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P L Zamorano

Publications and source records attributed to P L Zamorano.

13 recordsLinked to original sources
PubMed2004

MAGUKs in synapse assembly and function: an emerging view.

Neuronal morphogenesis, synaptogenesis and synaptic plasticity are fundamental aspects of nervous system development. Much of our current understanding of how each of these processes contributes to the establishment and maintenance of neural circuitry has come from a molecular description of specific classes of key molecules. With regard to synapse assembly and function, a family of membrane-associated guanylate kinase homologs (MAGUKs) have emerged as central organizers of multicomponent protein signaling complexes. In particular MAGUKs appear to play fundamental roles in the transport, anchoring and signaling of specific subclasses of synaptic receptors and ion channels. In this review, we will focus on the role that subfamilies of MAGUKs play during the formation, maintenance and plasticity of the vertebrate central nervous system glutamatergic synapse.

Animals↗
PubMed2001

Unwebbing the presynaptic web.

The release of neurotransmitter from nerve terminals occurs at a specialized region of the presynaptic plasma membrane called the active zone. A dense matrix of proteins associated with the active zone, called the presynaptic web, is thought to play a fundamental role in defining these neurotransmitter release sites. In this issue of Neuron, Phillips et al. have identified conditions for the biochemical purification of the presynaptic web and show that the web is comprised of proteins involved in the docking, fusion, and recycling of synaptic vesicles.

Animals↗
PubMed2001

The dynamics of SAP90/PSD-95 recruitment to new synaptic junctions.

SAP90/PSD-95 is thought to be a central organizer of the glutamatergic synapse postsynaptic reception apparatus. To assess its potential role during glutamatergic synapse formation, we used GFP-tagged SAP90/PSD-95, time lapse confocal microscopy, and cultured hippocampal neurons to determine its dynamic recruitment into new synaptic junctions. We report that new SAP90/PSD-95 clusters first appeared at new axodendritic contact sites within 20-60 min of contact establishment. SAP90/PSD-95 clustering was rapid, with kinetics that fit a single exponential with a mean time constant of approximately 23 min. Most new SAP90/PSD-95 clusters were found juxtaposed to functional presynaptic boutons as determined by labeling with FM 4-64. No evidence was found for the existence of discrete transport particles similar to those previously reported to mediate presynaptic active zone cytoskeleton assembly. Instead, we found that SAP90/PSD-95 is recruited to nascent synapses from a diffuse dendritic cytoplasmic pool. Our findings show that SAP90/PSD-95 is recruited to nascent synaptic junctions early during the assembly process and indicate that its assimilation is fundamentally different from that of presynaptic active zone components.

Animals↗
PubMed1999

Regulation of leptin gene expression and secretion by steroid hormones.

Previous work has shown that 17 beta-estradiol is the primary ovarian signal regulating body weight and adiposity, although its mechanisms of action remain unclear. We hypothesized that 17 beta-estradiol could enhance leptin levels as a mechanism of its anorectic effects. Administration of 5 microg 17 beta-estradiol subcutaneously (s.c.) for 2 days significantly elevated leptin mRNA levels in adipose tissue as compared to vehicle controls (P < 0.003). A time-course administration of estrogen showed increased mRNA levels in adipose tissue between 6 and 12 h after estrogen injection as compared to vehicle controls (P < 0.03). Corresponding to the increased leptin mRNA levels at 6 and 12 h, elevated plasma leptin levels were observed at 12 h after estrogen administration as compared to controls (P < 0.05). Administration of progesterone (1 mg/rat) after estradiol injection did not enhance the elevated leptin mRNA levels in adipose tissue. Serum leptin levels from cycling rats did not differ significantly between metestrous and proestrous animals. In conclusion, the present studies demonstrate that 17 beta-estradiol can regulate leptin gene expression and secretion in the female rat, thus providing a better understanding of the possible anorectic effect of estrogens.

Animals↗
PubMed1997

Expression and localization of the leptin receptor in endocrine and neuroendocrine tissues of the rat.

The obese gene (ob) product, leptin, has recently been shown to be produced by adipocytes and to circulate in the plasma acting as a hormone to modulate appetite and metabolism. Intriguingly, the ob/ob mutant female mouse, which does not produce an active form of leptin due to a mutation of the ob gene, has been shown to be acyclic and sterile. This sterility can be reversed by treatment with recombinant leptin, but not by diet restriction--suggesting that leptin is required for normal reproductive function. The mechanism(s) whereby leptin modulates reproductive function are unknown; however, it is possible that leptin could directly regulate reproductive tissues. To determine whether endocrine and neuroendocrine tissues could be targets for leptin action, we examined whether these tissues express the leptin receptor mRNA by utilizing reverse-transcription polymerase chain reaction (RT-PCR) analysis in selected tissues from the male and female rat. The results revealed that the leptin receptor mRNA transcript is highly expressed in the ovary, uterus and testis, moderately expressed in the hypothalamus and anterior pituitary, with low to no expression in the adrenal. The RT-PCR results were confirmed by Northern analysis. Furthermore, immortalized GnRH (GT1-7 and NLT) neurons and ovarian granulosa cells were also demonstrated by RT-PCR analysis to express the leptin receptor, suggesting that GnRH neurons and steroid-producing cells of the ovary could be targets for leptin action. Immunohistochemical studies revealed dense immunolocalization of the leptin receptor in the choroid plexus, and interestingly, in the arcuate nucleus/median eminence of the female rat--a key sit in the control of feeding and reproduction. Finally, treatment of the ob/ob mouse with recombinant leptin (0.15 mg/kg/day x 2 weeks) was found to markedly upregulate side chain cleavage and 17 alpha-hydroxylase mRNA levels in the ovary, demonstrating that leptin, acting either through a direct or indirect mechanism, can regulate gene expression in reproductive tissues.

Animals↗
PubMed1996

Quantitative RT-PCR for neuroendocrine studies. A minireview.

Determination of mRNA levels of specific genes is becoming increasingly important as a measure of gene expression. With the recent advent of RT-PCR, the sensitivity for mRNA determination has been increased dramatically, and this technique is becoming widely used in neuroendocrine studies which involve small tissue samples and/or isolated nuclei. Nevertheless, the exact procedure for reliable quantification of RT-PCR has been widely debated. This minireview attempts to assimilate the available literature on the RT-PCR technique and discuss the various approaches commonly used to obtain quantitative results using the technique. An example from our laboratory of the use of RT-PCR for the measurement of several gene products in the same sample using exogenous internal standards is also provided. Particular attention is paid to the choice of endogenous vs. exogenous internal standards, the length of the transcript of the standard and its relationship to the target sequence being amplified, the amplification pattern of the target gene and internal standard, the reproducibility of the method, and the overall usefulness and suitability of RT-PCR for neuroendocrine studies.

Base Sequence↗
PubMed1996

Decreased gonadotropin-releasing hormone neurosecretory response to glutamate agonists in middle-aged female rats on proestrus afternoon: a possible role in reproductive aging?

Before becoming acyclic, middle-aged rats display an attenuated LH surge and a decreased number of activated GnRH neurons. The present study examined whether the decreased activation of GnRH neurons in middle-aged rats could be due to defective glutamate neurosignaling in the hypothalamus. Arcuate nucleus/median eminence (ARC/ME) fragments were isolated from young (2-month-old) and middle-aged (9- to 11-month-old) rats at 1700 h on proestrus and incubated in vitro with or without the specific glutamate agonists, D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (1 mM), kainate (1 mM), and N-methyl-D-aspartate (NMDA; 50 mM). The results showed that basal GnRH release was similar in the two age groups. In contrast, stimulated GnRH release by D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, kainate, and NMDA was significantly attenuated in middle-aged vs. young rats. KCl stimulation at the end of the experiments confirmed the viability of all ARC/ME fragments. Quantitative reverse transcriptase-PCR revealed that messenger RNA levels for the major NMDA receptor subunit (NMDAR1) were significantly lower in the preoptic area and ARC/ME of the middle-aged rat on proestrus afternoon. As a whole, these findings suggest that a defect in hypothalamic glutamate neurosignaling may be an important mechanism leading to age-related defects in LH secretion and acyclicity in female animals.

Aging↗
PubMed1995

Localization of the N-methyl-D-aspartate R1 receptor subunit in specific anterior pituitary hormone cell types of the female rat.

N-methyl-D-aspartate (NMDA), a specific agonist of the NMDA-type glutamate receptor, has been shown to stimulate the release of several anterior pituitary hormones when administered in vivo. The primary site of action of NMDA has been suggested to be at the level of the hypothalamus via the control of hypothalamic releasing factors. However, recent studies have demonstrated that NMDA can exert stimulatory effects directly upon anterior pituitary cells perifused in vitro, suggesting that the anterior pituitary may also be a site of action for glutamate. Hence, the purpose of the present study was to determine whether the NMDA R1 receptor subunit is co-localized in specific hormone-secreting cells of the anterior pituitary. To achieve this aim, immunohistochemical studies were performed using an antibody specific for the NMDA R1 receptor subunit to stain sections from the pituitaries of steroid-treated ovariectomized immature rats. Double-immunohistochemistry was employed to demonstrate co-localization. The results of the studies revealed that moderate immunoreactive NMDA R1 receptor staining was observed in the anterior and intermediate lobe of the pituitary with little staining observed in the posterior lobe. Double-immunohistochemistry using antibodies for the NMDA R1 receptor subunit and specific anterior pituitary hormones revealed that the NMDA R1 receptor subunit is co-localized in many cell types of the anterior pituitary including LH (11.2%), FSH (9%), GH (4.6%), TSH (5.7%) and PRL cells (8%). Only one ACTH cell was found to co-localize the NMDA R1 receptor subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Regulation of anterior pituitary gonadotropin subunit mRNA levels during the preovulatory gonadotropin surge: a physiological role of progesterone in regulating LH-beta and FSH-beta mRNA levels.

In a previous study we demonstrated that in the ovariectomized estrogen-primed immature rat, progesterone induced a gonadotropin surge while the gonadotropin mRNA subunit levels were either suppressed or unaltered. This observation has now been confirmed using more frequent time points. Progesterone administered at 0900 h was found to suppress LH-beta mRNA levels at 1300, 1400, and 0800 h the next day, with no subsequent effects at 1000, 1200 or 1600 h. FSH-beta mRNA levels were unaffected by progesterone except for a slight elevation at 1400 h and a suppression at 0800 h. Progesterone was either suppressive or had no effect on alpha mRNA levels. Since elevations in LH-beta and FSH-beta mRNA levels were observed in the cycling rat, the observed differences in the ovariectomized estrogen-primed rat could be due to a higher basal synthesis occurring due to ovariectomy. This was indeed the case because LH-beta and FSH-beta mRNA levels were 3.7- and 42.7-fold higher in such animals as compared to intact estrogen-primed rats. In contrast to the ovariectomized estrogen-primed rats, in intact estrogen-primed rats LH-beta mRNA levels were increased at 1000 h and FSH-beta mRNA levels were increased at 1000, 1200 and 1300 h after the administration of progesterone. In pregnant mare's serum gonadotropin-primed immature rats, LH-beta, FSH-beta and alpha-subunit mRNA levels were significantly elevated at 1800 and 2000 h, paralleling the serum LH and FSH surge. The progesterone antagonist RU486 (0.2 and 1.0 mg) significantly reduced serum LH and FSH levels at 2000 h. The lower dose reduced LH-beta and alpha-subunit mRNA levels at 2000 h and FSH-beta mRNA levels at 1800 h. The higher dose caused an increase in LH-beta mRNA levels at 1200 and 1800 h and a decrease in FSH-beta mRNA levels at 1800 and 2000 h. In conclusion, the present study provides evidence that preovulatory progesterone plays an important role in the increase in FSH-beta mRNA levels as well as the release of LH and FSH during the normal preovulatory gonadotropin surge. This relationship appears to be dependent on the ongoing rate of synthesis because this does not occur in the ovariectomized estrogen-primed rat in which synthesis is at a high basal level. Furthermore, the correlation with FSH appears to be tighter as compared to LH.

Animals↗
PubMed1993

Steroid hormone effects on NMDA receptor binding and NMDA receptor mRNA levels in the hypothalamus and cerebral cortex of the adult rat.

Previous work has demonstrated that N-methyl-D-aspartate (NMDA) is capable of stimulating luteinizing hormone release in a variety of species. Interestingly, the ability of NMDA to stimulate luteinizing hormone release is significantly compromised in castrated male and female rats as compared to intact animals. The purpose of the present study was to determine if a difference exists in the number or affinity of NMDA receptors in the hypothalamus of intact or castrated adult male and female rats and whether steroid replacement has any effect on NMDA receptor binding. NMDA receptor mRNA levels were also determined in the respective models. The cerebral cortex was used as a control to check for specificity of any observed differences. The number of NMDA binding sites in the hypothalamus was found to be approximately 25% of that found in the cerebral cortex and the equilibrium association constant was similar in both tissues. In the female rat, neither ovariectomy nor ovariectomy with estrogen pellet replacement or estrogen and progesterone injections altered NMDA receptor binding or the equilibrium association constant in the hypothalamus or cerebral cortex as compared to intact controls. Similar to the case in the female, NMDA receptor binding in the hypothalamus and cerebral cortex of male rats did not change after castration or after treatment with testosterone propionate. Neither ovariectomy nor ovariectomy with estradiol replacement brought about any change in the NMDA receptor mRNA levels in the hypothalamus. However, in the cerebral cortex ovariectomy with estrogen replacement brought about a small but significant increase in NMDA receptor mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Amino-5-phosphonovalerate↗
PubMed1992

gamma-Aminobutyric acid-opioid interactions in the regulation of gonadotropin secretion in the immature female rat.

Previous studies in the male rat have demonstrated that GABA acting via GABAB receptors can abolish naloxone-induced LH secretion. The purpose of the present study was to determine if an analogous situation exists in the female rat. Naloxone administered to ovariectomized immature rats had no effect on LH release. In contrast, naloxone potently stimulated LH release in estrogen-primed ovariectomized immature rats. Elevation of endogenous brain levels of GABA by administering amino-oxyacetic acid, an inhibitor of GABA catabolism, prevented the naloxone-stimulated release of LH. This effect appeared to be both GABAA and GABAB receptor mediated, since exogenous administration of either muscimol (GABAA agonist) or baclofen (GABAB agonist) prevented the naloxone-induced release of LH. Neither GABA agonist had any effect on LHRH-stimulated LH release in vivo, suggesting that their effect was specific and achieved at the level of the CNS. In contrast to its inhibitory effect on naloxone-stimulated LH and FSH release, muscimol increased basal LH and FSH release in vivo and from hemipituitaries incubated in vitro, while having no effect on LHRH release from mediobasal hypothalamic and preoptic area fragments in vitro. Thus, under conditions of basal LH release, activation of GABAA receptors in the anterior pituitary can actually lead to enhanced LH secretion. Finally, naloxone-stimulated LH release was found to be inhibited by the alpha 1- and alpha 2-adrenergic blockers, prazosin and yohimbine, suggesting that naloxone-stimulated LH release is mediated via catecholamine neurotransmission involving alpha 1- and alpha 2-adrenergic receptor activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Excitatory amino acid receptors and puberty.

Glutamate is an important excitatory signal in the hypothalamus for the steroid-mediated preovulatory gonadotropin surge. Steroids may exert this action by regulating glutamate receptor levels or glutamate release, or both. Work in our laboratory found no changes in NMDA and kainate receptor binding in the hypothalamus of castrated or castrated plus steroid-replaced male and female rats. Likewise, we found that NMDA and kainate binding did not change over the onset of puberty in the female rat. A competitive quantitative RT-PCR assay using exogenous internal standards was used to measure NMDAR1, GluR1, and beta-actin mRNAs levels. NMDAR1 and GluR1 expression was examined in the preoptic hypothalamic area and in the medial basal hypothalamus at Postnatal Days 10, 15, 20, 25, 30, 32, 34, 36, 40, and 63. A transient increase in GluR1 mRNA levels in the preoptic hypothalamic area was observed on Day 20, with all other time points showing comparable levels. NMDAR1 levels in the POA and medial basal hypothalamus did not change significantly at any of the time points; in contrast, however, AMPA receptor binding levels were increased in the hypothalamus at the time of puberty in the female rat. Thus, in addition to the previously reported elevation of glutamate release rates in the hypothalamus at the time of puberty, AMPA receptors may also be elevated and play a role in mediating glutamate regulatory effects on the timing of puberty in the female rat.

Animals↗