Estrogenic activity assessment of environmental chemicals using in vitro assays: identification of two new estrogenic compounds.

Environmental chemicals with estrogenic activities have been suggested to be associated with deleterious effects in animals and humans. To characterize estrogenic chemicals and their mechanisms of action, we established in vitro and cell culture assays that detect human estrogen receptor [alpha] (hER[alpha])-mediated estrogenicity. First, we assayed chemicals to determine their ability to modulate direct interaction between the hER[alpha] and the steroid receptor coactivator-1 (SRC-1) and in a competition binding assay to displace 17ss-estradiol (E(2)). Second, we tested the chemicals for estrogen-associated transcriptional activity in the yeast estrogen screen and in the estrogen-responsive MCF-7 human breast cancer cell line. The chemicals investigated in this study were o,p'-DDT (racemic mixture and enantiomers), nonylphenol mixture (NPm), and two poorly analyzed compounds in the environment, namely, tris-4-(chlorophenyl)methane (Tris-H) and tris-4-(chlorophenyl)methanol (Tris-OH). In both yeast and MCF-7 cells, we determined estrogenic activity via the estrogen receptor (ER) for o,p'-DDT, NPm, and for the very first time, Tris-H and Tris-OH. However, unlike estrogens, none of these xenobiotics seemed to be able to induce ER/SRC-1 interactions, most likely because the conformation of the activated receptor would not allow direct contacts with this coactivator. However, these compounds were able to inhibit [(3)H]-E(2) binding to hER, which reveals a direct interaction with the receptor. In conclusion, the test compounds are estrogen mimics, but their molecular mechanism of action appears to be different from that of the natural hormone as revealed by the receptor/coactivator interaction analysis.

The PPARgamma agonist pioglitazone modifies the vascular sodium-angiotensin II relationship in insulin-resistant rats.

Glitazones are efficient insulin sensitizers that blunt the effects of angiotensin II (ANG II) in the rat. Sodium chloride is another important modulator of the systemic and renal effects of ANG II. Whether glitazones interfere with the interaction between sodium and the response to ANG II is not known. Therefore, we investigated the effects of pioglitazone on the relationship between sodium and the systemic and renal effects of ANG II in rats. Pioglitazone, or vehicle, was administered for 4 wk to 8-wk-old obese Zucker rats. Animals were fed a normal-sodium (NS) or a high-sodium (HS) diet. Intravenous glucose tolerance tests, systemic and renal hemodynamic responses to ANG II, and the renal ANG II binding and expression of ANG II type 1 (AT(1)) receptors were measured. The results of our study were that food intake and body weight increased, whereas blood pressure, heart rate, filtration fraction, and insulin levels decreased significantly with pioglitazone in obese rats on both diets. Pioglitazone blunted the systemic response to ANG II and abolished the increased responsiveness to ANG II induced by a HS diet. Pioglitazone modified the renal hemodynamic response to changes in salt intake while maintaining a lower filtration fraction with ANG II perfusion. These effects were associated with a decrease in the number and expression of the AT(1) receptor in the kidney. In conclusion, these data demonstrate that the peroxisome proliferator-activated receptor-gamma agonist pioglitazone modifies the physiological relationship between sodium chloride and the response to ANG II in insulin-resistant rats.

Vascular Effects of RWJ-676070, a Selective Combined V-1a/V-2 Vasopressin Receptor Antagonist

In recent years, several vasopressin antagonists have been developed that block V-1 receptors either selectively or nonselectively.(1,2) To date, one combined V-1/V-2 antagonist (primarily a V-2 antagonist, as determined on the basis of human receptor binding data), conivaptan, has been approved for the treatment of euvolemic hyponatremia.(3,4) We have previously shown that the vascular properties of a vasopressin V-1 antagonist can be investigated safely and reliably in healthy subjects. We used the measurement of skin blood flow after intradermic injection of exogenous arginine vasopressin on a skin area prevasodilated with calcitonin gene-related peptide (CGRP).(3,5) This technique enables the documentation of the dose-dependent effects of vasopressin or vasopressin antagonists. In this study, we have characterized the V-1a pharmacodynamic profile of increasing doses of RWJ-676070, a new orally active dual V-1a/V-2 receptor antagonist, in healthy subjects.(5)

Pharmacokinetic evaluation of losartan.

Introduction: Blockade of the renin-angiotensin system is one of the major therapeutic strategies in the management of patients with essential hypertension, congestive heart failure and diabetic as well as non-diabetic renal diseases. As the first angiotensin II receptor blocker (ARB) on the market, losartan belongs to the most frequently prescribed ARB. Area covered : The present review examines the pharmacokinetics of losartan with a special discussion on the dose of losartan that should be used in clinical practice to obtain the maximal benefits of the drug. Readers are provided with arguments suggesting that the dose of 50 mg losartan is probably too low and that losartan should preferably be prescribed at the dose of 100 mg/day or higher. Expert opinion : Losartan is an effective antagonist of angiotensin II AT(1) receptors which has been shown to provide important clinical benefits in patients with hypertension, congestive heart failure and renal diseases. Losartan should be prescribed at the dose of 100 mg/day and the use of higher doses should be reconsidered in future studies to improve its clinical efficacy.

The role of the NKG2D receptor for tumor immunity.

Natural killer (NK) cells have originally been identified based on their capacity to kill transformed cells in a seemingly non-specific fashion. Over the last 15 years, knowledge on receptor ligand systems used by NK cells to specifically detect transformed cells has been accumulating rapidly. One of these receptor ligand systems, the NKG2D pathway, has received particular attention, and now serves as a paradigm for how the immune system is able to gather information about the health status of autologous host cells. In addition to its significance on NK cells, NKG2D, as well as other NK cell receptors, play significant roles on T cells. This review aims at summarizing recent insights into the regulation of NKG2D function, the control over NKG2D ligand expression and the role of NKG2D in tumor immunity. Finally, we will discuss first attempts to exploit NKG2D function to improve immunity to tumors.

Blood pressure, cardiac, and renal responses to salt and deoxycorticosterone acetate in mice: role of Renin genes.

Several studies have demonstrated that mice are polymorphic for the number of renin genes, with some inbred strains harboring one gene (Ren-1(c)) and other strains containing two genes (Ren-1(d) and Ren-2). In this study, the effects of 1% salt and deoxycorticosterone acetate (DOCA)/salt were investigated in one- and two-renin gene mice, for elucidation of the role of renin in the modulation of BP, cardiac, and renal responses to salt and DOCA. The results demonstrated that, under baseline conditions, mice with two renin genes exhibited 10-fold higher plasma renin activity, 100-fold higher plasma renin concentrations, elevated BP (which was angiotensin II-dependent), and an increased cardiac weight index, compared with one-renin gene mice (all P < 0.01). The presence of two renin genes markedly increased the BP, cardiac, and renal responses to salt. The number of renin genes also modulated the responses to DOCA/salt. In one-renin gene mice, DOCA/salt induced significant renal and cardiac hypertrophy (P < 0.01) even in the absence of any increase in BP. Treatment with losartan, an angiotensin II AT(1) receptor antagonist, decreased BP in two-renin gene mice but not in one-renin gene mice. However, losartan prevented the development of cardiac hypertrophy in both groups of mice. In conclusion, these data demonstrate that renin genes are important determinants of BP and of the responses to salt and DOCA in mice. The results confirm that the Ren-2 gene, which controls renin production mainly in the submaxillary gland, is physiologically active in mice and is not subject to the usual negative feedback control. Finally, these data provide further evidence that mineralocorticoids promote cardiac hypertrophy even in the absence of BP changes. This hypertrophic process is mediated in part by the activation of angiotensin II AT(1) receptors.

Intraneuronal angiotensinergic system in rat and human dorsal root ganglia.

To elucidate the local formation of angiotensin II (Ang II) in the neurons of sensory dorsal root ganglia (DRG), we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of protein renin, Ang II, Substance P and calcitonin gene-related peptide (CGRP) in the rat and human thoracic DRG. Quantitative real time PCR (qRT-PCR) studies revealed that rat DRG expressed substantial amounts of Ang-N- and ACE mRNA, while renin mRNA as well as the protein renin were untraceable. Cathepsin D-mRNA and cathepsin D-protein were detected in the rat DRG indicating the possibility of existence of pathways alternative to renin for Ang I formation. Angiotensin peptides were successfully detected with high performance liquid chromatography and radioimmunoassay in human DRG extracts. In situ hybridization in rat DRG confirmed additionally expression of Ang-N mRNA in the cytoplasm of numerous neurons. Intracellular Ang II staining could be shown in number of neurons and their processes in both the rat and human DRG. Interestingly we observed neuronal processes with angiotensinergic synapses en passant, colocalized with synaptophysin, within the DRG. In the DRG, we also identified by qRT-PCR, expression of Ang II receptor AT(1A) and AT(2)-mRNA while AT(1B)-mRNA was not traceable. In some neurons Substance P and CGRP were found colocalized with Ang II. The intracellular localization and colocalization of Ang II with Substance P and CGRP in the DRG neurons may indicate a participation and function of Ang II in the regulation of nociception. In conclusion, these results suggest that Ang II may be produced locally in the neurons of rat and human DRG and act as a neurotransmitter.

Substance P-induced skin inflammation is not modulated by a single dose of sitagliptin in human volunteers.

Substance P (SP), an undecapeptide belonging to the tachykinin family, is released during the activation of sensory nerves, and causes vasodilation, edema and pain through activation of tissular Neurokinin 1 receptors. SP proinflammatory effects are terminated by angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP), while the aminopeptidase dipeptidylpeptidase IV (DPPIV) can also play a role. The aim of this randomized, crossover, double-blind study was to assess the cutaneous vasoreactivity (flare and wheal reaction, burning pain sensation) to intradermal injection of ascending doses of SP in six volunteers receiving a single therapeutic dose of the DPPIV inhibitor sitagliptin or a matching placebo. Cutaneous SP challenges produced the expected, dose-dependent flare and wheal response, while eliciting mild to moderate local pain sensation with little dose dependency. However, no differences were shown in the responses observed under sitagliptin compared with placebo, while the study would have been sufficiently powered to detect a clinically relevant increase in sensitivity to SP. The results of this pilot study are in line with proteolytic cleavage of SP by ACE and NEP compensating the blockade of DPPIV to prevent an augmentation of its proinflammatory action.

Receptor occupancy vs. induction of Na+-K+-ATPase and Na+ transport by aldosterone.

In the urinary bladder of the toad Bufo marinus aldosterone (between 0.8 and 100 nM) stimulates Na+ transport [half-maximal induction concentration (K1/2) = 6.5 nM]. At low hormone concentrations (0.8-8 nM), the increase of Na+ transport between 0.75 and 2.5 h is accompanied by a fall in transepithelial resistance (R). Higher hormone concentrations (30-800 nM) induce an additional resistance-independent fraction of Na+ transport within 2.5-8 h. From 6 h on, aldosterone (between 0.2 and 20 nM) stimulates in the same tissue the biosynthesis rate of the alpha- and beta-subunits of Na+-K+-ATPase (K1/2 = 3 and 1.5 nM, respectively). New pump synthesis is thus not a prerequisite for the early mineralocorticoid response but might be linked to the late transport event. The mineralocorticoid response is usually ascribed to interaction with the higher affinity type 1 receptor. In the present study we show, however, that at least 55% of the overall Na+ transport response is linked to nuclear occupation of the lower affinity type 2 receptors [dissociation constant (Kd) = 50 nM, maximum number of binding sites (Nmax) = 315 fmol/mg protein]. Distinct aldosterone effects, such as the fall in R and the increase in Na+-K+-ATPase synthesis, are more closely related to occupation of type 1 receptors (Kd = 0.3 nM, Nmax = 23 fmol/mg protein). At maximal induction of these latter parameters, only about 20% of type 2 receptors are occupied. These results suggest that both types of aldosterone receptors are involved in the mediation of the full mineralocorticoid response: type 1 in the early and late and type 2 particularly in the late tissue response.

The effects of losartan on renal function in the newborn rabbit.

The low GFR of newborns is maintained by various factors including the renin-angiotensin system. We previously established the importance of angiotensin II in the newborn kidney, using the angiotensin-converting enzyme inhibitor perindoprilat. The present study was designed to complement these observations by evaluating the role of angiotensin-type 1 (AT(1)) receptors, using losartan, a specific AT(1)-receptor blocker. Increasing doses of losartan were infused into anesthetized, ventilated, newborn rabbits. Renal function and hemodynamic variables were assessed using inulin and para-aminohippuric acid clearances as markers of GFR and renal plasma flow, respectively. Losartan 0.1 mg/kg slightly decreased mean blood pressure (-11%) and increased diuresis (+22%). These changes can be explained by inhibition of the AT(1)-mediated vasoconstrictive and antidiuretic effects of angiotensin, and activation of vasodilating and diuretic AT(2) receptors widely expressed in the neonatal period. GFR and renal blood flow were not modified. Losartan 0.3 mg/kg decreased mean blood pressure significantly (-15%), probably by inhibiting systemic AT(1) receptors. GFR significantly decreased (-25%), whereas renal blood flow remained stable. The decrease in filtration fraction (-21%) indicates predominant efferent vasodilation. At 3 mg/kg, the systemic hypotensive effect of losartan was marked (mean blood pressure, -28%), with decreased GFR and renal blood flow (-57% and -51%, respectively), a stable filtration fraction, and an increase in renal vascular resistance by 124%. The renal response to this dose can be considered as reflex vasoconstriction of afferent and efferent arterioles, rather than specific receptor antagonism. We conclude that under physiologic conditions, the renin-angiotensin is critically involved in the maintenance of GFR in the immature kidney.

Cutting edge: stimulation with the cognate self-antigen induces expression of the Ly49A receptor on self-reactive T cells which modulates their responsiveness.

NK cell self-tolerance is maintained by inhibitory receptors specific for MHC class I molecules. Inhibitory NK receptors are also expressed on memory CD8 T cells but their biological relevance on T cells is unclear. In this study, we describe the expression of the Ly49A receptor on a subset of autoreactive T cells which persist in mice double-transgenic for the lymphocytic choriomeningitis virus-derived peptide gp33 and a TCRalphabeta specific for the gp33. No Ly49A-expressing cells are found in TCRalphabeta single-transgenic mice, indicating that the presence of the autoantigen is required for Ly49A induction. Direct evidence for an Ag-specific initiation of Ly49A expression has been obtained in vitro after stimulation of autoreactive TCRalphabeta T cells with the cognate self-Ag. This expression of Ly49A substantially reduces Ag-specific activation of autoreactive T cells. These findings thus suggest that autoantigen-specific induction of inhibitory NK cell receptors on T cells may contribute to peripheral self-tolerance.

Role of Myotonic Dystrophy Protein Kinase [DMPK] in Glucose Homeostasis and Muscle Insulin Action

Myotonic dystrophy 1 (DM1) is caused by a CTG expansion in the 3′-unstranslated region of the DMPK gene, which encodes a serine/threonine protein kinase. One of the common clinical features of DM1 patients is insulin resistance, which has been associated with a pathogenic effect of the repeat expansions. Here we show that DMPK itself is a positive modulator of insulin action. DMPK-deficient (dmpk−/−) mice exhibit impaired insulin signaling in muscle tissues but not in adipocytes and liver, tissues in which DMPK is not expressed. Dmpk−/− mice display metabolic derangements such as abnormal glucose tolerance, reduced glucose uptake and impaired insulin-dependent GLUT4 trafficking in muscle. Using DMPK mutants, we show that DMPK is required for a correct intracellular trafficking of insulin and IGF-1 receptors, providing a mechanism to explain the molecular and metabolic phenotype of dmpk−/− mice. Taken together, these findings indicate that reduced DMPK expression may directly influence the onset of insulin-resistance in DM1 patients and point to dmpk as a new candidate gene for susceptibility to type 2-diabetes.

Role of Myotonic Dystrophy Protein Kinase [DMPK] in Glucose Homeostasis and Muscle Insulin Action

Myotonic dystrophy 1 (DM1) is caused by a CTG expansion in the 3′-unstranslated region of the DMPK gene, which encodes a serine/threonine protein kinase. One of the common clinical features of DM1 patients is insulin resistance, which has been associated with a pathogenic effect of the repeat expansions. Here we show that DMPK itself is a positive modulator of insulin action. DMPK-deficient (dmpk−/−) mice exhibit impaired insulin signaling in muscle tissues but not in adipocytes and liver, tissues in which DMPK is not expressed. Dmpk−/− mice display metabolic derangements such as abnormal glucose tolerance, reduced glucose uptake and impaired insulin-dependent GLUT4 trafficking in muscle. Using DMPK mutants, we show that DMPK is required for a correct intracellular trafficking of insulin and IGF-1 receptors, providing a mechanism to explain the molecular and metabolic phenotype of dmpk−/− mice. Taken together, these findings indicate that reduced DMPK expression may directly influence the onset of insulin-resistance in DM1 patients and point to dmpk as a new candidate gene for susceptibility to type 2-diabetes.

New perspectives on the melanocortins and their cardiovascular effects: Potential implications for the treatment of cardiovascular diseases with melanocortin analogues

The melanocortin peptides, including melanocyte-stimulating hormones, α-, β- and γ-MSH, are derived from the precursor peptide proopiomelanocortin and mediate their biological actions via five different melanocortin receptors, named from MC1 to MC5. Melanocortins have been implicated in the central regulation of energy balance and cardiovascular functions, but their local effects, via yet unidentified sites of action, in the vasculature, and their therapeutic potential in major vascular pathologies remain unclear. Therefore, the main aim of this thesis was to characterise the role of melanocortins in circulatory regulation, and to investigate whether targeting of the melanocortin system by pharmacological means could translate into therapeutic benefits in the treatment of cardiovascular diseases such as hypertension. In experiments designed to elucidate the local effects of α-MSH on vascular tone, it was found that α-MSH improved blood vessel relaxation via a nitric oxide (NO)-dependent mechanism without directly contracting or relaxing blood vessels. Furthermore, α-MSH was shown to regulate the expression and function of endothelial NO synthase in cultured human endothelial cells via melanocortin 1 receptors. In keeping with the vascular protective role, pharmacological treatment of mice with α-MSH analogues displayed therapeutic efficacy in conditions associated with vascular dysfunction such as obesity. Furthermore, α-MSH analogues elicited marked diuretic and natriuretic responses, which together with their vascular effects, seemed to provide protection against sodium retention and blood pressure elevation in experimental models of hypertension. In conclusion, the present results identify novel effects for melanocortins in the local control of vascular function, pointing to the potential future use of melanocortin analogues in the treatment of cardiovascular pathologies.

Chemokines, lymphocytes, and HIV

Chemokines are members of a family of more than 30 human cytokines whose best-described activities are as chemotactic factors for leukocytes and that are presumed to be important in leukocyte recruitment and trafficking. While many chemokines can act on lymphocytes, the roles of chemokines and their receptors in lymphocyte biology are poorly understood. The recent discoveries that chemokines can suppress infection by HIV-1 and that chemokine receptors serve, along with CD4, as obligate co-receptors for HIV-1 entry have lent urgency to studies on the relationships between chemokines and lymphocytes. My laboratory has characterized Mig and Crg-2/IP-10, chemokines that are induced by IFN-g and that specifically target lymphocytes, particularly activated T cells. We have demonstrated that the genes for these chemokines are widely expressed during experimental infections in mice with protozoan and viral pathogens, but that the patterns of mig and crg-2 expression differed, suggesting non-redundant roles in vivo. Our related studies to identify new chemokine receptors from activated lymphocytes resulted in the cloning of STRL22 and STRL33. We and others have shown that STRL22 is a receptor for the CC chemokine MIP-3a, and STRL22 has been re-named CCR6. Although STRL33 remains an orphan receptor, we have shown that it can function as a co-receptor for HIV-1 envelope glycoproteins, and that it is active with a broader range of HIV-1 envelope glycoproteins than the major co-receptors described to date. The ability of STRL33 to function with a wide variety of envelope glycoproteins may become particularly important if therapies are instituted to block other specific co-receptors. We presume that investigations into the roles of chemokines and their receptors in lymphocyte biology will provide information important for understanding the pathogenesis of AIDS and for manipulating immune and inflammatory responses for clinical benefit