Activation of sterol ester hydrolase of bovine corpus luteum by N6,O2′-dibutyryl cyclic adenosine 3′∶5′-phosphate

The direct activation of sterol ester hydrolase (E.C. 3.1.1.13) in homogenates of bovine corpus luteum by N⁶O^2′·dibutyryl cyclic adenosine 3′∶5′-phosphate, (dibutyryl cAMP), adenosine triphosphate (ATP), and Mg²⁺ has been demonstrated. Variability in the extent of activation by the additions was minimized by homogenization of the tissue in 5 mM Mg²⁺. Baseline sterol ester hydrolase activity was primarily associated with the 105,000 × g soluble fraction, and significant activation of the enzyme preparation preincubated with dibutyryl cAMP, ATP and Mg²⁺ occurred within the first 15 min, prior to addition of substrate. A requirement for protein kinase in the system was demonstrated by blocking the cofactor-dependent enzyme activation with commercial protein kinase inhibitor.     

Regulation of rat liver microsomal cholesterol ester hydrolase by reversible phosphorylation

The regulation of neutral cholesterol ester hydrolase activity by changes in its phosphorylation state was studied in rat liver microsomes. Treatment with cAMP-dependent protein kinase resulted in increased enzyme activity, which was further enhanced by the addition of cAMP and MgATP. Consistent activations were also achieved with MgCl₂ and MgATP, the magnesium effect being abolished by ethylenediaminetetraacetic acid and adenosine triphosphate. Cholesterol ester hydrolase was activated twofold by free calcium and Ca²⁺/calmodulin; this latter effect was blocked by the chelator ethyleneglycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid and the calmodulin antagonist trifluoperazine. The phosphatase inhibitors pyrophosphate and glycerophosphate led to marked and dose-dependent increases in esterase activity, whereas okadaic acid elicited no effect. Furthermore, pyrophosphate and okadaic acid did not change the increases in enzyme activity promoted by Ca²⁺, Ca²⁺/calmodulin, Mg²⁺ and MgATP. Cholesterol ester hydrolase was inactivated in a concentration-dependent manner by nonspecific alkaline phosphatases. In cAMP-dependent protein kinase/cAMP- or Ca²⁺/calmodulin-activated microsomes, a time-dependent loss of activation in cholesteryl oleate hydrolysis was caused by alkaline phosphatase. These findings suggest that microsomal cholesterol ester hydrolase is activated through cAMP and Ca²⁺/calmodulin phosphorylation, whereas enzyme deactivation is dependent on phosphatase action.     

Cupric ion-dependent inhibition of lysosomal acid cholesteryl ester hydrolase in the presence of hydroxylamine

In the presence of hydroxylamine or ascorbic acid, the inhibitory effects of Cu²⁺ on lysosomal acid cholesteryl ester hydrolase (acid CEH) partially purified from rat liver were studied. Hydroxylamine stimulated the inhibition of acid CEH activity by Cu²⁺ but not that by Zn²⁺, Fe²⁺, Co²⁺, Mn²⁺, Ca²⁺, Mg²⁺ and Hg²⁺. This Cu²⁺-dependent inhibition of acid cholesterol ester hydrolase (CEH) activity was completely prevented by ethylenediamine tetraacetic acid (EDTA), EGTA and o-phenanthroline, a chelator with a stability constant for Cu²⁺, and also by sulfhydryl agents and cytoplasmic reducing agents such as cysteine, glutathione and mercaptoethanol. In addition, the stimulative effects of hydroxylamine on Cu²⁺-dependent inhibition were maintained even after preincubation of Cu²⁺ with hydroxylamine. On the other hand, ascorbic acid was found to replace the stimulation by hydroxylamine of the Cu²⁺-dependent inhibition of acid CEH activity but the effects of ascorbic acid progressively became smaller with prolongation of the preincubation time. Moreover, addition of chemical radical scavengers to the reaction mixture did not prevent the Cu²⁺-dependent inhibition of acid CEH activity in the presence of ascorbic acid. These results suggest that Cu²⁺ causes inhibition of lysosomal acid CEH activity through the formation of Cu¹⁺ in a reductive medium.     

Cholesterol metabolism in the rat lactating mammary gland: The role of cholesteryl ester hydrolase

An acid cholesteryl ester hydrolase activity associated with a fraction containing mitochondria and lysosomes from rat lactating mammary glands was found to have a pH optimum of 5.0. Its sedimentation pattern was closely related to that of the lysosomal enzyme markers acid phosphatase and β-glucuronidase, suggesting that the activity is associated with the lysosomes. The enzyme was strongly inhibited by Cu²⁺, but was inhibited little by other divalent metal ions. Acid cholesteryl ester hydrolase activity was almost completely abolished byp-hydroxymercuribenzoate, but this effect was reversed in the presence of an equimolar concentration of reduced glutathione (GSH), indicating that the enzyme requires free sulfhydryl groups for activity. These properties are similar to those of acid, lysosomal cholesteryl ester hydrolases found in other tissues. Acid cholesteryl ester hydrolase activity was 8–14 fold higher in mammary tissue from lactating as compared to virgin rats. Neutral cholesteryl ester hydrolase activities associated with the microsomal and cytosolic subcellular fractions were also increased in lactating glands, but to a lesser extent. In addition, a 2-fold increase in the activities of both the acid and microsomal neutral enzymes was seen during the first few days of lactation, while the cytosolic neutral activity remained constant. These results suggest that mammary gland cholesteryl ester hydrolases have a role in the regulation of cholesterol metabolism in mammary cells, and in the provision of cholesterol for secretion into milk.     

Reversible ATP-dependent inactivation of adipose diacylglycerol acyltransferase

Diacylglycerol acyltransferase from rat adipose tissue is shown to be inactivated by 30 to 40% upon incubation with adenosine 5′-triphosphate (ATP) and Mg²⁺. The activity responsible for this inactivation is associated with the cytosolic fraction, specific for ATP, prevented when ATP is substituted by β,γ-methylene-ATP, and partially blocked by 1 mM ethylenediaminetetraacetate or 40 mM NaF, but not by inhibitors of adenosine 3′,5′-cyclic-monophosphate (cAMP)-dependent protein kinase and/or protein kinase C (PKC). The cytosolic activity cannot be mimicked by (cAMP)-protein kinase nor by PKC. Inactivated diacylglycerol acyltransferase from ATP/cytosol-treated microsomes can be reactivated by incubation with partially purified protein phosphate from rat liver, and can be inactivated again by further addition of ATP in the presence of cytosol. The results suggest the existence in adipose tissue of a protein kinase other than cAMP-protein kinase or PKC, which may be involved in the regulation of triacylglycerol synthesis.     

Temperature lability and cAMP-dependent protein kinase activation of cholesteryl ester hydrolase as a function of age in developing rat testis

Cholesteryl ester hydrolase (CEH) was measured at 32°C and 37°C, and with and without cofactors for stimulation of cyclic AMP-dependent protein kinase, in 104,000×g supernatants from rats aged 14–365 days. Activity at the two temperatures was also partially resolved by cation exchange FPLC. Total specific activity of CEH was relatively constant, with or without addition of cofactors, from 14 to 47 days, during which time temperature labile CEH was a very small fraction of total CEH activity. At later times, 51–150 days, activity was increased as much as two-fold, both with and without cofactors, with most of the increase occurring in the temperature labile fraction. Activation of temperature stable and temperature labile activities, where present, by protein kinase cofactors could be demonstrated in all age groups, but was highly variable as a function of age and protein concentration used in the assay. Apparent induction of temperature labile activity over the interval 47–51 days coincides with reported increases in testosterone synthesis and first appearance of spermatozoa in the testis. This and other lines of evidence suggest unique roles for these enzymes in regulation of availability of free cholesterol for testosterone and membrane synthesis, respectively.     

Synergistic enhancement of the antiproliferative activity ofcis-diamminedichloroplatinum(II) by the ether lipid analogue BM41440, an inhibitor of protein kinase C

The new phospholipid analogue 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine inhibits the phospholipid-calcium-dependent protein kinase, partially purified from Walker carcinoma cells with a K_i value of 0.56 μM. The compound inhibits the phorbol ester stimulated phosphorylation of the ribosomal protein S6 indicating that the depression of Ca²⁺-phospholipid-dependent protein kinase by the alkyl phospholipid also occurs in intact cells. The dose effect curve for the inhibition of cell proliferation by 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine in Walker cells exhibits a close correlation to the dose effect curve for the depression of Ca²⁺-phospholipid-dependent protein kinase activity. Although alternative mechanisms cannot be excluded, the data suggest that the growth inhibitory activity of 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine correlates with the inhibition of Ca²⁺-phospholipid-dependent protein kinase. The antiproliferative activity of 3-hexadecylmercapto-2-methoxymethyl-propyl-1-phosphocholine is synergistically enhanced bycis-diamminedichloroplatinum(II).     

Inhibition of the hormone-sensitive lipase in adipose tissue by long-chain fatty acyl coenzyme A

The effects of free fatty acids and fatty acyl esters of coenzyme A and carnitine on the activity of a hormone-sensitive lipase preparation made from pigeon adipose tissue were determined. Oleic acid (100 μM) resulted in a 40% inhibition of lipase activity A more potent inhibition of lipase activity was seen with long-chain fatty acyl CoA compounds. The concentration required for half-maximal inhibition with oleoyl CoA and palmitoyl CoA was 25–40 μM, whereas palmitoyl carnitine stimulated lipase activity. Activated lipase preparations (preincubated with Mg²⁺, ATP, cyclic AMP and protein kinase) were 4–6 times more sensitive to inhibition by oleoyl CoA than were nonactivated preparations. An increase in cellular levels of fatty acyl coenzyme A could, therefore, contribute to the feedback inhibition of lipolysis in adipose tissue.     

Effects of hexadecylphosphocholine on protein kinase C and TPA-induced differentiation of HL60 cells

Several structural analogs of alkylphosphocholine (APC) were studied for their effects on protein kinase C (PKC) and 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited biochemical and cellular events in HL60 cells. Hexadecylphosphocholine (He-PC₂), the APC prototype, inhibited PKC competitively with respect to phosphatidylserine and noncompetitively with respect to CaCl₂, both with an apparent K_i of about 15 μM. Inhibition of PKC by He-PC₂ was selective, since cyclic AMP dependent protein kinase and Ca²⁺/calmodulin dependent protein kinase II were relatively unaffected. He-PC₂ inhibited TPA-induced depletion of PKC and TPA-stimulated phosphorylation of cellular proteins in HL60 cells. TPA-induced differentiation of HL60 cells was also inhibited by He-PC₂, and this inhibition was synergistic or additive to the effects of 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), a PKC inhibitor. The present findings are consistent with the hypothesis that inhibition of PKC might be related, in part, to the antineoplastic effect of He-PC₂ and ether lipid analogs such as ET-18-OCH₃ (1-octadecyl-2-methyl-glycero-3-phosphocholine).     

A Novel GUCA1A Variant Associated with Cone Dystrophy Alters cGMP Signaling in Photoreceptors by Strongly Interacting with and Hyperactivating Retinal Guanylate Cyclase

Guanylate cyclase-activating protein 1 (GCAP1), encoded by the GUCA1A gene, is a neuronal calcium sensor protein involved in shaping the photoresponse kinetics in cones and rods. GCAP1 accelerates or slows the cGMP synthesis operated by retinal guanylate cyclase (GC) based on the light-dependent levels of intracellular Ca²⁺, thereby ensuring a timely regulation of the phototransduction cascade. We found a novel variant of GUCA1A in a patient affected by autosomal dominant cone dystrophy (adCOD), leading to the Asn104His (N104H) amino acid substitution at the protein level. While biochemical analysis of the recombinant protein showed impaired Ca²⁺ sensitivity of the variant, structural properties investigated by circular dichroism and limited proteolysis excluded major structural rearrangements induced by the mutation. Analytical gel filtration profiles and dynamic light scattering were compatible with a dimeric protein both in the presence of Mg²⁺ alone and Mg²⁺ and Ca²⁺. Enzymatic assays showed that N104H-GCAP1 strongly interacts with the GC, with an affinity that doubles that of the WT. The doubled IC₅₀ value of the novel variant (520 nM for N104H vs. 260 nM for the WT) is compatible with a constitutive activity of GC at physiological levels of Ca²⁺. The structural region at the interface with the GC may acquire enhanced flexibility under high Ca²⁺ conditions, as suggested by 2 μs molecular dynamics simulations. The altered interaction with GC would cause hyper-activity of the enzyme at both low and high Ca²⁺ levels, which would ultimately lead to toxic accumulation of cGMP and Ca²⁺ in the photoreceptor outer segment, thus triggering cell death.     

Effect of magnesium deficiency on Δ6 desaturase activity and fatty acid composition of rat liver microsomes

Experimental Mg²⁺ deficiency was induced in a group of rats by feeding them a Mg²⁺-deficient diet for 23 days. They were pair-fed to compare with a control group of rats fed a Mg²⁺-sufficient diet. In the Mg²⁺-deficient group the plasma total cholesterol and triglyceride levels were increased while HDL-cholesterol was decreased. In the Mg²⁺-deficient group the plasma level of thiobarbituric acid reacting substances (TBARS) used as a measure for lipid peroxidation was increased. The increase was attributed to the increased cytosolic Ca²⁺ in Mg²⁺-deficiency which can cause: 1) increase of hydro and endoperoxide levels as a consequence of the increase of arachidonic acid release and eicosanoid synthesis in Mg²⁺-deficiency, and 2) inhibition of the mitochondrial respiratory activity and activation of Ca²⁺-dependent proteases which may activate the conversion of xanthine dehydrogenase to xanthine oxidase which generates active O₂ species. In the Mg²⁺-deficient group, the fatty acid composition of the liver microsomes indicated a slower rate of conversion of linoleic acid to arachidonic acid which was consistent with the decrease of Δ6 desaturase activity in liver microsomes of Mg²⁺-deficient rats as measuredin vitro. The decrease of Δ6 desaturase activity was attributed to the lower concentration of actual enzyme molecules as a result of the decreased rate of protein synthesis in Mg²⁺-deficiency. The possible effects of the increased catecholamine release in Mg²⁺-deficiency are discussed.     

Modulation by sphingosine of substrate phosphorylation by protein kinase C in bovine mammary gland

The effect of sphingosine on the phosphorylation of endogenous proteins by protein kinase C (PKC) was investigated in bovine mammary gland. Several proteins were shown to be substrates for PKC in both cytosolic and total particulate fractions by phosphorylation in the absence or presence of 1-oleoy-2-acetyl-sn-glycerol, phosphatidylserine (PS) and Ca²⁺. At concentrations of 83 μM or less, sphingosine inhibited phosphorylation of several substrates for PKC in both fractions. Phosphorylation of cytosolic 36 kDa, 21 kDa and particulate 36 kDa proteins was particularly sensitive to sphingosine. Cytosolic 97 kDa phosphorylation (which was enhanced by Ca²⁺ alone) was also sensitive to sphingosine. The inhibition was reversed by excess addition of lipid cofactors, particularly PS, but not by Ca²⁺. At higher concentrations (167 and 417 μM), in addition to the inhibition seen at lower concentrations, sphingosine stimulated phosphorylation of several proteins, including cytosolic 19 kDa and particulate 53 kDa, which were not detected in the absence of sphingosine. The sphingosine-induced phosphorylation disappeared with excess addition of PS, but not with addition of Ca²⁺. The results point toward the importance of the interaction of sphingosine with membrane phospholipids in the signal transduction pathway mediated by PKC-dependent phosphorylation in bovine mammary gland.     

The stability of lactic acid production in resting suspensions of Lactobacillus delbrueckii

The stability of resting glycolytic activity (RGA) i.e. lactic acid production from glucose, in non-growing suspensions of Lactobacillus delbrueckii was examined. Non-growing conditions were imposed by removal of essential nutrients or by the addition of antibiotics. The stability of RGA was reduced in fast-grown chemostat cells. Mg²⁺, reducing agents, the rigorous exclusion of oxygen and a constant supply of energy source gave a five-fold increase in RGA and a 25-fold increase in stability in cells deprived of a nitrogen source. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity severely limited RGA after about 8 h of incubation. No evidence of excessive ATP accumulation or ADP depletion was found. Addition of chloramphenicol, actinomycin D or mitomycin C decreased RGA stability over a 24-h period. Addition of yeast extract and tryptone to a mixture containing phosphate, Mg²⁺, thioglycollate and chloramphenicol gave a three-fold increase in RGA which lasted for about 1 h. Mg²⁺ is believed to stimulate the glycolytic sequence directly and oxygen probably inhibits GAPDH by interaction with the -SH groups of the enzyme. Tryptone and yeast extract probably stimulate pyruvate kinase activity, and this enzyme is also absolutely dependent on fructose-1, 6-diphosphate for activity.     

Phospholipid synthesizing enzymes of dermatophytes: II. Characterization of choline kinase

Choline kinase was located in the cytosolic fractions of the filamentous, pathogenic fungi,Microsporum gypseum andEpidermophyton floccosum. A broad pH optima (6.0–9.0) was observed for theM. gypseum enzyme, but theE. floccosum enzyme was active at pH 8.4 as well as 10.5, the activity being higher at pH 8.4. Enzyme from both dermatophytes had Km value of 3.3×10^?4 M for choline; however, for ATP, it was 6.6×10^?4 M and 12.6×10^?4 M forM gypseum andE. floccosum, respectively. Choline kinase of both dermatophytes showed SH-group requirement. TheM. gypseum choline kinase was inhibited to a greater extent by Mn²⁺, Ca²⁺ and Ba²⁺ than was theE. floccosum enzyme. In comparison to other nucleotides, ATP was the most effective phosphate donor for phosphorylating choline in both dermatophytes. Higher concentrations of ATP inhibited the enzyme inM. gypseum as well asE. floccosum. Phosphorylcholine inhibited the choline kinase activity from both fungi, whereas phosphoethanolamine and glycerol 3-phosphate were stimulatory.     

Effect of ATP on the microsomal desaturation of unsaturated fatty acids

The effect of ATP on the microsomal desaturation of linoleic acid to γ-linolenic acid was studied in a system in vitro with the following results: (1) preincubation of rat liver microsomes with ATP alone in N₂ or in the presence of CoA and Mg⁺⁺ followed by subsequent incubation with 1-¹⁴C-linoleic acid plus NADH in O₂ resulted in enhancement of 1-¹⁴C-linoleic acid desaturation when compared with control samples in which no preincubation was performed; (2) the preincubation of the microsomes with ATP, Mg⁺⁺ and CoA in the presence of 1-¹⁴C-linoleic acid decreased the desaturation of the labeled acid to γ-linolenic acid upon subsequent incubation with NADH, as a consequence of incorporation of the acid into the microsomal lipids; (3) the increase of linoleic acid desaturation depended on the ATP concentration during preincubation and followed a sigmoidal curve. It was specific for ATP, and neither GTP, CTP, ADP nor AMP produced a similar effec. However, GTP or CTP could replace ATP as a cofactor in the microsomal desaturation of free linoleic acid to γ-linolenic, suggesting that directly or indirectly they may activate conversion of the free acid to linoleyl-CoA; (4) preincubation of microsomes with ATP activated the acylation of CoA. However, this activation showed no quantitative correlation with enhancement of the desaturation reaction; (5) addition of ATP also stimulated conversion of linoleyl-CoA to γ-linolenic acid. This enhancement was not related to inhibition of the linoleyl-CoA hydrolase; (6) however, in spite of these results, preincubation with ATP did not increase the initial velocity of linoleic acid or linoleyl-CoA desaturation; (7) preincubation of microsomes with ATP also increased the 6-desaturation of oleic acid and α-linolenic acid but did not increase the 9-desaturation of plamitic and stearic acid.     

Cholesterol ester hydrolase activity in mammary tissue of the lactating rat

Cholesterol ester hydrolase activity has been studied in mammary glands of rats. Subcellular fractionation of the glands obtained in mid-lactation indicated that around 80% of the recovered activity was associated with particulate fractions. Two distinct cholesterol ester hydrolase activities were identified, one with an optimum pH of 7.5–9.0 and the second (approximately 5% of the total activity) with a more acidic pH optimum. Although the neutral cholesterol ester hydrolase had some properties in common with the lipoprotein lipase in mammary tissue, it was shown to be a separate entity by several criteria. Its activity could be increased following treatment with Mg-ATP and cAMP-dependent protein kinase, suggesting identity with the hormone sensitive lipase of adipose tissue. The cholesterol ester hydrolase activity in mammary glands just after parturition was greater than in glands obtained either from late-pregnant or midlactating animals. The subcellular distribution of the neutral cholesterol ester hydrolase suggested that it may have a different function to the neutral cholesterol ester hydrolase of adrenals and other tissues. Nevertheless the fact that the activity of the enzyme can be modulated by cAMP-dependent protein kinase suggests the possibility that hormonal control of this enzyme may be involved in the regulation of cholesterol metabolism in the mammary gland.     

Derivatives of Di-O-octanoylglycerol and mono-O-octylglycerol as modulators of protein kinase C and diacylglycerol kinase activities

Twelve analogs of 1,2-di-O-octanoylglycerol modified at C-3 and three quaternaryN-alkyl-ammonium derivatives of glycerol were synthesized. The compounds were testedin vitro as potential modulators of the calcium activated, phospholipid dependent protein kinase C (PKC) and diacylglycerol (DAG) kinase activities in order to understand the molecular interactions of these enzymes with their natural activators, inhibitors, or substrates. PKC activity was assayed by measuring histone H₁ phosphorylation, and the compounds synthesized were tested either in the presence (inhibitors) or in the absence (activators) of 1,2-di-O-octanoyglycerol analogs with the phosphatidylserine/Ca²⁺ mixture. DAG kinase activity was measured by the incorporation of phosphate into 1,2-di-O-oleoyl-sn-glycerol in the presence of the various analogs synthesized. In regard to PKC activity, the assays revealed that 1,2-di-O-octanoylglycerol analogs are inactive when modified at C-3 with groups which do not permit hydrogen bonding. Under our conditions, di-O-octanoylthioglycerol, which has been reported as inactive, was able to activate PKC in the presence of phosphatidylserine. It has been shown to give a synergistic activation with diacylglycerol and had no affinity for the phorbol ester receptor binding site, suggesting thatO-octanoylthioglycerol interacts with the enzyme at a different site from the phorbol ester receptor binding site. PKC and DAG kinase activities are inhibited byN-alkyl-ammonium compounds (IC₅₀ 24 μM) only when either two 8-carbon alkyl or acyl chains are present at the 1- and 2-positions of the glycerol backbone. The fact that these compounds have a strong effect on the binding of [³H]phorbol 12,13-dibutyrate to protein kinase C, and also inhibit DAG kinase, may suggest binding to the DAG site of the regulatory domain of PKC.     

A protein tyrosine kinase associated with the ATP-dependent inactivation of adipose diacylglycerol acyltransferase

The activity that has been previously reported to reversibly inactivate adipose glycerolphosphate acyltransferase (GPAT) and diacylglycerol acyltransferase (DGAT)in vitro in the presence of ATP is shown here to be partially purified from adipose tissue with an apparent molecular weight of 68 kDa. The activity responsible for inactivating DGAT is associated with a kinase activity as determined by phosphate incorporation both into microsomal proteins and into a synthetic tyrosine-containing peptide as substrate for protein tyrosine kinase. Two microsomal polypeptides of 53 and 69 kDa are major substrates of this kinase. Both DGAT inactivating and kinase activities assayed from the purified sample have been found to be insensitive to the Ser/Thr kinase inhibitor H-7 while being sensitive to genistein and tyrphostin-25. A crude protein phosphatase preparation from liver was capable of reversing the effects of both activities. The purified sample was also shown to inactivate GPAT in the presence of ATP. These results suggest that a protein tyrosine kinase, in concert with a protein tyrosine phosphatase, may regulate the activities of DGAT and GPAT by a phosphorylation-dephosphorylation mechanism.     

Magnesium and Morphine in the Treatment of Chronic Neuropathic Pain–A Biomedical Mechanism of Action

The effectiveness of opioids in the treatment of neuropathic pain is limited. It was demonstrated that magnesium ions (Mg²⁺), physiological antagonists of N-methyl-D-aspartate receptor (NMDAR), increase opioid analgesia in chronic pain. Our study aimed to determine the molecular mechanism of this action. Early data indicate the cross-regulation of µ opioid receptor (MOR) and NMDAR in pain control. Morphine acting on MOR stimulates protein kinase C (PKC), while induction of NMDAR recruits protein kinase A (PKA), leading to a disruption of the MOR-NMDAR complex and promoting functional changes in receptors. The mechanical Randall-Selitto test was used to assess the effect of chronic Mg²⁺ and morphine cotreatment on streptozotocin-induced hyperalgesia in Wistar rats. The level of phosphorylated NMDAR NR1 subunit (pNR1) and phosphorylated MOR (pMOR) in the periaqueductal gray matter was determined with the Western blot method. The activity of PKA and PKC was examined by standard enzyme immunoassays. The experiments showed a reduction in hyperalgesia after coadministration of morphine (5 mg/kg intraperitoneally) and Mg²⁺ (40 mg/kg intraperitoneally). Mg²⁺ administered alone significantly decreased the level of pNR1, pMOR, and activity of both tested kinases. The results suggest that blocking NMDAR signaling by Mg²⁺ restores the MOR-NMDAR complex and thus enables morphine analgesia in neuropathic rats.     

Effect of 25-hydroxycholesterol on cholesteryl ester formation in Caco-2 cells

Incubation of Caco-2 cells, a human intestinal cell line, with 25-hydroxycholesterol (25-HOC) markedly enhanced cellular cholesteryl ester formation determined by incorporation of [¹⁴C]oleic acid into intracellular cholesteryl [¹⁴C]oleate. The stimulation by 25-HOC of cholesteryl ester formation was suppressed by staurosporine, a kinase inhibitor, but not by cycloheximide or actinomycin D. The specific activity of microsomal acyl-coenzyme A:cholesterol acyltransferase (ACAT) increased two-fold in cells treated with 10 μM 25-HOC for 5 h. ACAT activity decreased when microsomes were incubated without sodium fluoride, a phosphatase inhibitor, but the decrease in ACAT activity in cells stimulated with 25-HOC was more pronounced. The results suggest that protein phosphorylation may be involved in the stimulation of cholesteryl ester formation by 25-HOC in Caco-2 cells.