Central alpha(2) adrenergic receptors and cholinergic-induced salivation in rats

Salivation induced by intraperitoneal (i.p.) injections of pilocarpine (cholinergic agonist) is reduced by intracerebroventricular (i.c.v.) injections of moxonidine (alpha(2) adrenergic and imidazoline receptor agonist). In the present study, we investigated the involvement of central alpha(2) adrenergic receptors in the inhibitory effect of i.c.v. moxonidine on i.p. pilocarpine-induced salivation. Male Holtzman rats with stainless steel cannula implanted into the lateral ventricle (LV) were used. Saliva was collected using pre-weighted small cotton balls inserted into the animal's mouth under ketamine (100 mg kg(-1)) anesthesia. Salivation was induced by i.p. injection of pilocarpine (4 mu mol kg(-1)). Pilocarpine-induced salivation was reduced by i.c.v. injection of moxonidine (10 nmol) and enhanced by i.c.v. injections of either RX 821002 (160 nmol) or yohimbine (320 nmol). The inhibitory effect of i.c.v. moxonidine on pilocarpine-induced salivation was abolished by prior i.c.v. injections of the alpha(2) adrenergic receptor antagonists, RX 821002 (160 nmol) or yohimbine (160 and 320 nmol). The alpha(1) adrenergic receptor antagonist prazosin (320 nmol) injected i.c.v. did not change the effect of moxonidine on pilocarpine-induced salivation. The results suggest that moxonidine acts on central alpha(2) adrenergic receptors to inhibit pilocarpine-induced salivation, and that this salivation is tonically inhibited by central alpha(2) adrenergic receptors. (C) 2002 Elsevier B.V. All rights reserved.

Moxonidine and central alpha(2) adrenergic receptors in sodium intake

Central injections of the alpha(2) adrenergic/imidazoline receptor agonist moxonidine inhibit water and NaCl intake in rats. In the present study, we investigated the possible involvement of central alpha(2) adrenergic receptors on the inhibitory effect of moxonidine in 0.3 M NaCl intake induced by 24 h sodium depletion. Male Holtzman rats with stainless-steel cannulas implanted into the lateral ventricle (LV) were used. Sodium depletion was produced by the treatment with the diuretic furosemide (20 mg/kg of body weight) injected subcutaneously + 24 h of sodium-deficient diet. Intracerebroventricular (icv) injections of moxonidine (20 nmol/l mul) reduced sodium depletion-induced 0.3 M NaCl intake (6.6 +/- 1.9 ml/120 min vs. vehicle: 12.7 +/- 1.7 ml/120 min). Pre-treatment with the alpha(2) adrenoreceptor antagonists RX 821002 (80 nmol/l mul), SK&F 86466 (640 nmol/l mul) and yohimbine (320 nmol/3 mul) injected icv abolished the inhibitory effect of icv moxonidine on sodium depletion-induced 0.3 M NaCl intake (13.3 +/- 1.4, 15.7 +/- 1.7 and 11.8 +/- 2.2 ml/120 min, respectively). The results show that the activation of alpha(2) adrenoreceptors is essential for the inhibitory effect of central moxonidine on sodium depletion-induced NaCl intake. (C) 2003 Elsevier B.V. All rights reserved.

Dissociation between the circulating renin-angiotensin system and angiotensin II receptors in central losartan-induced hypertension

Losartan, an AT1 angiotensin II (ANG II) receptor non-peptide antagonist, induces an increase in mean arterial pressure (MAP) when injected intracerebroventricularly (icv) into rats. The present study investigated possible effector mechanisms of the increase in MAP induced by icv losartan in unanesthetized rats. Male Holtzman rats (280-300 g, N = 6/group) with a cannula implanted into the anterior ventral third ventricle received an icv injection of losartan (90 µg/2 µl) that induced a typical peak pressor response within 5 min. In one group of animals, this response to icv losartan was completely reduced from 18 ± 1 to 4 ± 2 mmHg by intravenous (iv) injection of losartan (2.5-10 mg/kg), and in another group, it was partially reduced from 18 ± 3 to 11 ± 2 mmHg by iv prazosin (0.1-1.0 mg/kg), an alpha1-adrenergic antagonist (P<0.05). Captopril (10 mg/kg), a converting enzyme inhibitor, injected iv in a third group inhibited the pressor response to icv losartan from 24 ± 3 to 7 ± 2 mmHg (P<0.05). Propranolol (10 mg/kg), a ß-adrenoceptor antagonist, injected iv in a fourth group did not alter the pressor response to icv losartan. Plasma renin activity and serum angiotensin-converting enzyme activity were not altered by icv losartan in other animals. The results suggest that the pressor effect of icv losartan depends on angiotensinergic and alpha1-adrenoceptor activation, but not on increased circulating ANG II.

Maternal Thyroid and Glucocorticoid Hormone Interactions in Larval Fish Development, and Their Applications in Aquaculture

Thyroid hormones (THs) have long been known to have regulatory roles in the differentiation and maturation of vertebrate embryos, beginning with the knowledge that hormones of maternal origin are essential for human fetal central nervous and respiratory system development. Precise measurements of circulating THs led to insights into their critically important actions throughout vertebrate growth and development, initially with amphibian metamorphosis and including embryogenesis in fishes. Thyroid cues for larval fish differentiation are enhanced by glucocorticoid hormones, which promote deiodinase activity and thereby increase the generation of triiodothyronine (T-3) from the less bioactive thyroxin (T-4). Glucocorticoids also induce the expression of thyroid hormone receptors in some vertebrates. Maternally derived thyroid hormones and cortisol are deposited in fish egg yolk and accelerate larval organ system differentiation until larvae become capable of endogenous endocrine function. Increases in the T-3/T-4 ratio during larval development may reflect the regulatory importance of maternal thyroid hormones. Experimental applications of individual hormones have produced mixed results, but treatments with combinations of thyroid and corticoid hormones consistently promote larval fish development and improve survival rates. The developmental and survival benefits of maternal endocrine provisioning are increased in viviparous fishes, in which maternal/larval chemical contact is prolonged. Treatments with exogenous thyroid and corticoid hormones consistently promote development and reduce mortality rates in larval fishes, with potential hatchery-scale applications in aquaculture.

Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GR alpha and ER beta in lung tissue. Allergic airway inflammation caused reduction in mtGR alpha, mtER beta, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GR alpha and ER beta in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.

The major central endocannabinoid directly acts at GABA(A) receptors

GABA(A) receptors are the major ionotropic inhibitory neurotransmitter receptors. The endocannabinoid system is a lipid signaling network that modulates different brain functions. Here we show a direct molecular interaction between the two systems. The endocannabinoid 2-arachidonoyl glycerol (2-AG) potentiates GABA(A) receptors at low concentrations of GABA. Two residues of the receptor located in the transmembrane segment M4 of β(2) confer 2-AG binding. 2-AG acts in a superadditive fashion with the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC) and modulates δ-subunit-containing receptors, known to be located extrasynaptically and to respond to neurosteroids. 2-AG inhibits motility in CB(1)/CB(2) cannabinoid receptor double-KO, whereas β(2)-KO mice show hypermotility. The identification of a functional binding site for 2-AG in the GABA(A) receptor may have far-reaching consequences for the study of locomotion and sedation.

TIME DEPENDENT ALTERATIONS IN CENTRAL NERVOUS SYSTEM DOPAMINE RECEPTORS INDUCED BY DOPAMINE AGONIST TREATMENT

Levodopa, the precursor of dopamine, is currently the drug of choice in the treatment of Parkinson's disease. Recently, two direct dopamine agonists, bromocriptine and pergolide, have been tested for the treatment of Parkinson's disease because of reduced side effects compared to levodopa. Few studies have evaluated the effects of long-term treatment of dopamine agonists on dopamine receptor regulation in the central nervous system. Thus, the purpose of this study was to determine whether chronic dopamine agonist treatment produces a down-regulation of striatal dopamine receptor function and to compare the results of the two classes of dopaminergic drugs.^ Levodopa with carbidopa, a peripheral decarboxylase inhibitor, was administered orally to rats whereas bromocriptine and pergolide were injected intraperitoneally once daily. Several neurochemical parameters were examined from 1 to 28 days.^ Levodopa minimally decreased striatal D-1 receptor activity but increased the number of striatal D-2 binding sites. Levodopa increased the V(,max) of tyrosine hydroxylase (TH) in all brain regions tested. Protein blot analysis of striatal TH indicated a significant increase in the amount of TH present. Dopamine-beta-hydroxylase (DBH) activity was markedly decreased in all brain regions studied and mixing experiments of control and drug-treated cortices did not show the presence of an increased level of endogenous inhibitors.^ Bromocriptine treatment decreased the number of D-2 binding sites. Striatal TH activity was decreased and protein blot analysis indicated no change in TH quantity. The specificity of bromocriptine for striatal TH suggested that bromocriptine preferentially interacts with dopamine autoreceptors.^ Combination levodopa-bromocriptine was administered for 12 days. There was a decrease in both D-1 receptor activity and D-2 binding sites, and a decrease in brain HVA levels suggesting a postsynaptic receptor action. Pergolide produced identical results to the combination levodopa-bromocriptine studies.^ In conclusion, combination levodopa-bromocriptine and pergolide treatments exhibited the expected down-regulation of dopamine receptor activity. In contrast, levodopa appeared to up-regulate dopamine receptor activity. Thus, these data may help to explain, on a biochemical basis, the decrease in the levodopa-induced side effects noted with combination levodopa-bromocriptine or pergolide therapies in the treatment of Parkinson's disease. ^

Most central nervous system D2 dopamine receptors are coupled to their effectors by Go

We reported previously that Go-deficient mice develop severe neurological defects that include hyperalgesia, a generalized tremor, lack of coordination, and a turning syndrome somewhat reminiscent of unilateral lesions of the dopaminergic nigro-striatal pathway. By using frozen coronal sections of serially sectioned brains of normal and Go-deficient mice, we studied the ability of several G protein coupled receptors to promote binding of GTPγS to G proteins and the ability of GTP to promote a shift in the affinity of D2 dopamine receptor for its physiologic agonist dopamine. We found a generalized, but not abolished reduction in agonist-stimulated binding of GTPγS to frozen brain sections, with no significant left–right differences. Unexpectedly, the ability of GTP to regulate the binding affinity of dopamine to D2 receptors (as seen in in situ [35S]sulpiride displacement curves) that was robust in control mice, was absent in Go-deficient mice. The data suggest that most of the effects of the Gi/Go-coupled D2 receptors in the central nervous system are mediated by Go instead of Gi1, Gi2, or Gi3. In agreement with this, the effect of GTP on dopamine binding to D2 receptors in double Gi1 plus Gi2- and Gi1 plus Gi3-deficient mice was essentially unaffected.

Glucocorticoid and progestin receptors are differently involved in the cooperation with a structural element of the mouse mammary tumor virus promoter.

We have previously characterized a regulatory element located between -294 and -200 within the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). This element termed AA element cooperates with the glucocorticoid response elements (GREs) for glucocorticoid activation. Here we show that in a MMTV LTR wild type context, the deletion of this element significantly reduces both glucocorticoid and progestin activation of the promoter. Deletion of the two most distal GREs forces the glucocorticoid receptor (GR) and the progestin receptor (PR) to bind the same response elements and results in a dramatic decrease in the inducibility of the MMTV promoter by the two hormones. The simultaneous deletion of the two distal GREs and of the AA element abolishes completely the glucocorticoid-induced activation of the promoter. In contrast it restores a significant level of progestin-induced activation. This different effect of the double deletion on glucocorticoid- and progestin-induced MMTV promoter activation is not cell specific because it is also observed, and is even stronger, when either GR or PR is expressed in the same cell line (NIH 3T3). This is the first description of a mutated MMTV promoter that, although retaining GREs, is activated by progestins and not by glucocorticoids. This suggests a different functional cooperation between protein(s) interacting with the AA element and GR or PR. Cotransfections with constructs containing wild-type or mutated MMTV LTR with either PR lacking its C-terminal domain or GR/PR chimeras in which the N-terminal domains have been exchanged demonstrate that the N-terminal domains of the receptors specify the different behavior of GR and PR regarding the AA element.

Identification of GABA receptors in chick cornea

Purpose: The cornea has an important role in vision, is highly innervated and many neurotransmitter receptors are present, e.g., muscarine, melatonin, and dopamine receptors. γ-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the retina and central nervous system, but it is unknown whether GABA receptors are present in cornea. The aim of this study was to determine if GABA receptors are located in chick cornea. Methods: Corneal tissues were collected from 25, 12-day-old chicks. Real time PCR, western blot, and immunohistochemistry were used to determine whether alpha1 GABAA, GABAB, and rho1 GABAC receptors were expressed and located in chick cornea. Results: Corneal tissue was positive for alpha1 GABAA and rho1 GABAC receptor mRNA (PCR) and protein (western blot) expression but was negative for GABAB receptor mRNA and protein. Alpha1 GABAA and rho1 GABAC receptor protein labeling was observed in the corneal epithelium using immunohistochemistry. Conclusions: These investigations clearly show that chick cornea possesses alpha1 GABAA, and rho1 GABAC receptors, but not GABAB receptors. The purpose of the alpha1 GABAA and rho1 GABAC receptors in cornea is a fascinating unexplored question.

Investigation of Gamma-aminobutyric acid (GABA) A receptors genes and migraine susceptibility

Background Migraine is a neurological disorder characterized by recurrent attacks of severe headache, affecting around 12% of Caucasian populations. It is well known that migraine has a strong genetic component, although the number and type of genes involved is still unclear. Prior linkage studies have reported mapping of a migraine gene to chromosome Xq 24–28, a region containing a cluster of genes for GABA A receptors (GABRE, GABRA3, GABRQ), which are potential candidate genes for migraine. The GABA neurotransmitter has been implicated in migraine pathophysiology previously; however its exact role has not yet been established, although GABA receptors agonists have been the target of therapeutic developments. The aim of the present research is to investigate the role of the potential candidate genes reported on chromosome Xq 24–28 region in migraine susceptibility. In this study, we have focused on the subunit GABA A receptors type ε (GABRE) and type θ (GABRQ) genes and their involvement in migraine. Methods We have performed an association analysis in a large population of case-controls (275 unrelated Caucasian migraineurs versus 275 controls) examining a set of 3 single nucleotide polymorphisms (SNPs) in the coding region (exons 3, 5 and 9) of the GABRE gene and also the I478F coding variant of the GABRQ gene. Results Our study did not show any association between the examined SNPs in our test population (P > 0.05). Conclusion Although these particular GABA receptor genes did not show positive association, further studies are necessary to consider the role of other GABA receptor genes in migraine susceptibility.

Progesterone, glucocorticoid, but not estrogen receptor mRNA is altered in breast cancer stroma

Our laboratory has previously found that anti-mitogenic nuclear receptor mRNA is elevated in late stage tumours and this study was performed to scrutinize the possibility of cancer-stroma crosstalk using hormone signaling in these tissues. RNA levels in stromal tissue were examined for the estrogen α, estrogen β, androgen, progesterone and glucocorticoid nuclear receptors by a semi-quantitative PCR. Significant differences in expression between the cancer stroma and control tissue were seen, analyzing for both cancer grade and estrogen receptor status. Stroma and control tissue were significantly different for the progesterone and glucocorticoid nuclear receptors (p = 5.908 × 10−7 and 2.761 × 10−5, respectively). Glucocorticoid receptor also showed a significant increase to mRNA levels in the stroma of estrogen receptor negative tumours (p = 5.85 × 10−5). By contrast, the estrogen receptors α and β, those most closely associated with breast tissue growth, showed no significant change in mRNA (p = 0.372 and 0.655, respectively). Androgen receptor mRNA also remained unaffected (p = 0.174).

Association of estrogen receptor and glucocorticoid receptor gene polymorphisms with sporadic breast cancer

We have utilized a cross-sectional association approach to investigate sporadic breast cancer. Polymorphisms in 2 candidate genes, ESRalpha and GRL, were examined in an unrelated breast cancer-affected and age-matched control population. Several polymorphic regions within the ESRalpha gene have been identified, and some alleles of these polymorphisms have been found to occur at increased levels in breast-cancer patients. Additionally, variations in GRL have the potential to disrupt cell transcription and may be associated with cancer formation. We analyzed 3 polymorphisms, from codons 10 (TCT to TCC), 325 (CCC to CCG) and 594 (ACA to ACG) of ESRalpha, and a highly polymorphic dinucleotide repeat, D5S207, located within 200 kb of the GRL. When allelic frequencies of the codon 594 (exon 8) ESR polymorphism were compared between affected and unaffected populations, a significant difference was observed (p = 0.005). Results from the D5S207 dinucleotide repeat located near GRL also indicated a significant difference between the tested case and control populations (p = 0.001). Allelic frequencies of the codon 10 and codon 325 ESR polymorphisms were not significantly different between populations (p = 0.152 and 0.181, respectively). Our results indicate that specific alleles of the ESR gene (alpha subtype) and a marker for the GRL gene locus are associated with sporadic breast-cancer development in the tested Caucasian population and justify further investigation of the role of these and other nuclear steroid receptors in the etiology of breast cancer.

Localization of Mineralocorticoid Receptors at Mammalian Synapses

In the brain, membrane associated nongenomic steroid receptors can induce fast-acting responses to ion conductance and second messenger systems of neurons. Emerging data suggest that membrane associated glucocorticoid and mineralocorticoid receptors may directly regulate synaptic excitability during times of stress when adrenal hormones are elevated. As the key neuron signaling interface, the synapse is involved in learning and memory, including traumatic memories during times of stress. The lateral amygdala is a key site for synaptic plasticity underlying conditioned fear, which can both trigger and be coincident with the stress response. A large body of electrophysiological data shows rapid regulation of neuronal excitability by steroid hormone receptors. Despite the importance of these receptors, to date, only the glucocorticoid receptor has been anatomically localized to the membrane. We investigated the subcellular sites of mineralocorticoid receptors in the lateral amygdala of the Sprague-Dawley rat. Immunoblot analysis revealed the presence of mineralocorticoid receptors in the amygdala. Using electron microscopy, we found mineralocorticoid receptors expressed at both nuclear including: glutamatergic and GABAergic neurons and extra nuclear sites including: presynaptic terminals, neuronal dendrites, and dendritic spines. Importantly we also observed mineralocorticoid receptors at postsynaptic membrane densities of excitatory synapses. These data provide direct anatomical evidence supporting the concept that, at some synapses, synaptic transmission is regulated by mineralocorticoid receptors. Thus part of the stress signaling response in the brain is a direct modulation of the synapse itself by adrenal steroids.