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V D Ramirez

Publications and source records attributed to V D Ramirez.

At least 19 recordsLinked to original sources
PubMed2001

Binding of estrogen and progesterone-BSA conjugates to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the effects of the free steroids on GAPDH enzyme activity: physiological implications.

In this study rat brain solubilized plasmalemma-microsomal fractions (B-P3) or cytosolic fractions were applied to P-3-BSA (progesterone linked to BSA at C-3 position) and E-6-BSA (17beta-estradiol linked to BSA at C-6 position) affinity columns. It is interesting that a 37 kDa protein was retained by both columns which was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by N-terminal sequencing. The 37 kDa protein (GAPDH) was not retained by either a control BSA conjugated affinity column or a corticosterone-BSA affinity column. E-6-BSA bound to GAPDH with higher binding affinity than P-3-BSA or T-3-BSA (testosterone linked to BSA at C-3 position) affinity columns. In addition, the binding of 17beta-E-6-BSA to GAPDH was impeded by free estrogen (17beta-estradiol) completely. Binding studies of E-6-BSA and P-3-BSA to commercial GAPDH from rabbit skeletal muscle using radiolabeled ligand binding assays revealed that P-3-BSA had 10x lower GAPDH binding affinity than E-6-BSA. Next, the effects of estrogen and progesterone on GAPDH activity were studied. Rapid and significant increases in V(max) and changes in K(m) were observed by the addition of 10 nM estradiol, whereas 100 nM progesterone decreased only V(max) significantly. Testosterone, corticosterone, 17alpha-estradiol, and diethylstilbestrol did not affect the enzyme activity. The results indicate that GAPDH is a target site for 17beta-estradiol and progesterone and suggest possible roles in the regulation of cellular metabolism and synaptic remodeling in which GAPDH has been reported to be involved.

Amino Acid Sequence↗
PubMed2000

Inhibition of mitochondrial proton F0F1-ATPase/ATP synthase by polyphenolic phytochemicals.

Mitochondrial proton F0F1-ATPase/ATP synthase synthesizes ATP during oxidative phosphorylation. In this study, we examined the effects of several groups of polyphenolic phytochemicals on the activity of the enzyme. Resveratrol, a stilbene phytoalexin that is present in grapes and red wine, concentration-dependently inhibited the enzymatic activity of both rat brain and liver F0F1-ATPase/ATP synthase (IC(50) of 12 - 28 microM). Screening of other polyphenolic phytochemicals using rat brain F0F1-ATPase activity resulted in the following ranking potency (IC(50) in parenthesis): piceatannol (8 microM)>resveratrol (19 microM)=(-)epigallocatechin gallate (17 microM)>(-)epicatechin gallate, curcumin (45 microM)>genistein=biochanin A=quercetin=kaempferol=morin (55 - 65 microM)>phloretin=apigenin=daidzein (approx. 100 microM). Genistin, quercitrin, phloridzin, (+)catechin, (+)epicatechin, (-)epicatechin and (-)epigallocatechin had little effect at similar concentrations. Tannic acid, theaflavins (tea extract) and grape seed proanthocyanidin extract (GSPE) had IC(50) values of 5, 20 and 30 microg ml(-1), respectively. Several monophenolic antioxidants and non-phenolic compounds were ineffective at concentrations of 210 microM or higher. The inhibition of F0F1-ATPase by resveratrol and genistein was non-competitive in nature. The effects of polyphenolic phytochemicals were additive. Both resveratrol and genistein had little effect on the Na(+)/K(+)-ATPase activity of porcine cerebral cortex, whereas quercetin had similar inhibitory potency as for F0F1-ATPase. In conclusion, the ATP synthase is a target for dietary phytochemicals. This pharmacological property of these phytochemicals should be included in the examination of their health benefits as well as potential cytotoxicity.

Animals↗
PubMed2000

Electron microscopic visualization of membrane-mediated uptake and translocation of estrogen-BSA:colloidal gold by hep G2 cells.

Previously, we have identified a membrane-mediated uptake and translocation of 1,3,5(10)-estratrien-3, 17beta-diol 6-(O-carboxymethyl)oxime:(125)I-labeled BSA (E6(125)I-BSA) in vivo in immature female rat liver from the plasma membrane (P3 fraction) to the mitochondria and/or lysosomes (P2 fraction). To further investigate this unique effect, current experiments have involved the use of 1,3,5(10)-estratrien-3, 17beta-diol 17-hemisuccinate:( 125)I-BSA (E17(125)I-BSA) to demonstrate the presence of binding sites and translocation of the ligand in human hepatoblastoma (Hep G2) cells. In addition, an estrogen-BSA:colloidal gold conjugate, E17 BSA:Au, was used to directly visualize this uptake in Hep G2 cells. Hep G2 cells displayed high-affinity, stereospecific binding of E17(125)I-BSA. This same ligand was also translocated from the P3 fraction to the P2 fraction. In contrast, (125! )I-BSA was minimally removed from the culture medium. Electron micrographs of Hep G2 cells labeled with E17 BSA:Au demonstrated uptake of this ligand by clathrin-coated pits, indicative of receptor-mediated endocytosis. Furthermore, this ligand was also found in larger vesicles and multivesicular bodies, suggesting the involvement of the compartment of uncoupling of receptor and ligand (CURL), but never in the nucleus. As early as 30 min post-exposure, the ligand could be viewed in organelles, many of which had vesiculated interiors, resembling rounded, vesiculated mitochondria. Labeled BSA was detected mainly in the extracellular compartment, with few multivesicular bodies containing the labeled BSA. The translocation of E17 BSA:Au was virtually eliminated by 100 nM unlabeled E17 BSA or free 17beta-estradiol, but not 17alpha-E6 BSA, 17alpha-estradiol or P6 BSA, and also by exposure of the cells to reduced temperature. These experiments are the first t! o visually demonstrate membrane binding and specific uptake of an estrogen-containing ligand while allowing the intracellular structures responsible to be seen. Furthermore, they identify a potentially new pathway of receptor-mediated endocytosis; namely, the shuttling of estrogens to the mitochondria, in addition to the classical lysosomal pathway.

Analysis of Variance↗
PubMed1999

Cellular localization of dopamine-releasing protein (DARP) in rat C6 glioma and primary mesencephalic cell cultures.

Dopamine-releasing protein (DARP) is a multisubunit protein shown to have dramatic effects on development, recovery, and function of the rat catecholaminergic (CA) system. This study details efforts to determine if glial cells are responsible for the production of DARP in the central nervous system (CNS). Enzyme-linked immunosorbent assays (ELISA), Western blotting, and immunocytochemical techniques were employed to measure DARP levels and identify DARP immunoreactive proteins in rat C6 glioma cells and medium, respectively. ELISA analysis of serum-free C6 culture media revealed a maximal concentration of DARP by culture day 1. However, ELISA analysis of C6 cultures grown in F-12K/serum medium revealed that maximal levels of DARP were detected on culture day 6 with a 108% increase in DARP immunoreactivity from culture day 1. These values were determined using a polyclonal antibody generated against DARP-36aa (anti-DARP-36aa), a synthetic peptide with dopamine (DA) releasing activity, and anti-DARP B9-B10, a monoclonal antibody generated against partially purified DARP. Western blot analysis revealed that anti-DARP B9-B10 recognized proteins of approximately 60, 50, and 45 kDa in C6 cell homogenates while anti-DARP-36aa had immunoreactivity with the 60-kDa protein alone. Immunocytochemical studies demonstrated that anti-DARP-36aa and anti-DARP B9-B10 had strong immunoreactivity with proteins throughout the cytosol and in several processes of C6 cells. These results reveal that DARP is detected in glioma cells and secreted in a time-dependent fashion during culture. Primary rat mesencephalic cultures were also examined using immunocytochemistry. Incubation with DARP antibodies and antisera against glial fibrillary acidic protein (GFAP) revealed that DARP and GFAP immunoreactivity co-localized in primary mesencephalic cultures. However, the majority of DARP immunoreactivity was localized to cells without GFAP staining. These findings reveal that DARP is detected in astrocytes although the majority of DARP immunoreactivity is found in non-astrocyte type cells.

Animals↗
PubMed1999

Piceatannol, a stilbene phytochemical, inhibits mitochondrial F0F1-ATPase activity by targeting the F1 complex.

Piceatannol is a stilbene phytochemical from the seeds of Euphorbia lagascae, previously identified as an antileukemic principle. Piceatannol is considered an inhibitor of several tyrosine kinases. We recently reported that resveratrol, another stilbene phytoalexin from grape seeds, was an inhibitor of ATP synthase. Here, we demonstrated that piceatannol potently inhibited the rat brain mitochondrial F0F1-ATPase activity in both solubilized and submitochondrial preparations (IC50 of 8-9 microM), while having relatively small effect on the Na(+), K(+)-ATPase activity of porcine cerebral cortex (no effect up to 7 microM). Piceatannol inhibited the ATPase activity of the purified rat liver F1 with IC50 of about 4 microM, while resveratrol was slightly less active (IC50 of about 14 microM). Our results indicate that piceatannol and resveratrol inhibit the F-type ATPase by targeting the F1 sector, which is located to the inner membrane of mitochondria and plasma membrane of normal endothelial cells and several cancer cell lines. This mechanism could potentially contribute to the multiple effects of these chemopreventive phytochemicals.

Animals↗
PubMed1999

Rapid inhibition of rat brain mitochondrial proton F0F1-ATPase activity by estrogens: comparison with Na+, K+ -ATPase of porcine cortex.

Our earlier studies have identified oligomycin sensitivity-conferring protein (OSCP), a subunit of proton F0F1-ATPase/ATP synthase in the mitochondrial inner membranes, as a new estradiol binding protein. This finding suggests that mitochondrial ATPase/ATP synthase could be a potential target for estradiol or compounds with similar structures. Here, we report that estradiol and several other compounds inhibited F0F1-ATPase activity of detergent-solubilized rat brain mitochondrial preparations in a following decreasing order: diethylstilbestrol (half-inhibition concentration, IC50 of 10-25 microM) > alpha-zearalenol, 4-hydroxyestradiol (1C50 of 55 microM) >2-hydroxyestradiol (IC50 of 110 microM), 17beta-estradiol, 17alpha-estradiol > beta-zearalanol > estriol, testosterone, 16alpha-hydroxyestrone > corticosterone, progesterone, dehydroepiandrosterone, dehydroepiandrosterone 3-sulfate, cholesterol (less than 10% inhibition at 140 microM). On the other hand, Na+, K+ -ATPase of porcine cortex showed different sensitivity to the compounds tested above. At 70 microM, the rank of inhibitory potency in decreasing order was as follows: 2-hydroxyestradiol (IC50 of 70 microM) > diethylstilbestrol> 4-hydroxyestradiol > progesterone > alpha-zearalenol, while other compounds had little effect (less than 5%). The data indicate that the ubiquitous mitochondrial F0F1-ATPase is a specific target site for estradiol and related estrogenic compounds; however, under this in vitro condition, the effect seems to require pharmacological concentrations.

Animals↗
PubMed1999

Purification and identification of an estrogen binding protein from rat brain: oligomycin sensitivity-conferring protein (OSCP), a subunit of mitochondrial F0F1-ATP synthase/ATPase.

Early studies have suggested the presence in the central nervous system of possible estrogen binding sites/proteins other than classical nuclear estrogen receptors (nER). We report here the isolation and identification of a 23 kDa membrane protein from digitonin-solubilized rat brain mitochondrial fractions that binds 17beta-estradiol conjugated to bovine serum albumin at C-6 position (17beta-E-6-BSA), a ligand that also specifically binds nER. This protein was partially purified using affinity columns coupled with 17beta-E-6-BSA and was recognized by the iodinated 17beta-E-6-BSA (17beta-E-6-[125I]BSA) in a ligand blotting assay. The binding of 17beta-E-6-BSA to this protein was specific for the 17beta-estradiol portion of the conjugate, not BSA. Using N-terminal sequencing and immunoblotting, this 23 kDa protein was identified as the oligomycin-sensitivity conferring protein (OSCP). This protein is a subunit of the FOF1 (F-type) mitochondrial ATP synthase/ATPase required for the coupling of a proton gradient across the F0 sector of the enzyme in the mitochondrial membrane to ATP synthesis in the F1 sector of the enzyme. Studies using recombinant bovine OSCP (rbOSCP) in ligand blotting revealed that rbOSCP bound 17beta-E-6-[125I]BSA with the same specificity as the purified 23 kDa protein. Further, in a ligand binding assay, 17beta-E-6-[125I]BSA also bound rbOSCP and it was displaced by both 17beta-E-6-BSA and 17alpha-E-6-BSA as well as partially by 17beta-estradiol and diethylstilbestrol (DES), but not by BSA. This finding opens up the possibility that estradiol, and probably other compounds with similar structures, in addition to their classical genomic mechanism, may interact with ATP synthase/ATPase by binding to OSCP, and thereby modulating cellular energy metabolism. Current experiments are addressing such an issue.

Adenosine Triphosphatases↗
PubMed1998

The chemical protecting group concept applied in crosslinking of natural tissues with glutaraldehyde acetals.

This work describes the results of the controlled crosslinking of collagen matrices by glutaraldehyde based on a double protection strategy, glutaraldehyde acetals and lysine protonation due to the acidic conditions of acetal formation. Materials crosslinked by this approach were characterized by thermal stability comparable to those obtained by conventional procedures with mechanical properties expected for bioprosthesis manufacture and with a higher stability toward collagenase hydrolysis. The integrity of the microfibrillar structure was confirmed by optical and scanning electronic microscopy. The results indicate that the glutaraldehyde acetals procedure may be of potential use for the crosslinking of bovine pericardium used in the manufacture of bioprosthetic devices. Advantages may be related to the production of materials with homogeneous crosslinking distributions, associated with better definition in the nature of the chemical link that they introduce, due to a better distribution of glutaraldehyde within the tissue matrix before the crosslinking reaction is allowed to occur. As a result, materials with improved biological and mechanical properties are expected.

Acetals↗
PubMed1998

Rapid uptake and binding of estradiol-17beta-6-(O-carboxymethyl)oxime:125I-labeled BSA by female rat liver.

To investigate potential membrane-mediated responses to estrogen, a membrane-impermeant, radioiodinated, steroid-BSA conjugate--estradiol-17beta-6-(O-carboxymethyl)oxime:125I-labeled BSA (17beta-E-6-125I-BSA)--or related steroid conjugates, or 125I-BSA was injected into female Sprague-Dawley rats, and tissues were collected at varying times postinjection. The liver, adrenal, and spleen displayed the most prominent uptake of 17beta-E-6-125I-BSA, reaching a maximum of 43 times blood levels in sonicated liver samples at 5 min postinjection, but no uptake of 125I-BSA. Isolation of liver membranes by differential centrifugation showed that over 50% of recovered radioactivity was in association with microsomes and plasmalemma (P3 fraction) at 30 sec postinjection. By 60 min postinjection, over 75% of recovered radioactivity was in association with mitochondrial and lysosomal membranes (P2 fraction), and less than 10% remained in the P3 fraction. In vitro competition assays demonstrated two binding sites in liver P3 fractions. The spleen and liver also showed saturable binding in vivo. These data suggest the presence of at least one membrane-binding protein for estrogen in liver, adrenal, and spleen. Initial studies of affinity-purified liver P3 fractions using ligand blots indicated the presence of two binding proteins. These potential membrane estrogen-binding proteins may be involved in a very rapid shuttling of estrogen from the plasmalemma to mitochondria and lysosomes.

Animals↗
PubMed1998

Mapping of bovine pericardium: physical and histopathologic tests.

BACKGROUND AND AIMS OF THE STUDY: This study was performed to identify the physical and histopathologic characteristics of different sections of glutaraldehyde-tanned bovine pericardium. METHODS: Ten pericardial sacs were obtained from animals aged from 18 to 36 months. Physical tests included shrinkage and mechanical resistance (rupture, elongation, tenacity index). Collagen and elastic fibers were evaluated in Gomori's trichrome-stained sections, hematoxylin and eosin, by PAS and Verhoeff's method. Studied areas were proximal to the great arteries, and the right atrial, right ventricular, left ventricular and left atrial regions. RESULTS AND CONCLUSIONS: Results showed that bovine pericardium does not have enough regional differences to identify any single region for bioprosthesis manufacture. However, histopathology showed better preservation of collagen and elastic fibers in the right ventricular region, implying that this area is more adequate as bioprosthetic material.

Animals↗
PubMed1997

Demonstration of membrane estrogen binding proteins in rat brain by ligand blotting using a 17beta-estradiol-[125I]bovine serum albumin conjugate.

This paper describes a ligand blotting procedure to visualize membrane estrogen receptors/binding proteins immobilized on nitrocellulose membranes. Using 17beta-estradiol covalently linked with [125I]bovine serum albumin (BSA) at the C-6 position (17beta-E-6-[125I]BSA) as a ligand, three major binding proteins with molecular masses of approximately 23, 28, and 32 kDa were identified from crude synaptosomal fractions (P2) of female rat brains. The binding of 17beta-E-6-[125I]BSA to these proteins is selective for 17beta-estradiol because BSA had no effect, and 17alpha-E-6-BSA was at least two orders of magnitude less potent than 17beta-E-6-BSA in displacing the binding. In addition, [125I]BSA and 17alpha-E-6-[125I]BSA at similar concentrations did not bind to these proteins. Competition and saturation assays indicate that the binding affinity of 17beta-E-6-[125I]BSA for these proteins was in the range of 1-10 nM. These proteins are not contaminants from cytosolic or serum estrogen binding proteins since no corresponding protein bands were found in cytosolic fractions. Three additional protein bands with molecular masses of approximately 18, 40, and 130kDa were also detected, although inconsistently. The 23 and 40 kDa proteins seem to be concentrated in mitochondrial fractions (mP2), whereas the 28 and 32 kDa proteins are enriched in microsomal fractions (P3). Application of digitonin-solubilized P2 fractions to 17beta-estradiol-coupled affinity columns resulted in significant purification of the 23 kDa protein as shown by ligand blotting. This protein was later identified as oligomycin-sensitivity conferring protein (OSCP), as reported previously. These data indicate that specific estrogen binding proteins different from classical nuclear estrogen receptor (66 kDa) are present in the cellular membranes of the female rat brain. The ligand blotting technique described here would also be applicable for the identification of other membrane steroid binding proteins/receptors using similar radiolabelled steroid-BSA conjugates.

Animals↗
PubMed1996

Membrane sex-steroid receptors in the brain.

In this review we discuss evidence indicating the existence of specific binding receptor sites for progesterone (P), estrogen (E), and testosterone (T) in neural membranes. The review starts with the methodological approaches for studying receptor binding sites in neural membranes. Second, we examine the physiological and biochemical data supporting the concept of cognate membrane receptors for P, E, and T, the so-called mPR, mER, and mTR. Last, we present an overview emphasizing the concept of continuity of action of steroids on cells from the outer surface to nuclear events.

Animals↗
PubMed1996

Membrane receptors for estrogen, progesterone, and testosterone in the rat brain: fantasy or reality.

1. There are numerous circumstantial evidence supporting the concept that steroid hormones control cellular function by means other than the nuclear receptor steroid binding mechanism. It is the intent of this report to present evidence indicating that steroids bind to specific sites in neuronal membranes. 2. Some of the criteria to define steroid membrane receptors using steroid-BSA conjugates that can be radioiodinated to desired specific activity have been fulfilled for each of the three sex steroids using crude synaptosomal membrane preparations (P2 fractions) from the CNS of female and male rats. Ligand binding for each of the three steroids indicate high-affinity and high-capacity sites with distinct brain selectivity and stereospecificity. For example, 17 beta-E-6-[125I]BSA binds hypothalamic P2 fractions (HYP-P2) with an estimated Kd of about 3 +/- 0.7 nM (X +/- SE; n = 3), whereas the cerebellum P2 (CB-P2) fractions bind the ligand with a Kd of 34 +/- 7 nM and, a Bmax of 3 and 42 pmol/mg protein, respectively. Estrogen and testosterone binding fit best a one-single site, while progesterone binding sites can be best represented by a two-binding site, one high-affinity (Kd = 1-2 nM) and one low affinity (Kd = 62 nM), in CB-P2 fractions from intact adult female rat brain. Kinetics studies for T-3-[125I]BSA indicate that the estimated Kd of 30 +/- 2 nM for the olfactory bulb P2 fractions (OB-P2) from male rats is in good agreement with Kd values computed from Scatchard-derived data using the LIGAND algorithm. 3. 17 beta-E-6-[125I]BSA binding sites are stereospecific and appears to be present as early as 5 days of age in both the OB- and the CB-P2 fractions without changes during development. In contrast, P-6-[125I]BSA binding sites are practically absent during days 5 and 12 and appear by day 22. 4. Finally, membrane receptor molecules for estrogen and progesterone have been isolated and purified by affinity chromatography and characterized by PAGE and Western blot. Microsequencing of one of the membrane estrogen binding proteins indicates that the high-affinity site corresponds to the OSCP subunit of the proton ATP synthase. 5. It remains to be determined if P and T also bind to this complex enzyme or if they bind to other subunits of the family of proton ATPases. Overall the data indicate that steroid hormones conjugated to BSA are important tools to study the "reality of membrane steroid receptors."

Animals↗
PubMed1996

Steroids conjugated to bovine serum albumin as tools to demonstrate specific steroid neuronal membrane binding sites.

Our laboratory has pioneered the use of bovine serum albumin (BSA) linked to different positions of the ring structure of progesterone to investigate steroid-membrane interactions. The complex can be radioiodinated to demonstrate the existence of specific membrane progesterone binding sites in the rat brain. Not only are these progesterone complexes specific ligands, but they also elicit functional responses in the central nervous system (CNS), particularly in the corpus striatum (CS) where progesterone-BSA conjugates linked at C-3 and C-11 positions (P-3-BSA and P-11-BSA) alter amphetamine-evoked dopamine release. In this communication we will report our current studies that use radioiodinated progesterone-BSA conjugates (P-3-125I-BSA, P-6-125I-BSA, and P-11-125I-BSA) and estradiol-BSA conjugates linked at C-6 position (17 beta-E-6-125I-BSA and 17-E-6-BSA) to demonstrate the existence of specific membrane binding sites for progesterone and estrogen in several regions of the rat brain. In addition, initial studies to isolate and purify these membrane binding sites from digitonin-solubilized P2-membrane fractions by affinity chromatography are reported. The data indicate that these sites are part of a complex membrane receptor for either estrogen or progesterone, the so-called membrane estrogen receptor (mER) and the membrane progesterone receptor (mPR), respectively.

Animals↗
PubMed1996

Maintenance of hypothalamic GnRH release during lactation in the rat: a push-pull perfusion study.

The activity of the gonadotropin-releasing hormone (GnRH) pulse generator during lactation was assessed by direct determination of GnRH levels impinging upon the pituitary gland. Sprague-Dawley rats were implanted on day 15 of pregnancy with a push-pull perfusion cannula directed to the anterior pituitary. All implanted animals showed normal parturition, maternal behavior and lactation. Push-pull perfusions were performed in 15 rats suckling 11.0 +/- 0.8 pups (range 4-15) on day 7-20 of lactation and repeated on diestrous 1 after weaning in some of the same animals. GnRH content of the samples was assayed by RIA. GnRH pulses were clearly detected during lactation. Mean GnRH secretion rate was 1.9 +/- 0.3 pg/10 min (chi +/- SE, range between 0.5 and 3 pg/10 min) and interpulse interval was 37.5 +/- 1.7 min (range between 27 and 50 min). There was a significant decrease of about 19% in the interpulse interval after weaning. There was no significant difference in GnRH pulse amplitude nor in GnRH secretion rate between lactation and diestrous. These results demonstrate that nursing does not suppress the GnRH pulse generator in the rat.

Animals↗
PubMed1994

Unilateral deficits induced in rats by MPP+ are markedly reduced by an N-terminal peptide fragment of dopamine-releasing protein.

Dopamine-releasing protein (DARP) is a novel factor involved in the function and development of catecholaminergic systems. To test whether a peptide synthesized from the N-terminus of DARP (DARP-36aa) ameliorates deficits in nigrostriatal dopamine, rats were unilaterally lesioned with MPP+, 1 day before administration of DARP-36aa began. Striatal delivery of 1 microgram DARPP-36aa daily for 10 days elevated striatal dopamine (P < 0.01) and reduced amphetamine-induced rotations (P < 0.05) relative to controls, both indicating the protection or restoration of dopaminergic function.

1-Methyl-4-phenylpyridinium↗
PubMed1994

Isolation of DARP (dopamine-releasing protein) from fetal rat brain and effects of DARP immunoneutralization on fetal mesencephalic dopamine levels.

Recent work from this laboratory suggests a role for a novel dopamine-releasing protein (DARP) during rat fetal development. We have previously shown that intrafetal administration of an anti-DARP monoclonal antibody (DARP mAb) at Embryonic Day 17 (E17) induces fetal resorption in a dose-dependent manner (Kuhananthan et al., Mol. Cell. Neurosci., 2, 410-417). In this study we present evidence that (1) DARP is present in the rat brain at E17 and (2) in vivo immunoneutralization of DARP at E17 alters dopamine (DA) levels selectively in the prenatal mesencephalon. Enzyme-linked immunosorbent assay of supernatants of crude fetal (E17) brain homogenates using DARP mAb as a probe detects the presence of a DARP-like immunoreactive protein in the E17 rat brain. Purification of these homogenates with concanavalin A and immunoaffinity chromatography yielded a single 60-kDa protein that displayed dopamine-releasing activity in an in vitro superfusion assay. In addition, intrafetal administration of DARP mAb at E17 significantly elevated DA levels in the mesencephalon 24 and 48 h postinjection, while decreasing these levels 72 h postinjection. No changes in DA levels were detected in the diencephalon or in the telencephalon. These findings indicate that DARP is present in the rat brain at E17 and may play a role in the development of dopaminergic neurons of the mesencephalon.

Animals↗