Endothelin A receptor blockade improves endothelium-dependent relaxation in obese woman.

Hypertension in obesity is associated with increased insulin resistance, vascular mass and body mass index (BMI). The purpose of the study was to visualize endothelin-1 (ET-1) mediated constriction in arteries isolated from subcutaneous adipose tissue from obese hypertensive women previously operated by gastric bypass. Functional studies were conducted in a microvascular myograph. Expressed as percentage of contraction elicited by 124 mM KCl concentration-response curves for ET-1 were shifted leftward in arteries from obese hypertensive patients compared to healthy normotensive subjects. The vasodilator response to the ET-1 antagonist BQ123 (1 microM) was significantly higher in arteries from obese hypertensive patients (p<0.001). BQ123 induced relaxation was inhibited by NO synthase inhibitor L-NAME (0.1 nM). Preincubation with BQ123 enhanced the relaxation induced by acetylcholine (ACh; 0.1 nM - 0.1 mM) (p<0.001), but not that induced by NO donor sodium nitroprusside (SNP; 0.1 nM - 0.1 mM), in arteries from obese hypertensive patients. The present study show that hypertension yet prevail after gastric bypass surgery and the ET(A) receptor antagonist BQ123 may be a useful tool in reducing blood pressure in obese hypertensive patients.

Non-canonical HIF-2α function drives autonomous breast cancer cell growth via an AREG-EGFR/ErbB4 autocrine loop.

Tumor progression is intrinsically tied to the clonal selection of tumor cells with acquired phenotypes allowing to cope with a hostile microenvironment. Hypoxia-inducible factors (HIFs) master the transcriptional response to local tissue hypoxia, a hallmark of solid tumors. Here, we report significantly longer patient survival in breast cancer with high levels of HIF-2α. Amphiregulin (AREG) and WNT1-inducible signaling pathway protein-2 (WISP2) expression was strongly HIF-2α-dependent and their promoters were particularly responsive to HIF-2α. The endogenous AREG promoter recruited HIF-2α in the absence of a classical HIF-DNA interaction motif, revealing a novel mechanism of gene regulation. Loss of AREG expression in HIF-2α-depleted cells was accompanied by reduced activation of epidermal growth factor (EGF) receptor family members. Apparently opposing results from patient and in vitro data point to an HIF-2α-dependent auto-stimulatory tumor phenotype that, while promoting EGF signaling in cellular models, increased the survival of diagnosed and treated human patients. Our findings suggest a model where HIF-2α-mediated autocrine growth signaling in breast cancer sustains a state of cellular self-sufficiency, thereby masking unfavorable microenvironmental growth conditions, limiting adverse selection and improving therapy efficacy. Importantly, HIF-2α/AREG/WISP2-expressing tumors were associated with luminal tumor differentiation, indicative of a better response to classical treatments. Shifting the HIF-1/2α balance toward an HIF-2-dominated phenotype could thus offer a novel approach in breast cancer therapy.

Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2.

Endosialin is a transmembrane glycoprotein selectively expressed in blood vessels and stromal fibroblasts of various human tumours. It has been functionally implicated in angiogenesis, but the factors that control its expression have remained unclear. As insufficient delivery of oxygen is a driving force of angiogenesis in growing tumours, we investigated whether hypoxia regulates endosialin expression. Here, we demonstrate that endosialin gene transcription is induced by hypoxia predominantly through a mechanism involving hypoxia-inducible factor-2 (HIF-2) cooperating with the Ets-1 transcription factor. We show that HIF-2 activates the endosialin promoter both directly, through binding to a hypoxia-response element adjacent to an Ets-binding site in the distal part of the upstream regulatory region, and indirectly, through Ets-1 and its two cognate elements in the proximal promoter. Our data also suggest that the SP1 transcription factor mediates responsiveness of the endosialin promoter to high cell density. These findings elucidate important aspects of endosialin gene regulation and provide a rational frame for future investigations towards better understanding of its biological significance.

Interactions of polybrominated diphenyl ethers with the aryl hydrocarbon receptor pathway.

Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants that have been in use as additives in various consumer products. Structural similarities of PBDEs with other polyhalogenated aromatic hydrocarbons that show affinity for the aryl hydrocarbon receptor (AhR), such as some polychlorinated biphenyls, raised concerns about their possible dioxin-like properties. We studied the ability of environmentally relevant PBDEs (BDE-47, -99, -100, -153, -154, and -183) and the "planar" congener BDE-77 to bind and/or activate the AhR in stably transfected rodent hepatoma cell lines with an AhR-responsive enhanced green fluorescent protein (AhR-EGFP) reporter gene (H1G1.1c3 mouse and H4G1.1c2 rat hepatoma). 7-Ethoxyresorufin-O-deethylation (EROD) was used as a marker for CYP1A1 activity. Dose- and bromination-specific inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced responses was measured by their ability to inhibit the induction of AhR-EGFP expression and EROD activity. Individual exposure to these PBDEs did not result in any increase in induction of AhR-EGFP or CYP1A1 activity. The lower brominated PBDEs showed the strongest inhibitory effect on TCDD-induced activities in both cell lines. While the highest brominated PBDE tested, BDE-183, inhibited EROD activity, it did not affect the induction of AhR-EGFP expression. Similar findings were observed after exposing stably transfected human hepatoma (xenobiotic response element [XRE]-HepG2) cells to these PBDEs, resulting in a small but statically significant agonistic effect on XRE-driven luciferase activity. Co-exposure with TCDD resulted again in antagonistic effects, confirming that the inhibitory effect of these PBDEs on TCDD-induced responses was not only due to direct interaction at receptor level but also at DNA-binding level. This antagonism was confirmed for BDE-99 in HepG2 cells transiently transfected with a Gal4-AhR construct and the corresponding Gal4-Luc reporter gene. In addition, a chromatin immunoprecipitation assay further confirmed that BDE-99 could bind to the AhR and activate the AhR nuclear translocation and dioxin responsive element (DRE) binding in the context of the CYP1A1 promoter. However, the transactivation function of the BDE-99-activated AhR seems to be very weak. These combined results suggest that PBDEs do bind but not activate the AhR-AhR nuclear translocator protein-XRE complex.

Structure and expression of the Ah receptor repressor gene.

The aryl hydrocarbon receptor (AhR) repressor (AhRR) gene has been isolated and characterized from a mouse genomic library. The gene is distributed as 11 exons in a total length of about 60 kilobase pairs. Fluorescence in situ hybridization analysis has shown that the AhRR gene is located at mouse chromosome 13C2, at rat chromosome 1p11.2, and at human chromosome 5p15.3. The AhRR gene has a TATA-less promoter and several transcription start sites. In addition, putative regulatory DNA sequences such as xenobiotic responsive element (XRE), GC box, and NF-kappaB-binding sites have been identified in the 5'-upstream region of the AhRR gene. Transient transfection analyses of HeLa cells with reporter genes that contain deletions and point mutations in the AhRR promoter revealed that all three XREs mediated the inducible expression of the AhRR gene by 3-methylcholanthrene treatment, and furthermore, GC box sequences were indispensable for a high level of inducible expression and for constitutive expression. Moreover, by using gel mobility shift assays we were able to show that the AhR/Arnt heterodimer binds to the XREs with very low affinity, which is due to three varied nucleotides outside the XRE core sequence. We have also shown that Sp1 and Sp3 can bind to the GC boxes. Finally, both transient transfection analysis and gel mobility shift assay revealed that the AhRR gene is up-regulated by a p65/p50 heterodimer that binds to the NF-kappaB site when the cells has been exposed to 12-O-tetradecanoylphorbol-13-acetate, and this inducible expression was further enhanced by cotreatment of 12-O-tetradecanoylphorbol-13-acetate and 3-methylcholanthrene.

Repression of dioxin signal transduction in fibroblasts. Identification Of a putative repressor associated with Arnt.

Heterodimeric complexes of basic helix-loop-helix/PAS transcription factors are involved in regulation of diverse physiological phenomena such as circadian rhythms, reaction to low oxygen tension, and detoxification. In fibroblasts, the basic helix-loop-helix/PAS heterodimer consisting of the ligand-inducible dioxin receptor and Arnt shows DNA-binding activity, and the receptor and Arnt are able to activate transcription when fused to a heterologous DNA-binding domain. However, fibroblasts are nonresponsive to dioxin with regard to induction mediated by the DNA response element recognized by the receptor and Arnt. Here we demonstrate that Arnt is associated with a fibroblast-specific factor, forming a complex that is capable of binding the dioxin response element. This factor may function as a repressor since negative regulation of target gene induction appears to be abolished by inhibition of histone deacetylase activity by trichostatin A. Finally, the negative regulatory function of this factor appears to be restricted for dioxin signaling since Arnt was able to mediate, together with hypoxia-inducible factor-1alpha, transcriptional activation in hypoxic cells. Taken together, these data suggest that fibroblast-specific inhibition of dioxin responsiveness involves recruitment by Arnt of a cell type- and signaling pathway-specific corepressor associated with a histone deacetylase.

Activation of hypoxia-inducible factor 1alpha: posttranscriptional regulation and conformational change by recruitment of the Arnt transcription factor.

In response to hypoxia the hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of a network of genes encoding erythropoietin, vascular endothelial growth factor, and several glycolytic enzymes. HIF-1 consists of a heterodimer of two basic helix-loop-helix PAS (Per/Arnt/Sim) proteins, HIF-1alpha and Arnt. HIF-1alpha and Arnt mRNAs are constitutively expressed and were not altered upon exposure of HeLa or HepG2 cells to hypoxia, suggesting that the activity of the HIF-1alpha-Arnt complex may be regulated by some as yet unknown posttranscriptional mechanism. In support of this model, we demonstrate here that Arnt protein levels were not increased under conditions that induce an hypoxic response in HeLa and HepG2 cells. However, under identical conditions, HIF-1alpha protein levels were rapidly and dramatically up-regulated, as assessed by immunoblot analysis. In addition, HIF-1alpha acquired a new conformational state upon dimerization with Arnt, rendering HIF-1alpha more resistant to proteolytic digestion in vitro. Dimerization as such was not sufficient to elicit the conformational change in HIF-1alpha, since truncated forms of Arnt that are capable of dimerizing with HIF-1alpha did not induce this effect. Moreover, the high affinity DNA binding form of the HIF-1alpha-Arnt complex was only generated by forms of Arnt capable of eliciting the allosteric change in conformation. In conclusion, the combination of enhanced protein levels and allosteric change by dimerization defines a novel mechanism for modulation of transcription factor activity.

Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor.

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the intracellular dioxin receptor mediate hypoxia and dioxin signalling, respectively. Both proteins are conditionally regulated basic helix-loop-helix (bHLH) transcription factors that, in addition to the bHLH motif, share a Per-Arnt-Sim (PAS) region of homology and form heterodimeric complexes with the common bHLH/PAS partner factor Arnt. Here we demonstrate that HIF-1 alpha required Arnt for DNA binding in vitro and functional activity in vivo. Both the bHLH and PAS motifs of Arnt were critical for dimerization with HIF-1 alpha. Strikingly, HIF-1 alpha exhibited very high affinity for Arnt in coimmunoprecipitation assays in vitro, resulting in competition with the ligand-activated dioxin receptor for recruitment of Arnt. Consistent with these observations, activation of HIF-1 alpha function in vivo or overexpression of HIF-1 alpha inhibited ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptor function on minimal reporter gene constructs. However, HIF-1 alpha- and dioxin receptor-mediated signalling pathways were not mutually exclusive, since activation of dioxin receptor function did not impair HIF-1 alpha-dependent induction of target gene expression. Both HIF-1 alpha and Arnt mRNAs were expressed constitutively in a large number of human tissues and cell lines, and these steady-state expression levels were not affected by exposure to hypoxia. Thus, HIF-1 alpha may be conditionally regulated by a mechanism that is distinct from induced expression levels, the prevalent model of activation of HIF-1 alpha function. Interestingly, we observed that HIF-1 alpha was associated with the molecular chaperone hsp90. Given the critical role of hsp90 for ligand binding activity and activation of the dioxin receptor, it is therefore possible that HIF-1 alpha is regulated by a similar mechanism, possibly by binding an as yet unknown class of ligands.

Differential effects of a topoisomerase I inhibitor on dioxin inducibility and high-level expression of the cytochrome P450IA1 gene.

The basic helix-loop-helix containing dioxin receptor mediates dioxin signal transduction. The ligand-activated receptor complex binds to specific sequences termed xenobiotic response elements and regulates transcription of target genes such as the gene for cytochrome P450IA1. This study demonstrates that induction of cytochrome P450IA1 and P450IB1 gene expression by a dioxin receptor ligand is repressed by camptothecin, an inhibitor of the topoisomerase I enzyme. However, a transiently transfected reporter construct under control of an xenobiotic response element-containing promoter was not affected by the topoisomerase inhibitor. In agreement with this observation, ligand-dependent activation of the dioxin receptor to its DNA-binding form is not altered by camptothecin as analyzed by electrophoretic mobility shift assay. Moreover, the inhibitory effect of camptothecin cannot be exerted once the P450IA1 gene has been activated. These results imply that topoisomerase I activity is necessary for the primary P450IA1 induction response, possibly involving dioxin-dependent alterations in chromatin structure of the P450IA1 promoter.

A tyrosine kinase-dependent pathway regulates ligand-dependent activation of the dioxin receptor in human keratinocytes.

Signal transduction by dioxin is mediated by the intracellular basic helix-loop-helix dioxin receptor which, in its ligand-activated state, binds to target DNA as a heteromeric complex with the partner factor Arnt. In contrast, the repressed form of the receptor is a complex with hsp90 which appears to maintain the receptor in an inducible conformation. In human keratinocytes dioxin receptor activation has previously been shown to depend on phosphorylation processes. To further dissect mechanisms regulating dioxin receptor function the importance of tyrosine phosphorylation was investigated by the use of specific tyrosine kinase inhibitors. Here we report that the inhibitor genistein inhibited dioxin-dependent induction of expression of the target gene cytochrome P-450IA1. This effect was rapid and reversible and did not lead to altered levels of dioxin receptor protein. Analyses of dioxin receptor or Arnt fusion proteins that function independently of one another showed that the target for genistein action was the dioxin receptor, and, more specifically, a region of the receptor harboring its ligand-binding domain. In addition, function of an unrelated transactivator, the glucocorticoid receptor, was inhibited by genistein while a truncated form lacking the ligand-binding domain was not. A common denominator between the ligand-binding domains of both receptors is their ability to interact with hsp90. Importantly, co-immunoprecipitation experiments showed that genistein inhibited ligand-induced release of hsp90 from the glucocorticoid receptor. Thus, the interaction of these transactivators with hsp90 may be regulated by a tyrosine kinase-dependent pathway.

Nonresponsiveness of normal human fibroblasts to dioxin correlates with the presence of a constitutive xenobiotic response element-binding factor.

Polychlorinated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzofuran (TCDF) have been shown to induce transcription of the cytochrome P-450IA1 gene by activating an intracellular receptor protein (the Ah- or dioxin receptor) to bind to specific DNA sequences, termed xenobiotic response elements (XREs). However, the expression and inducibility of the cytochrome P-450IA1 activity exhibit tissue-specific differences. With regard to the TCDF induction response, we have examined three human cell types of endodermal (the hepatoma cell line HepG2), ectodermal (normal keratinocytes), and mesodermal origin (normal fibroblasts). DNase I hypersensitivity analysis of the 5' flank and first intron of the P-450IA1 gene showed that in the nonresponsive fibroblasts the chromatin structure lacked open regions while in the two responsive cell types (keratinocytes and HepG2) several constitutive hypersensitive sites as well as TCDF-induced alterations in the chromatin structure could be detected. This observation might correlate with the fact that the XRE, in either the context of the P-450IA1 gene sequences or in front of a heterologous promoter, was inefficient in directing a TCDF induction response in fibroblasts. In in vitro DNA binding studies, the dioxin receptor was activated to a DNA-binding nuclear form in all three cell types. However, in fibroblast nuclear extracts two novel constitutive protein-XRE complexes were detected. The fibroblast factor(s) were immunochemically distinct from the receptor but exhibited indistinguishable DNA binding specificity. These data are compatible with a model where the P-450IA1 is noninducible in fibroblasts due to the presence of a putative repressor(s) which may compete effectively with the receptor for binding to the response element as indicated by in vitro DNA-binding off-rate experiments.

Cardiovascular effects of intrathecal administration of agents active at 5-hydroxytryptamine1-receptors in the rat: modulation by substance P and a substance P antagonist.

The intrathecal (i.th., T8-10) administration in conscious rats of the 5-hydroxytryptamine (5-HT)1A agonist 8-OH-DPAT (10 nmol), and to a lesser extent the 5-HT1B agonist CGS 12066B (6 nmol), resulted in a blood pressure reduction and a bradycardia. These responses were prevented by the i.th. pretreatment with substance P (SP) (6.5 nmol) and enhanced following pretreatment with the non-peptide SP antagonist CP-96,345 (10 nmol). The partial 5-HT1A agonist 8-MeO-CLEPAT (10 nmol) had by itself small cardiovascular effects. Following the pretreatment with SP, 8-MeO-CLEPAT caused a pressor response and a tachycardia whereas the opposite effects were observed following pretreatment with the SP antagonist. These results support the notion of a functional interaction between serotonergic and SP mechanisms at the level of the preganglionic sympathetic nerves in the spinal cord.

Cross-coupling of signal transduction pathways: the dioxin receptor mediates induction of cytochrome P-450IA1 expression via a protein kinase C-dependent mechanism.

Signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) is mediated by the intracellular dioxin receptor which, in its dioxin-activated state, regulates transcription of target genes encoding drug-metabolizing enzymes, such as cytochrome P-450IA1 and glutathione S-transferase Ya. Exposure of the dioxin receptor to dioxin leads to an apparent translocation of the receptor to the nucleus in vivo and to a rapid conversion of the receptor from a latent, non-DNA-binding form to a species that binds to dioxin-responsive positive control elements in vitro. This DNA-binding form of receptor appears to be a heterodimeric complex with the helix-loop-helix factor Arnt. In this study, we show that activation of the cytochrome P-450IA1 gene and minimal dioxin-responsive reporter constructs by the dioxin receptor was inhibited following prolonged treatment of human keratinocytes with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Inhibition of the receptor-mediated activation response was also achieved by treatment of the cells with a number of protein kinase inhibitors, one of which, calphostin C, shows selectivity for protein kinase C. Taken together, these data suggest that protein kinase C-dependent phosphorylation may play an essential role in the dioxin signaling pathway. This hypothesis is supported by the observation that pretreatment of the cells with 12-O-tetradecanoylphorbol-13-acetate inhibited the DNA-binding activity of the dioxin receptor in vivo. In vivo, the dioxin receptor was found to be a phosphoprotein. In vitro, dephosphorylation of the ligand-activated, heteromeric dioxin receptor form or dephosphorylation of the individual ligand-binding and Arnt receptor subunits inhibited the xenobiotic response element-binding activity. Moreover, dephosphorylation experiments with the individual receptor subunits prior to assembly of the xenobiotic response element-binding receptor form indicated that phosphorylation seemed to be important for the DNA-binding activity per se of the receptor, whereas Arnt appeared to require phosphorylation to interact with the receptor. Finally, a protein kinase C inhibitor-sensitive cytosolic catalytic activity that could restore the DNA-binding activity of the dephosphorylated dioxin receptor form was identified.

Contrasting cardiovascular responses from intrathecal administration of epinephrine and norepinephrine in conscious rats: role of alpha 1- and alpha 2-adrenoceptors.

In conscious rats, intrathecal (i.t.) administration of norepinephrine (NE) produced pressor responses, whereas i.t. epinephrine (Epi) caused depressor responses at low doses (0.1-1 microgram) and pressor responses at a higher dose (10 micrograms). Epi administered i.t. produced bradycardia; however, NE caused tachycardia at low doses and bradycardia at high doses. The cardiovascular responses were dissimilar to those observed after intravenous (i.v.) administration of these doses of NE and Epi. When [3H]NE or [3H]Epi (1.0 microgram, 10 mCi) was injected i.t., minimal radioactivity was detected in peripheral blood (PB) samples, indicating that the effects of i.t.-injected catecholamines on blood pressure (BP) and heart rate (HR) are due to stimulation of central spinal adrenoceptors and not to peripheral effects after leakage. Pretreatment with i.t. administration of the alpha 1-antagonist prazosin (1.0 microgram) attenuated pressor responses and tachycardia produced by i.t. NE (1.0 microgram), whereas i.t. pretreatment with the alpha 2-antagonist yohimbine (10 micrograms) counteracted depressor responses and bradycardia produced by i.t. Epi. Therefore, these spinally released catecholamines appear to produce opposite cardiovascular effects whereby sympathetic preganglionic neurons are excited by NE through spinal alpha 1-adrenoceptors and are inhibited by Epi through spinal alpha 2-adrenoceptors.

Substance P injection into the dorsal raphe increases blood pressure and serotonin release in hippocampus of conscious rats.

Microinjections of substance P (SP, 100 pmol) into the dorsal raphe nucleus (DRN) in conscious rats increased blood pressure and heart rate for 30-40 min. Concomitantly, the extracellular levels of 5-hydroxytryptamine (5-HT) in the ventral hippocampus, monitored by microdialysis, increased by 30% for 20 min compared with the vehicle control. Pretreatment with the 5-HT2 receptor antagonist, ritanserin (1 mg/kg i.v.), prevented the pressor response to SP but not the increase in heart rate. Pretreatment with the partial 5-HT1A receptor agonist, 8-methoxy-2-(N-2-chloroethyl-N-n-propyl)amino tetralin (8-MeO-CLEPAT, 10 micrograms/kg i.v.) prevented the increase in both blood pressure and heart rate. It is suggested that microinjections of SP into the DRN increase blood pressure through activation of serotonergic DRN neurons and that the postsynaptic receptor responsible for the pressor response is of the 5-HT2 type.

The stability of dioxin-receptor ligands influences cytochrome P450IA1 expression in human keratinocytes.

Three dioxin-receptor ligands were analyzed for their effect on cytochrome P450IA1 mRNA expression in normal human keratinocytes. Although a 2 h pulsed treatment with the receptor agonists 2,3,7,8-tetrachlorodibenzofuran (TCDF) and beta-naphthoflavone (BNF) gave the same maximal induction response, the effect of BNF was transient compared to effect of TCDF. This was most likely due to metabolism of BNF as exemplified by the fact that a P450IA1 enzyme suicide-inhibitor, 1-ethynylpyrene, could prolong the induction response following a short BNF treatment. The TCDF induction of a reporter gene construct under the control of the -1140 to +2435 part of the CYPIA1 gene transiently transfected into HK was effectively inhibited by the dioxin-receptor antagonist alpha-naphthoflavone (ANF). In addition, ANF inhibited the accumulation of TCDF-activated nuclear receptors with capacity to bind to a xenobiotic response element. Interestingly, ANF could also suppress already maximally induced P450IA1 mRNA levels. The data demonstrate that the stability of the ligand influences the long-term effects on gene expression and that the effect of stable ligands may be masked due to receptor antagonist presence. In addition, the results support the hypothesis that a constant low level of activated nuclear receptors is required to maintain induced P450IA1 expression.

Cardiovascular effects of intrathecal administration of substance P in the rat: interactions with serotonergic mechanisms.

Intrathecal (i.th.) administration of substance P (SP, 6.5 nmol) at the Th 8-10 level in conscious rats increased blood pressure (carotid artery), heart rate and plasma catecholamine concentrations. The responses were antagonized by the intravenous (i.v.) but not i.th. pretreatment with the 5-HT2-receptor antagonists ketanserin and ritanserin and intrathecally administered serotonin (5-HT, 10 micrograms). The pressor response and the increase in plasma noradrenaline concentrations were also antagonized by i.v. or i.th. pretreatment with the 5-HT1A-agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT). In contrast the pressor response to SP was facilitated by the 5-HT1A-antagonist 1-pindolol (i.v. or i.th). Pretreatment with SP (i.th) reduced the hypotensive response to i.v. 8-OH-DPAT. These results demonstrate functional interactions between SP and serotonergic mechanisms in the central system, but the precise location and nature were not elucidated.

Antihypertensive effects of chronic 5-hydroxytryptamine (5-HT2) receptor blockade with irindalone in the spontaneously hypertensive rat.

The present study was conducted to evaluate the antihypertensive effects of irindalone, a potent 5-HT2-receptor blocking agent with weak alpha 1-adrenoceptorblocking properties. Young spontaneously hypertensive rats (SHR) received irindalone in the food (daily intake estimated to 10 mg/kg) for 10 weeks and older SHR received irindalone subcutaneously for 2 weeks (3 mg/kg/day by osmotic pump). The indirect systolic blood pressure was measured each week (tail plethysmography) and at the end of the intervention periods the direct intraarterial blood pressure was measured in conscious rats. Subsequently a dose-response curve of pressor responses to phenylephrine was constructed in pithed rats. Chronic oral treatment with irindalone reduced the development of hypertension but influenced neither body weight nor heart weight. The subcutaneous treatment with irindalone reduced the blood pressure in relatively older SHR compared with controls. Pressor responses to phenylephrine were antagonized in rats receiving oral treatment. However, during subcutaneous treatment with irindalone the dose-response curve to phenylephrine was not influenced, suggesting that the blood pressure reduction was not directly related to a concomitant alpha 1-adrenoceptor blockade. These results demonstrate that irindalone effectively reduces the blood pressure in SHR and that no tolerance develops to its antihypertensive effects. Since the blood pressure reduction, at least following subcutaneous treatment, was not directly related to a concomitant alpha 1-blockade, it is suggested that the 5-HT2-receptor blockade may be of relevance for the antihypertensive effect.

Serum and extracellular calcium modulate induction of cytochrome P-450IA1 in human keratinocytes.

Culture conditions allowing for cytochrome P-450IA1 induction by 2,3,7,8-tetrachlorodibenzofuran (TCDF) in normal human keratinocytes (HK) were investigated. HK grown in serum-free low extracellular Ca2+ (0.1 mM) medium did not accumulate P-450IA1 mRNA in response to TCDF. If, however, the cultures were pretreated for more than 24 h with either serum or elevated extracellular Ca2+ (2.0 mM), induction of P-450IA1 was obtained by TCDF. Serum and elevated Ca2+ concentrations were found to be additive in this respect. When analyzing HK derived from five individuals, no apparent difference was found in the relative induction of P-450IA1 by increasing concentrations of TCDF, giving an EC50 of approximately 2 nM. The permissive effect of serum and elevated Ca2+ could be conferred to a reporter gene by the -1140 to +2435 part of the human CYPIA1 gene. Culture conditions allowing for P-450IA1 induction correlated with conditions that induced mRNA corresponding to the differentiation specific enzyme epidermal transglutaminase. This finding, together with the known differentiation promoting effects of serum and elevated Ca2+, suggest that terminal differentiation is necessary for P-450IA1 induction in HK by Ah receptor ligands.