scholarly journals Rapid decrease in the expression of 3-hydroxy-3-methylglutaryl-CoA reductase protein owing to inhibition of its rate of synthesis after Ca2+ mobilization in rat hepatocytes. Inability of taurolithocholate to mimic the effect

1991 ◽  
Vol 279 (2) ◽  
pp. 377-383 ◽  
Author(s):  
V A Zammit ◽  
A M Caldwell ◽  
M P Kolodziej
Keyword(s):  
Glycogen Phosphorylase ◽  
Rat Hepatocytes ◽  
Total Activity ◽  
Enzyme Synthesis ◽  
Enzyme Protein ◽  
Extracellular Medium ◽  
Hmg Coa Reductase ◽  
Cell Incubation ◽  
Formed Part ◽  
Coa Reductase

The mechanisms through which Ca2+ mobilization in rat hepatocytes results in the loss of total activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase [Zammit & Caldwell (1990) Biochem. J. 269, 373-379] were investigated. The loss of total activity was shown to be paralleled by an equal loss of immunoreactive HMG-CoA reductase protein after exposure of hepatocytes to optimal concentrations of vasopressin plus glucagon for 40 min. This loss of enzyme protein was due to an inhibition of enzyme synthesis; the rate of degradation was unaffected. Other Ca(2+)-mobilizing conditions (phenylephrine, glucagon, vasopressin added singly and A23187) also resulted in graded inhibition of synthesis of HMG-CoA reductase. These effects were accentuated by omission of Ca2+ from the cell incubation medium, suggesting that it is the depletion of an intracellular InsP3-sensitive pool of Ca2+ to which synthesis of HMG-CoA reductase is sensitive. In agreement with this we found that t-butylhydroxybenzoquinone, which inhibits the activity of the Ca(2+)-ATPase of the endoplasmic-reticular membrane, mimicked the action of Ca(2+)-mobilizing hormones. However, taurolithocholate, which transiently mobilizes Ca2+ from the same pool, was ineffective. All these effects on HMG-CoA reductase were accompanied by parallel inhibition of 35S incorporation from [35S]methionine into total protein, suggesting that inhibition of reductase synthesis formed part of a generalized response of the hepatocyte to Ca2+ mobilization. Inhibition of the rate of synthesis of HMG-CoA reductase was, however, more responsive to Ca2+ mobilization in the absence of added Ca2+ from the extracellular medium. The concentrations of vasopressin required to elicit the inhibition of synthesis of HMG-CoA reductase were of the same order as those that elicited activation of glycogen phosphorylase in hepatocytes.

scholarly journals The roles of different protein kinases and of calmodulin in the effects of Ca2+ mobilization on 3-hydroxy-3-methylglutaryl-CoA reductase activity in isolated rat hepatocytes

1991 ◽  
Vol 273 (2) ◽  
pp. 485-488 ◽  
Author(s):  
V A Zammit ◽  
A M Caldwell
Keyword(s):  
Protein Kinase ◽  
Reductase Activity ◽  
Rat Hepatocytes ◽  
Total Activity ◽  
Isolated Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Hmg Coa Reductase ◽  
Lysosomal Proteolysis ◽  
Dependent Protein ◽  
Coa Reductase

The roles of protein kinase C, Ca2+/calmodulin-dependent protein kinase and AMP-activated protein kinase in the phosphorylation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase induced by Ca2(+)-mobilizing conditions in isolated hepatocytes were investigated. Only partial evidence for the involvement of AMP-activated kinase was found. Antagonism of calmodulin action prolonged the decrease in expressed/total activity ratio induced by vasopressin plus glucagon. Protease inhibitors active against Ca2(+)-dependent cytosolic proteases or lysosomal proteolysis did not attenuate the loss of total HMG-CoA reductase induced by glucagon plus vasopressin, but calmodulin antagonists largely prevented this effect.

scholarly journals Direct demonstration that increased phosphorylation of 3-hydroxy-3-methylglutaryl-CoA reductase does not increase its rate of degradation in isolated rat hepatocytes

1992 ◽  
Vol 284 (3) ◽  
pp. 901-904 ◽  
Author(s):  
V A Zammit ◽  
A M Caldwell
Keyword(s):  
Rat Hepatocytes ◽  
Total Activity ◽  
Specific Rate ◽  
Ionophore A23187 ◽  
Isolated Rat Hepatocytes ◽  
Hmg Coa Reductase ◽  
Phosphorylation State ◽  
Direct Demonstration ◽  
Coa Reductase ◽  
Isolated Rat

Increased phosphorylation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase has been suggested to target the protein towards an increased rate of degradation. Our previous observations [Zammit & Caldwell (1990) Biochem. J. 269, 373-379] suggested that, although Ca(2+)-mobilizing hormones and other effectors can alter both the phosphorylation state of the enzyme and its total activity in isolated rat hepatocytes, there appears to be no causal correlation between the two parameters. In the present paper we set out to make direct measurements of the specific rate of degradation of 35S-labelled HMG-CoA reductase in hepatocytes treated with agents that produced very marked and prolonged increases in the degree of phosphorylation of the protein, through different mechanisms. Okadaic acid (which inhibits phosphatases 1 and 2A), fructose (which increases cellular AMP through its metabolism to fructose 1-phosphate) and the Ca2+ ionophore A23187 (which also raises cellular AMP through an unknown mechanism) were all unable to alter the rate of HMG-CoA reductase degradation. We conclude that the basal rate of degradation of HMG-CoA reductase is unaffected by its phosphorylation state and that a transiently increased degree of phosphorylation cannot be the mechanism through which mevalonate increases the rate of degradation of the enzyme in rat hepatocytes and other cell types.

scholarly journals Conditions that result in the mobilization and influx of Ca2+ into rat hepatocytes induce the rapid loss of 3-hydroxy-3-methylglutaryl-CoA reductase activity that is not reversed by phosphatase treatment

1990 ◽  
Vol 269 (2) ◽  
pp. 373-379 ◽  
Author(s):  
V A Zammit ◽  
A M Caldwell
Keyword(s):  
Enzyme Activity ◽  
Okadaic Acid ◽  
Reductase Activity ◽  
Total Activity ◽  
Ionophore A23187 ◽  
Hmg Coa Reductase ◽  
Pronounced Increase ◽  
Phosphorylation State ◽  
Total Enzyme Activity ◽  
Coa Reductase

We investigated the effects of conditions that induce Ca2+ mobilization from intracellular stores and Ca2+ influx into hepatocytes on the expressed and total (fully dephosphorylated) activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Vasopressin and phenylephrine when added alone had small or negligible effects on the phosphorylation state of the enzyme, as judged from the expressed/total activity ratio. However, when added in combination with glucagon, they elicited appreciable increases in the phosphorylation of the enzyme. Glucagon on its own had no effect either on phosphorylation state or on total HMG-CoA reductase activity during 40 min of incubation. Under conditions of sustained Ca2+ influx (i.e. vasopressin or phenylephrine plus glucagon), there was a marked loss of total HMG-CoA reductase activity. This effect was more pronounced when vasopressin was used; 50% of the enzyme activity was lost within 40 min. The involvement of Ca2+ in these effects was verified directly by the use of ionophore A23187. Its addition to hepatocytes resulted both in a very pronounced increase in the phosphorylation state of the enzyme and in the loss of 50% of the total activity within 30 min. There was no correlation between the ability of any set of conditions to increase the phosphorylation of the enzyme and the subsequent loss of total HMG-CoA reductase activity. The latter parameter appeared to be directly related, however, to the maintenance of prolonged Ca2+ influx, as indicated by the continued activation of glycogen phosphorylase, measured in the same cells. The lack of a causal relationship between increased phosphorylation and loss of total activity was demonstrated directly by studies in which okadaic acid was used to induce phosphorylation of HMG-CoA reductase in hepatocytes by inhibition of phosphatase 1 and 2A activities. This was not accompanied by any loss of total enzyme activity. Neither did okadaic acid enhance the loss of reductase induced by A23187 when the two agents were added together. It is concluded that altered Ca2+ fluxes in hepatocytes in vivo, under conditions of acute or chronic stress (such as may be associated with trauma or diabetes respectively), may be involved in the regulation of the expression of HMG-CoA reductase activity through alteration of enzyme concentration in the liver.

scholarly journals Role of reversible phosphorylation in mediating changes in the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase during pregnancy and lactation in the rat

1987 ◽  
Vol 248 (3) ◽  
pp. 993-996 ◽  
Author(s):  
R A Easom ◽  
V A Zammit
Keyword(s):  
Reductase Activity ◽  
Post Partum ◽  
Active Form ◽  
Total Activity ◽  
Female Rats ◽  
Hmg Coa Reductase ◽  
Pregnancy And Lactation ◽  
Coa Reductase

1. The expressed and total (completely dephosphorylated) activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase were measured in microsomal fractions isolated from cold-clamped liver samples from female rats in various stages of the reproductive cycle. 2. There was little change in total HMG-CoA reductase activity during pregnancy and early lactation, but after 2 days post partum there was a marked increase in total activity. 3. The expressed/total activity ratio of HMG-CoA reductase showed a profound decrease during the last 2 days of pregnancy. The fraction of the enzyme in the active form increased progressively during the first 2 days of lactation. 4. The combined effect of these changes was that the expressed activity of HMG-CoA reductase changed in parallel with the known changes in the hepatic rate of cholesterogenesis during pregnancy and lactation in vivo.

scholarly journals Diurnal variation in the fraction of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the active form in the mammary gland of the lactating rat

1986 ◽  
Vol 239 (2) ◽  
pp. 285-293 ◽  
Author(s):  
R A Smith ◽  
B Middleton ◽  
D W West
Keyword(s):  
Reductase Activity ◽  
Active Form ◽  
Total Activity ◽  
Maximum Activity ◽  
Covalent Modification ◽  
Light Phase ◽  
Hmg Coa Reductase ◽  
Enzyme Molecules ◽  
Coa Reductase ◽  
Homogenization Medium

‘Expressed’ and ‘total’ activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) were measured in freeze-clamped samples of mammary glands from lactating rats at intervals throughout the 24 h light/dark cycle. ‘Expressed’ activities were measured in microsomal fractions isolated and assayed in the presence of 100 mM-KF. ‘Total’ activities were determined in microsomal preparations from the same homogenates but washed free of KF and incubated with exogenously added sheep liver phosphoprotein phosphatase before assay. Both ‘expressed’ and ‘total’ activities of HMG-CoA reductase underwent a diurnal cycle, which had a major peak 6 h into the light phase and a nadir 15 h later, i.e. 9 h into the dark period. Both activities showed a secondary peak of activity (around 68% of the maximum activity) at the time of changeover from dark to light, with a trough in the value of the ‘expressed’ activity that was close to the nadir value. ‘Expressed’ activity was lower than ‘total’ at all time points, indicating the presence of enzyme molecules inactivated by covalent phosphorylation. Nevertheless the ‘expressed’/‘total’ activity ratio was comparatively constant and varied only between 43% and 75%. Immunotitration of enzyme activity, with antiserum raised in sheep against purified rat liver HMG-CoA reductase, confirmed the presence of both active and inactive forms of the enzyme and indicated that at the peak and nadir the variation in ‘expressed’ HMG-CoA reductase activity resulted from changes in the total number of enzyme molecules rather than from covalent modification. The sample obtained after 3 h of the light phase exhibited an anomalously low ‘total’ HMG-CoA reductase activity, which could be increased when Cl- replaced F- in the homogenization medium. The result suggests that at that time the activity of the enzyme could be regulated by mechanisms other than covalent phosphorylation or degradation.

The effects of pravastatin, an HMG-CoA reductase inhibitor, on cell viability and dna production of rat hepatocytes

Life Sciences ◽  
1996 ◽  
Vol 59 (14) ◽  
pp. 1103-1108 ◽  
Author(s):  
Takeaki Satoh ◽  
Hidehiko Isobe ◽  
Kusuo Ayukawa ◽  
Hironori Sakai ◽  
Hajime Nawata
Keyword(s):  
Cell Viability ◽  
Reductase Inhibitor ◽  
Rat Hepatocytes ◽  
Hmg Coa Reductase ◽  
Dna Production ◽  
Hmg Coa Reductase Inhibitor ◽  
Coa Reductase

scholarly journals Biogenesis of the crystalloid endoplasmic reticulum in UT-1 cells: evidence that newly formed endoplasmic reticulum emerges from the nuclear envelope.

1986 ◽  
Vol 102 (6) ◽  
pp. 2158-2168 ◽  
Author(s):  
R K Pathak ◽  
K L Luskey ◽  
R G Anderson
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Cholesterol Biosynthesis ◽  
Integral Membrane Protein ◽  
Enzyme Synthesis ◽  
Hmg Coa Reductase ◽  
Outer Nuclear Membrane ◽  
Sequence Of Events ◽  
Coa Reductase

The crystalloid endoplasmic reticulum (ER), a specialized smooth ER of the compactin-resistant UT-1 cell, is composed of multiple membrane tubules packed together in a hexagonal pattern. This membrane contains large amounts of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, an integral membrane protein that enzymatically regulates endogenous cholesterol biosynthesis. Using morphological and immunocytochemical techniques, we have traced the sequence of events in the biogenesis of this ER when compactin-withdrawn UT-1 cells, which do not have a crystalloid ER, are incubated in the presence of compactin. After 15 h of incubation in the presence of compactin, many cells had profiles of ER cisternae that were juxtaposed to the nuclear envelope and studded with ribosomes on their outer membrane. Both the outer nuclear membrane and the ER membrane contained HMG CoA reductase; however, there was little or no detectable enzyme in rough ER that was free in the cytoplasm. With longer times of incubation in the presence of compactin, these cells had lamellar stacks of smooth ER next to the nuclear envelope that contained HMG CoA reductase. Coordinate with the appearance of the smooth ER, crystalloid ER appeared in the same cell. Often regions of continuity were found between the membrane of the smooth ER and the membrane of the crystalloid ER tubules. These studies suggest that HMG CoA reductase is synthesized along the outer nuclear membrane and in response to increased enzyme synthesis, a membrane emerges from the outer nuclear membrane as smooth ER cisternae, which then transforms into crystalloid ER tubules.

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