Biomedical subjects
V D Ramirez
Publications and source records attributed to V D Ramirez.
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.
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."
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.
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.
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.
Immunocytochemical mapping of a novel dopamine-releasing protein in the rat brain.
This report partially describes, for the first time, the immunocytochemical distribution of a novel dopamine-releasing protein (DARP) in the male rat brain. This glycoprotein detected specifically with a monoclonal antibody against DARP follows a close distribution to that of catecholamines (CA) in areas in the CNS rich in either CA fibers or CA neuron cell groups as previously reported by others using a tyrosine hydroxylase (TH) marker. The striking similarities in the topographic arrangement of the DARP-positive reaction product and the TH-positive reaction product is another argument favoring the view that DARP is involved in the regulation of catecholamine neurons.
A specific membrane binding protein for progesterone in rat brain: sex differences and induction by estrogen.
Progesterone conjugated to bovine serum albumin (BSA) was used as a probe to study sex differences and the effects of hormonal status on binding of progesterone to crude synaptosomal membrane preparations (P2) derived from the mediobasal hypothalamic-anterior hypothalamic-preoptic area or the corpus striatum. Binding of 125I-labeled BSA linked to progesterone at the 11 position of the steroid (P-11-BSA) was decreased by competition with unlabeled P-11-BSA or P-3-BSA (in which progesterone is bound to BSA at the 3 position). P-3-BSA displayed higher affinity than P-11-BSA. Hypothalamic and striatal preparations from adult females show high specific binding (60-80%) to the progesterone-BSA conjugate. Specific binding was reduced more than 80% 14 days after ovariectomy. Estrogen treatment (10 micrograms per rat for 4 days) of 14-day ovariectomized rats restored specific binding to levels equivalent to intact females. In contrast, adult males displayed drastically reduced or no specific binding in either tissue. No specific binding was detected after orchidectomy. Estrogen treatment of orchidectomized animals induced specific binding sites similar to those in intact females. Additionally, an affinity probe was developed by linking primary amines on the P-3-BSA conjugate to agarose activated aldehydes in an AminoLink column. A digitoxin-solubilized fraction from female rat P2 cerebellum preparations yielded a single major band after affinity purification with an estimated molecular mass of 40-50 kDa in an SDS/PAGE system after silver stain. These results show a reversible sex difference in the specific binding of progesterone to synaptosomal membrane sites in the central nervous system of male and female rats which is dependent on estrogen.
Immunocytochemical localization of dopamine-releasing protein in the rat adrenal.
The mechanism by which adrenal medulla transplants to the brain of Parkinson's patients ameliorate the symptoms of this neurodegenerative disease is unknown. It has been postulated but not proven that these transplants secrete a neurotrophic factor(s) responsible for the activation of the dopamine (DA) neurons. DA-releasing protein (DARP) was identified by immunocytochemistry in cells of the adrenal gland but not in tissues such as the liver or spleen. In the adrenal gland the immunopositive DARP cells were distributed both in the cortex as well as in the medulla. In the former, the distribution was rather uniform covering a broad area of the zona glomerulosa and fasciculata without positive cells in the zona reticularis. In the adrenal medulla the DARP signal was found in segregated cells forming clusters similar to the homotypic distribution of norepinephrine and epinephrine cells. The immunopositive cells for DARP were oval or spindle in shape and the staining was localized in the cytoplasm surrounding the nucleus. In addition, in this paper we present evidence confirming that the adrenal gland is the source of a protein that after immunopurification in nanogram amounts releases DA from corpus striatum fragments superfused in vitro. These data clearly demonstrate the existence of DARP in the adrenal gland and support the hypothesis that the effectiveness of the adrenal medulla transplants to the brain of Parkinson's patients is in part due to the secretion of neurotrophic factors. We submit that DARP is one of such factors.
Age-dependent changes in olfactory-mediated behavioral investigations in the male rat.
In the present experiment male Sprague-Dawley rats at three different ages (young, middle-aged, and old) were tested in a series of olfactory-mediated behavioral tests. The amount of ano-genital investigation directed to receptive females by old rats was significantly reduced compared to that of young and middle-aged animals. In contrast, general body investigation of receptive females was similar among the three age groups. The amount of anogenital investigation directed to nonreceptive females or immature male rats indicated significant differences only between young and old rats, with values from middle-aged rats being intermediate and not significantly different from either of the two extreme groups. General body investigation of nonreceptive females and immature males did not differ among the three age groups. Old rats showed the best performance in a location of buried food test with latencies to locate food approximately one-half that obtained from young and middle-aged rats. Measures of general locomotor activity indicated significantly greater values from young compared to middle-aged and old rats, with the latter two showing very similar levels. These results demonstrate clear discriminatory differences in olfactory-mediated behavioral performance of the male rat as a function of the specific test and age of the animal. Overall, the results suggest that olfactory-mediated behaviors related to reproduction appear more sensitive to age-related declines, while behavior related to food procurement is more minimally affected.
Influences of castration and testosterone on spring to summer changes in release of luteinizing-hormone-releasing hormone in rabbits.
Push-pull cannulae were implanted toward the tuberal region of the hypothalamus in ten intact New Zealand male rabbits. In the first experiment, rabbits were perfused at different times after castration: 5-10 days (n = 10), 22-31 days (n = 9) and 50-64 days (n = 8). The release, mean amplitude and mean frequency of luteinizing-hormone-releasing hormone (LHRH) signals from 37 perfusions in ten animals were analysed in intact rabbits and at different times after castration. No significant changes in release of LHRH and in amplitude were observed, but the frequency was significantly higher 22-31 days after castration than in intact rabbits (intact: 0.86 +/- 0.12; castrated: 1.20 +/- 0.13 pulses h-1, P < 0.035; n = 9). In Expt 2, testosterone and placebo Silastic capsules were implanted in the castrated rabbits. Perfusions were performed in the following four periods, defined by season and time after testosterone and placebo implants: (i) spring; before implants, (ii) late spring; 0-2 weeks after implants, (iii) summer solstice; 2-4 weeks after implants and (iv) summer; 4-6 weeks after implants. Castrated rabbits were perfused during spring; castrated rabbits with testosterone capsule implants were perfused during late spring, around summer solstice and in summer and castrated rabbits with placebo implants were perfused during periods (iii) and (iv). Castrated animals with placebo implants showed no significant changes in mean LHRH release and amplitude, although the frequency was significantly higher around the summer solstice period than in castrated rabbits perfused in the spring. In castrated rabbits with testosterone implants LHRH release was significantly higher in late spring than around the summer solstice and in the summer. In addition, the concentrations of LHRH in late spring were significantly higher than those of intact and castrated animals. In contrast, mean LHRH amplitude and frequency did not change. Mean amount of LHRH released and amplitude in castrated rabbits with testosterone implants were significantly lower around the summer solstice than in late spring or summer and compared with intact animals around summer solstice and in castrated rabbits in early spring. These data demonstrate that there were no significant changes in the mean amplitude and release of LHRH after castration from 5 and up to 64 days in rabbits with hypothalamic push-pull cannulae, in contrast to the well established dramatic effect of castration on gonadotrophin concentrations. However, there was a small, but significant, increase in the mean frequency of LHRH pulses 22-31 days after castration compared with values from intact rabbits.(ABSTRACT TRUNCATED AT 400 WORDS)
Prolactin stimulates dopamine release from the rat corpus striatum in the absence of extra-cellular calcium.
Prolactin (PRL) increased basal dopamine (DA) release and attenuated amphetamine (AMPH)-stimulated DA release in vitro from rat corpus striatum in a concentration-dependent manner with 10(-5) M PRL being the most effective. The effects of PRL on DA release were enhanced in the absence of extracellular calcium. PRL at 10(-5) M did not alter the DA post-superfusion content of the striatal tissue. These results indicate that the stimulatory effect of PRL on basal DA release does not require extra-cellular calcium and the inhibitory effect on AMPH-stimulated DA release is not due to depletion of DA stores.
The positive feedback action of vasopressin on its own release from rat septal tissue in vitro is receptor-mediated.
The effect of arginine vasopressin (AVP) on its own septal release was evaluated using an in vitro superfusion procedure. As compared to basal release from septal fragments, pulses of synthetic AVP (15 pg/5 min) resulted in a 25-fold augmented release of endogenous AVP, indicating a positive feedback action. Both the basal and stimulated AVP release were significantly increased by 60 mM potassium and markedly reduced by omission of calcium. Preincubation of the septal fragments with the V2/V1 AVP receptor antagonist d(CH2)5 [D-Tyr (Et)2,Val4]AVP resulted in a dose-dependent inhibition of the positive feedback action of AVP which was nearly completely blocked at doses between 1.25 and 5 ng per 100 microliters incubation medium. As compared to this effect, the V1 antagonist d(CH2)5 Tyr (Me)2 AVP as well as oxytocin were significantly less potent. The results suggest that the positive feedback action of AVP on its own release from septal fragments is potassium-stimulated, calcium-dependent and mainly V2 receptor-mediated. The physiological significance of this phenomenon remains to be shown.
Modulatory effects of progesterone upon dopamine release from the corpus striatum of ovariectomized estrogen-treated rats are stereo-specific.
We have shown previously that progesterone linked with bovine serum albumin at position 3 of the progesterone molecule (P-3-BSA) was effective in modulating (both stimulating and inhibiting) amphetamine (AMPH)-evoked dopamine (DA) release in vitro from superfused corpus striatal (CS) tissue fragments of ovariectomized estrogen-primed rats. To examine the stereospecificity of this effect, progesterone linked with bovine serum albumin at position 11 (P-11-BSA) and deoxycorticosterone linked with bovine serum albumin at position 21 (DOC-BSA) were tested for their ability to modulate amphetamine-stimulated DA release in vitro when administered in either a pulsatile or a continuous infusion mode at different doses. The results demonstrate that a specific orientation of the P-BSA molecule is required for differential modulation of AMPH stimulated DA release from the CS of ovariectomized estrogen-treated rats, since DOC-BSA was ineffective and P-11-BSA showed only a stimulatory effect.
Changes in dopamine release in vitro from the corpus striatum of young versus aged rats as a function of infusion modes of L-dopa, potassium, and amphetamine.
In the present experiments we examined the effects of two different modes of infusion (two separate 10 min versus continuous infusions) of a depolarizing concentration of potassium (K+, 30 mM), amphetamine (AMPH, 10 microM), or L-DOPA (5 microM) upon dopamine (DA) release in vitro from superfused corpus striatal (CS) tissue fragments of young (2-4 months) and aged (18-24 months) male rats. The relative changes in DA release to two infusions of K+ and AMPH (R2/R1) were virtually identical for CS from both young and aged rats. In the case of L-DOPA, DA release from CS of young rats was markedly increased in response to the second compared to the first L-DOPA infusion (R2/R1 = 2.48 +/- 0.33, N = 7) and significantly greater than that of aged rats (R2/R1 = 1.24 +/- 0.17, N = 6). A continuous infusion of K+ and AMPH resulted in an overall greater amount of DA release from the CS of young versus aged rats. In contrast, DA release from CS of aged rats showed an overall more rapid and greater amount of DA release to continuous L-DOPA infusion than that from the CS of young rats. These results demonstrate age-dependent differences in DA release from the CS as a function of the infusion mode of specific secretagogues. Particularly interesting were the responses to L-DOPA where a pulsatile administration resulted in an overall greater amount of DA release from the CS of young rats while a continuous infusion produced a greater amount of DA release from the CS of aged rats.
In vivo models for the study of gonadotropin and LHRH secretion.
The objective of this paper is to describe recent data from my laboratory dealing with the in vivo control of LHRH secretion in male rabbits and rats using the technique of push-pull perfusion (PPP) that allows repetitive determinations of this neuropeptide in quasi-normal physiological conditions. In addition, we have applied this method to simultaneously measure LHRH and LH in freely behaving male rats bearing a push-pull cannula (PPC) in the anterior pituitary. A description of the validation of this technique and its potential use will be discussed as well as data indicating that castration in the male rat induces a significant increase in the LHRH and LH signals; however, following testosterone treatment, in spite of a clear return of LH output to intact levels, even higher levels of LHRH reaching the anterior pituitary were detected. Curiously, in the rabbit no changes in LHRH release were noticed with castration, but following testosterone treatment, a transient but robust 5-8-fold increase in LHRH release was noticed. In short, these studies have demonstrated the existence of apparently opposite rather than similar responses in the testicular control of the hypothalamic-hypophysial axis of the male rat as compared with those of the male rabbit.
Effect of mating behavior on luteinizing hormone-releasing hormone release in female rabbits as monitored with push-pull cannulae.
To examine the effect of mating behavior on luteinizing hormone-releasing hormone (LHRH) release, intact New Zealand female rabbits were implanted with push-pull cannulae (PPC) aimed at the tuberal region of the hypothalamus and perfused with modified Krebs-Ringer phosphate medium at 11-13 microliters/min. In the mating experiments, does (n = 10) were initially perfused for a control period of 60-170 min followed by a mating period (100-160 min) which included the introduction of the male rabbit for an average time period of 30 min. Two groups of LHRH release patterns were observed: positive and negative responders. In the positive LHRH responders (n = 5), a clear rapid increase in LHRH release following mounting by the male occurred with a significant increase in the mean LHRH release (1.83 +/- 0.33 to 3.27 +/- 0.80 pg/10 min, p less than 0.040), in the mean LHRH amplitude (1.97 +/- 0.46 to 4.33 +/- 1.29 pg, p less than 0.022) and in the amplitude of the largest LHRH pulse (2.13 +/- 0.43 to 7.58 +/- 3.65 pg, p less than 0.022). In the negative LHRH responders (n = 5), no changes in LHRH release were detected although all rabbits ovulated, with some becoming pregnant. It appears from histological analysis that the difference between these two patterns of responses following mating are due to different cannula placements. In the positive responders, the tip of the PPC was localized in the tuberal region whereas in the negative responders, the placements were more dorsal and, in some cases, anterior.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenergic mediation of the naloxone-induced GnRH release from hypothalami of ovariectomized, steroid-treated immature rats.
The present study examines the effect of naloxone on GnRH release in vitro under different steroid milieus. Naloxone (6.1 mumol/kg) administered 30 min before decapitation was highly effective in evoking GnRH release from superfused hypothalamic tissues derived from ovariectomized, estradiol- and progesterone-treated immature rats, while ineffective in altering GnRH release from intact, ovariectomized and vehicle- or estradiol-treated rats. To further explore the possible involvement of catecholamines in the naloxone-stimulated GnRH release, diethyldithiocarbamic acid (2.9 mmol/kg), an inhibitor of noradrenalin synthesis, was administered ip 30 min before naloxone injection into ovariectomized, estradiol- and progesterone-treated rats. Diethyldithiocarbamic acid markedly reduced the naloxone-evoked GnRH release, although it was ineffective in modifying the spontaneous release of GnRH. A blockade of alpha-adrenergic receptor with phenoxybenzamine significantly suppressed the naloxone-stimulated GnRH release, whereas treatment with propranolol, a beta-adrenergic receptor blocker, failed to alter GnRH release. The present data suggest that the endogenous opioid peptide may participate in the regulation of GnRH release under a particular steroid milieu, and the inhibitory action of endogenous opioid peptide seems to require the mediation of adrenergic neurotransmission, presumably through alpha-adrenergic receptor.