Adenosine 3',5'-monophosphate-binding capacity in small and large ovine luteal cells

The photoaffinity analog [32P]8-azidoadenosine cAMP ([32P]8-N3cAMP) was used to study available cAMP-binding sites on cAMP-dependent protein kinases in homogenates of ovine small (12-22 microns) and large (greater than 22 microns) luteal cells. Binding of the analogs to specific proteins was detected by autoradiography after sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and was quantitated by liquid scintillation counting. In homogenates of untreated small and large cells, only the type I isoenzyme of the regulatory subunit (R1) of protein kinase had a measurable number of available cAMP-binding sites. Small luteal cells were incubated for 2 h with increasing concentrations of ovine LH (oLH), cholera toxin, or forskolin, and media were collected for quantification of cAMP and progesterone. Cells were harvested and homogenized, and intracellular cAMP content and photoincorporation of [32P]8-N3cAMP into RI were measured. Treatment of small luteal cells with oLH, cholera toxin, and forskolin resulted in dose-dependent increases in cAMP in both the cells and the incubation media and in the progesterone content of the media. These increases were accompanied by a dose-dependent decrease in photoincorporation of [32P]8-N3cAMP into RI of small cells, which was correlated (P less than 0.05) with the increase in progesterone secretion. A time-dependent decrease (P less than 0.05) in photoincorporation in small cells incubated with oLH (100 ng/ml), cholera toxin (1000 ng/ml), or forskolin (5 microM) preceded an increase (P less than 0.05) in progesterone secretion by these cells. Large ovine luteal cells incubated with increasing concentrations of cholera toxin or forskolin demonstrated a dose-dependent decrease in photoincorporation of [32P]8-N3cAMP into RI. The time-dependent decrease (P less than 0.05) in photoincorporation in large cells incubated with cholera toxin (1000 ng/ml) or forskolin (5 microM) was not followed by enhanced progesterone secretion. These observations are consistent with a role for cAMP involvement in progesterone secretion by ovine small luteal cells and suggest a lack of cAMP involvement in progesterone synthesis by large cells.     

Differential effects of luteinizing hormone on intracellular free Ca2+ in small and large bovine luteal cells

The effect of LH on the intracellular free Ca2+ concentration ([Ca2+]i) was investigated in highly purified small and large bovine luteal cell populations. Luteal cells were obtained from midcycle corpora lutea dispersed with collagenase and separated by flow cytometry into large and small cells. Resting levels of Ca2+ were higher (P less than 0.05) in the large than small cells [314 +/- 25 nM (n = 5) vs. 186 +/- 13 nM (mean +/- SE; n = 13) for large and small cells, respectively]. LH rapidly increased [Ca2+]i in both small and large cells loaded with the fluorescent Ca2+ probe fura-2. In the small cells, [Ca2+]i was immediately increased 2- to 6-fold (from 176 +/- 8 to 468 +/- 8 nM; n = 5) after adding LH. The LH induced [Ca2+]i rise occurred in two phases: an initial peak due to intracellular Ca2+ mobilization and a secondary rise due to Ca2+ influx from extracellular sources. Preincubation of the small cells with EGTA reduced the initial phase and abolished the secondary rise in [Ca2+]. Both forskolin and 8-bromo-cAMP increased [Ca2+]i in the small cells. In contrast, only a single phase of [Ca2+]i rise was observed in LH-treated large cells, and the response was 1.5- to 2-fold greater than the resting Ca2+ levels [314 +/- 25 vs. 435 +/- 60 nM (n = 4), for resting vs. LH-treated values, respectively]. The addition of both LH and prostaglandin F2 alpha (PGF2 alpha) to the large cells resulted in increases in [Ca2+]i that were greater than those induced by each hormone separately (2.0-fold for LH and 2.7-fold for PGF2 alpha vs. 7- to 9-fold in the presence of both hormones). These findings demonstrate that LH induces rapid increases in intracellular [Ca2+]i that differ in magnitude and profile between the small and large bovine luteal cells. Furthermore, LH and PGF2 alpha interacted to promote increases in [Ca2+]i in the large cells, that were higher than the sum of [Ca2+]i induced by each hormone separately.     

Increased cellular cholesterol upregulates high density lipoprotein binding to rat luteal cells.

Rat luteal cells preferentially utilize cholesterol derived from high density lipoproteins (HDL) as a substrate for steroid hormone synthesis. The uptake of cholesterol from HDL by these cells is in contrast to nonsteroidogenic cells, which export cholesterol to HDL. A previous study demonstrated that HDL binding to luteal cell membranes was increased in conjunction with in vivo cholesterol depletion or cholesterol loading of the ovary induced by pharmacological agents. These results suggest a biphasic regulation of the HDL receptor in luteinized rat ovaries. In the present studies, the in vitro regulation of HDL binding in rat luteal cells by increased intracellular cholesterol was examined. Cultured luteal cells were incubated with increasing doses of low density lipoproteins (LDL) for 2 days after which the cellular sterol content and the effects on progesterone production and HDL binding were measured. As expected, the LDL treatment increased total cellular sterol content in a dose-dependent manner, resulting in a 2.1-fold increase over control at a dose of 1 mg LDL/ml. Increased cellular cholesterol was accompanied by a comparable increase in progesterone secretion. These results suggest that exogenous cholesterol was utilized by these cells. The LDL treatment also increased the binding of HDL to the cells in a dose-dependent manner to a maximum of 2.2-fold over control. The effect of increased cellular sterol on HDL binding was also examined using a more polar cholesterol derivative, 25-hydroxycholesterol. Cells were cultured for 2 days in media containing 0.3-40 micrograms/ml 25-hydroxycholesterol in the presence of 100 micrograms/ml aminoglutethimide, an inhibitor of cholesterol metabolism. The HDL binding to luteal cells exhibited dose-dependent up-regulation by 25-hydroxycholesterol with a 5.8-fold increase in binding at the maximum dose tested. Equilibrium binding studies using cells treated with 10 micrograms/ml 25-hydroxycholesterol revealed a 2.1-fold increase in the number of HDL binding sites on the luteal cells without affecting the binding affinity. From the results of this study, it is concluded that HDL binding in rat luteal cells is up-regulated by an increase in the intracellular cholesterol level.     

Morphological and biochemical characterization of small and large bovine luteal cells during pregnancy

Small (15–18 μm) and large (18–45 μm) luteal cells were obtained from bovine corpora lutea of pregnancy by centrifugal elutriation of enzymatically dispersed luteal cells. Small luteal cells accounted for about 85% and large luteal cells for 8–12% of total luteal cell population.Small luteal cells were characterized by a low cytoplasmic/nuclear ratio with cytoplasm containing mitochondria, lysosomes, lipid droplets, dense granules and endoplasmic reticulum. Large luteal cells possessed a higher cytoplasmic/nuclear ratio with cytoplasm containing more abundant mitochondria, lipid droplets, dense granules and lysosomes compared to small luteal cells. Some of the mitochondria were very long. Both small and large luteal cells contained scarce amounts of Golgi elements. Dense granules were found close to the nucleus in both cell types. The nucleus of both cell types was acentric, irregular in shape and contained a well-defined nucleolus. The highly condensed chromatin in small luteal cells was found at the nuclear periphery and in the central region. Dispersed chromatin was found throughout the nucleus with condensed chromatin at the nuclear periphery of large luteal cells. Macrophages and fibroblasts were occasionally found in small luteal cell preparations, but their morphology was quite distinct from both small and large luteal cells. Scanning electron microscopy revealed that the majority of the small and large luteal cells were spherical or slightly elongated in shape. Small luteal cells displayed the presence of blebs, ruffles and short microvilli. Large luteal cell surface contained ruffles and randomly distributed clusters of blebs of different sizes, predominantly spherical in shape with a smooth surface. Finger-like projections were also occasionally seen.Small luteal cells contained significantly lower amounts of protein, but the ratios between protein and DNA were similar in both cell types. The basal, human chorionic gonadotropin (hCG)- or cyclic AMP-st′imulated progesterone production, the apparent dissociation constants for [¹²⁵I]hCG binding and the apparent total number of available sites per cell were similar in small and large luteal cells. The activities of enzymes that are involved directly or indirectly in progesterone biosynthesis and those involved in general cellular metabolism and biosynthesis were also similar in small and large luteal cells with one exception. That is, the activities of 5′-nucleotidase and NADH cytochrome c reductase were significantly higher in small compared to large luteal cells.     

Effects of neutrophils in rat luteal cells.

Ovarian function may be modulated by cells of the immune system. We have investigated the role of neutrophils (polymorphonuclear leukocytes) on rat luteal cell function. Activated neutrophils inhibited LH-sensitive cAMP accumulation, which was dependent on neutrophil cell number. At a concentration of 10(6) neutrophils/ml and 10(5) luteal cells/ml, LH-stimulated cAMP accumulation was inhibited by 50%. The inhibitory effect of activated neutrophils was reversed by superoxide dismutase (SOD) and catalase. LH-stimulated progesterone production was also inhibited by activated neutrophils. Progesterone production by 10(5) luteal cells was inhibited approximately 20% in the presence of 10(6) activated neutrophils, and this inhibition was blocked by SOD and catalase. Conditioned medium from activated neutrophils also produced inhibitory effects on LH-stimulated cAMP accumulation and progesterone production, which could be reversed by SOD and catalase. The phosphodiesterase inhibitor isobutylmethylxanthine had no significant effect on the inhibition of cAMP accumulation by conditioned medium from activated neutrophils. Luteal cells loaded with a fluorescent indicator for determining intracellular reactive oxygen species (dichlorofluorescein diacetate) showed increased fluorescence in the presence of activated neutrophils. No increase in fluorescence occurred in the absence of neutrophils or in the presence of SOD and catalase. These studies demonstrate that reactive oxygen species produced by activated neutrophils can enter the luteal cell and cause antigonadotropic effects. Although the experimental model used in the present studies may not be truly physiological, the data demonstrate that neutrophils may play a role in functional and structural regression of the corpus luteum in the rat.     

Protein kinase A and C activities and endogenous substrates in ovine small and large luteal cells

Protein kinase A (cAMP-dependent) and C (calcium, phospholipid-dependent) activities were measured and in vitro phosphorylation of endogenous proteins by these kinases were observed by SDS-PAGE in 100,000 x g supernatant (soluble) fractions of ovine small (12-22 microns) and large (greater than 22 microns) luteal cells. No differences in stimulation (P less than 0.05) of A kinase activity between small and large cells were detected. Protein kinase C activity was stimulated (P less than 0.05) 2.9-fold in small cells but not significantly enhanced above basal (P greater than 0.05) in large cells. By direct comparison, greater stimulation (P less than 0.05) over basal of A versus C kinase (6.1- versus 2.9-fold) was measured in small cells. These stimulations were greater than those observed in large cells (A kinase, 4.8-fold; C kinase, 1.8-fold). Maximal specific activities of both kinases (per mg protein) were greater (P less than 0.05) in small than in large cells. Endogenous proteins that could serve as substrates for phosphorylation by A and C kinases differed between small and large cells. Phosphorylation of six proteins by A kinase was consistently greater in small than in large cells. One endogenous protein (37 kDa) appeared to serve as a preferred substrate for phosphorylation by A kinase in small cells and C kinase in large cells. One protein (81 kDa) was predominantly phosphorylated in large rather than small cells by a calcium-dependent, C kinase-independent mechanism. These results support the accepted role of cAMP via A kinase and a possible role for C kinase in regulating steroidogenesis in ovine small luteal cells. The inability of large cells to respond to cAMP with enhanced secretion of progesterone may be due to an unavailability of phosphoprotein substrates for A kinase. Furthermore, protein kinase C activity and available protein substrates display quantitative and qualitative differences between small and large cells. Differences in regulation of steroidogenesis between the cell types may be due to these observed differences.     

Sugar alcohols enhance calcium transport from rat small and large intestine epithelium in vitro

We compared the effect of a variety of sugar alcohols on calcium absorption from the rat small and large intestine in vitro. An Ussing chamber technique was used to determine the net transport of Ca across the epithelium isolated from the jejunum, ileum, cecum, and colon of rats. The concentration of Ca in the serosal and mucosal Tris buffer solution was 1.25 mM and 10 mM, respectively. The Ca concentration in the serosal medium was determined after incubation for 30 min and the net Ca absorption was evaluated. The addition of 0.1–200 mM erythritol, xylitol, sorbitol, maltitol, palatinit, or lactitol to the mucosal medium affected net Ca absorption in the intestinal preparations. Differences in Ca transport were observed between portions of the intestine, but not between sugar alcohols tested. We concluded that sugar alcohols directly affect the epithelial tissue and promote Ca absorption from the small and large intestine in vitro.     

Luteotropic regulation of dispersed rat luteal cells in early pregnancy.

Viable and functional luteal cells were prepared, using a combination of hyaluronidase, collagenase, and a low concentration of trypsin in a Dulbecco's modified Eagle medium containing 0.5% bovine serum albumin and 3.3 mM Ca++, from corpora lutea taken from 2-day pregnant rats. The viability and functional capacity of the dispersed cells were evaluated by electronmicroscopy and by measuring steroidogenic capicity during perifusion. Dispersed luteal cells previously exposed in vivo to biphasic prolactin (PRL) surges were found to respond during perifusion to as little as 0.5 ng/ml LH by increased steroid secretion. The net progesterone synthesis and secretion remained elevated over a time course of 2 1/2 hours perifusion, and the magnitude of the luteotropic stimulation was dose dependent on LH. However, luteotropic stimulation of LH could not be maintained beyond 2 1/2 h without renewed (in vitro) PRL exposure. PRL by itself maintained the low initial secretion rate of progesterone but demonstrated no stimulatory effect. Different steroidogenic responses were noted during the in vitro administration of LH alone and the administration of LH plus PRL. In the former case, the decreasing rate of progesterone secretion was accompanied by an increasing 20 alpha-dihydroprogesterone secretion, suggesting that luteal 20 alpha-hydroxysteroid dehydrogenase activity was not suppressed. In the latter case, progesterone secretion was maintained and 20 alpha-dihydroprogesterone secretion fell suggesting an inhibitory action by PRL against 20 alpha-hydroxysteroid dehydrogenase activity. Dispersed luteal cells, preincubated at 36 C in medium containing only PRL, retained viability and functional capacity in response to LH-PRL stimulation for periods of time up to 48 h. Preincubation with LH alone did not prolong cell viability.     

Effect of prolactin on LH receptor in rat luteal cells.

This investigation was undertaken to determine whether prolactin might act as a luteotropic hormone in the rat by increasing the receptor for LH. Immature female rats were treated with 5 SC injections of oFSH beginning at 0900 h on day 25; luteinization was induced with a single SC injection of oLH at 1500 h on day 27. In this model, LH-receptor activity, assessed by specific binding of [125I]iodohCG to ovarian membrane fractions, increased steadily (greater than 7-fold) from day 27 to day 33; specific [125I]iodoprolactin binding rose greater than 3-fold between days 27 and 31. Serum progesterone measured at 1500 h rose from greater than 10 ng/ml on day 27 to greater than 130 ng/ml on day 34. 2-Bromo-alpha-ergocryptine (CB-154, [EC]; 35 mug) given 2 X/day beginning at the time of LH injection, blocked nocturnal increases in serum prolactin measured at 0200 h on days 29, 30, and 31, and resulted in reduced binding of [125I]iodohCG on days 31 and 33; serum progesterone was similarly reduced on days 31 and 34 in EC-treated rats. Simultaneous treatment with EC and prolactin completely reversed the effects of EC with regard to both binding of [125A]iodohCG and serum progesterone. Changes in ovarian uptake of [125I]iodohCG in vivo were similar to those of the in vitro experiments described above. EC and/or prolactin treatment did not affect binding of [125I]iodoprolactin. In summary, the data indicate that LH in the absence of prolactin can induce the formation of corpora lutea which have an increased number of prolactin receptors but which have a few LH receptors and a reduced capacity to produce progesterone. If prolactin is present during the early luteinization process, corpora lutea develop an increased capacity to bind LH and to produce progesterone. It is possible that the increase in LH-receptor and progesterone production occur as independent events both mediated by the luteotropic action of prolactin. Alternatively, LH receptor might be limiting for LH stimulation of progesterone production.     

Probucol enhances cholesterol transport in cultured rat hepatocytes

Probucol has been investigated extensively in vivo and ex vivo. However, little information is available on direct treatment of cultured cells. Treatment of cultured hepatocytes by 1 to 10 microM probucol for 16 to 20 hours had no apparent deleterious effect on the cell, and in fact decreased lactic dehydrogenase leakage. Also, total cellular cholesterol was increased, cholesterol synthesis was decreased, and cholesterol esterification was increased. The increase in cellular cholesterol was not the result of increased lipoprotein uptake but appeared to be the result of an interaction between cell and lipoprotein in which the cell became enriched and the lipoprotein depleted in cholesterol. This type of change in lipoprotein composition after treatment is also observed clinically.     

Insulin action and characterization of insulin receptors in rat luteal cells

The effect of insulin (Ins) on luteal cell function was assayed and Ins binding was characterized in isolated collagenase-dispersed rat luteal cells. Ins stimulated progesterone production at concentrations over 10(-8) M, whereas human CG (hCG) produced maximal stimulation at 10(-11) M. Ins had an additive effect when it was added to the luteal cells in the presence of hCG in concentrations that produced submaximal stimulation. At 10(-9) M Ins the additive effect on hCG response became apparent (80% of maximal progesterone production). The maximal hCG response cannot be exceeded, even in the presence of high amounts of both hormones. hCG maximally stimulated cAMP production at 10(-10) M, whereas Ins neither stimulated cAMP production nor enhanced hCG response. Ins binding to luteal cells attained highest values after 30 min of incubation at 20 C. A curvilinear Scatchard plot was obtained and it was analyzed using a two-binding site model. The affinity constant values were 2.1 X 10(8) M-1 and 5.2 X 10(6) M-1 and the number of binding sites were 35,000 and 900,000 per cell, respectively. Bound Ins was not displaceable by hCG, human GH, human LH, epidermal growth factor, or ovine PRL. A protein moiety was essential for the receptor activity, since after trypsin digestion marked loss of binding was verified. Subcellular fractionation was performed and the highest binding was observed in the 105,000 X g pellet fraction. This study demonstrates that Ins binds specifically to rat luteal cells and stimulates steroidogenesis, adding support for the hypothesis that insulin acts directly upon the ovary.     

Effect of cholesterol transport inhibitors on steroidogenesis and plasma membrane cholesterol transport in cultured MA-10 Leydig tumor cells

These studies were directed toward understanding the cellular actions of inhibitor drugs that affect steroidogenesis and cholesterol transport. We investigated the microfilament inhibitor cytochalasin-D, the microtubule inhibitor colchicine, the calmodulin antagonist trifluoperazine, and the inhibitor of acidic vesicle function nigericin. We found that all of these compounds caused dose-dependent inhibition of progesterone synthesis in the MA-10 cells. Each compound also inhibited (Bu)2cAMP-stimulated pregnenolone synthesis, indicating that each inhibited a fundamental process required for steroidogenesis. Each compound was next evaluated for inhibitory actions on cholesterol transport to and from the plasma membrane. On the basis of inhibitor sensitivity, two different categories of cholesterol transport were defined. Transport of newly synthesized or low density lipoprotein-derived cholesterol from the cell interior to the plasma membrane was inhibitor insensitive. Plasma membrane cholesterol internalization, however, was sensitive to all of the inhibitors and did not result because of any drug effect on the acyl-coenzyme-A-cholesterol acyl transferase. Cycling of cholesteryl ester-derived cholesterol through the plasma membrane appeared to occur before its use for steroidogenesis. Thus, inhibition of plasma membrane internalization would prevent utilization of both plasma membrane cholesterol and cholesteryl ester-derived cholesterol, the two major substrate sources for steroid hormone synthesis. Consistent with this interpretation was the finding that inhibition of plasma membrane cholesterol internalization by each inhibitor paralleled the inhibitor's effect on steroidogenesis.     

Inhibition of gonadotropin action and progesterone synthesis by xanthine oxidase in rat luteal cells.

Hydrogen peroxide produces marked antigonadotropic and lytic actions in luteal cells, but the effects of superoxide, the archetypal oxygen radical, are unknown. Xanthine oxidase generates superoxide, and the activity of this enzyme, and purine substrate, are increased under ischemia, such as that seen at luteal regression. We therefore examined the actions of xanthine oxidase on luteal cells to assess the effects of this enzyme and the superoxide anion on luteal function. Xanthine oxidase, in the presence of hypoxanthine (50 microM), produced marked inhibition of LH-sensitive cAMP and progesterone production with complete inhibition at 25 mU/ml and half-maximal inhibition at about 5 mU/ml. These antigonadotropic actions of xanthine oxidase were rapid with maximal effects within 5 min, followed several minutes later by substantial depletion of ATP. Heat, superoxide dismutase, and catalase or catalase alone abolished the actions of xanthine oxidase. While depletion of ATP by xanthine oxidase was prevented by 3-amino-benzamide, an inhibitor of DNA repair, inhibition of cAMP and progesterone production was still evident. Xanthine oxidase also inhibited progesterone synthesis stimulated by 8-bromo-cAMP. Isobutylmethylxanthine, a cAMP phosphodiesterase inhibitor, did not reverse the inhibition of cAMP accumulation by xanthine oxidase, and the enzyme had no effect on LH receptor binding activity. Since catalase reversed the effects of xanthine oxidase, we conclude that superoxide was rapidly dismuted to hydrogen peroxide and mediated the antigonadotropic and antisteroidogenic actions of xanthine oxidase in luteal cells. The sensitivity of luteal cells to xanthine oxidase raises the possibility that this enzyme may serve as a significant source of hydrogen peroxide in the corpus luteum.     

Involvement of microtubules in lipoprotein degradation and utilization for steroidogenesis in cultured rat luteal cells

Cells isolated from superovulated rat ovaries metabolize low density lipoprotein (LDL) and high density lipoprotein (HDL) of human or rat origin and use the lipoprotein-derived cholesterol as a precursor for progesterone production. Under in vitro conditions, both lipoproteins are internalized and degraded in the lysosomes, although degradation of HDL is of lower magnitude than that of LDL. In this report we have examined the role of cellular microtubules in the internalization and degradation of human LDL and HDL in cultured rat luteal cells. The microtubule depolymerizing agents colchicine, podophyllotoxin, vinblastine, and nocodazole as well as taxol, deuterium oxide, and dimethyl sulfoxide, which are known to rapidly polymerize cellular tubulin into microtubules, were used to block the function of microtubules. When these antimicrotubule agents were included in the incubations, degradation of the apolipoproteins of [125I]iodo-LDL and [125I]iodo-HDL by the luteal cells was inhibited by 50-85% compared to untreated control values. Maximum inhibitory effects were observed when the cells were preincubated with the inhibitor for at least 4 h at 37 C before treatment with the labeled lipoprotein. Lipoprotein-stimulated progesterone production by luteal cells was also inhibited by 50% or more in the presence of antimicrotubule agents. However, basal and hCG-stimulated progesterone production were unaffected by these inhibitors. The binding of [125I]iodo-LDL and [125I]iodo-HDL to luteal cell plasma membrane receptors was not affected by the microtubule inhibitors. Although binding was unaffected and degradation was impaired in the presence of the inhibitors, there was no detectable accumulation of undegraded lipoprotein within the cells during the 24 h of study. From this study we conclude that the uptake and utilization of LDL and HDL by cultured rat luteal cells are mediated by cellular microtubules.     

Iron transport in the small intestinal cells

Book Reviews in this articles: Morphology of the Blood and Marrow in Clinical Practice. By Richard T. Silver . Atlas of Haematology. By G. A. Mc Donald , T. C. Dodds and B. Cruickshank . Leukemia and Lymphoma. Ed. by J. F. Holland , P. A. Miescher and E. R. Jaffe . Coagulation Intravasculaires. Disséminees et Localisées. By C. Raby . The Biochemistry of the Cells of Blood and Bone Marrow. By Joseph F. Seitz .     

小分子药物调控胆固醇逆向转运机制的研究进展

本文从小分子药物调控胆固醇逆向转运机制的视角,介绍胆固醇逆向转运关键蛋白参与小分子药物调控胆固醇外流和脂质代谢机制的最新研究进展,探讨不同化学结构类型小分子药物对介导胆固醇逆向转运不同药靶蛋白表达的影响,从药理学的研究角度提供开展胆固醇逆向转运相关研究新的思维模式。     

Differential processing of prodynorphin and proenkephalin in specific regions of the rat brain.

Prodynorphin-derived peptides [dynorphin A (Dyn A)-(1-17), Dyn A-(1-8), Dyn B, alpha-neo-endorphin, and beta-neo-endorphin] and proenkephalin-derived peptides [[Leu]enkephalin [( Leu]Enk) and [Met]enkephalin-Arg6-Gly7-Leu8 [( Met]Enk-Arg-Gly-Leu]) in selected brain areas of the rat were measured by specific radioimmunoassays. We report here that different regions of rat brain contain strikingly different proportions of the prodynorphin and proenkephalin-derived peptides. There is a molar excess of alpha-neo-endorphin-derived peptides over Dyn B and Dyn A-derived peptides in many brain areas. [Leu]Enk concentrations exceed those of [Met]Enk-Arg-Gly-Leu in certain brain areas such as the substantia nigra, dentate gyrus, globus pallidus, and median eminence (areas rich in dynorphin-related peptides). These results indicated that (i) there is differential processing of prodynorphin in different brain regions and (ii) [Leu]Enk may be derived from Dyn A or Dyn B (or both). In certain brain regions [Leu]Enk may derive from two separate precursors (prodynorphin and proenkephalin) in two distinct neuronal systems.