Acidic residues emulate a phosphorylation switch to enhance the activity of rat hepatic neutral cytosolic cholesterol esterase.

Site-directed mutagenesis of rat hepatic neutral cytosolic cholesteryl ester hydrolase (rhncCEH) was used to substitute acidic, basic or neutral amino acid residues for Ser506, required for activation by protein kinase A. The substitution of acidic Asp506 resulted in esterase activities with cholesteryl oleate, p-nitrophenylcaprylate (PNPC) and p-nitrophenylacetate (PNPA) equivalent to those of native rhncCEH with Ser506. The substitution of 2 acidic residues (Asp505/506), emulating the 2 negative charges of phosphoserine, resulted in a 10-fold greater cholesterol esterase activity than that of native rhncCEH, similar to the activity of rhncCEH treated with protein kinase A. In contrast to mutants with Ser506, protein kinase A did not increase the specific activities of mutants with Asp505/506. The substitution of basic (Lys506) or neutral (Asn506) residues abolished activity with cholesteryl oleate but not PNPC or PNPA. The substitution of neutral Gln for basic residues Lys496/Arg503 also abolished cholesterol esterase activity but not PNPC- and PNPA-esterase activities. These structure-activity relationships are modeled by homology with a recently reported crystal structure for the homologous human triacylglycerol hydrolase. The results suggest that the cholesterol esterase activity of carboxylesterases is enhanced by interactions between one or more basic residues on helix alpha16 (residues 485-503) and acidic groups at residues 505-506 in the adjacent surface loop.

Comparisons of steady-state anisotropy of the plasma membrane of living cells with different probes.

We have used an extended Perrin equation which was in agreement with literature data for steady-state anisotropy (rSS) for a wide variety of artificial and isolated biological membranes labeled with various probes (Van der Meer et al. (1986) Biochim. Biophys. Acta 854, 38-44 to obtain the static component (r infinity) for the intact plasma membranes of living cells. We show that lipid structural order parameters can be obtained for DPH and TMA-DPH in the plasma membranes of intact cells. We have examined the relationship between 'fractional limiting hindered anisotropy', r infinity/r0, which is related to the lipid structural order parameter, of DPH, TMA-DPH, DPHpPC, and a series of depth-dependent probes (n-(9-anthroyloxy) fatty acids, with n = 2-16), using data from 19 cell types. There was a linear relationship between r infinity/r0 values of DPH and TMA-DPH, but the relationship between either of these probes was non-linear with respect to DPHpPC or the series of fatty acid probes. The relationship between r infinity/r0 values of DPHpPC and the series of fatty acid probes was linear, suggesting that they not only undergo similar motions in the membrane, but also experience similar types of restriction to motion, a type which is different from that experienced by DPH and TMA-DPH. We show that for the plasma membranes of living cells, 'second degree' order parameters can be estimated for DPH and TMA-DPH, and propose that the parameter r infinity/r0, or the 'fractional limiting hindered anisotropy', analogous to a 'first degree' order parameter, can be estimated for DPHpPC and the depth-dependent fatty acid probes to evaluate the density of membrane packing.

Age-related changes in mRNA, protein and catalytic activity of hepatic neutral cholesterol ester hydrolase in male rats: evidence for transcriptional regulation.

Messenger RNA, protein mass and catalytic activity of hepatic neutral cholesteryl ester hydrolase (CEH) were measured in male Sprague-Dawley rats, aged 6, 8, 9.5, 12 and 24 weeks (wks). CEH mRNA increased 101% from 6 to 9.5 wks, corresponding to onset of puberty, and declined by 52% from 12 to 24 wks. CEH mass was highly correlated with mRNA levels at all ages, increasing 170% from 6 to 9.5 wks and declining 61% from 12 to 24 wks. CEH activity was highly correlated with mass and mRNA from 8-24 wks, but was greater at 6 wks than the activity predicted by the measured mass. In all age groups, activity was consistently increased by activation of endogenous protein kinase A and consistently inhibited by alkaline phosphatase, suggesting that age-related differences in catalytic activity were not due to differences in the level of enzyme phosphorylation. These data suggest transcriptional regulation and indicate an important role for CEH in cholesterol homeostasis in the developing rat.

Renal corticosterone 6 beta-hydroxylase in the spontaneously hypertensive rat.

Excess 6 beta-OH-corticosterone production by family 3A cytochromes P-450 may play a role in genesis of hypertension in the spontaneously hypertensive rat (SHR), by producing a renal defect in Na+ excretion. Renal cytochromes P-450 may be a causal factor in this genetic model. Since family 3A P-450 is present in rat kidney (collecting duct), the renal family 3A catalytic (6 beta-OHase) and immunoreactive activities were compared in SHR and normotensive control (Wistar-Kyoto; WKY) rats. Corticosterone 6 beta-hydroxylation is markedly higher in SHR than in WKY renal microsomal preparations. Western blot analysis with antibodies to rat and rabbit liver family 3A isoforms demonstrated related proteins. Densitometry revealed greater relative intensity of staining in SHR compared to WKY with both antibodies. Both antibodies inhibited corticosterone 6 beta-hydroxylation by SHR renal microsomes. Increased renal 6 beta-OH-corticosterone production by increased renal family 3A cytochromes P-450 may play a role in the blood pressure elevation in SHR.

Molecular cloning and expression of rat hepatic neutral cholesteryl ester hydrolase.

The 1923 bp cDNA for rat hepatic cholesteryl ester hydrolase (CEH) was cloned by screening a lambda gt11 expression library with an oligonucleotide containing the consensus active site sequence for cholesteryl esterases. Expression of a fusion protein, cross-reacting with antibody to the purified liver CEH, was demonstrated by Western blot analysis. The cDNA was sequenced and found to have only 44% homology with pancreatic CEH. Although unique, the cDNA sequence exhibited much greater overall homology with liver carboxylesterases, in both coding and 5'/3' non-coding regions. In Northern blot analysis, the cDNA hybridized with a single band from liver mRNA but not with pancreatic mRNA. The 1.7 kb coding sequence, predicting a 62 kDa protein, was cloned into an Escherichia coli expression system with an inducible promoter and into COS-7 cells. Both expression systems produced a protein which comigrated with liver CEH (66 kDa) on SDS-PAGE and immunoreacted with antibodies to liver CEH on Western blots. Whereas the prokaryotic system produced an inactive protein, expression in COS-7 cells was accompanied by a 5-fold increase in CEH activity and a corresponding increase in immunoreactive protein.

Over-expression of hepatic neutral cytosolic cholesteryl ester hydrolase in mice increases free cholesterol and reduces expression of HMG-CoAR, CYP27, and CYP7A1.

Hepatic neutral cytosolic cholesteryl ester hydrolase (hncCEH) is a key enzyme in the regulation of hepatic free cholesterol (FC). In examining the effects of over-expression of this enzyme on cholesterol homeostasis, mice were infected with a recombinant adenovirus construct (AdCEH) of the rat hncCEH cDNA driven by the human cytomegalovirus promoter. Cholesteryl esterase and p-nitrophenylcaprylate (PNPC) esterase activities were measured in liver postmitochondrial supernatants at 1, 3, 7, and 11 d after infection with AdCEH or a control virus expressing beta-galactosidase (AdbetaGAL). The PNPC esterase activity of AdCEH mice peaked threefold higher than controls on day 2, declining on subsequent days. In contrast, cholesteryl esterase peaked eightfold higher than controls on day 3, indicating a shift in substrate selectivity of hncCEH. Hepatic FC peaked at 144% of controls, 7 d postinfection. The mRNAs for cholesterol 7alpha-hydroxylase, sterol 27-hydroxylase, and HMG-CoA reductase decreased to 47, 46, and 58% of controls, respectively, on day 7, coinciding with peak FC concentrations. Coinciding with increased cholesteryl esterase activity, hepatic esterified cholesterol dropped precipitously from day 3 onward, to 11% of controls by day 11. Hepatic TAG levels also declined, consistent with the reported TAG lipase activity of hncCEH. These results demonstrate elevation of FC and depletion of cholesteryl esters by over-expression of hncCEH, which were resistant to compensatory responses by other enzymes of cholesterol homeostasis.

Hormone release by islet B cell-enriched and A and D cell-enriched populations prepared by flow cytometry.

Dispersed pancreatic islet cells were analyzed for their low forward angle light scatter using flow cytometry. The cells produced a distinct light scatter pattern which appeared to be a function of cell size and not cell granularity. RIA of hormone content of cells collected from different regions of the pattern revealed that glucagon- and somatostatin-containing cells were concentrated in regions of lower scatter intensity and that insulin-containing cells were more numerous in regions of higher intensity. Relative to the original cell suspension, these preparations were enriched 3-fold in glucagon and somatostatin content and 6-fold in insulin content. The function of intact islets, unsorted dispersed cells, and sorted dispersed cells was examined before and after 4 days of culture. Before culture, all of the dispersed cell populations had elevated basal secretion compared with intact islets and did not respond to stimulatory concentrations of glucose, arginine, or 3-isobutyl-1-methylxanthine. After culture for 4 days, basal secretion fell, and responsiveness returned. In both the A/D cell-enriched and the B cell-enriched cultured populations, the percentage of single cells was approximately 95%. The insulin release patterns from these populations were similar to those from intact islets and unsorted dispersed cells. Glucagon release from all of the dispersed cell populations far exceeded that from intact islets. This study suggests that the structural organization of islets influences A cell function, but a clear influence upon B cell function has not been demonstrated.

A corticosterone metabolite produced by A6 (toad kidney) cells in culture: identification and effects on Na+ transport.

A6 cells form typical tight epithelia when grown in culture on permeable supports and exhibit active Na+ transport [short circuit current (Isc)], which is stimulated by aldosterone and corticosterone. Previous studies demonstrated nuclear binding of polar corticosterone metabolites produced by the cells. This study was performed to determine whether sufficient quantities of the metabolite(s) are released into the medium of A6 cells for identification and to test for agonist activity on active Na+ transport. Cells were incubated in [3H]corticosterone (10(-8)-10(-4) M) for 24 h. Approximately 25-35% of the radiolabel, recovered in ethyl acetate extracts of medium, chromatographed on reverse phase HPLC as a single peak more polar than corticosterone. This derivative cochromatographed with 6 beta-hydroxycorticosterone (6 beta-OH-corticosterone) on HPLC and normal phase high performance TLC. Mass spectroscopy of 6 beta-OH-corticosterone and the unknown yielded 10 identical molecular ions, including the molecular ion with a mass to charge ratio of 362 corresponding to the mol wt of 6 beta-OH-corticosterone stimulated Isc in A6 epithelia with a time course typical of a steroid and an EC50 of 10(-6) M. The Isc induced by 6 beta-OH-corticosterone was equivalent to net Na+ flux, indicating active Na+ transport stimulation. At maximum effective concentrations of corticosteroids, 6 beta-OH-corticosterone plus aldosterone induced a greater Isc stimulation than aldosterone alone, suggesting that at least a portion of the effect of 6 beta-OH-corticosterone is mediated by a steroidal pathway other than that used by aldosterone. Also, corticosterone produced twice the Isc increase produced by aldosterone. Thus, 6 beta-OH-corticosterone may contribute to the enhanced corticosterone effect on Isc compared to aldosterone alone.

Altered membrane anisotropy gradients of plasma membranes of living peripheral blood leukocytes in aging and Alzheimer's disease.

Several reports have suggested that membrane rigidity, a term that refers to the relative motion of membrane constituents, is decreased in Alzheimer's Disease. Accordingly, a series of fluorescent membrane probes was used to evaluate the rigidity from the surface to the center of the outer hemi-leaflet of the plasma membrane of living neutrophils, monocytes and lymphocytes. Anisotropy, a parameter which increases with increasing membrane rigidity, was calculated from flow cytometric measurements of vertically and horizontally polarized components of the fluorescence emission of the probes. These preliminary experiments suggest that whereas membrane rigidity in certain regions of the plasma membrane of peripheral blood leukocytes is increased as expected in elderly controls, it is decreased in Alzheimer's disease.

Electronic sorting of granulocyte precursor cells from stimulated bone marrow.

A commerical cell sorter was used to obtain preparations of cells in various stages of granulocyte development from rabbit marrows stimulated by inflammatory response. Marrow cells were fractionated on density gradients of Ficoll/Hypaque and each fraction sorted using light scatter. Trial and error selection of appropriate gradient fractions and light scatter windows allowed sorting of early (blast cells, promyelocytes), intermediate (myelocytes, metamyelocytes) and late stage (band cells, polys) granulocytes with enhanced purity.

DNA analysis and sorting of viable mouse testis cells.

The dye Hoechst 33342 and a 2-parameter cell sorter have been used to measure DNA content in viable testis cells and to sort pachytene spermatocytes and round spermatids from adult mouse testis to virtually 100% homogeneity. Early diploid spermatogenic cells were enriched to 90%, a 10-fold purification. The capability for viable sorting of most testis cell types to homogeneity in numbers suitable for many biochemical applications is demonstrated.

Separation of resting and proliferating granulocytic precursors.

We have obtained cells in various stages of granulocytic development by a combination of isopycnic separation and electronic cell sorting. Not only were immature cells (blast cells, promyelocytes and myelocytes) separated from mature cells (bands and polys), but the immature cells were separated into proliferating (S + G2 + M) and resting (Go/G1) compartments of the cell cycle. This permits the study of the morphological and biochemical changes associated with development apart from those changes associated with proliferation.

Renal and hepatic family 3A cytochromes P450 (CYP3A) in spontaneously hypertensive rats.

Troleandomycin (TAO), a selective family 3A cytochromes P450 (CYP3A) inhibitor, decreases enhanced in vivo corticosterone 6 beta-hydroxylation and blood pressure in spontaneously hypertensive rats (SHR). Corticosterone 6 beta-hydroxylation was measured in liver and kidney microsomes, to determine ontogeny and the effect of TAO on CYP3A activity at the organ level. SHR kidney CYP3A activity increased from 4 to 8 weeks, stabilized at 11 and 16 weeks, and was much higher than in control (Wistar-Kyoto, WKY) rats at all ages. Hepatic activity showed less consistency in strain difference. TAO produced a relatively large decrease in renal CYP3A activity compared with liver. Although renal CYP3A mRNA was not present in sufficient quantity for detection by northern blot analysis of total RNA, its presence was demonstrated in SHR by reverse transcriptase-polymerase chain reaction amplification. Correlations between renal CYP3A activity and systolic blood pressure in SHR and WKY rats with variations in age, strain and drug treatment are consistent with the role of the enzyme in the pathogenesis of blood pressure elevation in SHR.

Decline in cholesteryl ester hydrolase activity of rat brain with progress of experimental allergic encephalomyelitis followed by rebound during recovery.

Cholesteryl ester hydrolase and cholesteryl esters were measured in brain homogenates from rats in various stages of experimental allergic encephalomyelitis (EAE), a model for multiple sclerosis, and in age-matched controls. Cholesteryl esters were elevated approximately 280-507% in EAE rats with clinical indices of 1-3, corresponding to increasing severity of symptoms, but were not significantly different from controls upon disappearance of symptoms during the recovery period (stage = R2). Cholesteryl ester hydrolase declined progressively 12-34% during stages 1-3, rebounded to a level 16% greater than controls during the initial stage of recovery (R1) and remained significantly elevated by 6% over controls upon disappearance of symptoms (R2). A highly significant (P less than 0.001) correlation of reduction in cholesteryl ester hydrolase activity with clinical index was confirmed by linear regression analysis (R = 0.91). It is concluded that a decline in cholesteryl ester hydrolase activity is an early event in EAE which leads to accumulation of cholesteryl esters and may play a role in induction and progression of this demyelinating disorder.

Activation of myelin-associated cholesteryl ester hydrolase in developing rat brain.

Short-term regulation of rat brain cholesteryl ester hydrolase (CEH) by protein kinases is described. CEH was activated 280-340% in the presence of Mg2(+)-ATP and this inhibition was partially abolished by rabbit skeletal muscle protein kinase inhibitor or chlorpromazine, a phospholipid interacting drug, suggesting the involvement of cAMP-dependent protein kinase and protein kinase C, respectively. However, the involvement of other kinases cannot be ruled out. In developing rat brain, CEH activity per unit brain weight closely correlated with myelination. During the premyelination period (5 days postnatal), significantly higher activation (P less than 0.001) of CEH was observed by cAMP-dependent protein kinase or protein kinase C, when compared to activation observed during the period of active myelination (20 days postnatal). These results indicate that CEH in rat brain is tightly regulated and closely related to myelination.

Distribution of long chain polyenoic acids among phospholipids of mouse testis.

Fatty acid composition of phospholipid (PL) classes was measured in mouse testis. Among the long-chain polyenoic acids (LCPA), 22:6 was found in highest concentration in phosphatidylethanolamine (PE), whereas percentages of 20:4 and 22:5 were not different in PE than in phosphatidycholine. Each PL class had a unique fatty acid composition which was also different from that of triglycerides and cholesteryl esters. Differential metabolisms of 22:5 and 22:6 suggest different roles for these fatty acids in mouse testis. Tissue-specific functions of LCPA in mouse spermatogenesis may be divided between 22:5 and 22:6.

Metabolism of arachidonate in rat testis: characterization of 26-30 carbon polyenoic acids.

Fatty acid methyl esters of long-chain polyenoic fatty acids (LCPA) from rat testis injected with [1-14C] arachidonate were analyzed and separated by reversed-phase high performance liquid chromatography (RP-HPLC). Earlier, all previously identified LCPA were prepared in high purity along with 4 previously unidentified fatty acids, which were further characterized by capillary gas chromatography (GC), catalytic hydrogenation and alkaline isomerization. Unidentified fatty acids proved to be 26:4, 26:5, 28:5 and 30:5. All of these LCPA incorporated 14C from arachidonate (20:4) to specific activities that were comparable to that of 20:4 and previously identified metabolites of 20:4 and much greater than specific activities of 18:1n-9 or 22:6n-3. LCPA were analyzed on a capillary GC system capable of resolving known cis-trans and positional isomers of the n-3, n-6, n-7 and n-9 families of unsaturated fatty acids. Log plots of isothermal retention times and normal plots of temperature programmed retention times were linear (r = 0.999) in carbon number when values for known and previously unidentified LCPA of 4 or 5 double bonds, respectively, were coplotted. Thus, the newly identified fatty acids belong to the n-6 family of fatty acids synthesized from arachidonic acid.

Rapid three-step purification of a hepatic neutral cholesteryl ester hydrolase which is not the pancreatic enzyme.

A rat liver cytosolic cholesteryl ester hydrolase (CEH) was purified 12,600-fold by ammonium sulfate precipitation, cation exchange chromatography and gel permeation high-performance liquid chromatography, with an overall yield of 20%. Its properties are compared to those of pancreatic CEH, with which it has sometimes been identified. Liver CEH exhibited a single silver stained band following SDS-polyacrylamide gel electrophoresis (Mr = 66 kDa), was activated by 0.5-10 mM taurocholate but was strongly inhibited by higher levels of taurocholate, which activate pancreatic CEH. Whereas bile salts are known to induce formation of a hexamer of pancreatic CEH, in the current study, 0.5 mM taurocholate dissociated a multimeric form of liver CEH to monomer. Liver CEH did not coelute with pancreatic CEH from cation exchange and chromatofocusing columns, exhibited no immunoreactivity with anti-rat pancreatic CEH IgG in Western blots, was not inhibited by anti-rat pancreatic CEH IgG and had a different amino acid composition from pancreatic CEH. In contrast to liver CEH, which is known to be activated by protein kinases A and C, pancreatic CEH was unaffected by cofactors for protein kinase A and was inhibited by cofactors for protein kinase C.

HPLC measurement of testicular long chain acyl-CoA synthetases with different substrate specificities.

Acyl-CoA synthetase activity with various long chain fatty acid substrates was measured in microsomes from rat testes, isolated spermatids and testes of hypophysectomized adult rats, using reversed-phase high performance liquid chromatography (HPLC). The spectrophotometric HPLC method produced results comparable to those of parallel radiometric assays and was highly specific for acyl-CoA products. At optimal pH and cofactor concentrations, specific activity from whole testis was similar for 18:1, 20:4 and 22:5 but somewhat lower for 16:0 over the substrate range 0.01-3.2 mM. Activity from spermatids or from testes of hypophysectomized rats was much lower with 22:5 than with 18:1 or 20:4, whereas activities with 18:1 and 20:4 were similar at all substrate concentrations. All substrates exhibited Michaelis-Menten type saturation kinetics and linear Lineweaver-Burke plots at lower substrate concentrations but inhibited activity at higher concentrations. Apparent values of KM for 16:0, 18:1 and 20:4 were more than twice that of 22:5, whereas both observed and calculated maximum velocities were similar for the four fatty acids. Differences in pseudokinetic parameters and differential expression of the testicular acyl-CoA synthetase activities with different fatty acids suggest the presence of multiple enzymes, at least one of which may be hormonally regulated.

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 degrees C and 37 degrees C, and with and without cofactors for stimulation of cyclic AMP-dependent protein kinase, in 104,000 X 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.