Defense reaction induced by a metabotropic glutamate receptor agonist microinjected into the dorsal periaqueductal gray of rats

The behavioral effects of trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), a metabotropic glutamate receptor (mGluR) agonist, or 0.9% (w/v) saline, injected into the dorsal periaqueductal gray (DPAG), was investigated. Male Wistar rats showed defense reactions characterized by jumps toward the top edges of the cages (saline = 0 vs t-ACPD = 6.0, medians P<0.05) and gallops (saline = 0 vs t-ACPD = 10.0, medians P<0.05) during the 60-s period after the beginning of the injection. In another experiment animals were placed inside an open arena for 5 min immediately after injection. Their behavior was recorded by a video camera and a computer program analyzed the videotapes. Eleven of fifteen rats injected with t-ACPD showed a short-lasting (about 1 min) flight reaction. No saline-treated animal showed this reaction (P<0.0005, chi-square test). The drug induced an increase in turning behavior (P = 0.002, MANOVA) and a decrease in the number of rearings (P<0.001, MANOVA) and grooming episodes (P<0.001, MANOVA). These results suggest that mGluRs play a role in the control of defense reactions in the DPAG.

Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor

Abnormal production of interferon alpha (IFN-a) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-a. IFN-a can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFN-a inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subunit 1 of the interferon-a receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis. Under reducing SDS-PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFN-aB on Daudi cells, but did not block IFN-aB activity at higher concentrations (>6.25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-aB on Hep 2/c cells challenged with encephalomyocarditis virus.

Acute AT1 receptor blockade does not improve the depressed baroreflex in rats with chronic renal hypertension

To assess the role of angiotensin II in the sensitivity of the baroreflex control of heart rate (HR) in normotensive rats (N = 6) and chronically hypertensive rats (1K1C, 2 months, N = 7), reflex changes of HR were evaluated before and after (15 min) the administration of a selective angiotensin II receptor antagonist (losartan, 10 mg/kg, iv). Baseline values of mean arterial pressure (MAP) were higher in hypertensive rats (195 ± 6 mmHg) than in normotensive rats (110 ± 2 mmHg). Losartan administration promoted a decrease in MAP only in hypertensive rats (16%), with no changes in HR. During the control period, the sensitivity of the bradycardic and tachycardic responses to acute MAP changes were depressed in hypertensive rats (~70% and ~65%, respectively) and remained unchanged after losartan administration. Plasma renin activity was similar in the two groups. The present study demonstrates that acute blockade of AT1 receptors with losartan lowers the MAP in chronic renal hypertensive rats without reversal of baroreflex hyposensitivity, suggesting that the impairment of baroreflex control of HR is not dependent on an increased angiotensin II level.

Excitatory amino acid receptor blockade within the caudal pressor area and rostral ventrolateral medulla alters cardiovascular responses to nucleus raphe obscurus stimulation in rats

Pressor responses elicited by stimulation of the nucleus raphe obscurus (NRO) depend on the integrity of the rostral ventrolateral medulla (RVLM). Therefore, to test the participation of excitatory amino acid (EAA) receptors in the cardiovascular responses evoked by NRO stimulation (1 ms, 100 Hz, 40-70 µA, for 10 s), the EAA antagonist kynurenic acid (Kyn) was microinjected at different sites in the ventrolateral medullar surface (2.7 nmol/200 nl) of male Wistar rats (270-320 g, N = 39) and NRO stimulation was repeated. The effects of NRO stimulation were: hypertension (deltaMAP = +43 ± 1 mmHg, P<0.01), bradycardia (deltaHR = -30 ± 7 bpm, P<0.01) and apnea. Bilateral microinjection of Kyn into the RVLM, which did not change baseline parameters, almost abolished the bradycardia induced by NRO stimulation (deltaHR = -61 ± 3 before vs -2 ± 3 bpm after Kyn, P<0.01, N = 7). Unilateral microinjection of Kyn into the CVLM did not change baseline parameters or reduce the pressor response to NRO stimulation (deltaMAP = +46 ± 5 before vs +48 ± 5 mmHg after Kyn, N = 6). Kyn bilaterally microinjected into the caudal pressor area reduced blood pressure and heart rate and almost abolished the pressor response to NRO stimulation (deltaMAP = +46 ± 4 mmHg before vs +4 ± 2 mmHg after Kyn, P<0.01, N = 7). These results indicate that EAA receptors on the medullary ventrolateral surface play a role in the modulation of the cardiovascular responses induced by NRO stimulation, and also suggest that the RVLM participates in the modulation of heart rate responses and that the caudal pressor area modulates the pressor response following NRO stimulation.

A brain microdialysis study on 5-HT release in freely moving rat lines selectively bred for differential 5-HT1A receptor function

Breeding for high and low hypothermic responses to systemic administration of a serotonin1A (5-HT1A) receptor agonist (8-hydroxy-2-(di-n-propylamino)tetralin, 8-OH-DPAT) has resulted in high DPAT-sensitive (HDS) and low DPAT-sensitive (LDS) lines of rats, respectively. These lines also differ in several behavioral measures associated with stress. In the present microdialysis study we observed that basal 5-HT concentrations in the prefrontal cortex and dorsal hippocampus did not differ significantly between HDS and LDS rats. Thus, behavioral differences between the HDS and LDS lines might not be attributed to differences in basal 5-HT release. However, both lines had lower basal levels of 5-HT release than their randomly bred control group (random DPAT-sensitive, RDS) in the prefrontal cortex (mean ± SEM, pg/20 µl, was 3.0 ± 0.4 for LDS, 3.8 ± 0.3 for HDS and 6.4 ± 0.6 for RDS; F(2,59) = 5.8, P<0.005). The administration of (±)-fenfluramine (10 mg/kg) induced a greater increase in hippocampal 5-HT levels in HDS rats (500%) as compared with LDS (248%) or RDS (243%) rats (P<0.0001). There were no significant differences in the prefrontal cortex among lines, with a fenfluramine-induced 5-HT increase of about 900% in the three groups. This differential response to fenfluramine may be due to functional alterations of hippocampal 5-HT reuptake sites in the HDS line.

Cytotoxicity of chlorhexidine digluconate to murine macrophages and its effect on hydrogen peroxide and nitric oxide induction

Chlorhexidine, even at low concentrations, is toxic for a variety of eukaryotic cells; however, its effects on host immune cells are not well known. We evaluated in vitro chlorhexidine-induced cytotoxicity and its effects on reactive oxygen/nitrogen intermediate induction by murine peritoneal macrophages. Thioglycollate-induced cells were obtained from Swiss mice by peritoneal lavage with 5 ml of 10 mM phosphate-buffered saline, washed twice and resuspended (10(6) cells/ml) in appropriate medium for each test. Cell preparations contained more than 95% macrophages. The cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the presence of hydrogen peroxide (H2O2) and nitric oxide (NO) by the horseradish peroxidase-dependent oxidation of phenol red and Griess reaction, respectively. The midpoint cytotoxicity values for 1- and 24-h exposures were 61.12 ± 2.46 and 21.22 ± 2.44 µg/ml, respectively. Chlorhexidine did not induce synthesis or liberation of reactive oxygen/nitrogen intermediates. When macrophages were treated with various sub-toxic doses for 1 h (1, 5, 10, and 20 µg/ml) and 24 h (0.5, 1, and 5 µg/ml) and stimulated with 200 nM phorbol myristate acetate (PMA) solution, the H2O2 production was not altered; however, the NO production induced by 10 µg/ml lipopolysaccharide (LPS) solution varied from 14.47 ± 1.46 to 22.35 ± 1.94 µmol/l and 13.50 ± 1.42 to 20.44 ± 1.40 µmol/l (N = 5). The results showed that chlorhexidine has no immunostimulating activity and sub-toxic concentrations did not affect the response of macrophages to the soluble stimulus PMA but can interfere with the receptor-dependent stimulus LPS.

Maternal aggression in Wistar rats: effect of 5-HT2A/2C receptor agonist and antagonist microinjected into the dorsal periaqueductal gray matter and medial septum

The objective of the present study was to assess the role of the 5-HT2A/2C receptor at two specific brain sites, i.e., the dorsal periaqueductal gray matter (DPAG) and the medial septal (MS) area, in maternal aggressive behavior after the microinjection of either a 5-HT2A/2C receptor agonist or antagonist. Female Wistar rats were microinjected on the 7th postpartum day with the selective agonist alpha-methyl-5-hydroxytryptamine maleate (5-HT2A/2C) or the antagonist 5-HT2A/2C, ketanserin. The agonist was injected into the DPAG at 0.2 (N = 9), 0.5 (N = 10), and 1.0 µg/0.2 µl (N = 9), and the antagonist was injected at 1.0 µg/0.2 µl (N = 9). The agonist was injected into the medial septal area (MS) at 0.2 (N = 9), 0.5 (N = 7), and 1.0 µg/0.2 µl (N = 6) and the antagonist was injected at 1.0 µg/0.2 µl (N = 5). For the control, saline was injected into the DPAG (N = 7) and the MS (N = 12). Both areas are related to aggressive behavior and contain a high density of 5-HT receptors. Non-aggressive behaviors such as horizontal locomotion (walking) and social investigation and aggressive behaviors such as lateral threat (aggressive posture), attacks (frontal and lateral), and biting the intruder were analyzed when a male intruder was placed into the female resident's cage. For each brain area studied, the frequency of the behaviors was compared among the various treatments by analysis of variance. The results showed a decrease in maternal aggressive behavior (number of bites directed at the intruder) after microinjection of the agonist at 0.2 and 1.0 µg/0.2 µl (1.6 ± 0.7 and 0.9 ± 0.3) into the DPAG compared to the saline group (5.5 ± 1.1). There was no dose-response relationship with the agonist. The present findings suggest that the 5-HT2A/2C receptor agonist has an inhibitory effect on maternal aggressive behavior when microinjected into the DPAG and no effect when microinjected into the MS. Ketanserin (1.0 µg/0.2 µl) decreased locomotion when microinjected into the DPAG and MS, but did not affect aggressive behavior. We interpret these findings as evidence for a specific role of 5-HT2A/2C receptors in the DPAG in the inhibition of female aggressive behavior, dissociated from those on motor activity.

Estrogen receptor 1 gene polymorphisms and coronary artery disease in the Brazilian population

We examined the association of three established single nucleotide polymorphisms, IVS1-397T>C, IVS1-351A>G, and +261G>C, in the ESR1 gene with the prevalence and severity of coronary atherosclerosis in a southern Brazilian population of European ancestry. Three hundred and forty-one subjects (127 women and 214 men) with coronary artery disease (CAD) were classified as having significant disease (CAD+ patient group) when they showed 60% or more luminal stenosis in at least one coronary artery or major branch segment at angiography; patients with 10% or less luminal stenosis were considered to have minimal CAD (CAD- patient group). The control sample consisted of 142 subjects (79 women and 63 men) without significant disease, in whom coronary angiography to rule out the presence of asymptomatic CAD was not performed. The polymorphisms were investigated by polymerase chain reaction followed by restriction analyses. In the male sample, the +261G>C*C allele was more frequent in CAD+ than CAD- subjects (8 versus 1%, P = 0.024). Homozygosity for the C allele of the IVS1-397T>C polymorphism was also significantly associated with increased CAD severity (OR: 2.99; 95% CI = 1.35-6.63; P = 0.007). In agreement with previous findings, these results suggest that the IVS1-397T>C*C allele was associated with CAD severity independent of gender, whereas the association of the +261G>C variant with CAD was observed in males only. The relation between ESR1 variation and CAD may influence clinical decisions such as the use of hormone therapy, and additionally will be helpful to identify the genetic susceptibility determinants of cardiovascular disease development.

Biphasic modulation of insulin receptor substrate-1 during goitrogenesis

Insulin receptor substrate-1 (IRS-1) is the main intracellular substrate for both insulin and insulin-like growth factor I (IGF-I) receptors and is critical for cell mitogenesis. Thyrotropin is able to induce thyroid cell proliferation through the cyclic AMP intracellular cascade; however, the presence of either insulin or IGF-I is required for the mitogenic effect of thyroid-stimulating hormone (TSH) to occur. The aim of the present study was to determine whether thyroid IRS-1 content is modulated by TSH in vivo. Strikingly, hypothyroid goitrous rats, which have chronically high serum TSH levels (control, C = 2.31 ± 0.28; methimazole (MMI) 21d = 51.02 ± 6.02 ng/mL, N = 12 rats), when treated with 0.03% MMI in drinking water for 21 days, showed significantly reduced thyroid IRS-1 mRNA content. Since goiter was already established in these animals by MMI for 21 days, we also evaluated IRS-1 expression during goitrogenesis. Animals treated with MMI for different periods of time showed a progressive increase in thyroid weight (C = 22.18 ± 1.21; MMI 5d = 32.83 ± 1.48; MMI 7d = 31.1 ± 3.25; MMI 10d = 33.8 ± 1.25; MMI 14d = 45.5 ± 2.56; MMI 18d = 53.0 ± 3.01; MMI 21d = 61.9 ± 3.92 mg, N = 9-15 animals per group) and serum TSH levels (C = 1.57 ± 0.2; MMI 5d = 9.95 ± 0.74; MMI 7d = 10.38 ± 0.84; MMI 10d = 17.72 ± 1.47; MMI 14d = 25.65 ± 1.23; MMI 18d = 35.38 ± 3.69; MMI 21d = 31.3 ± 2.7 ng/mL, N = 9-15 animals per group). Thyroid IRS-1 mRNA expression increased progressively during goitrogenesis, being significantly higher by the 14th day of MMI treatment, and then started to decline, reaching the lowest values by the 21st day, when a significant reduction was detected. In the liver of these animals, however, a significant decrease of IRS-1 mRNA was detected after 14 days of MMI treatment, a mechanism probably involved in the insulin resistance that occurs in hypothyroidism. The increase in IRS-1 expression during goitrogenesis may represent an important event associated with the increased rate of cell mitosis promoted by TSH and indicates that insulin and IGF-I are important co-mitogenic factors in vivo, possibly acting through the activation of IRS-1.

Effect of 5-HT1B receptor agonists injected into the prefrontal cortex on maternal aggression in rats

Serotonin (5-HT1B) receptors play an essential role in the inhibition of aggressive behavior in rodents. CP-94,253, a 5-HT1B receptor agonist, can reduce aggression in male mice when administered directly into the ventro-orbitofrontal (VO) prefrontal cortex (PFC). The objective of the current study was to assess the effects of two selective 5-HT1B receptor agonists (CP-94,253 and CP-93,129), microinjected into the VO PFC, on maternal aggressive behavior after social instigation in rats. CP-94,253 (0.56 µg/0.2 µL, N = 8, and 1.0 µg/0.2 µL, N = 8) or CP-93,129 (1.0 µg/0.2 µL, N = 9) was microinjected into the VO PFC of Wistar rats on the 9th day postpartum and 15 min thereafter the aggressive behavior by the resident female against a male intruder was recorded for 10 min. The frequency and duration of aggressive and non-aggressive behaviors were analyzed using ANOVA and post hoc tests. CP-93,129 significantly decreased maternal aggression. The frequency of lateral attacks, bites and pinnings was reduced compared to control, while the non-aggressive behaviors and maternal care were largely unaffected by this treatment. CP-94,253 had no significant effects on aggressive or non-aggressive behaviors when microinjected into the same area of female rats. CP-93,129, a specific 5-HT1B receptor agonist, administered into the VO PFC reduced maternal aggressive behavior, while the CP-94,253 agonist did not significantly affect this behavior after social instigation in female rats. We conclude that only the 5-HT1B receptor agonist CP-93,129 administered into the VO PFC decreased aggression in female rats postpartum after social instigation.

Is there an association between T102C polymorphism of the serotonin receptor 2A gene and urinary incontinence?

The regulation of bladder function is influenced by central serotonergic modulation. Several genetic polymorphisms related to serotonin control have been described in the literature. T102C polymorphism of the serotonin receptor 2A gene (5-HT2A) has been shown to be associated with certain diseases such as non-fatal acute myocardial infarction, essential hypertension, and alcoholism. In the present study, we examined the association between 5-HT2A gene polymorphism and urinary incontinence in the elderly. A case-control study was performed in 298 elderly community dwellers enrolled in the Gravataí-GENESIS Project, Brazil, which studies gene-environmental interactions in aging and age-related diseases. Clinical, physical, biochemical, and molecular analyses were performed on volunteers. 5-HT2A genotyping was determined by PCR-RFLP techniques using the HpaII restriction enzyme. The subjects had a mean age of 68.05 ± 6.35 years (60-100 years), with 16.9% males and 83.1% females. The C allele frequency was 0.494 and the T allele frequency was 0.506. The CC genotype frequency was 21.78%, the CT genotype frequency was 55.24% and the TT genotype frequency was 22.98%. We found an independent significant association between the TT genotype (35.7%) and urinary incontinence (OR = 2.06, 95%CI = 1.16-3.65). Additionally, urinary incontinence was associated with functional dependence and systolic hypertension. The results suggest a possible genetic influence on urinary incontinence involving the serotonergic pathway. Further investigations including urodynamic evaluation will be performed to better explain our findings.

Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate

The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25ºC in the presence of dimethylsulfoxide. Although glutamyl (E) and aspartyl (D) residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40% dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351) phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351) moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate) and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate).

The polymorphism of the serotonin-2A receptor T102C is associated with age

Epidemiological investigations suggest that T102C polymorphism of gene 5-HT2A may be associated with mean life span because diseases and behaviors related to this polymorphism, such as schizophrenia, suicide, aggression, and addiction, may potentially shorten mean life span. A sample of 687 individuals without previous neuropsychiatric disease was genotyped and separated into 3 groups according to their gender and age: 14-45 years old, 46-64 years old and 65-100 years old. Molecular genotyping was performed using the technique of polymerase chain reaction followed by restriction fragment length polymorphism using HpaII restriction enzyme. 5-HT2A genotype frequencies were: TT = 21.5% (148), CC = 16.6% (114) and TC = 61.9% (425) and allele frequencies were T = 52.5% and C = 46.5%. Significant differences were found between mean age of the TT genotype carriers (60.27 ± 12.60 years) and TC genotype carriers (56.80 ± 13.18 years) of T102C polymorphism of gene 5-HT2A (P = 0.026) as well as the age groups (P = 0.012). Carriers of genotype TT were older than the other two genotypes, whereas carriers of genotype CC had an intermediate age compared with TT and CC subjects. The present results demonstrate an association between T102C polymorphism of gene 5-HT2A and age. Our results suggest that T102C polymorphism of gene 5-HT2A is associated with mean life span, and thus this gene becomes a possible candidate for the group of adaptive genes to meat consumption proposed in the literature. Further studies should be conducted in order to elucidate this association.

Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 µCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

NMDA receptor blockade alters the intracellular distribution of neuronal nitric oxide synthase in the superficial layers of the rat superior colliculus

Nitric oxide (NO) is a molecular messenger involved in several events of synaptic plasticity in the central nervous system. Ca2+ influx through the N-methyl-D-aspartate receptor (NMDAR) triggers the synthesis of NO by activating the enzyme neuronal nitric oxide synthase (nNOS) in postsynaptic densities. Therefore, NMDAR and nNOS are part of the intricate scenario of postsynaptic densities. In the present study, we hypothesized that the intracellular distribution of nNOS in the neurons of superior colliculus (SC) superficial layers is an NMDAR activity-dependent process. We used osmotic minipumps to promote chronic blockade of the receptors with the pharmacological agent MK-801 in the SC of 7 adult rats. The effective blockade of NMDAR was assessed by changes in the protein level of the immediate early gene NGFI-A, which is a well-known NMDAR activity-dependent expressing transcription factor. Upon chronic infusion of MK-801, a decrease of 47% in the number of cells expressing NGFI-A was observed in the SC of treated animals. Additionally, the filled dendritic extent by the histochemical product of nicotinamide adenine di-nucleotide phosphate diaphorase was reduced by 45% when compared to the contralateral SC of the same animals and by 64% when compared to the SC of control animals. We conclude that the proper intracellular localization of nNOS in the retinorecipient layers of SC depends on NMDAR activation. These results are consistent with the view that the participation of NO in the physiological and plastic events of the central nervous system might be closely related to an NMDAR activity-dependent function.