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S Azhar

Publications and source records attributed to S Azhar.

At least 109 records · Page 6Linked to original sources
PubMed1987

Calcium-activated, phospholipid-dependent protein kinases from rat liver: subcellular distribution, purification, and characterization of multiple forms.

Three forms of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) were extensively purified from rat liver homogenate. Subcellular fractionation analysis indicated that the majority (approximately 85%) of the activity was associated with particulate fractions of the liver. Among these, the microsomal and nuclear fractions accounted for approximately 63% and approximately 10% of total activity. The remaining 15% of protein kinase C was recovered in the soluble fraction following differential centrifugation. It was also found that most of the membrane-associated protein kinase C was latent, with 4-6-fold stimulation with detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, octyl beta-glucoside, or Triton X-100. The activity of both the bound form and the soluble enzyme was enhanced by the addition of Ca2+ and phosphatidylserine, when histone H1 was used as substrate. The bound protein kinase C activity was dissociated by homogenization of liver in buffer containing ethylene glycol bis(beta-aminoethyl ether)-N,-N,N',N'-tetraacetic acid, ethylenediaminetetraacetic acid, and various proteolytic inhibitors, and the solubilized extract was used to purify multiple forms of the enzyme. The purification procedure sequentially utilized (NH4)2SO4 fractionation, ion-exchange chromatography on DEAE-cellulose, gel permeation chromatography on Fractogel TSK HW-55 (F), ion-exchange chromatography on hydroxylapatite, gel permeation chromatography on Ultrogel AcA34, and affinity chromatography on polyacrylamide-immobilized phosphatidylserine. On hydroxylapatite columns, protein kinase C activity was resolved into three isoenzymic forms designated C-I, C-II, and C-III. The molecular weights of the three isoenzymic forms were in the range of 208,000-225,000 as shown by chromatography on calibrated Ultrogel AcA34 columns and sucrose density gradient centrifugation. Furthermore, all three isoenzymes demonstrated a single peak with a sedimentation coefficient (s20.w) in the range of 9.0-9.2. However, with polyacrylamide gel electrophoresis, all the forms showed a single protein component with average molecular weight of 64K, suggesting that the native isoenzymes may be composed by subunits. Finally, all three isoenzymes exhibited nearly identical enzymatic properties.

Amino Acids↗
PubMed1987

Age-related changes in rat muscle glycogen synthase activity.

This study was designed to evaluate effects of aging on glycogen synthase activity in rat skeletal muscle. Total enzyme activity was shown to be significantly, (p less than .001) lower in tensor fascia latae, biceps femoris, and soleus muscle obtained from 24-month-old compared with 2-month-old rats. Similarly, values for the active form of enzyme were significantly lower, (p less than .001) in all three muscle types of 24-month-old compared with 2-month-old rats. This age-related decline in glycogen synthase activity was not due to a reduction in the affinity of the enzyme for its activator (glucose-6-phosphate) and was independent of the concentration of substrate (UDP-glucose) in the assay system. Because similar age-related changes were seen when enzyme activity was expressed per milligram of muscle protein or per gram of muscle tissue, the fall in enzyme activity was not a simple function of an age-related decline in muscle mass. Glycogen levels also were reduced significantly in tensor fascia latae, biceps femoris, and soleus of 24-month-old rats compared with 2-month-old rats, p less than .001. These results document an age-related change in a key enzyme regulating glycogen metabolism in muscle.

Aging↗
PubMed1987

Opposite effect of human chorionic gonadotropin on placental and ovarian synthesis of androstenedione.

In the present studies, we have investigated the role of human chorionic gonadotropin on the biosynthesis of androgens by placentas and corpora lutea of the pregnant rat. We first sought to compare the effect of hCG on placental and ovarian secretion of androstenedione in vivo. For this purpose either 1.5 IU hCG or vehicle was administered to pregnant rats twice on days 12 and 13 and once on the morning of day 14. Blood was obtained from either the ovarian or the uterine vein. After hCG administration, levels of androstenedione secreted in the ovarian vein increased dramatically, whereas those in the uterine vein declined significantly. To establish that changes in androgen levels in the uterine and ovarian veins are due to changes in biosynthetic activity and also to compare the action of hCG on placentas and corpora lutea, tissues were dissected out from rats treated with 0, 1.5, or 9 IU hCG twice daily and incubated in vitro. hCG administration increased the capacity of luteal cells to synthesize androstenedione de novo by approximately 100% and concomitantly decreased placental secretion of androstenedione by approximately 75%. Addition of high density lipoprotein to the medium enhanced both basal and hCG-stimulated androstenedione production by luteal tissues but had no effect on either basal or hCG-inhibited androstenedione biosynthesis by the placenta. To determine which step in the placental biosynthesis of androstenedione is inhibited by increased levels of LH, we determined the effect of hCG administration, on cholesterol biosynthesis and storage, synthesis of progesterone substrate, and the activities of 17 alpha-hydroxylase/17,20-lyase. hCG did not affect the activities of the rate limiting cholesterogenic enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase, placental content of cholesterol and cholesteryl ester, or the placental production of progesterone. However, hCG did cause a substantial decrease in the activity of 17 alpha-hydroxylase/17,20-lyase enzyme(s); responsible for the conversion of progesterone to androgen. In summary, results of the present investigation demonstrate that increases in LH activity in the circulation act on two different steroidogenic glands to either enhance or reduce androgen biosynthesis. hCG stimulates luteal secretion of androstenedione and simultaneously inhibits placental production of this steroid.(ABSTRACT TRUNCATED AT 400 WORDS)

Aldehyde-Lyases↗
PubMed1987

The influence of age on steroidogenic enzyme activities of the rat adrenal gland: enhanced expression of cholesterol side-chain cleavage activity.

The ability of isolated adrenocortical cells to secrete corticosterone in response to ACTH challenge declines as rats age, but the site or mechanism(s) of this impairment is still unknown. To test the functionality of steroidogenic capacity per se, we measured the key enzyme activities involved in corticosterone biosynthesis. We also measured the mitochondrial cytochrome P-450 content and nonsteroidogenic enzymes specific for subcellular fractions. Mitochondria and microsomal fractions were isolated from the adrenals of 2-, 12-, and 18-month-old animals and used for various enzyme measurements. Mitochondrial side-chain cleavage enzyme activity (nanomoles per min mg protein-1) increased from a mean of 0.43 +/- 0.06 in 2-month-old rats to 1.26 +/- 0.11 and 1.51 +/- 0.06 in 12- and 18-month old rats, respectively. After incubation with 5-cholesten-3 beta,25-diol (25-hydroxycholesterol; 25 micrograms/ml) side-chain cleave activity rose to 5.0 +/- 0.6, 12.4 +/- 1.2, and 16 +/- 1.4 nmol min-1 mg protein-1 in adrenal mitochondrial fractions from 2-, 12-, and 18-month-old rats, respectively. In contrast, mitochondrial cytochrome P-450 content did not vary with advancing age. Microsomal delta 5-3 beta-hydroxysteroid dehydrogenase-delta 5-delta 4-isomerase activities were similar in 2- and 12-month-old rats, but 21-hydroxylase (nanomoles per min mg protein-1) activity was significantly increased in 12-month-old rats (2-month-old, 5.2 +/- 0.2; 12-month-old, 7.7 +/- 0.5). Finally, mitochondrial 11 beta-hydroxylase was comparable in both age groups. In addition, activities of mitochondrial nonsteroidogenic enzymes, such as monoamine oxidase, amytal insensitive NADH cytochrome c reductase, cytochrome c oxidase, succinate dehydrogenase, and malate dehydrogenase, did not change with age. It appears from the evidence presented that the activities of the steroidogenic enzymes are not responsible for the diminished capacity in corticosterone production seen with aging in the rat.

Adrenal Glands↗
PubMed1987

Effects of aging on cholesterol content and cholesterol-metabolizing enzymes in the rat adrenal gland.

Previous studies from this laboratory have documented a progressive decline in basal and ACTH-stimulated corticosterone production in isolated adrenocortical cells as rats age. In the current study we examined the possibility that the aging process exerts this effect by interfering with the mechanism(s) by which cholesterol is processed and/or synthesized by the adrenal gland. Freshly excised adrenals from 2-, 5-, 12-, and 18-month-old rats were used for the measurement of cholesteryl ester, free cholesterol, cholesteryl esterases, and acyl co-enzyme A (CoA)-cholesterol acyltransferase activities as well as key enzymes involved in cholesterol biosynthesis. The results showed that cholesteryl ester content increased in a linear manner with advancing age, while neutral cholesteryl esterase activity decreased steadily until at 18 months of age it reached 40% that of 2-month-old control rats. In contrast, lysosomal acid cholesteryl esterase did not change with age, and acyl CoA: Cholesterol acyltransferase showed only a 33% decrease at 12 months of age. The activity of 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, decreased steadily with advancing age, and at 18 months of age, activity was only half of that in 2-month-old control rats. In contrast, the activities of other enzymes involved in the de novo synthesis of cholesterol, namely acetoacetyl CoA thiolase and HMG CoA synthase, were similar in 2- and 12-month-old rats, while mevalonate kinase activity was significantly lower in the 12-month-old rats. After depletion of plasma lipoprotein cholesterol by 4-aminopyrazolo-[3,4-d]pyrimidine, the intraadrenal cholesteryl ester content in young and aged animals fell significantly. Furthermore, such treatment enhanced the activities of all of the cholesterol de novo synthetic enzymes examined. In addition, HMG CoA synthase and HMG CoA reductase activities rose to much greater levels in both young and old rats compared to acetoacetyl CoA thiolase and mevalonate kinase. Finally, markedly higher activities of HMG CoA reductase were observed in 12- and 18-month-old rats after 4-aminopyrazolo-[3,4-d]pyrimidine treatment. Similar results were seen using 17 alpha-ethinyl estradiol to deplete cholesterol and adrenal sterol ester stores. The metabolism of endogenous cholesterol and exogenous hydroxysterols (which bypass the cAMP-dependent transport of endogenous cholesterol to mitochondrial side-chain cleavage enzyme complex) to corticosterone by collagenase-dispersed adrenocortical cells isolated from rats of various ages were also studied.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Glands↗
PubMed1986

The effect of colchicine on cholesterol processing by the progesterone-producing cells of the luteinized ovary.

Previously, we indicated that luteal cells from colchicine-treated superovulatory (luteinized) rats show decreased capacity for progesterone production. The current study investigates the possibility that colchicine exerts this effect by interfering with the mechanism by which cholesterol is processed and/or synthesized by luteal cells. To this end, animals were treated with saline or colchicine after which the luteinized ovary or isolated luteal cells were assayed for their cholesterol content, their ability to synthesize cholesterol endogenously, or their ability to utilize lipoprotein-delivered cholesterol for the production of progesterone. The results show that animals treated with colchicine show a number of changes in luteal cell cholesterol metabolism: namely a 60% decline in stored cholesterol, a 3-fold rise in the activity of the cholesterol synthesizing enzyme HMG CoA reductase (although no change occurs in other cholesterol metabolizing enzymes), and a 3-fold rise in the capacity of the cells to incorporate precursor [14C]acetate into cholesterol. On the other hand, cells of animals treated with colchicine or cells treated with colchicine under in vitro circumstances are unable to fully utilize cholesterol provided by high density lipoproteins (HDL): this occurs despite the fact that the binding of HDL particles to luteal cells is quite normal after colchicine treatment. These findings are consistent with the view that a primary effect of colchicine on luteal cell progesterone production is in preventing the normal uptake of HDL-cholesterol.

Animals↗
PubMed1986

Uptake of low density lipoproteins by rat tissues. Special emphasis on the luteinized ovary.

The aim of this study was to determine how luteal cells of the hormone-primed (luteinized) ovary process low density lipoproteins (LDL). Ovary uptake of perfused 125I-LDL was assessed by tissue levels of radioactivity; the distribution of LDL protein in cells was assessed on autoradiograms of the fixed tissue; and the level of stimulation of steroidogenesis, as well as degradation of LDL protein, was assessed on effluent perfusion samples. Human LDL ligand used in these studies was rigorously defined biochemically and physiologically. Homologous (rat) LDL was used as a special ligand control. Other tissue controls included the use of perfused or in vivo-infused luteinized ovaries from animals pretreated to reduce circulating lipoprotein levels, perfused ovaries from a second hormone-primed model, perfused liver from estrogen-treated rats, and isolated and cultured cells from the same ovarian tissues used in the perfusion experiments. The results show that perfused LDL promptly stimulates steroidogenesis. However, the labeled protein moiety of the LDL is not interiorized by the luteal cells, nor is there evidence of LDL protein degradation in the effluent samples. In contrast, internalization of the ligand occurs when luteal cells are incubated with the ligand in vitro. We have observed also that uptake of the 125I-LDL by the ovary can be displaced equally well by excess unlabeled LDL or HDL3. Overall, these experiments suggest that in the intact luteinized ovary, LDL binds to the same sites on the cell surface where HDL "binds," and that LDL cholesterol must be obtained by these steroid hormone-producing cells by a mechanism that does not require internalization of the intact lipoprotein particle.

Animals↗
PubMed1986

Evidence that insulin deficiency in the rat has disparate effects on fructose 2,6-bisphosphate levels in muscle and liver.

The level of fructose 2,6-bisphosphate (F2,6P2), a potent stimulator of 6-phosphofructo-1-kinase and inhibitor of fructose 1,6-bisphosphatase, was measured in three different muscle types (tensor fascia latae, biceps femoris, and soleus) and in the liver of normal and diabetic rats. The mean (+/- SEM) content of F2,6P2 (nanomoles per g tissue) varied among the three types of skeletal muscle in normal rats, with the biceps femoris having the highest (0.97 +/- 0.15) and the soleus the lowest (0.57 +/- 0.03) levels. However, these differences were unrelated to simultaneous estimates of skeletal muscle activity of 6-phosphofructo-1-kinase activity. The total concentration of F2,6P2 was more than 8-fold higher (8.5 +/- 0.9) in the liver, and this value fell to 5.3 +/- 0.8 (P less than 0.05) after the induction of diabetes with streptozotocin. In contrast, F2,6P2 levels did not fall in skeletal muscle of rats with streptozotocin-induced diabetes, and the concentration actually increased. Thus, the fall in hepatic F2,6P2 concentration associated with insulin deficiency was not observed in skeletal muscle.

Animals↗
PubMed1986

Differential effects of aging on adrenocorticotropin receptors, adenosine 3'5'-monophosphate response, and corticosterone secretion in adrenocortical cells from Sprague-Dawley rats.

This study examined the effect of aging on adrenal cell function in Sprague-Dawley rats, as judged by the binding of iodinated Phe2, Nle4-ACTH-(1-38) to the adrenal cells, and the ability of the cells to respond to ACTH stimulation by the production of cAMP and corticosterone in vitro. Collagenase-dispersed adrenal cells obtained from 2-, 12-, and 18-month-old-rats were used. The maximum corticosterone concentration after incubation with ACTH was significantly lower (P less than 0.001) in the 12-month-old (40 +/- 7 ng/micrograms DNA) and 18-month-old rats (28 +/- 3 ng/micrograms DNA) compared to that in 2-month-old controls (102 +/- 9 ng/micrograms DNA). The ED50 values of ACTH-induced corticosterone production measured in the cell suspension were similar in 2- and 12-month-old groups (30 pg/ml), and the diminished production of corticosterone in the 12-and 18-month-old rats persisted after incubation with N6,O2-dibutyryl cAMP. Neither the number nor the binding affinity of adrenal receptors for [125I]I-Tyr23,Phe2,Nle4-ACTH-(1-38) changed from 2-12 months of age. Furthermore, increases in concentrations of intra- and extracellular cAMP after ACTH stimulation were not significantly different in the 2-, 12-, and 18-month-old groups. Similarly, adrenal hydrolysis of cAMP by low and high Km phosphodiesterases did not change significantly with advancing age. These results provide strong evidence that there is a diminished capacity for corticosterone production with aging in the rat, and that the site of the defect lies distal to binding of trophic hormone to its receptor and to the production of its secondary messenger. Finally, an age-related decline in adrenal steroidogenic capacity could be viewed as a counterregulatory mechanism invoked in old rats to compensate, at least partially, for elevated plasma ACTH and corticosterone concentrations.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Effects of antimicrotubule agents on phospholipid metabolism in rat hepatic subcellular membranes.

Treatment of animals with antimicrotubule drugs has been shown to cause a perplexing variety of cellular changes which, theoretically, could be the result of changes in endomembrane biosynthesis, composition or flow. In the current study we have focused on this possibility by identifying antimicrotubule drug-induced changes in the phospholipid metabolism of hepatic subcellular membranes. Young adult rats were pretreated with radiolabeled [32 P]orthophosphate for 12 hr, and subsequently given saline, colchicine (2.5 mg/kg body wt) or vinblastine (20 mg/kg body wt) for 4 additional hr. Afterwards, the livers were homogenized, and separate microsomal and Golgi membrane fractions were prepared and subjected to phospholipid extraction and identification using two-dimensional thin-layer chromatography. The results show that colchicine and vinblastine given in vivo caused specific, rapid and in some cases, dramatic changes in phospholipid turnover in different membrane fractions of rat liver. The drugs specifically increased labeling of phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-biphosphate and decreased the radioactivity associated with phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in all fractions examined. In contrast, the antimicrotubule drugs produced a differential effect on the labeling pattern of sphingomyelin and lysophosphatidylcholine, i.e. they stimulated labeling of these phospholipids in microsomes, produced no changes in heavy Golgi fractions, and markedly increased their labeling in light Golgi fractions. These data suggest that antimicrotubule drugs restrict the incorporation of certain precursor phospholipids into forming membranes but do not affect the subsequent metabolism of these phospholipids. At the same time, the drugs appear to retard the flow of membranes from one cellular compartment to another.

Animals↗
PubMed1985

Regulation of luteal cell 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by estradiol.

Recent studies have suggested that estradiol or androgen precursor may stimulate steroidogenesis in the luteal cell by modulating intracellular sterol availability and metabolism. This investigation was performed to examine the effect of estradiol on de novo synthesis of cholesterol. Pregnant rats hypophysectomized and hysterectomized on Day 12 were treated for 72 h with either estradiol or testosterone. De novo cholesterol synthesis was determined by measurement of the specific activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis, in microsome-enriched preparations of luteal tissue and incorporation of [14C] acetate into cholesterol by corpora lutea incubated in vitro. Estradiol or testosterone treatment caused a 4- to 5-fold stimulation of luteal cholesterol biosynthesis, as measured by these techniques. NaF, an inhibitor of phosphatase which blocks the conversion of the inactive enzyme to the active form, reduced the HMG CoA reductase activity to 30% in corpora lutea obtained from either steroid or vehicle-treated rats. However, an increase in enzyme activity of comparable magnitude by steroids was observed whether microsomes were isolated with or without NaF. The effect of estradiol appears to be enzyme-specific, since it failed to affect the microsomal marker, NADPH-cytochrome c reductase. Since the cholesteryl ester content of corpora lutea falls in response to steroid treatment, rats were treated with 4-aminopyrazolo-[3,4d]pyrimidine (4-APP) to deplete cellular cholesterol content.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗
PubMed1984

Gonadotropin modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in desensitized luteinized rat ovary.

These studies were done to examine the effect of gonadotropin on rat luteal 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity (the rate-limiting step in cholesterol biosynthesis) in ovaries of pregnant mare's serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG) primed rats. Administration of hCG stimulated HMG CoA reductase activity in a time- and dose-dependent manner: significant increases were noted within 4 h, with maximum effects (30-40-fold increases) seen 24 h after hCG (25 IU) administration. This effect was specific in that only LH, of several hormones tested, was as effective as hCG in stimulating HMG CoA reductase activity, and no change in the activity of either liver microsomal HMG CoA reductase or luteal microsomal NADPH-cytochrome c reductase was seen after hCG. The gonadotropin-induced increase in HMG CoA reductase activity seemed to be due to a net increase in enzyme activity, not to a change in the phosphorylated/dephosphorylated state of the enzyme. Pretreatment of animals with aminoglutethimide, an inhibitor of the conversion of cholesterol to steroid (pregnenolone), prevented the hCG-induced rise in HMG CoA reductase activity, whereas treatment with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP), which depletes cellular cholesterol content, led to striking increases in enzyme activity. However, the combined effects of 4-APP and hCG were additive, suggesting that the stimulating effect of hCG on HMG CoA reductase activity is not entirely due to a depletion of cellular sterol content of luteinized ovaries. Similarly, cholesteryl ester and cholesterol syntheses as measured by [14C]acetate conversion were also increased by hCG and 4-APP treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1984

Amelioration of streptozotocin-induced diabetes in the rat by exercise-training: role of muscle glycogenolytic and glycolytic enzyme activity.

Streptozotocin-induced insulin deficient rats allowed to run at will had significantly (P less than 0.001) lower mean (+/- SEM) plasma glucose levels (12.1 +/- 0.9 vs 22.6 +/- 1.7 mM/l) than did equally insulin deficient sedentary rats. Muscle glycogen phosphorylase and synthase activities were similar in exercise-trained and sedentary diabetic rats, and were unchanged from control values. In contrast, muscle phosphofructokinase activity was reduced (P less than 0.001) in sedentary rats with insulin deficiency, and the defect was significantly reversed (P less than 0.01) when such rats were allowed to run spontaneously. These results are consistent with the view that the ability of exercise-training to attenuate the magnitude of hyperglycemia in streptozotocin-induced insulin deficiency is associated with an effect on a key regulatory enzyme in the glycolytic pathway.

Animals↗
PubMed1984

Insulin resistance in older rats.

Insulin-stimulated glucose utilization was estimated in vivo in 1.5-, 4-, and 12-mo-old rats with an insulin suppression test wherein the height of the steady-state plasma glucose ( SSPG ) concentration, at similar steady-state plasma insulin levels, provides a direct reflection of the efficiency of insulin-stimulated glucose disposal. In parallel studies, the effect of age on in vitro insulin-stimulated glucose uptake was assessed in perfused hindlimb preparations. In addition, changes in the activity of enzymes that regulate muscle glycolysis, glycogenesis, and glycogenolysis were determined in isolated soleus muscle. The results indicated that rats got heavier as they became older, and changes in weight were associated with parallel increases in mean (+/- SE) SSPG concentrations as rats grew from 1.5 (56 +/- 3 mg/dl) to 4 (172 +/- 6 mg/dl) to 12 mo of age (194 +/- 8 mg/dl). The age-related decline in in vivo insulin action was associated with a reduction in insulin action on muscle, and maximal insulin-stimulated glucose uptake by perfused hindlimbs of 12-mo-old rats was approximately 50% of the value seen with perfused hindlimbs from 1.5-mo-old rats. Soleus muscle enzyme activity also varied with age, with significant increases in glycogen synthase and decreases in glycogen phosphorylase documented. Furthermore, muscle glycogen phosphorylase activity, which fell during an insulin infusion in 1.5-mo-old rats, did not change when 12-mo-old rats were infused at comparable insulin levels. Finally, glycogen content was significantly increased (P less than 0.01) in soleus muscle from 12-mo-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1984

Morphological evidence that high density lipoproteins are not internalized by steroid-producing cells during in situ organ perfusion.

Although it is clear that high density lipoproteins (HDL) can support steroidogenesis in several rat cell systems, questions still arise as to how HDL are processed by cells. In particular, it is not yet clear whether HDL are internalized by a pathway similar to that used for low density lipoproteins. This issue was examined in the present study using the luteinized ovaries of hormone-primed immature rats in an in situ perfusion system. Ovaries were perfused for 2-120 min with 125I-labeled human or rat HDL and processed for autoradiographic studies at the light and electron microscopic level, or homogenized and used for isolation of subcellular membranes. The results show that the luteal cells of this tissue bind both human and rat HDL with great specificity. Moreover, the intact HDL particle does not appear to be internalized by the luteal cell during the period of perfusion: i.e., the protein moiety of the labeled HDL remains associated with the plasma membrane at all times. Evidence from the autoradiographs suggest, however, that with time, an increasing proportion of the plasma membrane-bound protein is associated with inverted microvilli, which are embedded within the cytoplasm and make close contact with structures of the interior of the cell. We speculate that HDL-cholesterol may be transferred at such sites.

Animals↗
PubMed1984

Regulation of luteal cell lipoprotein receptors, sterol contents, and steroidogenesis by estradiol in the pregnant rat.

Although estradiol has been found to possess receptors in the luteal cell and to stimulate progesterone synthesis, its mechanism of action in the corpus luteum remains completely unknown. To determine whether estradiol modulates cellular uptake of lipoprotein substrate and intracellular cholesterol utilization, pregnant rats were hypophysectomized and hysterectomized on day 12 to reduce the luteal content of estradiol. They were treated with either 100 micrograms estradiol daily or with a 1-cm capsule filled with testosterone, which maintained luteal estradiol at levels found in intact pregnant rats. Blood was obtained 24, 48, and 72 h later for progesterone and cholesterol measurement. At 72 h, rats were killed, and corpora lutea (CL) were isolated for measurement of cholesteryl ester, free cholesterol, and [125I]iodo high density lipoprotein [( 125I]iodo-HDL)- and [125I]iodo-hCG-binding activities. In vivo treatment with estradiol or testosterone increased serum progesterone concentrations from 35 +/- 7 ng/ml in vehicle-treated rats to 128 +/- 21 and 118 +/- 16, respectively, and luteal weight from 2.1 +/- 0.2 mg/CL to 3.9 +/- 0.3 and 4.0 +/- 0.3 within 72 h. However, steroid treatment did not induce a change in luteal cell number, since the content of DNA per CL remained similar in all groups. It also did not modify levels of serum cholesterol. [125I]Iodo-HDL binding in luteal cells increased from 3.2 +/- 0.3 pg/cell in vehicle-treated rats to 9.9 +/- 0.8 and 7.9 +/- 0.6 after estradiol or testosterone treatment, while the luteal cell content of cholesteryl ester declined from 12.5 +/- 2.0 to 7.7 +/- 0.5 and 8.4 +/- 0.9 micrograms/CL, respectively. Thus, estradiol or testosterone increases luteal cell size but not cell number, depletes cholesteryl ester, and enhances HDL receptor content and progesterone synthesis. These results suggest that one possible mechanism by which estradiol and testosterone stimulate luteal cell steroidogenesis is by increasing the delivery of cholesterol substrate through a receptor-mediated process and by enhancing cholesterol utilization.

Animals↗
PubMed1983

Effect of antimicrotubule agents on terminal glycosyltransferases and other enzymes associated with rat liver subcellular fractions.

Previous studies have shown that anti microtubule agents disrupt Golgi complexes in hepatocytes and other cells, causing breakdown or vesiculation of Golgi cisternal membranes. Whether this change in the structure of the Golgi membranes is associated with changes in Golgi membrane function is not known. The present study was initiated to investigate this issue; i.e., to determine whether anti-microtubule agents that cause structural changes in Golgi membranes in vivo would, at the same time, affect characteristic enzyme functions of Golgi membranes. To this end, colchicine was given to young rats in vivo and various hepatic subcellular membranes were subsequently isolated and utilized for enzyme assays. Initially it was shown that colchicine (2.5 mg/kg body wt.) given for 5h significantly decreased the activities of the Golgi membrane associated enzymes galactosyl-, sialyl- and N-acetylglucosaminyl-transferases. More detailed experiments indicated that low doses of colchicine (0.8 mg/kg body wt.), although less effective than higher doses, decreased the activities of the terminal glycosylating enzymes maximally at 5h, with partial and complete recovery at 12 and 24h respectively. Treatment in vivo of rats with vinblastine (20 mg/kg body wt.) for 5h mimicked the action of colchicine. Two microsomal glycosylating enzymes (mannosyl and N acetylglucosaminyl transferases) were unaffected by the treatment with colchicine, as were various hepatic 'marker' enzymes such as 5' nucleotidase, glucose 6 phosphatase and succinate: 2-(p iodophenyl)-3-(p nitrophenyl)-5-phenyltetrazolium reductase (succinate dehydrogenase; EC 1.3.99.1), which were found to be enriched in plasma membrane, endoplasmic-reticulum and mitochondrial-membrane fractions respectively. These results show that anti-microtubule agents specifically suppress the activity of Golgi-associated glycosyltransferases in liver. Although it seems likely that these changes are related to the previously observed structural changes in hepatocyte Golgi complexes after colchicine treatment, to what extent the results are linked to the interaction of colchicine with microtubule protein remains to be clarified.

Animals↗