline-stimulated increase in cyclic AMP. 7. The effect of ionophore A23187 on obelin luminescence and on cyclic AMP formation in the ‘ghosts’ was markedly decreased by sealing EGTA inside the ‘ghosts’. 8. It was concluded that cyclic AMP formation inside sealed pigeon erythrocyte ‘ghosts’ could be inhibited by more than 50% by free Ca2+ concentrations in the range 1–10 micrometer.

scholarly journals The activation of Na+-dependent efflux of Ca2+ from liver mitochondria by glucagon and β-adrenergic agonists

1983 ◽  
Vol 210 (2) ◽  
pp. 463-472 ◽  
Author(s):  
T P Goldstone ◽  
R J Duddridge ◽  
M Crompton
Keyword(s):  
Cyclic Amp ◽  
Liver Mitochondria ◽  
Ionophore A23187 ◽  
Adrenergic Agonists ◽  
Adrenergic Agonist ◽  
Perfusion Medium ◽  
Beta Adrenergic ◽  
Beta Adrenergic Agonist

The Na+-induced efflux of Ca2+ from liver mitochondria was activated by tissue pretreatment with 1 microM-adrenaline, 1 microM-isoprenaline, 10 nM-glucagon and 100 microM-cyclic AMP when 10 mM-lactate plus 1 mM-pyruvate were present in the perfusion medium. Infusion of the alpha 1-adrenergic agonist, phenylephrine (10 microM), was ineffective. The activation induced by the beta-adrenergic agonist, isoprenaline, was maximal after infusion of agonist for 2 min. The isoprenaline-induced activation was very marked (120-220%), with about 7 nmol of intramitochondrial Ca2+/mg of protein, but was not evident with greater than 15 nmol of Ca2+/mg. Ca2+ efflux in the absence of Na+ and in the presence of the Ca2+ ionophore A23187 was not affected by isoprenaline pretreatment over the range 6-23 nmol of internal Ca2+/mg. With 10 mM-lactate plus 1 mM-pyruvate in the perfusion medium, glucagon and isoprenaline infusion increased tissue cyclic AMP content about 8-fold and 3-fold respectively. With 10 mM-pyruvate alone, neither glucagon nor isoprenaline caused a significant increase in cyclic AMP. Omission of lactate also abolished the ability of glucagon, but not of isoprenaline, to activate the Na+-induced efflux of Ca2+. The data indicate that cyclic AMP may mediate the activation caused by glucagon, but provide no evidence that cyclic AMP is an obligatory link in the beta-adrenergic-induced activation.

scholarly journals Calcium metabolism and amylase release in rat parotid acinar cells

1976 ◽  
Vol 160 (3) ◽  
pp. 547-564 ◽  
Author(s):  
P Kanagasuntheram ◽  
P J Randle
Keyword(s):  
Cyclic Amp ◽  
Molecular Mechanisms ◽  
Acinar Cells ◽  
Ionophore A23187 ◽  
Free Medium ◽  
Dibutyryl Cyclic Amp ◽  
Trypan Blue Exclusion ◽  
Amylase Release ◽  
Parotid Acinar Cells ◽  
Rat Parotid

1. A method is described for the isolation of rat parotid acinar cells by controlled digestion of the gland with trypsin followed by collagenase. As judged by Trypan Blue exclusion, electron microscopy, water, electrolyte and ATP concentrations and release of amylase and lactate dehydrogenase, the cells are morphologically and functionally intact. 2. A method was developed for perifusion of acinar cells by embedding them in Sephadex G-10. Release of amylase was stimulated by adrenaline (0.1-10μM), isoproternol (1 or 10 μM), phenylephrine (1 μM), carbamoylcholine (0.1 or 1 μM), dibutyryl cycle AMP (2 MM), 3-isobutyl-1-methylxanthine (1mM) and ionophore A23187. The effects of phenylephrine, carbamoylcholine and ionophore A23187 required extracellular Ca2+, whereas the effects of adrenaline and isoproterenol did not. 3. The incorporation of 45Ca into parotid cells showed a rapidly equilibrating pool (1-2 min) corresponding to 15% of total Ca2+ and a slowly equilibrating pool (> 3h) of probably a similar dimension. Cholinergic and α-adrenergic effectors and ionophore A23187 and 2,4-dinitrophenol increased the rate of incorporation of 45Ca into a slowly equilibrating pool, whereas β-adrenergic effectors and dibutyryl cyclic AMP were inactive. 4. The efflux of 45Ca from cells into Ca2+-free medium was inhibited by phenylephrine and carbamoylcholine and accelerated by isoproterenol, adrenaline (β-adrenergic effect), dibutyryl cyclic AMP and ionophore A23187. 5. A method was developed for the measurement of exchangeable 45Ca in mitochondria in parotid pieces. Incorporation of 45Ca into mitochondria was decreased by isoproterenol, dibutyryl cyclic AMP or 2,4-dinitrophenol, increased by adrenaline, and not changed significantly by phenylephrine or carbamoylcholine. Release of 45Ca from mitochondria in parotid pieced incubated in a Ca2+-free medium was increased by isoproterenol, adrenaline, dibutyryl cyclic AMP or 2,4-dinitrophenol and unaffected by phenylephrine or carbamoylcholine. 6. These findings are compatible with a role for Ca2+ as a mediator of amylase-secretory responses in rat parotid acinar cells, but no definite conclusions about its role can be drawn in the absence of knowledge of the molecular mechanisms involved, their location, and free Ca2+ concentration in appropriate cell compartment(s).

scholarly journals Insulin and tri-iodothyronine induce glucokinase mRNA in primary cultures of neonatal rat hepatocytes

1990 ◽  
Vol 271 (3) ◽  
pp. 585-589 ◽  
Author(s):  
M R Narkewicz ◽  
P B Iynedjian ◽  
P Ferre ◽  
J Girard
Keyword(s):  
Hormonal Regulation ◽  
Time Course ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Neonatal Rat ◽  
Free Medium ◽  
Ontogenic Development ◽  
Lactate And Pyruvate ◽  
Dose Dependent

Glucokinase (EC 2.7.1.2) first appears in the liver of the rat 2 weeks after birth and increases after weaning on to a high-carbohydrate diet. We investigated the hormonal regulation of glucokinase (GK) mRNA in primary cultures of hepatocytes from 10-12-day-old suckling rats. GK mRNA was undetectable in such cells after 48 h of culture in serum-free medium devoid of hormones. Addition of insulin or tri-iodothyronine (T3) to the medium resulted in induction of GK mRNA. The effects of insulin and T3 were dose-dependent and additive. Dexamethasone alone did not induce GK mRNA, but enhanced the response to insulin and decreased the response to T3. Induction of GK mRNA by insulin was not affected when the medium glucose concentration was varied between 5 and 15 mM, nor when culture was conducted in glucose-free medium supplemented with lactate and pyruvate or galactose. The time course of initial accumulation of GK mRNA in response to insulin was characterized by a lag of 12 h and an induction plateau reached after 36 h. If hepatocytes were then withdrawn from insulin for 24 h and subsequently subjected to a secondary stimulation by insulin, GK mRNA re-accumulated with much faster kinetics and reached the fully induced level within 8 h. Both primary and secondary responses to insulin were abolished by actinomycin D. These results provide insight into the role of hormonal stimuli in the ontogenic development of hepatic glucokinase.

scholarly journals Epidermal growth factor mimics insulin effects in rat hepatocytes

1986 ◽  
Vol 239 (3) ◽  
pp. 523-530 ◽  
Author(s):  
F Bosch ◽  
B Bouscarel ◽  
J Slaton ◽  
P F Blackmore ◽  
J H Exton
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cyclic Amp ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Rat Hepatocytes ◽  
Maximum Effect ◽  
Similar Time ◽  
Isolated Rat Hepatocytes ◽  
Epidermal Growth

Epidermal growth factor (EGF) mimicked the effect of insulin to activate glycogen synthase and stimulate glycogen synthesis in isolated rat hepatocytes. Both agents required glucose (greater than 5 mM) and had similar time courses of action. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were 9 nM and 4 nM respectively. Combinations of the two agents produced additive responses. EGF also resembled insulin in its ability to inhibit the effects of 0.1-1.0 nM-glucagon on cyclic AMP and glycogen phosphorylase in hepatocytes. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were approx. 5 nM and 0.5 nM respectively. EGF and insulin inhibited phosphorylase activation by exogenous cyclic AMP, and inhibited cyclic AMP accumulation induced by forskolin. They also inhibited phosphorylase activation provoked by phenylephrine, but not by vasopressin. EGF added alone rapidly activated phosphorylase and increased cytosolic [Ca2+], but the effects were no longer apparent at 5 min and were smaller than those of vasopressin. Insulin did not induce these changes. In hepatocytes previously incubated with myo-[3H]inositol, EGF did not significantly increase myo-inositol 1,4,5-trisphosphate. However, its ability to increase cytosolic [Ca2+] was blocked by neomycin, an inhibitor of phosphatidylinositol bisphosphate hydrolysis. It is concluded that some, but not all, of the effects of EGF in liver are strikingly similar to those exerted by insulin, suggesting that these agents may have some similar mechanisms of action in this tissue.

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