Co-administration of conjugated linoleic acid and rosiglitazone increases atherogenic co-efficient and alters isoprenaline-induced vasodilatation in rats fed high fat diet.

The cis(c)-9, trans(t)-11 (c9,t11) and t10,c12 isomers of conjugated linoleic acid (CLA) have been reported as agonists of peroxisome proliferator-activated receptor (PPAR) and beneficial in lipidemia and glycemia. However, it is unclear whether CLA isomers enhance or antagonize effects of conventional drugs targeting PPAR. Male Sprague-Dawley rats were fed high fat diet (HFD) for 8 weeks and treated without or with CLA, rosiglitazone or both for 4 weeks. Oral glucose tolerance and surrogate markers of insulin resistance were not significantly different for all treatments compared to untreated normal diet (ND) or HFD group, except lipoprotein levels. The combination of CLA and rosiglitazone had suppressed levels of low and high density lipoproteins (46 % and 25 %, respectively), compared to HFD-alone. Conversely, the atherogenic co-efficient of the animals received HFD or HFD+rosiglitazone+CLA was 2-folds higher than ND, HFD+rosiglitazone or HFD+CLA. Isolated aortic rings from the combined CLA and rosiglitazone treated animals were less sensitive to isoprenaline-induced relaxation among endothelium-denuded aortas with a decreased efficacy and potency (R(max)=53+/-4.7 %; pEC50=6+/-0.2) compared to endothelium-intact aortas (R(max)=100+/-9.9 %; pEC50=7+/-0.2). Our findings illustrate that the combination of CLA and rosiglitazone precede the atherogenic state with impaired endothelium-independent vasodilatation before the onset of HFD-induced insulin resistance.

Contractile function of smooth muscle retained after overnight storage.

The functional responses of different overnight-stored in vitro tissues are not clearly described in any animal model. The influence of overnight storage in an animal model may vary between tissue types. We employed Sprague-Dawley rat as our animal model and investigated the functional changes of rat aorta, trachea, bronchus and bladder that were used (i) immediately after surgical removal (denoted as fresh) and (ii) after storage in aerated (95% O2, 5% CO2) Krebs-Ringer bicarbonate solution at 4 °C for 24 h (denoted as stored). The aorta ring was pre-contracted with phenylephrine, and the functional response of the tissue was investigated using isoprenaline, forskolin and carbachol. Carbachol was also used to increase the tone in trachea, bronchus rings and bladder strips. A clear reduced function of endothelium, with a minor if any effect in the smooth muscle function in rat aorta was observed after overnight storage. The contractile response of overnight-stored rat airway (trachea and bronchus) and bladder smooth muscles remained unchanged. Among all tested tissues, only bronchus showed a reduced response rate (only 40% responded) after storage. In vitro rat tissues that are stored in Krebs solution at 4 °C for 24 h can still be used to investigate smooth muscle responses, however, not endothelium-mediated responses for aorta. The influence of overnight storage on different tissues from an animal model (Sprague-Dawley rat in our study) also provides an insight in maximising the use of sacrificed animals.

Acalypha wilkesiana extracts induce apoptosis by causing single strand and double strand DNA breaks.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Acalypha wilkesiana have been used empirically by traditional healers in Southwest Nigeria together with other plants as a powder mixture to treat patients with breast tumours and inflammation. AIM OF THE STUDY: There is an increasing interest among researchers in searching for new anticancer drugs from natural resources, particularly plants. This study aimed to investigate the anticancer properties of Acalypha wilkesiana extracts and the characteristics of DNA damage against brain and lung cancer cells. MATERIALS AND METHODS: The antiproliferative activity of Acalypha wilkesiana extracts (ethyl acetate, hexane, and ethanol) was examined on human glioma (U87MG), human lung carcinoma (A549), and human lung fibroblast (MRC5) cells. RESULTS: Cell viability MTT assay revealed that ethyl acetate extract of the plant possessed significant antiproliferative effects against both U87MG (GI(50)=28.03 ± 6.44 µg/ml) and A549 (GI(50)=89.63 ± 2.12 µg/ml) cells (p value<0.0001). The hexane extract was found to exhibit crucial antiproliferative effects on U87MG (GI(50)=166.30 ± 30.50 µg/ml) (p value<0.0001) but not on A549 cells. Neither plant extract possessed noticeable antiproliferative effects on the non-cancerous MRC5 cells (GI(50)>300 µg/ml). The ethanol extract showed no antiproliferative effects on any cell line examined. Haematoxylin & Eosin (H & E) staining and single cell gel electrophoresis (SCGE) comet assay confirmed that plant extract-treated cells underwent apoptosis and not necrosis. SCGE comet assays confirmed that plant extracts caused both single strand (SSB) and double strand (DSB) DNA breaks that led to the execution of apoptosis. CONCLUSION: The extracts (especially ethyl acetate and hexane) of Acalypha wilkesiana possess valuable cytotoxic effects that trigger apoptosis in U87MG and A549 cancer cells through induction of DNA SSBs and DSBs.

Molecular and pharmacological evidence for MT1 melatonin receptor subtype in the tail artery of juvenile Wistar rats.

1. In this study reverse transcriptase-polymerase chain reaction (RT-PCR) has been used to identify mt1 and MT2 receptor mRNA expression in the rat tail artery. The contributions of both receptors to the functional response to melatonin were examined with the putative selective MT2 receptor antagonists, 4-phenyl-2-propionamidotetraline (4-P-PDOT) and 2-benzyl-N-pentanoyltryptamine. In addition, the action of melatonin on the second messenger cyclic AMP was investigated. 2. Using RT-PCR, mt1 receptor mRNA was detected in the tail artery from seven rats. In contrast MT2 receptor mRNA was not detected even after nested PCR. 3. At low concentrations of the MT2 selective ligands, neither 10 nM 4-P-PDOT (pEC50=8.70+/-0.31 (control) vs 8.73+/-0.16, n=6) nor 60 nM 2-benzyl-NV-pentanoyltryptamine (pEC50= 8.53+/-0.20 (control) vs 8.83+/-0.38, n = 6) significantly altered the potency of melatonin in the rat tail artery. 4. At concentrations non-selective for mt1 and MT2 receptors. 4-P-PDOT (3 microM) and 2-benzyl-N-pentanoyltryptamine (5 microM) caused a significant rightward displacement of the vasoconstrictor effect of melatonin. In the case of 4-P-PDOT, the estimated pKB (6.17+/-0.16, n=8) is similar to the binding affinity for mt1 receptor. 5. Pre-incubation with 1 microM melatonin did not affect the conversion of [3H]-adenine to [3H]-cyclic AMP under basal condition (0.95+/-0.19% conversion (control) vs 0.92+/-0.19%, n=4) or following exposure to 30 microM forskolin (5.20+/-1.30% conversion (control) vs 5.35+/-0.90%, n=4). 6. Based on the above findings, we conclude that melatonin receptor on the tail artery belongs to the MT1 receptor subtype, and that this receptor is probably independent of the adenylyl cyclase pathway.

Rilmenidine reveals differences in the pharmacological characteristics of prejunctional alpha2-adrenoceptors in the guinea-pig, rat and pig.

1. The alpha2A and alpha2D-adrenoceptor subtypes are thought to be species homologs most easily differentiated on the basis of the potency of antagonists. In the present study we have compared the effect of rilmenidine with two other selective alpha2-adrenoceptor agonists, UK-14304 (5-bromo-6- [2-imidazolin-2-ylamino]-quinoxaline) and clonidine, against electrically-evoked contractions in five isolated preparations from the rat, guinea-pig and pig, and, where possible, determined the receptor subtype involved. 2. UK-14034, clonidine and rilmenidine produced concentration-dependent inhibition of the electrically-evoked contractions of the rat isolated vas deferens and tail artery and the guinea-pig ileum. These inhibitory effects were reversed by the selective alpha2-adrenoceptor antagonist, RX-811058 (1 microM), except in the rat tail artery preparations where the remaining neurogenic response was inhibited; evidence for the involvement of 'innervated' alpha2-adrenoceptors. Both clonidine and UK-14304 produced concentration-dependent inhibition of responses in the porcine isolated tail artery and urinary bladder but clonidine was markedly less efficacious in these preparations. In contrast, rilmenidine failed to inhibit the neurogenic contractions in either preparation. 3. Although rilmenidine failed to elicit a detectable response in either the porcine isolated tail artery or urinary bladder, it (10 microM and 30 microM, respectively) competitively antagonised the inhibitory effects of UK-14304 with an estimated dissociation constant of (pK(B)) 5.82 and 5.93, respectively. 4. Prazosin (1 microM) failed to alter the effect of UK-14304 against neurogenic contractions in the porcine isolated urinary bladder, while rauwolscine (pK(B) 8.87) was 10 fold more potent than phentolamine (pK(B) 7.56). On the other hand, phentolamine (pK(B) 8.42) was only marginally more potent than rauwolscine (pK 8.05) against clonidine-induced inhibition of electrically-evoked contractions of the guinea-pig isolated ileum. This pharmacological evidence with antagonists supports the presence of alpha2D-adrenoceptors in the rat and guinea-pig and the alpha2A-adrenoceptors in the pig. 5. We have demonstrated that rilmenidine, unlike clonidine and UK-14304, is devoid of any agonist activity at prejunctional alpha2A-adrenoceptors in the pig, but is an efficacious agonist at alpha2D-adrenoceptors in the rat and guinea-pig.

Studies on the vasoconstrictor action of melatonin and putative melatonin receptor ligands in the tail artery of juvenile Wistar rats.

1. In this study we compared the vasoconstrictor activity of melatonin in rat isolated tail artery using two different recording systems, the Halpern pressure myograph and the Halpern-Mulvany wire myograph, with the view to determining a reliable method for obtaining pharmacological data on vascular melatonin receptors. In addition, we characterized the melatonin receptor in this preparation, using analogues of melatonin, and examined the activity of various naphthalenic derivatives with biological activity in non-vascular models of melatonin receptors. 2. Using the Halpern pressure myograph, cumulative addition of melatonin (0.1 nM to 1 microM) produced direct vasoconstriction (19.3+/-6.4% reduction in lumen diameter, n=5) in five of 11 pressurized segments, with pEC50 of 9.14+/-0.17. Similarly, non-cumulative application of melatonin caused vasoconstriction (19.7+/-4.6% reduction in lumen diameter, n=7) in seven of 20 preparations examined with pEC50 of 8.74+/-0.26. The selective alpha2-adrenoceptor agonist, UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate), produced vasoconstriction in all 'melatonin-insensitive' preparations. 3. Melatonin (0.1 nM to 1 microM) failed to elicit isometric contractions of tail artery segments in the Halpern wire myograph, but produced concentration-dependent potentiation of electrically-evoked, isometric contractions (maximum effect of 150-200% enhancement) when applied either noncumulatively (seven of seven preparations) or cumulatively (four of seven preparations). The pEC50 value of melatonin (non-cumulative) was 8.50+/-0.10 (n=7) which was not different from that obtained in the pressure myograph. All further experiments were conducted using a non-cumulative protocol against electrically-evoked, isometric contractions. 4. Based on the pEC50 values for the melatonin analogues examined, the pharmacological profile for the enhancement of electrically-evoked contractions was 2-iodomelatonin > 6-chloromelatonin > or = (-)-AMMTC > or = S21634 > or = melatonin > or = S20098 > S20242 > or = S20304 > 6-hydroxymelatonin > S20932 > (+)-AMMTC > N-acetyl-5-HT. Our data suggests the vascular receptor belongs to the MEL1-like subtype. All the indole-based analogues of melatonin, 2-iodomelatonin, (-)-AMMTC, (+)-AMMTC, S20932, 6-chloromelatonin, 6-hydroxymelatonin and N-acetyl-5-HT, behaved as full agonists. All the naphthalenic derivatives examined, S21634, S20098, S20242 and S20304 behaved as partial agonists relative to melatonin. 5. The naphthalenic-based antagonists, S20928 and S20929, did not modify electrically-evoked, isometric contractions of the tail artery, but produced a parallel, rightward displacement of the melatonin concentration-response curve. Based upon the effect of 1 microM S20928 and S20929, the estimated pK(B) values for these antagonists were 7.18+/-0.25 (n=4) and 7.17+/-0.25 (n=5), respectively. 6. We demonstrated that enhancement of electrically-evoked, isometric contractions of the rat isolated tail artery (using the Halpern-Mulvany wire myograph) is a simple and reproducible model for assessing the activity of putative agonists, partial agonists and antagonists at vascular melatonin receptors. Pharmacological characterization of the receptor suggests the presence of a MEL1-like subtype.