Ontogeny of androgen receptors in fetal guinea pig brain

Sexual differentiation of the guinea pig brain is androgen dependent. To understand the cellular mechanisms of androgen action, we studied the ontogeny of cytosolic (ARc) and nuclear (ARn) androgen receptors in the brains and anterior pituitaries of fetal, neonatal, and adult guinea pigs. Using cytosol from the hypothalamus-preoptic area-amygdala-septum of 60- to 65-day fetuses and nuclear preparations from 6-day-old neonates treated with testosterone propionate, validation studies revealed an AR with an apparent Kd of 1.9 +/- 1.1 (mean +/- SEM, n = 3) x 10(-10) M (ARc) and 3.4 +/- 3.2 (n = 3) x 10(-10) M (ARn). The cytosolic receptors were highly specific for androgens. After assay validation, AR content was determined from specific brain regions of fetuses obtained on Days 30, 40, 50, and 59 of gestation and on Days 6 and 120 postpartum. ARc differed significantly (p less than 0.05) between brain regions and times of gestation, but no sex differences were apparent. In contrast, ARn showed little difference between tissues or with gestational age, but there were significant differences between males and females, especially in late gestation and early postnatal life, with males having greater ARn binding (p less than 0.05). These data demonstrate the presence of ARc and ARn in the fetal brain and pituitary gland during the critical period of sexual differentiation (Days 30-37 of gestation), thus establishing the identity of cellular structures involved in androgen action.     

Effects of exogenous steroids on androgen receptors in fetal guinea pig brain

We treated pregnant guinea pigs on Day 50 of gestation with 10 mg testosterone propionate (TP), obtaining fetuses 2, 4, 8, or 18 h later as well as after 5 days of treatment. In a second group of pregnant guinea pigs, dihydrotestosterone propionate (DHTP), estradiol benzoate (E2B), progesterone (P), or cortisol was given 2 h before obtaining fetuses. Although TP treatment elevated fetal serum T (p less than 0.05), brain cytosolic androgen receptor (ARc) content was unchanged in fetuses of either sex. In female fetuses, nuclear androgen receptors (ARn) increased 10-fold in medial-basal hypothalamus (MBH) and preoptic area (POA) at 2 and 4 h (respectively) after treatment, while fetal male ARn content was unchanged. Maternal injection of other steroids (E2B, P, or cortisol, but not DHTP) significantly increased these hormones in the fetus 2 h later (p less than 0.05). Only androgens affected fetal androgen receptor (AR) content. While TP increased ARn in female MBH, DHTP decreased ARc in fetal anterior pituitary of both sexes. In this latter case, a metabolite of DHT may mediate the effects. We conclude that T crosses the guinea pig placenta and activates ARn in POA and MBH of female fetuses; male ARn appear to be maximally occupied by endogenous T. Steroids of other classes do not induce AR responses in fetal guinea pig brain. These AR changes may represent an initial cellular mechanism in brain sexual differentiation.     

Aromatase is pre-synaptic and sexually dimorphic in the adult zebra finch brain

Oestrogens organize and activate circuits within the vertebrate central nervous system. Oestrogen synthesis occurs via the expression of aromatase, a P450 enzyme detected in microsomes and more recently in pre-synaptic boutons. Synaptic aromatase has only been described in brain regions that express aromatase in many subcellular compartments, so its function remains poorly understood. To more thoroughly study the role of oestrogen synthesis at synaptic terminals, we examined the ultrastructural compartmentalization of aromatase in the zebra finch; a species in which high aromatase activity can be measured in brain areas that do not contain somal aromatase. Here, we report the presence of aromatase in pre-synaptic boutons in the hippocampus and the high vocal centre brain areas with low and undetectable somal aromatase, respectively, in addition to areas with abundant somal aromatase such as the preoptic area and caudomedial nidopallium. At these brain areas, males had more total synapses, more aromatase pre-synaptic boutons and importantly, the proportion of total synaptic profiles that expressed aromatase was significantly higher in males relative to females. Aromatase-positive pre-synaptic boutons were always observed innervating aromatase-negative post-synaptic elements. We conclude that oestrogen may be provided to discrete oestrogen-sensitive targets by synaptic aromatization. Further, some targets may be exposed to more oestrogen in males. The expression of aromatase in individual synapses of projection neurons represents a unique mechanism of neuroendocrine action. Neurons with steroidogenic capability may modulate distant targets with the specificity of axonal innervation.     

Aromatase activity in individual day-11 pig blastocysts

Blastocysts were flushed from both uterine horns of 10 gilts on Day 11 of pregnancy. In these gilts 15.1 +/- 2.3 (mean +/- s.d.) corpora lutea were present and 12.7 +/- 3.1 spherical blastocysts were recovered. In all the gilts variation in blastocyst diameter was observed. Aromatase activity was measurable in 118 of 121 examined blastocysts and varied from 0.005 to 19.330 pmol [1 beta-3H]androstenedione converted into 3H2O in 20 min (mean 1.31). This variation in aromatase activity reflected a difference between and within gilts. Of the total variation between all blastocysts, 67% was due to differences between gilts. A positive exponential relationship existed between blastocyst diameter and aromatase activity, but this relationship was different between gilts (P less than 0.0001). The observed variation in aromatase activity may be caused by differences in germ layer differentiation of the blastocysts.     

Enhanced serotonin-stimulated adenylate cyclase activity in membranes from adult guinea pig hippocampus

We have developed an assay for serotonin (5-HT) stimulation of adenylate cyclase activity in membranes from adult guinea pig hippocampus. The response to 5-HT is concentration-dependent, with an EC50 of 0.01 microM, a shallow slope, and mean maximal stimulation of 90% over basal activity. The response to 5-HT is GTP-dependent and additive to the maximal stimulation by histamine. Micromolar concentrations of the known 5-HT receptor agonists, tryptamine and 5-methoxytryptamine, also stimulate cAMP production in this system, and their effect is not additive to that elicited by a maximal concentration of 5-HT. These results are consistent with the hypothesis that the response to 5-HT is elicited through a distinct receptor coupled to adenylate cyclase; the magnitude and the reproducibility of the 5-HT response in this system should make it useful for receptor classification. To examine the effect of prior exposure to endogenous 5-HT on the responsiveness of the system, we assayed 5-HT stimulation of enzyme activity in membranes prepared from animals 25-27 hrs after treatment with a single injection of reserpine (5 mg/kg, i.p.). The mean maximal stimulation of adenylate cyclase by 5-HT was increased to 150% over basal activity with no effect on the EC50 or slope of the 5-HT concentration-response curve. Reserpine pretreatment did not affect basal activity or histamine-stimulated adenylate cyclase activity. These results are discussed in the context of a hypothesis that endogenous 5-HT normally exerts a desensitizing effect on its receptors in situ.     

Creutzfeldt-Jakob infection increases adenylate cyclase activity in specific regions of guinea pig brain

Creutzfeldt-Jakob disease is a slow, infectious, progressive neurological disorder which results in human dementia. Synaptic membranes from various brain regions of guinea pigs infected with Creutzfeldt-Jakob disease show increased guanyl nucleotide- or 5-hydroxytryptamine-mediated activation of adenylate cyclase. This increased enzyme activity appears due, primarily, to facilitated 'coupling' between the GTP-binding protein which stimulates adenylate cyclase (GNs) and the catalytic moiety of that enzyme rather than increased sensitivity to 5-hydroxytryptamine. It is possible that this phenomenon is due to direct effects of the Creutzfeldt-Jakob infectious agent, or a pathological product resulting from that agent, upon synaptic membrane adenylate cyclase.     

AM630 is a competitive cannabinoid receptor antagonist in the guinea pig brain

AM 630 has been demonstrated to be a cannabinoid receptor antagonist in the mouse brain and vas deferens. Conversely, it was recently reported that AM630 acts as a cannabinoid agonist in the guinea pig ileum. This research was designed to determine whether the difference in the action of AM630 is species specific. Studies conducted in guinea pig brain reveal that AM630 antagonizes the stimulatory effect of the cannabinoid agonist WIN 55,212-2 on [³⁵S]GTPγS binding suggesting that difference in AM630 activity in different tissues is not due to species Variation.     

Estrone sulfatase activity in guinea pig tissues

Estrone sulfatase activity is widespread in guinea pig tissues. Whole homogenates of adult testis. uterus. lung, adrenal, amnion, ovary, chorion, small intestine, placenta, spleen, kidney and liver exhibit approximately descending order of specific activity. Certain properties, including pH requirement, lack of inhibition by inorganic sulfate and magnitude of estimated K_mvalues, are similar to that for arylsulfatase C of rat liver. Of the subcellular fractions prepared from guinea pig tissues, microsomes exhibit the highest specific activity although considerable enzyme activity remains associated with large cellular fragments sedimenting at 750 g. The sulfatase activity is readily inhibited by inorganic phosphate even when substrate concentration satisfies zero order kinetics. Rat liver arylsulfatase C is not inhibited under these conditions. Sensitivity of the guinea pig enzyme activity to inhibition by a variety of steroids and related compounds, is markedly less than for rat liver. Diethylstilbestrol (DES) strongly inhibits the rat liver enzyme but has little effect on the guinea pig liver system. Guinea pig testicular activity is suppressed to a degree intermediate between these extremes by increasing DES concentration. In guinea pig lung. kidney, and possibly liver, elevated fetal enzyme activities decrease from neonatal to adult life. Teslicular activity appears to follow the opposite trend. Uterine enzyme activity is not markedly affected by pregnancy.     

Localisation of a particulate luliberin hydrolysing activity in microsomal membranes of guinea pig brain

A particulate luliberin hydrolysing enzyme has been described for guinea pig brain. Examination of subcellular fractions generated under different conditions indicated that particulate luliberin hydrolysing activity was most closely associated with the microsomal marker, rotenone-insensitive NADH cytochrome C reductase. The results obtained indicate that luliberin hydrolysing activity is not associated with synaptosomal membrane preparations and that such luliberin hydrolysing activity as is observed in synaptosomal membranes is probably the result of contamination by microsomes. The enzyme could be released from microsomes by Triton X-100 treatment and the solubilised enzyme was found to be inhibited by puromycin and sulphydryl reagents but to be unaffected by phosphoramidon, captopril, phenylmethyl sulphonyl fluoride and by chelating agents except 1,10-phenanthroline.     

Dextromethorphan binding sites in the guinea pig brain

1. Dextromethorphan (DM), a dextrorotatory nonopioid antitussive, binds to specific high-affinity sites in the central nervous system. These sites are distinct from the opioid and other known neurotransmitter receptor sites. Antitussives such as carbetapentane and caramiphen also bind to DM sites with a nanomolar affinity. 2. The anticonvulsant drugs phenytoin and ropizine produce an allosteric enhancement of the binding of [3H]DM to guinea pig brain. DM, carbetapentane, and caramiphen also are efficacious anticonvulsant agents in the rat maximal electroshock seizures test, and DM enhances the anticonvulsant effects of phenytoin (PHT). 3. These results suggest that drugs that bind to the DM sites could be used alone as anticonvulsants or in combination with PHT to lower its effective dose and reduce its side effects. 4. The investigation of the DM binding sites may help to open new approaches for the treatment of convulsive disorders and to explain further some of the molecular mechanisms of neutronal excitability.     

Radiation-induced edema is dependent on cyclooxygenase 2 activity in mouse brain

Cerebrovascular dysfunction, characterized by compromise of the blood-brain barrier and formation of cerebral edema, is common during the acute period after brain irradiation and may contribute to delayed pathology (e.g. vascular collapse, white matter necrosis) that leads to functional deficits. Another response of normal brain tissue to radiation is the induction of inflammatory markers, such as cytokine expression and glial activation. In particular, radiation-induced neuroinflammation is associated with an elevation in cyclooxygenase 2 (COX2), one of two isoforms of the obligate enzyme in prostanoid synthesis and the principal target of non-steroid anti-inflammatory drugs. Since prostanoids serve as autocrine and paracrine mediators in numerous physiological and pathological processes, including vasoregulation, we investigated COX2 protein expression and COX2-mediated prostanoid production in radiation-induced cerebral edema in male C57/BL6 mice. We found that radiation induces COX2 protein that is accompanied by specific increases in prostaglandin E(2) and thromboxane A(2) within 4 and 24 h after brain irradiation. Furthermore, we showed that treatment with NS-398, a selective COX2 inhibitor, attenuated prostanoid induction and edema formation. These results suggest that radiation-induced changes in vascular permeability are dependent on COX2 activity, implicating this enzyme and its products as targets for potential therapeutic treatment/protection from the effects of radiation on normal brain tissue.     

Interleukin 1 reduces opioid binding in guinea pig brain

Interleukin 1 (IL1) is a macrophage-derived polypeptide which signals neurons in the preoptic-anterior hypothalamus to initiate fever and the acute-phase glycoprotein response. Recently, increases in cerebrospinal fluid and hypothalamic levels of β-endorphin have been reported during endotoxin (LPS)- and IL1-induced fevers, suggesting that this opioid may participate in the modulation of IL1 effects in the CNS. In this study, we investigated whether purified (human) IL1 influences the specific binding of three prototypic opioid agonists (2-D-alanine-5-L-methionineamide, DAME; (−)-ethylketocyclazocine, EKC; dihydromorphine, DHM) and one antagonist (naloxone) to opioid receptor-enriched membrane preparations in cerebral cortex, hypothalamus, midbrain, pons, medulla, and cerebellum of guinea pig brain. IL1 reduced the binding of these ligands to their receptors during a 30-min incubation. The extent of IL1 inhibition of a given ligand for its binding sites varied according to the brain region; within some regions, the extent of this inhibition also varied with the four ligands tested. But in cortex the effect of IL1 on the specific binding of DHM is dose-dependent. Similar results were obtained with crude homologous IL1. S. enteritidis endotoxin, suspended in pyrogen-free saline at concentrations from 4 to 36 μg/ml, did not inhibit the binding of these opioid ligands to their receptors in any brain region. These results indicate that IL1 interacts with the opiate receptors in guinea pig brain. This interaction, moreover, is not limited to the hypothalamus alone, the primary site of the pyrogenic action of IL1, but also occurs in other brain regions.