Receptor-mediated Actions Research Articles

Minireview: Vitamin D: Is There a Role in Extraskeletal Health?

In recent years, vitamin D has received increased attention due to the resurgence of vitamin D deficiency and rickets in developed countries together with the identification of extraskeletal vitamin D receptor-mediated actions, suggesting unexpected benefits of vitamin D in health and diseases. Although there is increased awareness of the importance of vitamin D, the role of vitamin D in extraskeletal health has been a matter of debate. In this review, we will summarize what is known and indicate the questions that remain and need to be addressed.

Dual Functions of Prostaglandin D2 in Murine Contact Hypersensitivity via DP and CRTH2

Prostaglandin D2 (PGD2) exerts its effects through two distinct receptors: the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and the D prostanoid (DP) receptor. Our previous study demonstrated that CRTH2 mediates contact hypersensitivity (CHS) in mice. However, the function of DP receptor remains to be fully established. In this study, we examine the pathophysiological roles of PGD2 using DP-deficient (DP(-/-)) and CRTH2/DP-deficient (CRTH2(-/-)/DP(-/-)) mice to elucidate receptor-mediated PGD2 action in CHS. We observed profound exacerbation of CHS in DP(-/-) mice. CRTH2(-/-)/DP(-/-) mice showed similar exacerbation, but to a lesser extent. These symptoms were accompanied by increased production of interferon-γ and IL-17. The increase in IL-17 producing γδ T cells was marked and presumably contributed to the enhanced CHS. DP deficiency promoted the in vivo migration of dendritic cells to regional lymph nodes. A DP agonist added to DCs in vitro was able to inhibit production of IL-12 and IL-1β. Interestingly, production of IL-10 in dendritic cells was elevated via the DP pathway, but it was lowered by the CRTH2 pathway. Collectively, PGD2 signals through CRTH2 to mediate CHS inflammation, and conversely, DP signals to exert inhibitory effects on CHS. Thus, we report opposing functions for PGD2 that depend on receptor usage in allergic reactions.

Estrogen action and prostate cancer

Early work on the hormonal basis of prostate cancer focused on the role of androgens, but more recently estrogens have been implicated as potential agents in the development and progression of prostate cancer. In this article, we review the epidemiological, laboratory and clinical evidence that estrogen may play a causative role in human prostate cancer, as well as rodent and grafted in vivo models. We then review recent literature highlighting potential mechanisms by which estrogen may contribute to prostate cancer, including estrogenic imprinting and epigenetic modifications, direct genotoxicity, hyperprolactinemia, inflammation and immunologic changes, and receptor-mediated actions. We discuss the work performed so far separating the actions of the different known estrogen receptors (ERs), ERα and ERβ, as well as G-protein-coupled receptor 30 and their specific roles in prostate disease. Finally, we predict that future work in this field will involve more investigations into epigenetic changes, experiments using new models of hormonal dysregulation in developing human prostate tissue, and continued delineation of the roles of the different ER subtypes, as well as their downstream signaling pathways that may serve as therapeutic targets.

Dual blockade of the renin‐angiotensin system with angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs)

Dual blockade of the renin‐angiotensin system with angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs)

Role of dopamine D3 and serotonin 5-HT1A receptors in l-DOPA-induced dyskinesias and effects of sarizotan in the 6-hydroxydopamine-lesioned rat model of Parkinson’s disease

Sarizotan, a 5-HT(1A) agonist with additional affinity for D(3) and D(4) receptors, has been demonstrated to have anti-dyskinetic effects. The mechanism by which these effects occur is not clear. Using unilateral 6-hydroxydopamine-lesioned rats that received chronic intraperitoneal (ip) administration of L: -3,4-dihydroxyphenylalanine (L: -DOPA) we investigated the involvement of D(3) and 5-HT(1A) receptors in the effects of sarizotan on contraversive circling and abnormal involuntary movements (AIMs). Before sensitization by chronic L: -DOPA treatment (12.5 with 3.25mg/kg benserazide ip, twice daily for 21days), no effect of the selective D(3) agonist, PD128907 (1 or 3mg/kg ip), or the selective D(3) antagonist, GR103691 (0.5 or 1.5mg/kg ip), was observed. Treatment with sarizotan (1 or 5mg/kg ip) dose-dependently inhibited the L: -DOPA-induced contraversive turning and AIMs. In co-treatment with the 5-HT(1A) antagonist, WAY100635 (1mg/kg ip), sarizotan failed to affect this behaviour, confirming the prominent 5-HT(1A) receptor-mediated mechanism of action. In the presence of PD128907 (3mg/kg ip), the effects of sarizotan on contraversive turning, locomotive dyskinesia and axial dystonia, but not on orolingual and forelimb dyskinesia, were blocked. On its own, PD128907 had no effect on the behavioural effects of L: -DOPA except that it tended to reduce orolingual and forelimb dyskinesia. GR103691 had no effect on its own or in combination with sarizotan. These data identify an involvement of D(3) receptors in the action of sarizotan on some, but not all L: -DOPA-induced motor side effects. This selective involvement is in contrast to the more general involvement of 5-HT(1A) receptors in the anti-dyskinetic effects of sarizotan.

Androgens: they don’t just make a man out of you

Hyperandrogenism is known to be associated with polycystic ovarian (or ovary) syndrome. In this disease, ovarian follicles proliferate in a dysregulated fashion – in part due to excess androgen signaling within the ovary itself – leading to anovulation and infertility. Intriguingly, while excess androgen signaling in the ovary promotes unregulated follicle growth, a certain level of androgen signaling appears to be required for normal follicular development. In fact, deficient androgen signaling in the ovary might lead to premature ovarian failure, another cause of female infertility. This article discusses various in vitro and animal models being used to study the pathophysiology of hyper- and hypoandrogenism, focusing on the role of androgens and androgen receptor-mediated actions in normal and pathologic ovarian physiology.

Endothelin ETB Receptor Is Involved in Sex Differences in the Development of Balloon Injury-Induced Neointimal Formation

The purpose of this study was to evaluate the involvement of endothelin (ET)(B) receptor-mediated action in the sex differences in balloon injury-induced neointimal formation using the spotting-lethal rat, which carries a naturally occurring deletion in its ET(B) receptor gene. Male and female ET(B)-deficient and wild-type rats underwent balloon injury of the carotid artery. In the wild-type rats, the neointima/media ratio was significantly lower in females than in males, but this sex difference was attenuated by ovariectomy and restored by treatment with 17β-estradiol (20 μg/kg/day). In the ET(B)-deficient rats, the neointima/media ratio of the male and female rats was markedly increased to the same level, and this increase was not affected by ovariectomy or 17β-estradiol treatment. Treatment with (+)-(5S,6R,7R)-2-butyl-7-[2-((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxyphenyl)cyclopenteno[1,2-b]pyridine-6-carboxylic acid (J-104132) (10 mg/kg/day), an ET(A)/ET(B) dual receptor antagonist, markedly decreased the neointima/media ratio of the male wild-type rats and the male and female ET(B)-deficient rats, but not the female wild-type rats. In addition, 2R-(4-propoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N-(2,6-diethylphenyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (A-192621) (30 mg/kg/day), a selective ET(B) receptor antagonist, abolished the sex difference of balloon injury-induced neointimal formation. 2R-(4-methoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (ABT-627) (10 mg/kg/day), a selective ET(A) receptor antagonist, and J-104132 (10 mg/kg/day) markedly decreased the neointima/media ratio to the same extent in males but not intact females. These results indicate that the sex difference in balloon injury-induced neointimal formation was abolished by genetic ET(B) receptor deficiency or its pharmacological blockade. The lack of a vasoprotective effect of estrogen and the augmentation of ET(A) receptor-mediated action seem to be responsible for the abolition of sex differences in the ET(B) receptor-inhibited condition.

GABA A receptors in VTA mediate the morphine-induced release of ascorbic acid in rat nucleus accumbens

Local perfusion of morphine produces increased levels of extracellular ascorbic acid (AA) in the nucleus accumbens (NAc) of freely moving rats. However, the pathways that regulate morphine-induced AA release in the NAc are unclear. In the present study, we used high performance liquid chromatography with electrochemical detection (HPLC–ECD) to examine the effects of intra-ventral tegmental area (VTA) administration of a GABA A agonist and antagonist on morphine-induced increases in AA of the NAc. Also, using high performance liquid chromatography with fluorescent detection (HPLC–FD) and HPLC–ECD, the releases of γ-aminobutyric acid (GABA) and dopamine (DA) in the NAc induced by intra-VTA administration of a GABA A agonist and antagonist were also investigated. The results obtained showed that morphine (1 mM), locally perfused into the NAc, significantly increased AA release in the NAc and also GABA release. Intra-VTA infusion of bicuculline (150 ng/rat), a GABA receptor antagonist, not only abolished the enhanced extracellular AA and GABA levels produced by local perfusion of morphine but also decreased the basal release of extracellular GABA and increased the basal release of extracellular DA in the NAc. Muscimol (100 ng/rat), a GABA receptor agonist, affected the basal release of GABA and DA, but not the basal AA levels, or the morphine-induced changes in AA and GABA levels. These findings suggest that the GABA A receptors in the VTA play an important role in the modulation of morphine-induced AA release in the NAc, and the effect of morphine on AA release in the NAc is partially regulated by the GABA A receptor-mediated action of DA afferents from the VTA.

Testosterone induces activation of porcine primordial follicles in vitro.

The mechanism underlying primordial follicle activation is poorly understood. In this study, in-vitro culture and subsequent xenotransplantation were conducted to determine whether testosterone promotes the activation of porcine primordial follicles. Prepubertal porcine ovarian cortical strips containing primordial follicles were cultured in the presence of testosterone for 7 days, and subsequently transplanted to immunodeficient mice for 2 months. After culture and transplantation, development of follicles was examined histologically. The presence of androgen receptors in oocytes was assessed by use of western blot and immunohistochemical analyses. Testosterone at 10-6 m induced the primordial follicle transition to the intermediate (19 ± 4%) and primary (3 ± 1%) stages after 7-day culture, while 56 ± 5% of primordial follicles remained in the initial pool. Higher concentrations, above 10-5 m, or lower concentrations, below 10-6 m, did not induce follicle transition to the primary stage. After 7-day culture with 10-6 m testosterone, ovarian cortical strips were transplanted to immunodeficient mice. Some of the follicles developed to the secondary (15 ± 3%) and antral (10 ± 3%) stages, whereas 44 ± 7% of primordial follicles remained in the initial pool. In the culture experiment, estradiol-17β (10-7-10-5 m) had no significant effect on follicle activation. The androgen receptor antagonist, cyproterone acetate, inhibited the stimulatory effect of testosterone on primordial follicle activation, suggesting an androgen receptor-mediated action of testosterone. Western blot and immunohistochemical analyses revealed that androgen receptors were present in the oocytes of primordial follicles. These results suggest that testosterone at 10-6 m promotes the activation of porcine primordial follicles in vitro through the androgen receptors in the oocytes.

Macrophage Migration Inhibitory Factor in the Paraventricular Nucleus Plays a Major Role in the Sympathoexcitatory Response to Salt

Central hyperosmotic stimulation (HS) evokes increases in sympathetic nerve activity mediated by activation of angiotensin type 1 receptors in the hypothalamic paraventricular nucleus (PVN). Macrophage inhibitory migration factor (MIF) is an intracellular inhibitory regulator of angiotensin type 1 receptor-mediated actions of angiotensin II within neurons of the PVN. MIF mediates its actions via its intrinsic thiol-protein oxidoreductase activity. We demonstrate that intracerebroventricular injection of hypertonic saline into Sprague-Dawley rats elicits a significant (≈112%) increase in MIF mRNA expression in the PVN. Next, we evaluated the effect of viral-mediated expression of either MIF or [C60S]-MIF (which lacks thiol-protein oxidoreductase activity) in the PVN on the sympathoexcitation evoked by HS. We used a decorticate, arterially perfused in situ preparation of male Wistar rats (60 to 80 g). HS was induced by raising perfusate osmolality from 290 to 380 milliosmoles for 40 seconds. Seven to 10 days before experiments, rats were injected bilaterally (500 nL per side) with 0.9% saline (control) or with adenoassociated virus to express MIF, [C60S]-MIF, or enhanced green fluorescent protein in the PVN. HS produced sympathoexcitation in both the 0.9% saline and enhanced green fluorescent protein groups (sympathetic nerve activity increase of +27±4% and +25±4%, respectively; P<0.05), an effect that was not observed in the MIF group (+4±5%). Conversely, the HS-induced increase in sympathetic nerve activity was potentiated in the [C60S]-MIF group (+45±6%; P<0.05). We propose that MIF acting within the PVN is a major counterregulator of HS-induced sympathoexcitation, an effect that depends on thiol-protein oxidoreductase activity.

Serotonin-induced contraction in isolated intestine from a teleost fish (Carassius auratus): characterization and interactions with melatonin

Serotonin (5-HT) plays a critical role in several gastrointestinal functions in vertebrates. In teleosts lacking enterochromaffin cells, intestinal 5-HT originates from serotonergic enteric neurons. In the present study, the foregut of a stomachless teleost, the goldfish (Carassius auratus), was used to evaluate the in vitro effect of 5-HT on fish intestinal motility. We also studied the role of melatonin (MEL), an indoleamine sharing the biosynthetic pathway with 5-HT, as regulator of serotonergic activity. An organ bath system, with longitudinal strips from the goldfish intestinal bulb attached to an isometric transducer was used to record foregut smooth muscle contractions. Concentration-dependent curves of the contractile response exerted by 5-HT and its agonists, 5-methoxytryptamine (5-MT) and 5-carboxamidotryptamine (5-CT), suggest a receptor-mediated action, supported by the blockade by a general 5-HT antagonist, methysergide. The 5-HT-induced contraction was abolished in the presence of atropine, revealing the involvement of cholinergic transmission in gut actions of 5-HT. Furthermore, MEL inhibited the contractile effect of 5-HT and its agonists by up to 50%, which was counteracted by MEL antagonists. We can provisionally propose that at least two different 5-HT receptor subtypes are involved in fish intestinal motility, a 5-HT₄-like (5-MT-preferring) and a 5-HT₇-like (5-CT- and fluphenazine-sensitive) receptor. In summary, our results indicate that 5-HT regulates the contractile activity of goldfish foregut through specific receptors located in cholinergic neurons, and that MEL can modulate these serotonergic actions through high-affinity membrane receptors.

Involvement of Adenosine Signaling in Controlling the Release of Ghrelin from the Mouse Stomach

Ghrelin, a potent orexigenic hormone released from the stomach, is important in regulating energy metabolism. Abnormal ghrelin levels are associated with eating disorders and metabolic diseases. However, factors involved in the regulation of ghrelin release remain unclear. Here, we examined the involvement of adenosine signaling in the control of ghrelin release from the perfused mouse stomach. Adenosine stimulated ghrelin release concentration-dependently, and the A(2A) receptor-selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) and 2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH 58261) abolished the increased release. The A(2A) receptor-selective agonist 2-p-(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680) augmented ghrelin release concentration-dependently, whereas the A(1) receptor-selective agonist 2-chloro-N(6)-cyclopentyladenosine inhibited ghrelin release. In A(2A) receptor knockout mice, adenosine inhibited ghrelin release, and the A(1) receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked this inhibition. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride increased ghrelin release in wild-type and A(1) receptor knockout mice but not in A(2A) receptor knockout mice. Colocalization of ghrelin immunoreactivity with A(1) and A(2A) receptor immunoreactivities in the gastric nerve fibers were observed. Colocalization was also detected for ghrelin and A(1) receptor immunoreactivities in the gastric mucosa. Blockade of neural activities with tetrodotoxin abolished the stimulatory effect of adenosine on ghrelin release. In conclusion, adenosine exerts predominantly a tonic A(2A) receptor-mediated stimulatory action on gastric ghrelin release, whereas an A(1) receptor-mediated inhibitory action is also apparent when the tonic excitatory effect was removed.

C5a Receptor Targeting in Neointima Formation After Arterial Injury in Atherosclerosis-Prone Mice

Receptor binding of complement C5a leads to proinflammatory activation of many cell types, but the role of receptor-mediated action during arterial remodeling after injury has not been studied. In the present study, we examined the contribution of the C5a receptor (C5aR) to neointima formation in apolipoprotein E-deficient mice employing a C5aR antagonist (C5aRA) and a C5aR-blocking monoclonal antibody. Mice fed an atherogenic diet were subjected to wire-induced endothelial denudation of the carotid artery and treated with C5aRA and anti-C5aR-blocking monoclonal antibody or vehicle control. Compared with controls, neointima formation was significantly reduced in mice receiving C5aRA or anti-C5aR-blocking monoclonal antibody for 1 week but not for 3 weeks, attributable to an increased content of vascular smooth muscle cells, whereas a marked decrease in monocyte and neutrophil content was associated with reduced vascular cell adhesion molecule-1. As assessed by immunohistochemistry, reverse transcription polymerase chain reaction, and flow cytometry, C5aR was expressed in lesional and cultured vascular smooth muscle cells, upregulated by injury or tumor necrosis factor-alpha, and reduced by C5aRA. Plasma levels and neointimal plasminogen activator inhibitor-1 peaked 1 week after injury and were downregulated in C5aRA-treated mice. In vitro, C5a induced plasminogen activator inhibitor-1 expression in endothelial cells and vascular smooth muscle cells in a C5aRA-dependent manner, possibly accounting for higher vascular smooth muscle cell immigration. One-week treatment with C5aRA or anti-C5aR-blocking monoclonal antibody limited neointimal hyperplasia and inflammatory cell content and was associated with reduced vascular cell adhesion molecule-1 expression. However, treatment for 3 weeks failed to reduce but rather stabilized plaques, likely by reducing vascular plasminogen activator inhibitor-1 and increasing vascular smooth muscle cell migration.

Signal transduction of receptor-mediated antiproliferative action of melatonin on human prostate epithelial cells involves dual activation of Gαs and Gαq proteins

Melatonin has been shown to inhibit the proliferation of malignant and transformed human prostate epithelial cells by transcriptional up-regulation of p27(Kip1) expression via MTNR1A receptor-mediated activation of protein kinase A (PKA) and protein kinase C (PKC) in parallel. Given that melatonin MTNR1A receptor is a G protein-coupled receptor, this study was conducted to identify the specific G proteins that mediate the antiproliferative action of melatonin on human prostate epithelial cells. In 22Rv1 and RWPE-1 cells, knockdown of either Gα(s) or Gα(q) , but not Gα(i2) expression by RNA interference, abrogated the effects of melatonin on p27(Kip1) and cell proliferation. Conversely, cellular overexpression of activated mutants of Gα(s) and Gα(q) in 22Rv1 and RWPE-1 cells mimicked the effects of melatonin on prostate epithelial cell antiproliferation by increasing p27(Kip1) expression through downstream activation of PKA and PKC in parallel. Moreover, melatonin or 2-iodomelatonin induced elevation of adenosine-3',5'-cyclic monophosphate (cAMP) in 22Rv1 and RWPE-1 cells. The effects of 2-iodomelatonin on cAMP were blocked by the nonselective MTNR1A/MTNR1B receptor antagonist luzindole but were not affected by the selective MTNR1B receptor antagonist 4-phenyl-2-propionamidotetraline (4-P-PDOT). Furthermore, knockdown of Gα(s) mitigated the stimulatory effects of 2-iodomelatonin on cAMP. Collectively, the data demonstrated, for the first time, functional coupling of MTNR1A receptor to Gα(s) in cancerous or transformed human cells expressing endogenous melatonin receptors. Our results also showed that dual activation of Gα(s) and Gα(q) proteins is involved in the signal transduction of MTNR1A receptor-mediated antiproliferative action of melatonin on human prostate epithelial cells.

Abstract 4607: Novel role of PARP4 in hormone-dependent breast cancer progression and metastasis

Abstract Estrogen/estrogen receptor-mediated actions play an important role in regulating a variety of biological process including development, homeostasis and breast cancer progression. Even though systemic hormone therapies that either block local estrogen production by aromatase inhibitors (AI) or block actions of estrogen/estrogen receptor (ER) by antiestrogens (AE) is well tolerated, the development of resistance to these agents is still a major concern. Since multiple signaling pathways involved in ER activation, combination of endocrine and non endocrine agents that block these signaling pathways may delay the onset of resistance to hormonal therapy. Recent studies from our laboratory have identified Poly(ADP-ribose) polymerase 4(PARP4) as a novel ER co-regulator. We have investigated with hormone resistant breast cancer cells to demonstrate whether blocking of PARP4 results in resensitization to hormone therapies. PARP comprise of a family of enzymes which catalyses poly(ADPribosyl) action of DNA-binding proteins. PARP-1, the best characterized member, however PARP4 is an understudied protein with numerous capabilities. PARP4 has both N-terminal PXXP and PPXXP motifs which are known to interact with SH3 domain of c-Src as well as LXXLL motif which interacts with estrogen receptor (ER). We show that PARP4 is widely expressed in breast cancer cells (MCF-7, MDA-MB-231, ZR-75, and SKBR3) and its expression is significantly higher in these cells compared to non malignant MCF-10A cells. To understand the important role of PARP4-ER axis in hormonal resistance, metastasis and to test whether blocking of PARP4 mediated actions provide a therapeutic advantage to treat endocrine resistant breast tumors, we have used hormone resistant breast cancer cells that over-express PARP4 or and treated with PARP4 siRNA nanoparticles. The PLGA-siRNA nanoparticles were designed multifunctionally using chemical conjugation. The nanoparticles were characterized physiochemically. Our studies show that PARP4 functionally interacts with ER. As demonstrated by immunoprecipitation, PARP4 interacts with ER upon ligand (E2)-mediated stimulation. PARP4 induces cell cycle progression, local aromatase activity and promotes in vitro tumorigenic potential of breast cancer cells. siRNA nanoparticles of PARP4 inhibit the metastatic incidence in vivo. Our study also shows that PARP4-ER axis enhances multiple signaling cross-talk leading to hormonal independence/resistance and metastasis. Our results suggests that targeting PARP4 may provide a therapeutic advantage to treat endocrine resistant tumors and blocking the development of resistance to hormonal therapeutic agents and as well as decrease the metastatic potential in therapy resistant breast tumors. (Supported by NIH/NCI P30 CA 54174, RRT) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4607.

Genetic Effect of Endothelin and Angiotensin Receptor Blockade on Renal Injury in Salt‐Sensitive and Salt‐Resistant Hypertension

Proteinuria was inhibited by angiotensin receptor antagonists with a variable response. It remained unclear whether failure of some experimental models of hypertension to respond to treatment with the angiotensin antagonists may be due to genetic background. To investigate genetic effects on renal injuries in response to endothelin‐A blocker (ABT‐627), endothelin‐B blocker (A‐192621), and angiotensin receptor blocker (valsartan), we compared deoxycorticosterone acetate (DOCA)‐salt rats and spontaneously hypertensive rats (SHR) with the genetic control Wistar‐Kyoto rats. Drugs were initiated after hypertension was established. Compared with controls, arterial pressure increased in both strains and was not decreased after 4 weeks’ drug intervention. In both hypertensive strains, renal creatinine clearance decreased, and urinary excretion of protein, interstitial fibrosis, and TGF‐β mRNA levels were significantly increased. In vehicle‐treated DOCA‐salt rats, such damage was overcome by ABT‐627 and substantially blunted by valsartan, but not with A‐192621. The improvement degree of proteinuria in DOCA‐salt rats was significantly greater in rats treated by ABT‐627 than those treated by valsartan. In SHR, valsartan, but not ABT‐627, can significantly attenuate the renal injury. These results strongly support the important role of genetic predisposition in different renal responses to treatments. In DOCA‐salt rats, endothelin‐A receptor– and angiotensin receptor‐mediated action plays an important role in the pathogenesis of renal damage. On the other hand, only angiotensin receptor antagonist is useful for treatment in SHR.

A Novel Role for Protein Inhibitor of Activated STAT (PIAS) Proteins in Modulating the Activity of Zimp7, a Novel PIAS-like Protein, in Androgen Receptor-mediated Transcription

The PIAS proteins (protein inhibitor of activated STAT) were originally identified as inhibitors of the JAK-STAT pathway. Subsequently, their roles on transcriptional regulation have been identified in modulation of the androgen receptor (AR) and other nuclear hormone receptor-mediated actions. Zimp7, also named Zmiz2, is a novel PIAS-like protein and functions as a transcriptional co-activator. In this study, we demonstrate an interaction between Zimp7 and PIAS proteins with higher preference for PIAS3. A modified mammalian one-hybrid assay showed that the NH(2)-terminal proline-rich domain of Zimp7 and the region spanning amino acids 321-486 of PIAS3 were the primary interaction segments. The interaction between Zimp7 and PIAS3 proteins was further confirmed by in vitro protein pull-down and co-immunoprecipitation assays with both exogenous and endogenous proteins. Expression of exogenous PIAS3 further enhances Zimp7-mediated augmentation of AR transcription. Knockdown of the endogenous PIAS3 protein using a specific PIAS3 small hairpin RNA reduced the augmentation of Zimp7 on AR-mediated transcription. Co-localization of Zimp7 and PIAS3 proteins was observed in the nuclei of cells by immunostaining. Exogenous PIAS3 expression enhances the stability of the Zimp7 protein. Using chromatin immunoprecipitation assays, we showed that PIAS3 is involved in the AR- and Zimp7-formed protein complex(es) in the AR downstream target promoter to facilitate androgen-induced transcription. Finally, we further demonstrated that loss of Zimp7 significantly impaired PIAS3-mediated enhancement on AR activity in mouse Zimp7 null (zimp7(-/-)) embryonic fibroblasts. Taken together, these results demonstrate a novel interaction between PIAS and PIAS-like proteins and elucidate a novel regulatory mechanism for PIAS proteins in AR-mediated transcription.

In vivo imaging of dopamine receptors in a model of temporal lobe epilepsy

Alterations in dopamine neurotransmission in animal models of epilepsies have been frequently demonstrated using invasive neuroscience or ex vivo techniques. We aimed to test whether corresponding alterations could be detected by noninvasive in vivo brain imaging with positron emission tomography (PET) in the chronic phase of the rat pilocarpine model of temporal lobe epilepsy. Six pilocarpine-treated Wistar rats exhibiting spontaneous recurrent seizures and nine control rats were studied with PET using [(18)F]-fallypride, a high-affinity dopamine D(2/3) receptor ligand. Parametric images of [(18)F]-fallypride specific binding were calculated using a reference tissue method, and the two groups were contrasted by whole-brain voxel-based analysis implemented in statistical parametric mapping (SPM5). Dopamine D(2/3) receptor availability was 27% lower in the bilateral anterior caudate-putamen of pilocarpine-treated rats as compared to controls (p < 0.05), but binding was unaffected in other striatal or extrastriatal regions. The finding of substantially reduced availability of dopamine D(2/3) receptors in the anterior caudate-putamen of rats during the chronic phase of the pilocarpine model is in agreement with results of invasive (microinjection, microdialysis) animal studies that have revealed increased dopamine tonus and a D(2/3) receptor-mediated anticonvulsant action of dopamine in the anterior segment of the rat striatum. The present PET approach could be prospectively applied for monitoring dopamine receptor changes longitudinally, that is, at different phases of the epileptogenic process, and opens perspectives for testing dopaminergic agents as potential antiepileptogenic drugs.

A Novel Substituted Piperazine, CM156, Attenuates the Stimulant and Toxic Effects of Cocaine in Mice

Cocaine is a highly abused drug without effective pharmacotherapies to treat it. It interacts with sigma (sigma) receptors, providing logical targets for the development of medications to counteract its actions. Cocaine causes toxic and stimulant effects that can be categorized as acute effects such as convulsions and locomotor hyperactivity and subchronic effects including sensitization and place conditioning. In the present study, 3-(4-(4-cyclohexylpiperazin-1-yl)butyl)benzo[d]thiazole-2(3H)-thione (CM156), a novel compound, was developed and tested for interactions with sigma receptors using radioligand binding studies. It was also evaluated against cocaine-induced effects in behavioral studies. The results showed that CM156 has nanomolar affinities for each of the sigma receptor subtypes in the brain and much weaker affinities for non-sigma binding sites. Pretreatment of male Swiss-Webster mice with CM156, before administering either a convulsive or locomotor stimulant dose of cocaine, led to a significant attenuation of these acute effects. CM156 also significantly reduced the expression of behavioral sensitization and place conditioning evoked by subchronic exposure to cocaine. The protective effects of CM156 are consistent with sigma receptor-mediated actions. Together with previously reported findings, the data from CM156 and related sigma compounds indicate that sigma receptors can be targeted to alleviate deleterious actions of cocaine.

Developmental effect of antenatal exposure to betamethasone on renal angiotensin II activity in the young adult sheep

Antenatal corticosteroids may have long-term effects on renal development which have not been clearly defined. Our objective was to compare the responses to intrarenal infusions of ANG II in two groups of year-old, male sheep: one group exposed to a clinically relevant dose of betamethasone before birth and one not exposed. We wished to test the hypothesis that antenatal steroid exposure would enhance renal responses to ANG II in adult life. Six pairs of male sheep underwent unilateral nephrectomy and renal artery catheter placement. The sheep were infused for 24 h with ANG II or with ANG II accompanied by blockade of the angiotensin type 1 (AT(1)) or type 2 (AT(2)) receptor. Baseline mean arterial blood pressure among betamethasone-exposed sheep was higher than in control animals (85.8 +/- 2.2 and 78.3 +/- 1.0 mmHg, respectively, P = 0.003). Intrarenal infusion of ANG II did not increase systemic blood pressure (P >/= 0.05) but significantly decreased effective renal plasma flow and increased renal artery resistance (P < 0.05). The decrease in flow and increase in resistance were significantly greater in betamethasone- compared with vehicle-exposed sheep (betamethasone P < 0.05, vehicle P >/= 0.05). This effect appeared to be mediated by a heightened sensitivity to the AT(1) receptor among betamethasone-exposed sheep. Sodium excretion initially decreased in both groups during ANG II infusion; however, a rebound was observed after 24 h. AT(1) blockade was followed by a significant rebound after 24 h in both groups. AT(2) blockade blunted the 24-h rebound effect among the vehicle-exposed sheep compared with the betamethasone-exposed sheep. In conclusion, antenatal corticosteroid exposure appears to modify renal responsiveness to ANG II by increasing AT(1)- and decreasing AT(2) receptor-mediated actions particularly as related to renal blood flow and sodium excretion.