Brain Res. 1999 Mar 6;821(1):50-9.
Cardiovascular effects of catecholamines injected into the DBB of rats, influence of urethane anaesthesia and local colchicine.

Abdelmalek A, Ayad G, Thornton SN.

C.N.R.S. U.R.A. 9054, Neurobiologie des Regulations, College de France, 11 Place Marcelin Berthelot, 75231, Paris Cedex 05, France.

In a previous publication it was shown that 1 microgram colchicine injected into the diagonal band of Broca (DBB) produced a significant decrease in femoral artery blood pressure (and/or volume) measured in urethane-anaesthetised rats. In order to test if the central catecholamines were involved in this effect, guide cannulae were implanted in the DBB and a catheter in the femoral artery. On-line pressure recordings were taken before during and after alpha1, alpha2 and beta adrenoreceptor agonists and antagonists were injected into the region of the DBB of non-anaesthetised and urethane anaesthetised male Wistar rats with and without injection of colchicine. Arterial pressure was significantly increased in the non-anaesthetised rats (114.6+/-2.6 n=11 vs. 149.3+/-3.3 mmHg n=12, p<0.01) yet significantly reduced (82.0+/-3.9 n=11 vs. 63.8+/-4.5 mmHg n=12, p<0.01) in the urethane treated rats by the alpha2 agonist clonidine. The alpha2 antagonist yohimbine blocked these effects in both preparations. In contrast, the beta adrenoreceptor agonist isoprenaline produced a significant decrease in arterial pressure in both preparations (107.7+/-3.9 n=11 vs. 85.9+/-4.0 mmHg n=12, p<0.01) (102.6+/-6.7 n=11 vs. 81.7+/-3.4 mmHg n=12, p<0.01) and this effect was blocked by the beta antagonist propranolol. Colchicine injected into the DBB abolished the effects of the alpha2 agonist and antagonist in the non-anaesthetised but not the anaesthetised rats. The responses to the beta agonist and antagonist were not greatly affected by the colchicine in the non-anaesthetised rats whereas in the anaesthetised rat beta agonist injection tended to totally depress arterial pressure. These results suggest that the sympathetic nervous system in the DBB plays a significant role in the central control of arterial pressure and that the alpha2 component is significantly affected by the state of anaesthesia. 1999 Elsevier Science B.V.


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Anal Biochem. 1999 Mar 15;268(2):270-7.
A continuous fluorescence assay for the study of P-glycoprotein-mediated drug efflux using inside-out membrane vesicles.

Bebawy M, Morris MB, Roufogalis BD.

Department of Pharmacy, University of Sydney, Sydney, New South Wales, 2006, Australia.

A fluorimetric procedure for assaying the transport activity of P-glycoprotein (P-gp) using a membrane vesicle model has been developed. In this assay methylene blue is incorporated into inside-out vesicles prepared from human acute lymphoblastic leukemic cells resistant to 100 ng. ml-1 vinblastine (VBL100) and their sensitive controls. The fluorescence of a fluorescent derivative of colchicine (fluorescein-colchicine) is quenched as the probe is transported across the vesicle membrane. The fluorescein-colchicine transport was found to be dependent on the presence of P-glycoprotein, required ATP, and was inhibited by vanadate and the reversal agent, verapamil, in a dose-dependent manner. Furthermore, the transport was competed against by the P-gp substrates, vinblastine and methotrexate. The transport of fluorescein-colchicine by P-gp was found to be cooperative (n = 1. 23). The assay is rapid, requires small amounts of sample, and removes the need for the radioactive procedures used in the past. The assay should find use in characterizing the transport kinetics of P-gp, for examining and optimizing combinations of chemotherapeutics, and for examining the effects of reversal agents and substrates which potentially compete for transport with the fluorescent substrate probe. Other possible applications include examining P-gp-mediated transport properties of purified P-gp in reconstituted systems. 1999 Academic Press.


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Toxicol Appl Pharmacol. 1999 Mar 15;155(3):215-26.
ATP-Dependent colchicine transport by human erythrocyte glutathione conjugate transporter.

Awasthi S, Singhal SS, Pandya U, Gopal S, Zimniak P, Singh SV, Awasthi YC.

Department of Internal Medicine, The University of Texas Medical Branch at Galveston, Galveston, Texas, 77555-1067, USA. sawasthtmb.edu

We have recently demonstrated mutually inhibitory ATP-dependent transport of dinitrophenyl-S-glutathione (DNP-SG) and doxorubicin by DNP-SG ATPase purified from human erythrocyte membranes (S. Awasthi et al., 1998a,b). Our previous studies indicate a broad substrate specificity for this transport mechanism, including some P-glycoprotein substrates. Present studies were carried out to determine whether colchicine (COL), a classical P-glycoprotein substrate, could be transported by purified human erythrocyte DNP-SG ATPase reconstituted in artificial liposomes. We also investigated whether leukotriene C4 (LTC4), an endogenous proinflammatory glutathione-conjugate derived from arachidonic acid, would inhibit colchicine transport. Uptake of COL was compared in proteoliposomes reconstituted with the purified DNP-SG ATPase as well as control liposomes in the presence or absence of ATP. Increased colchicine uptake was observed upon addition of ATP to proteoliposomes, but not control liposomes. Uptake was linear with respect to the amount of vesicle protein used. Sensitivity to osmolarity was consistent with intravesicular COL accumulation. The ATP-dependent colchicine uptake was sensitive to temperature in a manner consistent with a protein-mediated transport process with activation energy of 7.3 kcal/mol. Time-dependent COL uptake by proteoliposomes in the presence of ATP was consistent with a single compartment model with an apparent rate constant of 0.21 +/- 0.02 min-1. Kinetic studies indicated a saturable behavior with respect to ATP (Km 2.3 +/- 0.7 mM) and colchicine (Km 4.3 +/- 0.2 microM). LTC4 was found to be a competitive inhibitor of COL transport (Kis 16.4 microM). Since DNP-SG ATPase is present in many tissues, it may play an important role in determining colchicine accumulation in cells. Increased LTC4 would tend to increase cellular COL accumulation. 1999 Academic Press.


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Clin Exp Pharmacol Physiol. 1999 Mar;26(3):230-5.
Reduction of human recombinant type II phospholipase A2 and prostaglandin F2 alpha release by microtubule depolymerizing agents.

Munns MJ, King RG, Rice GE.

Perinatal Research Centre, Royal Women's Hospital, Carlton, Victoria, Australia.

1. The present study examines the effects of the microtubule depolarizing agent colchicine on secretory type II phospholipase A2 (PLA2) function in Chinese hamster ovary (CHO) cells that specifically overexpress human type II PLA2 and the effect of both colchicine and tubulazole on the release of type II PLA2 and prostaglandin (PG) F2 alpha from human placental explants. 2. Significant suppression by colchicine (0.01-10 mumol/L) of PLA2 activity (P < 0.00001), immunoreactive type II PLA2 (irPLA2; P < 0.00001) and PGF 2 alpha release (P < 0.01) was observed in medium from overexpressing CHO cells. These effects were significantly reduced (P < 0.0001) in the presence of 10 mumol/L taxol, an agent that prevents depolymerization of microtubules. The addition of 30 mumol/L arachidonic acid significantly reduced (P < 0.0001) the inhibition of PGF2 alpha production in CHO cell lines. 3. The addition of 1 mumol/L colchicine to human placental explants for 24 h significantly reduced irPLA2 (P < 0.00001) and PGF2 alpha production (P < 0.00001). Similarly, 1 mumol/L tubulazole significantly blocked irPLA2 (P < 0.001) and PGF2 alpha (P < 0.0001). 4. At 10 mumol/L, taxol significantly reduced irPLA2 inhibition by colchicine (n = 8; P < 0.05) and tubulazole (n = 8; P < 0.05). Similarly, taxol significantly reduced the reduction in PGF2 alpha production caused by colchicine (P < 0.001) and by tubulazole (P < 0.001). 5. These results suggest that integrity of the microtubule system is required for PLA2 function and the subsequent production of pro-inflammatory mediators.


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