We studied the effect of verapamil on Pgp expression (Pgp) in MDR human leukemia cell lines, K562/ADR and CEM VLB100. In the K562/ADR cell line, addition of verapamil to the culture medium (15 microM concentration) resulted in a 3-fold decrease in Pgp expression after 72 hr exposure. The effect of verapamil was reversible, and Pgp expression reached the level of untreated controls 24 hr after discontinuation of verapamil. Similar results were obtained with the human vinblastine-resistant cell line, CEM VLB100. On the contrary, no effect on Pgp expression was observed when the cells were treated with nifedipine or diltiazem (2 other calcium-channel blockers), even at doses that inhibited cell proliferation. The level of Pgp mRNA in the presence of verapamil was measured by Northern blot and was also decreased 2-fold (with the maximum reached within 24 hr), suggesting a transcriptional or post-transcriptional mechanism for verapamil. We further established that the effect of verapamil on Pgp expression led to an increase in DNR and VLB accumulation and cytotoxicity. These results suggest that verapamil acts specifically on Pgp expression in these drug-selected leukemic cells. The identification of a potentially novel mechanism of action may provide new insights as to how chemosensitization may be more effectively applied in vivo.

1 Although the actions of angiotensin II (Ang II) on renal haemodynamics appear to be mediated by activation of the AT 1 receptor subtype, AT 2 binding sites have also been evidenced in the adult kidney vasculature. As NO is known to mask part of the renal eects of vasoconstrictor drugs, we queried whether the Ang II-induced vasoconstrictions could occur via multiple receptor subtypes during inhibition of NO synthesis. We explored the eect of AT 1 and AT 2 receptor (AT-R) antagonists on Ang II-induced pressure increases during NO synthase or soluble guanylyl cyclase inhibition in rat isolated kidneys perfused in the presence of indomethacin at constant¯ow in a single-pass circuit. 2 In the absence of NO blockade, the AT 1 -R antagonist L-158809 (500 nM) antagonized the Ang IIinduced vasoconstrictions, while the AT 2 -R antagonist PD-123319 (500 nM) had no eect. 3 Perfusing kidneys in the presence of either NO synthase inhibitors, L-NAME (100 mM) or L-NOARG (1 mM), or soluble guanylyl cyclase inhibitor, LY-83583 (10 mM), signi®cantly increased both molar pD 2 (from 9.40+0.25 to 10.36+0.11) and E max values (from 24.9+3.1 to 79.9+4.9 mmHg) of the concentration ± response curve for Ang II-induced vasoconstriction. 4 In the presence of L-NAME, 500 nM L158809 abolished the Ang II-induced vasoconstrictions whatever the concentration tested. On the other hand, 500 nM PD-123319 reversed the left shift of the concentration ± response curve for Ang II (molar pD 2 value 9.72+0.13) leaving E max value unaected (91.3+7.6 mmHg). 5 In the presence of L-NAME, the potentiated vasoconstriction induced by 0.1 nM and the augmented vasoconstriction induced by 10 nM Ang II were fully inhibited in a concentration-dependent manner by L-158809 (0.05 ± 500 nM). By contrast, PD-123319 (0.5 ± 500 nM) did not aect the 10 nM Ang II-induced vasoconstriction and concentration-dependently decreased the 0.1 nM Ang II-induced vasoconstriction plateauing at 65% inhibition above 5 nM antagonist. 6 Similar to PD-123319, during NO blockade the AT 2 -R antagonist CGP-42112A at 5 nM decreased by 50% the 0.1 nM Ang II-induced vasoconstriction and at 500 nM had no eect on 10 nM Ang II-induced vasoconstriction. 7 In conclusion, the renal Ang II-induced vasoconstriction, which is antagonized only by AT 1 -R antagonist in the presence of endogenous NO, becomes sensitive to both AT 1 -and AT 2 -R antagonists during NO synthesis inhibition. While AT 1 -R antagonist inhibited both L-NAME-potentiated and -augmented components of Ang II-induced vasoconstriction, AT 2 -R antagonists inhibited only the L-NAME-potentiated component.

An increasing body of evidence appears to implicate the lipid bilayer of multidrug resistant (MDR) cells with P-glycoprotein activity. Several cationic amphiphilic drugs (CADs) have been extensively described as modulators of MDR. These same agents are also known to (1) inhibit lysosomal acid sphingomyelinase (ASmase), a phospholipid degrading enzyme, and/or (2) induce phospholipidosis in animal tissues or cultured cell lines. In this report, we randomly selected 17 CADs and evaluated their potency in modulating MDR in the murine MDR P388/ADR leukemia cell line. We compared these results with their ability to inhibit ASmase and observed a significant dose-dependent linear relationship (95% central confidence interval), between ASmase inhibition and MDR reversal. This approach permitted us to identify three new modestly potent chemosensitizers: trimipramine, desipramine, and mianserine. Modulation of MDR was not cell line specific, since CADs at 10 microM increased doxorubicin (DOX) and vinblastine (VBL) (but not methotrexate, MTX) cytotoxicity in both P388/ADR and the human MDR cell lines MES-SA/Dx5 and K562/R7, but not in the parental drug-sensitive cells. Although all chemosensitizing CADs at 10 microM significantly increased Rhodamine-123 (Rho-123) accumulation in the human leukemia MDR cell line K562/R7 and most presented significant displacement of the photoaffinity labelling probe iodoarylazidoprazosin, no correlation between these observations and the ability of CADs to sensitize MDR cells to DOX and VBL was found. In conclusion, our study strongly suggests that the chemosensitizing potency of agents such as CADs may be due to a dual mechanism of action: direct antagonism of P-gp activity and indirect modulation of P-gp activity through the disruption of cellular lipid metabolism.

The purpose of this study was to determine whether administration of doxorubicin (DOX) as a continuous infusion or a bolus injection resulted in similar leukemic cell drug concentration in patients with refractory chronic lymphocytic leukemia (CLL). This study was carried out on five patients with refractory CLL, with DOX administered either as a bolus injection (35 mg/m2; CHOP protocol) or as a constant-rate infusion for a period of 96 h (9 mg/m2 per day; VAD protocol). The two types of drug administration were used alternatively with the same patient. Plasma and cellular DOX concentration were determined using high-performance liquid chromatography. Peak plasma DOX levels were higher after the bolus injection than after continuous administration (1509 +/- 80 ng/ml vs 11.6 +/- 1.8 ng/ml, respectively), whereas the plasma area under the curve (AUC) levels were similar. Maximum DOX cellular concentrations were 8629 +/- 2902 ng/10(9) cells (bolus injection) and 2745 +/- 673 ng/10(9) cells (96 h infusion). The cellular AUC after the bolus injection was 2.85 times greater than that observed after continuous administration. This difference was due to a higher cellular peak level followed by a relatively prolonged retention of the drug, with a loss of only 25% in the first 24 h following. These findings demonstrated that in CLL the cellular DOX exposure can be notably modified by the method of drug administration, with higher drug intracellular concentrations being achieved after bolus administration than with the infusion schedule.

1 We showed earlier that NO inhibition caused a left-shift and augmented E max of the concentrationresponse curve of AT 1 -mediated (angiotensin II)-induced vasoconstrictions (AII-VC) in the rat kidney. The 0.01 ± 0.1 nM AII-VC unmasked by the potentiating e ect of NO inhibition, were sensitive not only to AT 1 (L158809), but also to AT 2 receptor (PD123319) antagonists. We now demonstrate the role of endothelium and eicosanoids in the NO-masked AT 1 /AT 2 -mediated component of the AII-VC in isolated indomethacin-perfused kidneys of the rat. 2 L-NAME increased 0.1 nM AII-VC 7.2 fold. Pretreatment of the kidneys with factor VIII antibody/ complement or with the detergent CHAPS to damage endothelium, decreased carbachol-induced vasodilatation and blunted by 60 and 30% respectively, the enhancement of AII-VC during NO inhibition. 3 L-NAME also increased 3 mM noradrenaline (NA)-induced vasoconstriction (NA-VC) 8.1 fold. In contrast to AII-VC, endothelium damage was without e ect on the enhancement of NA-VC by L-NAME, suggesting a dominant role of endothelium-derived NO in the enhancement of NA-VC. 4 During NO inhibition, ETYA (2 mM; an inhibitor of all arachidonic acid derived pathways) and anaphto¯avone (10 mM; an inhibitor of the cytochrome P450 isozymes), decreased by 85% the 0.1 nM AII-VC. 5 In conclusion, during NO inhibition, the AT 1 -mediated constriction to low concentrations of AII, which is sensitive to AT 2 antagonists, depends on intact endothelium, and can be blocked by inhibition of eicosanoid synthesis. The results suggest that the AII-mediated vasoconstriction through AT 1 receptors is potentiated in the absence of NO, by the release of eicosanoids from the endothelium through AT 2 receptors.