Evidence that ET-1, but not ET-3 and S6b, ET(A)-receptor mediated contractions in isolated rat mesenteric arteries are modulated by co-activation of ET(B) receptors.

The effects of agonists with endothelin (ET) ET(A)-receptor activity have been analysed in relation to their interaction with ET(B) receptors in rat mesenteric arteries. ET-1, sarafotoxin 6b (S6b) and ET-3 induced large, slow-onset and sustained contractions whereas S6c induced weak transient contractions. However, following pre-contraction with U46619 and subsequent relaxation with forskolin, the effect of S6c was amplified, indicating a potential for powerful ET(B)-receptor mediated contraction. The selective ET(A)-receptor antagonist, FR139317, produced parallel rightward shifts of ET-1, S6b and ET-3 concentration-effect curves indicating that the contractions were mediated by ET(A) receptors. However, the corresponding FR139317 pK(B) values were significantly different between the agonists. As expected FR139317 had no effect on S6c responses. Pre-treatment with S6c to desensitize ET(B) receptors, increased ET-1 potency and the pK(B) value for FR139317. In contrast, neither the potency of S6b and ET-3 nor the pK(B) values for FR139317 estimated using these agonists were affected by ET(B)-receptor desensitization. Segments pre-contracted with submaximal concentrations of S6b and ET-3, but not ET-1, rapidly relaxed following wash-out or FR139317 administration. The results indicate that the small contractile response to selective ET(B) receptor activation, barely detectable under standard bioassay conditions, is greatly amplified when adenylate cyclase activity is elevated. Moreover, the response to ET(A) receptor activation by ET-1, but not ET-3 and S6b, is significantly modified by co-activation of ET(B) receptors. This interaction has a significant effect on the apparent affinity of ET(A)-receptor selective antagonists when ET-1 is used as agonist and decreases the potency of ET-1.     

Placental passage of olomoucine II,but not purvalanol A,is affected by p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and multidrug resistance-associated proteins (ABCCs)

1.?Purine cyclin-dependent kinase inhibitors have recently been recognised as promising candidates for the treatment of various cancers. While pharmacodynamic properties of these compounds are relatively well understood, their pharmacokinetics including possible interactions with placental transport systems have not been characterised to date.

2.?In this study, we investigated transplacental passage of olomoucine II and purvalanol A in rat focusing on possible role of p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and/or multidrug resistance-associated proteins (ABCCs). Employing the in situ method of dually perfused rat term placenta, we demonstrate transplacental passage of both olomoucine II and purvalanol A against the concentration gradient in foetus-to-mother direction. Using several ATP-binding cassette (ABC) drug transporter inhibitors, we confirm the participation of ABCB1, ABCG2 and ABCCs transporters in the placental passage of olomoucine II, but not purvalanol A.

3.?Transplacental passage of olomoucine II and purvalanol A from mother to foetus is significantly reduced by active transporters, restricting thereby foetal exposure and providing protection against harmful effects of these xenobiotics. Importantly, we demonstrate that in spite of their considerable structural similarity, the two molecules utilise distinct placental transport systems. These facts should be kept in mind when introducing these prospective anticancer candidates and/or their analogues into the clinical area.     

The immune system of geriatric mice is modulated by estrogenic endocrine disruptors (diethylstilbestrol, alpha-zearalanol, and genistein): effects on interferon-gamma

The immune system is a potential target for estrogenic endocrine disrupters. To date, there is limited information on whether estrogenic endocrine disruptors modulate the immune system of aged individuals. To address this issue, groups of 74-week-old mice were given nine oral doses of selected estrogenic endocrine disrupters: diethylstilbestrol (DES, 3 microg/100 g bw), alpha-zearalanol (0.5 mg/100 g bw), or genistein (0.15 mg/100 g bw) in corn oil, or corn oil alone, over 2.5 weeks. Both developmental (thymus) and mature (spleen) lymphoid organs were affected, although specific effects varied with the chemical. DES significantly decreased thymocyte numbers. However, relative percentages of thymocyte subsets were not altered. While splenic cellularity and percentages of T and B cells were unchanged, splenocytes from DES-exposed mice had significantly decreased ability to proliferate in response to Concanavalin-A (Con-A). Con-A-activated splenocytes from mice treated with genistein or alpha-zearalanol had decreased levels of interferon-gamma (IFNgamma) protein in their culture supernatants compared to similar cultures from oil-treated mice. RT-PCR analysis of Con-A-activated splenocytes revealed that the expression of IFNgamma gene is altered by DES or genistein treatment. Together, these results suggest that estrogenic endocrine disruptors modulate the immune system of aged mice.     

Docetaxel (Taxotere) is not metabolized by recombinant human CYP1B1 in vitro, but acts as an effector of this isozyme.

The objective of this study was to determine whether recombinant human cytochrome P450 1B1 (rhCYP1B1) metabolizes the anticancer agent docetaxel (Taxotere) in vitro. First, the catalytic activities of Supersomes-expressed rhCYP1B1 toward 17beta-estradiol and of rhCYP3A4 toward docetaxel in our conditions were determined. Second, [(14)C]docetaxel at 0.1 and 1 microM was incubated with rhCYP1B1 in the presence of NADPH up to 60 min. No metabolism of docetaxel was detected. Third, several activators of P450 isoenzymes were added to docetaxel incubations with rhCYP1B1, such as 2-chloro 3-pyridine 3-yl 5,6,7,8-tetrahydroindolizine 1-carboxamide, alpha-naphthoflavone, and organic solvents. Again, no metabolism of docetaxel was detected. As a forth step, 10 incubation factors were tested at two levels each in 16 different combinations, using a fractional factorial statistical experimental design. Docetaxel was not metabolized by rhCYP1B1 under any of the combinations. As a final step, the effect of docetaxel on the rhCYP1B1-mediated 7-ethoxyresorufin O-deethylase (EROD) activity was studied, to evaluate if docetaxel can bind to CYP1B1. Alpha-Naphthoflavone (1 microM), a CYP1B1 inhibitor, totally inhibited the EROD activity. Docetaxel at 3, 10, and 30 microM did not show major effects on EROD activity. At 100 microM, docetaxel increased EROD activity by 3.8-fold. Additionally, it was shown that 7-epidocetaxel, which is in equilibrium with docetaxel as a minor compound in solutions, was a potent activator of rhCYP1B1, with a >7-fold increase of EROD activity at 10 microM. In conclusion, docetaxel was not metabolized by recombinant human CYP1B1 in vitro, under any of the conditions tested. Docetaxel was shown to bind to recombinant human CYP1B1 and to act as an effector of this enzyme.     

Antioxidant effects of angiotensin-converting enzyme (ACE) inhibitors: free radical and oxidant scavenging are sulfhydryl dependent, but lipid peroxidation is inhibited by both sulfhydryl- and nonsulfhydryl-containing ACE inhibitors.

With an assay that generates free radicals (FR) through photooxidation of dianisidine sensitized by riboflavin, 4 x 10(-5) M captopril, epicaptopril (SQ 14,534, captopril's stereoisomer), zofenopril, and fentiapril [all sulfhydryl (-SH)-containing angiotensin-converting enzyme (ACE) inhibitors] were shown effective scavengers of nonsuperoxide free radicals whereas non-SH ACE inhibitors were not. Captopril was a more effective FR scavenger at pH 5.0 than at pH 7.5. Captopril (2 x 10(-5) M) also scavenged the other toxic oxygen species hydrogen peroxide and singlet oxygen and inhibited microsomal lipid peroxidation. Finally, captopril reduced the amount of superoxide anion-radical detected after neutrophils in whole blood were activated with zymosan, probably by inhibiting leukocyte superoxide production.     

Metabolic disruption in male mice due to fetal exposure to low but not high doses of bisphenol A (BPA): Evidence for effects on body weight,food intake,adipocytes, leptin,adiponectin, insulin and glucose regulation

Exposure to bisphenol A (BPA) is implicated in many aspects of metabolic disease in humans and experimental animals. We fed pregnant CD-1 mice BPA at doses ranging from 5 to 50,000 μg/kg/day, spanning 10-fold below the reference dose to 10-fold above the currently predicted no adverse effect level (NOAEL). At BPA doses below the NOAEL that resulted in average unconjugated BPA between 2 and 200 pg/ml in fetal serum (AUC_0–24 h), we observed significant effects in adult male offspring: an age-related change in food intake, an increase in body weight and liver weight, abdominal adipocyte mass, number and volume, and in serum leptin and insulin, but a decrease in serum adiponectin and in glucose tolerance. For most of these outcomes non-monotonic dose–response relationships were observed; the highest BPA dose did not produce a significant effect for any outcome. A 0.1-μg/kg/day dose of DES resulted in some but not all low-dose BPA outcomes.     

Persistent (>24h) long-term depression in the dentate gyrus of freely moving rats is not dependent on activation of NMDA receptors, L-type voltage-gated calcium channels or protein synthesis

Hippocampal long-term depression (LTD) comprises a persistent reduction of synaptic strength that is typically induced by low frequency stimulation (LFS). Although LTD has been described for the dentate gyrus in vitro, this phenomenon in the dentate gyrus of the intact animal is less well understood. In the current study, we investigated the contribution of NMDA receptors, L-type voltage gated calcium channels and protein synthesis to LFS-induced LTD in the dentate gyrus of freely moving rats. Animals were implanted with electrodes to enable chronic measurement of evoked potentials from medial perforant path-dentate gyrus synapses. LTD persisted for at least 24h, and was unaffected by prior treatment with the NMDA receptor antagonists AP5 or ifenprodil, which, in contrast, prevented LTP. Neither the L-type voltage-gated calcium channel antagonist, methoxyverapamil, nor the protein translation inhibitors, anisomycin or emetine had an effect on the profile of LTD. Our results suggest that NMDA receptors and L-type voltage-gated calcium channels are not involved in the induction of LTD in the dentate gyrus in vivo. Intriguingly, persistent LTD can be established without the synthesis of new proteins, suggesting that in the dentate gyrus, alternative mechanisms exist for the sustainment of enduring LTD.     

Protein kinase calpha but not p44/42 mitogen-activated protein kinase, p38, or c-Jun NH(2)-terminal kinase is required for intercellular adhesion molecule-1 expression mediated by interleukin-1beta: involvement of sequential activation of tyrosine kinase, nuclear factor-kappaB-inducing kinase, and IkappaB kinase 2

IL-1beta induced an increase in ICAM-1 expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C inhibitor (D609) attenuated IL-1beta-induced ICAM-1 expression. IL-1beta produced an increase in PKC activity and this effect was abolished by D609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited IL-1beta-induced response. TPA, a PKC activator, stimulated ICAM-1 expression as well, this effect being inhibited by tyrosine kinase inhibitors. Treatment of cells with IL-1beta resulted in stimulation of p44/42 MAPK, p38, and JNK. However, neither the mitogen activated protein kinase kinase inhibitor PD 98059 nor the p38 inhibitor SB 203580 affected IL-1beta-induced ICAM-1 expression. NF-kappaB DNA-protein binding and ICAM-1 promoter activity were enhanced by IL-1beta and these effects were inhibited by tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity as well, these effects being inhibited by tyrosine kinase inhibitors. Dominant-negative PKCalpha, NIK, or IKK2, but not IKK1 mutant, inhibited IL-1beta- or TPA-induced ICAM-1 promoter activity. IKK activity was stimulated by either IL-1beta or TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. Taken together, IL-1beta activates phosphatidylcholine-specific phospholipase C and induces activation of PKCalpha and protein tyrosine kinase, resulting in the stimulation of NIK, IKK2, and NF-kappaB in the ICAM-1 promoter, then initiation of ICAM-1 expression. However, activation of p44/42 MAPK, p38, and JNK is not involved.