MAB3077 Sigma-AldrichAnti-G Protein Giα-2 Antibody, clone L5

The mouse vomeronasal organ (VNO) has a pivotal role in chemical communication. The vomeronasal sensory neuroepithelium consists of distinct populations of vomeronasal sensory neurons (VSNs). A subset of VSNs, with cell bodies in the basal part of the basal layer, coexpress Vmn2r G-protein-coupled receptor genes with H2-Mv genes, a family of nine nonclassical class I major histocompatibility complex genes. The in vivo, physiological roles of the H2-Mv gene family remain mysterious more than a decade after the discovery of combinatorial H2-Mv gene expression in VSNs. Here, we have taken a genetic approach and have deleted the 530 kb cluster of H2-Mv genes in the mouse germline by chromosome engineering. Homozygous mutant mice (ΔH2Mv mice) are viable and fertile. There are no major anatomical defects in their VNO and accessory olfactory bulb (AOB). Their VSNs can be stimulated with chemostimuli (peptides and proteins) to the same maximum responses as VSNs of wild-type mice, but require much higher concentrations. This physiological phenotype is displayed at the single-cell level and is cell autonomous: single V2rf2-expressing VSNs, which normally coexpress H2-Mv genes, display a decreased sensitivity to a peptide ligand in ΔH2Mv mice, whereas single V2r1b-expressing VSNs, which do not coexpress H2-Mv genes, show normal sensitivity to a peptide ligand in ΔH2Mv mice. Consistent with the greatly decreased VSN sensitivity, ΔH2Mv mice display pronounced deficits in aggressive and sexual behaviors. Thus, H2-Mv genes are not absolutely essential for the generation of physiological responses, but are required for ultrasensitive chemodetection by a subset of VSNs.

Heterotrimeric G proteins play a key role in membrane-mediated cell-signalling and hormonal regulation. Our earlier studies gave evidence of G protein subunit Galpha(i2) being under hormonal regulation in human in vivo. In this study, we used immortalized human oviduct epithelial cell line OE-E6/E7 as a model to study the hormonal regulation of Galpha(i2). We aimed at clarifying whether estradiol or progesterone could individually regulate the expression of Galpha(i2) and its potential signalling partners. Furthermore, we aimed to investigate which sex hormone receptors could potentially mediate the gene regulation in OE-E6/E7 cell line. OE-E6/E7 cells were cultured for 5 days with different concentrations of estradiol or progesterone. Quantitative real-time polymerase chain reaction (Q-PCR) was performed using cDNA of the hormone-treated cells to reveal any changes in gene expression. The presence of potential receptor targets in these cells was studied using PCR. Our data clearly showed that low concentrations of estradiol up-regulated the expression of Galpha(i2) gene and down-regulated the expression of membrane progesterone receptor mPRalpha gene in OE-E6/E7 cell line. Progesterone had no significant effect on Galpha(i2) gene expression, but it caused up-regulation of mPRalpha gene expression. In conclusion, it appears that sex hormones regulate the expression of Galpha(i2) and mPRalpha genes in a reverse manner in OE-E6/E7 cells. Our results suggest that estrogen receptor ERbeta mediates the regulatory effects of estradiol in these cells.

Although alterations in adenylate cyclase (AC) activity and somatostatin (SRIF) receptor density have been reported in Alzheimer's disease, the effects of amyloid beta-peptide (Abeta) on these parameters in the hippocampus are unknown. Our aim was to investigate whether the peptide fragment Abeta(25-35) can affect the somatostatinergic system in the rat hippocampus. Hence, Abeta(25-35) was injected intracerebroventricularly (i.c.v.) to Wistar rats in a single dose or infused via an osmotic minipump connected to a cannula implanted in the right lateral ventricle during 14 days. The animals were decapitated 7 or 14 days after the single injection and 14 days after chronic infusion of the peptide. Chronic i.c.v. infusion of Abeta(25-35) decreased SRIF-like immunoreactive content without modifying the SRIF receptor density, SRIF receptor expression, or the Gialpha(1), Gialpha(2), and Gialpha(3) protein levels in the hippocampus. This treatment, however, caused a decrease in basal and forskolin-stimulated AC activity as well as in the capacity of SRIF to inhibit AC activity. Furthermore, the protein levels of the neural-specific AC type I were significantly decreased in the hippocampus of the treated rats, whereas an increase in the levels of AC V/VI was found, with no alterations in type VIII AC. A single i.c.v. dose of Abeta(25-35) exerted no effect on SRIF content or SRIF receptors but induced a slight decrease in forskolin-stimulated AC activity and its inhibition by SRIF. Because chronic Abeta(25-35) infusion impairs learning and memory whereas SRIF facilitates these functions, the alterations described here might be physiologically important given the decreased cognitive behavior previously reported in Abeta-treated rats.

In the CNS, the heterotrimeric G protein Galphai2 is a minor Galpha subunit with restricted localization in the ventricular regions including the ependymal cilia. The localization of Galphai2 is conserved in cilia of different tissues, suggesting a particular role in ciliary function. Although studies with Galphai2-knockout mice have provided information on the role of this Galpha subunit in peripheral tissues, its role in the CNS is largely unknown. We used intracerebroventricular (icv) antisense administration to clarify the physiological role of Galphai2 in the ventricular system.High resolution MRI studies revealed that continuous icv-infusion of Galphai2-specific antisense oligonucleotide caused unilateral ventricular dilatation that was restricted to the antisense-receiving ventricle. Microscopic analysis demonstrated ependymal cell damage and loss of ependymal cilia. Attenuation of Galphai2 in ependymal cells was confirmed by immunohistochemistry. Ciliary beat frequency measurements on cultured ependymal cells indicated that antisense administration resulted in ciliary stasis.Our results establish that Galphai2 has an essential regulatory role in ciliary function and CSF homeostasis.

Melatonin is known to increase neuronal activity in the hippocampus, an effect contrary to that of somatostatin (somatotropin release-inhibiting factor, SRIF). Thus, the aim of this study was to investigate whether the somatostatinergic system is implicated in the mechanism of action of melatonin in the rat hippocampus. One group of rats was injected a single dose of melatonin [25 microg/kg subcutaneously (s.c.)] or saline containing ethanol (0.5%, s.c.) and killed 5 hr later. Melatonin significantly decreased the SRIF-like immunoreactivity levels and induced a significant decrease in the density of SRIF receptors as well as in the dissociation constant (Kd). SRIF-mediated inhibition of basal and forskolin-stimulated adenylyl cyclase activity was markedly decreased in hippocampal membranes from melatonin-treated rats. The functional activity of Gi proteins was similar in hippocampal membranes from melatonin-treated and control rats. Western blot analyses revealed that melatonin administration did not alter Gialpha1 or Gialpha2 levels. To determine if the changes observed were related to melatonin-induced activation of central melatonin receptors, a melatonin receptor antagonist, luzindole, was administered prior to melatonin injection. Pretreatment with luzindole (10 mg/kg, s.c.) did not alter the melatonin-induced effects on the above-mentioned parameters and luzindole, alone, had no observable effect. The present results demonstrate that melatonin decreases the activity of the SRIF receptor-effector system in the rat hippocampus, an effect which is apparently not mediated by melatonin receptors. As SRIF exerts an opposite effect to that of melatonin on hippocampal neuronal activity, it is possible that the SRIFergic system could be implicated in the mechanism of action of melatonin in the rat.

If melatonin or its analogs are to be used therapeutically in humans, their chronic effects on responsiveness of melatonin target cells need to be assessed. We have previously demonstrated that acute melatonin treatment regulates the somatostatinergic system in the rat hippocampus. In the present study, we have investigated the effects of subchronic and chronic daily treatment with melatonin on the somatostatinergic system in the rat hippocampus. Male Wistar rats (200-250 g) were injected with melatonin (25 microg/kg body weight, subcutaneously) daily for 4, 7 or 14 days and sacrificed 24 h after the last injection. Melatonin administration for 4 days induced a decrease in the hippocampal somatostatin (SRIF)-like immunoreactivity content as well as a decrease in the number of SRIF receptors and an increase in their apparent affinity. The decreased number of SRIF receptors in the melatonin (4 days)-treated rats was associated with a decreased capacity of SRIF to inhibit both basal and forskolin-stimulated adenylyl cyclase activity. These melatonin-induced effects reversed to control values after 7 or 14 days of treatment. Hippocampal membranes from control and melatonin-treated rats showed similar Gi and Gs activities. Melatonin treatment altered neither the functional Gi activity nor the Gialpha 1 or Gialpha 2 levels at any of the time periods studied. The present results suggest that chronic exposure to melatonin results in a tolerance of the hippocampus to this hormone.

The effect of Gly-Pro-Glu (GPE) on the somatostatinergic system of the temporal cortex in amyloid beta-peptide (Abeta) treated rats was investigated. Intracerebroventricular Abeta25-35 administration for 14 days (300 pmol/day) to ovariectomized rats produced a marked reduction in somatostatin (SRIF) content, SRIF receptor density and reduced the inhibitory effect of SRIF on adenylyl cyclase activity. I.p. injection of three doses (300 microg) of GPE on days 0, 6 and 12 resulted in a partial recovery of the parameters affected by Abeta25-35 administration. These results indicate that GPE may have an in vivo effect protecting the temporal cortical somatostatinergic system from Abeta insult.

Melatonin and somatostatin are known to exert similar effects on locomotor activity. We have previously demonstrated that acute melatonin treatment regulates somatostatin receptor function in the rat frontoparietal cortex. However, the effects of subchronic and chronic melatonin treatment on the somatostatin receptor-G protein-adenylyl cyclase system in the rat frontoparietal cortex are unknown. Melatonin was administered subcutaneously at a daily dose of 25 microg/kg for 4 days, 1 wk or 2 wk. Twenty-four hours after the last injection, the animals were sacrificed. Melatonin did not alter the somatostatin-like immunoreactivity content in the frontoparietal cortex from control and melatonin-treated rats during any of the previously indicated periods. Four days of melatonin administration induced both an increase in the number of [(125)I]-Tyr11-somatostatin receptors and a decrease in the affinity of somatostatin for its receptors in frontoparietal cortical membranes. The increased number of somatostatin receptors in the melatonin-treated rats was associated with an increased capacity of somatostatin to inhibit basal and forskolin-stimulated adenylyl cyclase activity. Melatonin administration for 4 days induced a higher adenylyl cyclase activity both under basal conditions and after direct stimulation of the enzyme with forskolin. No significant differences were observed in the function of Gi proteins in the 4-day melatonin-treated rats. Western blot analyses showed that the 4-day melatonin treatment reduced Gialpha(2) levels, without altering the amount of Gialpha(1). These melatonin-induced changes reverted to control values after 7 or 14 days of treatment. Altogether, the present findings suggest that subchronic melatonin treatment modulates the somatostatin receptor/effector system in the rat frontoparietal cortex.

Since melatonin (N-acetyl-5-methoxytryptamine) decreases locomotor activity and rearing and increases grooming behavior in a similar manner as somatostatin (SRIF), we examined if melatonin could induce these changes through somatostatinergic neurotransmission in the rat frontoparietal cortex. Male Wistar rats (200-250 g) received a single injection of melatonin (25 microg/kg per day) subcutaneously (s.c.) and were sacrificed 5 hr later. Melatonin treatment increased the number of 125I-Tyr11-SRIF receptors in frontoparietal cortical membranes without any changes in the dissociation constant (Kd). The capacity of SRIF to inhibit basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity was increased in melatonin-treated rats as compared to the control animals. Melatonin administration also induced a lower AC activity, both under basal conditions and after stimulation of the enzyme via stimulatory guanine nucleotide-binding proteins (Gs), or directly with FK. Functional inhibitory guanine nucleotide-binding protein (Gi) activity was increased in frontoparietal cortical membranes from melatonin-treated rats when compared to controls. Western blot analyzes showed that melatonin administration did not alter the amount of the Gialpha1, or Gialpha3 subunits, but reduced Gialpha2 levels in frontoparietal cortical membranes. No significant changes in SRIF-like immunoreactivity content and SRIF mRNA levels were detected in this brain area after melatonin treatment. Administration of the melatonin receptor antagonist luzindole (10 mg/kg, s.c.) 30 min before melatonin injection did not change the melatonin-induced effects on the SRIF receptor effector system. In conclusion, the present results show that acute melatonin administration increases the activity of the SRIF receptor effector system and decreases Gialpha2 levels in the rat frontoparietal cortex. In addition, the coupling of Gs to AC is disturbed by melatonin.

5-Oxo-eicosatetraenoic acid (5-oxoETE) stimulated human neutrophil (PMN) and eosinophil chemotaxis, PMN hexose uptake, and PMN membrane GTP/GDP exchange. Pertussis toxin (PT), a blocker of heterotrimeric G proteins (GP), completely inhibited these responses, but proved far less effective on the same responses when elicited by leukotriene B4, C5a, FMLP, platelet-activating factor, IL-8, or RANTES chemotactic factors. 5-OxoETE also specifically bound to the membrane preparations that conducted GTP/GDP exchange. This binding was down-regulated by GTPgammaS, but not ADPgammaS, and displaced by 5-oxoETE analogues, but not by leukotriene B4, lipoxin A4, or lipoxin B4. Finally, PMN expressed PT-sensitive GP alphaiota2 and PT-resistant GP alphaq/11- and alpha13-chains; eosinophils expressed only alphai2 and alphaq/11. We conclude that 5-oxoETE activates granulocytes through a unique receptor that couples preferentially to PT-sensitive GP. The strict dependency of this putative receptor on PT-sensitive GP may underlie the limited actions of 5-oxoETE, compared with other CF, and help clarify the complex relations between receptors, GP, cell signals, and cell responses.