W. McLean Grogan

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.

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.

Regulation of hepatic neutral cholesteryl ester hydrolase by hormones and changes in cholesterol flux

To understand molecular events in regulation of hepatic neutral cholesteryl ester hydrolase (EC3.1.1.13; CEH), catalytic activity, protein mass, and mRNA levels were measured in rats with various perturbations of hepatic cholesterol metabolism. Cholesterol feeding decreased activity (56 ± 2%), mass (44 ± 2%), and mRNA (14 ± 3%). The cholesterol precursor mevalonate also decreased activity (42 ± 6%), mass (76 ± 3%), and mRNA (23 ± 16%). Inhibition of cholesterol biosynthesis by lovastatin increased activity (65 ± 12%) and mRNA (31 ± 24%). Stimulation of cholesterol efflux by chronic biliary diversion increased activity (138 ± 34%), mass (29 ± 7%), and mRNA (146 ± 28%). Chenodeoxycholate feeding decreased activity (46 ± 6%) and mRNA (26 ± 12%). These data suggest rational regulation of CEH in response to changes in cholesterol flux through the liver. In primary hepatocytes, steady-state mRNA markedly decreased during 72-h cultures and addition of l-thyroxine and dexamethasone synergistically maintained mRNA levels near control values. Lovastatin increased mRNA levels by 103 ± 15%. Taurocholate and phorbol 12-myristate 13-acetate suppressed mRNA (61 ± 4% and 49 ± 13%, respectively), suggesting that protein kinase C mediated effects of bile acids on CEH mRNA levels. These data suggest regulation of CEH by hormones and signal transduction in addition to changes in cholesterol flux.To understand molecular events in regulation of hepatic neutral cholesteryl ester hydrolase (EC3.1.1.13; CEH), catalytic activity, protein mass, and mRNA levels were measured in rats with various perturbations of hepatic cholesterol metabolism. Cholesterol feeding decreased activity (56 +/- 2%), mass (44 +/- 2%), and mRNA (14 +/- 3%). The cholesterol precursor mevalonate also decreased activity (42 +/- 6%), mass (76 +/- 3%), and mRNA (23 +/- 16%). Inhibition of cholesterol biosynthesis by lovastatin increased activity (65 +/- 12%) and mRNA (31 +/- 24%). Stimulation of cholesterol efflux by chronic biliary diversion increased activity (138 +/- 34%), mass (29 +/- 7%), and mRNA (146 +/- 28%). Chenodeoxycholate feeding decreased activity (46 +/- 6%) and mRNA (26 +/- 12%). These data suggest rational regulation of CEH in response to changes in cholesterol flux through the liver. In primary hepatocytes, steady-state mRNA markedly decreased during 72-h cultures and addition of L-thyroxine and dexamethasone synergistically maintained mRNA levels near control values. Lovastatin increased mRNA levels by 103 +/- 15%. Taurocholate and phorbol 12-myristate 13-acetate suppressed mRNA (61 +/- 4% and 49 +/- 13%, respectively), suggesting that protein kinase C mediated effects of bile acids on CEH mRNA levels. These data suggest regulation of CEH by hormones and signal transduction in addition to changes in cholesterol flux.

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 incorporated14C 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 knowncis-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.

Separation and differential activation of rat liver cytosolic cholesteryl ester hydrolase, triglyceride lipase and retinyl palmitate hydrolase by cholestyramine and protein kinases

Cholesteryl ester hydrolase (CEH), triacylglycerol lipase (TGL) and retinyl palmitate hydrolase (RPH) were measured in 104,000 ×g supernatants from rat liver under optimal conditions for measurement of cytosolic CEH. Similar levels of hydrolytic activity were seen with oil droplet dispersions of cholesteryl oleate, trioleoylglycerol and retinyl palmitate. No cytosolic TGL activity was seen with substrate presented in the triton-albumin emulsion used for measurement of lipoprotein lipase-like TGL associated with hepatic plasma membrane. Cytosolic CEH, TGL and RPH were differentially partially purified by both ammonium sulfate precipitation and anion exchange fast protein liquid chromatography (FPLC). Of the three activities, only CEH was stimulated by cholestyramine feeding and by activators of protein kinases A and C. All three activities were inhibited by alkaline phosphatase treatment, although to different degrees. It is concluded that these activities are catalyzed by at least three differentially regulated enzymes with a high degree of specificity for their respective substrates.

Renal corticosterone 6β-hydroxylase in the spontaneously hypertensive rat

Abstract Excess 6β-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 (6β-OHase) and immunoreactive activities were compared in SHR and normotensive control (Wistar-Kyoto; WKY) rats. Corticosterone 6β-hydroxulation 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 6β-hydroxylation by SHR renal microsomes. Increased renal 6β-OH-corticosterone production by increased renal family 3A cytochromes P-450 may play a role in the blood pressure elevation in SHR.

Fatty acid synthesis in isolated spermatocytes and spermatids of mouse testis

In vitro incorporation of [1-14C] acetate into fatty acids and lipid classes of spermatocytes, round spermatids and condensing spermatids enriched by Staput 1×g sedimentation was measured by thin layer and gas radiochromatography. All three cell fractions showed the full range of de novo synthetase, elongation and desaturase activities necessary for biosynthesis of fatty acids characteristic of mouse testis, but synthesis of fatty acids of >16 carbons declined with progressive stages of differentiation. The magnitudes and patterns of distribution of fatty acid synthesis in the germinal cells were similar to those of whole testis incubated in vitro or injected in vivo with [14C] acetate. On the other hand, complex lipid synthesis was much more variable and incorporation into triacylglycerol was generally much lower in dispersed germinal cells than in whole testis in vitro or in vivo. Cells remained viable throughout the 15-hr incubation. Thus, isolated germinal cells are fully capable of synthesizing their constituent fatty acids, including the long-chain polyenoic acids which they accumulate, but the intratubular environment or association with Sertoli cells may be necessary for maintenance of adequate complex lipid synthesis.

Biosynthesis of long-chain polyenoic acids from arachidonic acid in cultures of enriched spermatocytes and spermatids from mouse testis.

Primary spermatocytes (PS), round spermatids (RS) and condensing spermatids (CS) from mouse testes were enriched on Sta-Put 1×g density gradients and cultured for 22 or 44 hr in the presence of [1-14C]arachidonate. Mass and radioactivity were measured by gas radiochromatography of constituent fatty acids of the various complex lipid classes fractionated by thin layer chromatography. Patterns and levels of incorporation were compared with those of whole testis, both in vitro and in vivo. The 20∶4, 22∶4, 22∶5, 24∶4 and 24∶5 of the germinal cells contained levels of radioactivity in each lipid class which were consistent with an important role for the germinal cells in long-chain polyenoic acid (LCPA) metabolism. Cells which represented later stages of spermatogenesis (RS, CS) incorporated much higher percentages and absolute amounts of radioactivity into the fatty acids derived from 20∶4 by elongation-desaturation pathways than did PS or whole testis in vitro. These differences were most pronounced in triacylglycerol of CS. Distributions of mass and radioactivity among lipid classes suggest synthesis of triacylglycerol by CS with a high degree of specificity for 22 or 24 carbon LCPA at thesn-3 position.

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 Alzheimers 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 Alzheimers disease.

Temperature sensitivity of cholesteryl ester hydrolases in the rat testis.

Cholesteryl ester hydrolase (CEH) (EC 3.1.1.13) activity was assayed in the 104,000×g supernatant (S104) of rat and mouse testes and livers at various temperatures between 27 C and 44 C. The CEH activity in the testis dropped from 44 pmol [4-14C] cholesteryl oleate hydrolyzed/hr/mg protein to 14 pmol hydrolyzed/hr/mg protein (a 68% decrease) between testicular and abdominal temperatures (32 C and 37 C, respectively) in the rat. This decrease in activity is essentially a reversible phenomenon. CEH from the testis S104 was stabilized in 10 mM EDTA and was purified by HPLC size exclusion. These steps did not alter the temperature effect previously noted. The temperature effect on the testicular CEH was demonstrated in vivo by assaying the enzyme following unilateral cryptorchidism. The HPLC purification yielded 3 peaks of CEH activity from the testicular S104. The 28,000 MW peak was found to be temperature insensitive while the 70,000 and 420,000 MW peaks were temperature labile. The liver CEH of both species remained relatively constant over the range 32—37 C. CEH is a potential regulator of both steroidogenesis and membrane composition in the testis and its temperature lability may suggest a unique regulatory mechanism responsible for impaired spermatogenesis seen with elevated testicular temperatures.