Ragnhild Jaeger

Leukotriene C 4 (LTC 4 ) does not share a cellular efflux mechanism with cGMP: characterisation of cGMP transport by uptake to inside-out vesicles from human erythrocytes

The transport of cGMP out of cells is energy requiring and has characteristics compatible with an ATP-energised anion pump. In the present study a model with inside-out vesicles from human erythrocytes was employed for further characterisation of the cGMP transporter. The uptake of leukotriene C(4) (LTC(4)), a substrate for multidrug resistance protein (MRP), was concentration-dependently inhibited by the leukotriene antagonist MK571 (IC(50)=110+/-20 nM), but cGMP was unable to inhibit LTC(4) uptake. Oxidised glutathione (GSSG) and glutathione S-conjugates caused a concentration-dependent inhibition of [(3)H]cGMP uptake with IC(50) of 2200+/-700 microM for GSSG, 410+/-210 microM for S-(p-nitrobenzyl)glutathione and 37+/-16 microM for S-decylglutathione, respectively. Antioxidants such as reduced glutathione and dithiothreitol did not influence transport for concentrations up to 100 microM, but both inhibited cGMP uptake with approx. 25% at 1 mM. The cGMP pump was sensitive to temperature without activity below 20 degrees C. The transport of cGMP was dependent on pH with maximal activity between pH 8.0 and 8.5. Calcium caused a concentration-dependent inhibition with IC(50) of 43+/-12 microM. Magnesium gave a marked activation in the range between 1 and 20 mM with maximum effect at 10 mM. The other divalent cations, Mn(2+) and Co(2+), were unable to substitute Mg(2+), but caused some activation at 1 mM. EDTA and EGTA stimulated cGMP transport concentration-dependently with 50% and 100% above control at 100 microM, respectively. The present study shows that the cGMP pump has properties compatible with an organic anion transport ATPase, without affinity for the MRP substrate LTC(4). However, the blockade of the cGMP transporter by glutathione S-conjugates suggests it is one of several GS-X pumps.

Renal and hepatic toxicity after high-dose 7-hydroxymethotrexate in the rat

To examine directly the hepatic and renal toxicity of 7-hydroxymethotrexate (7-OH-MTX) without interference of the parent compound methotrexate (MTX), we purified and gave 100 mg/kg 7-OH-MTX to rats, a dose resulting in serum levels of 7-OH-MTX comparable with those achieved in the clinic after the administration of high-dose MTX (HD-MTX). After only 5 h, the 7-OH-MTX-treated rats demonstrated 2.6-fold increases in serum creatinine values and 2-fold elevations in serum aspartate aminotransferase (ASAT) levels as compared with the controls. Morphologic evidence of toxicity, however, was apparent only in the kidneys. Intraluminal cellular debris containing membranous material and deteriorated organelles was seen, but no precipitate of the delivered drug. The peak serum concentration of 7-OH was up to 939 μM, and concentrations of 7-OH-MTX declined triphasically, showing at1/2α value of 2.45 min, at1/2β value of 30.5 min, and a terminal half-life (t1/2γ) of 240 min. The total clearance value was 14.5 ml min−1 kg, and the postdistributional volume of distribution (Vβ) was 5070 ml/kg. Our results may indicate a direct toxic effect of 7-OH-MTX on kidney and liver cells.

Urinary levels of cyclic guanosine monophosphate (cGMP) in patients with cancer of the uterine cervix: a valuable prognostic factor of clinical outcome?

Changes in urinary cyclic nucleotide levels have been reported in patients with various types of cancers. The present study was conducted to relate changes in urinary levels of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) to the clinical outcome of 11 patients treated for cancer of the uterine cervix. Urine was sampled for 24 h before and 3 months after primary treatment. The levels of cGMP increased in all the patients (n = 5) who relapsed within the observation period of 39 months. 4 of these patients showed an increased cGMP/cAMP ratio. In the patients without relapse (n = 6), the cGMP levels decreased, whereas the cGMP/cAMP ratios were unchanged. No marked changes in the levels of cAMP were observed for either of the groups. The measurement of urinary cGMP levels seems to be a valuable tool in the follow-up of patients with cancer of the uterine cervix.

Interactions with the protein binding of 7-hydroxy-methotrexate in human serum in vitro

7-Hydroxy-methotrexate (7-OH-MTX), the major extracellular methotrexate (MTX) metabolite, is 90-95% bound in human serum, with albumin (HSA) as the major binding protein. Reports of an interaction with concomitantly administered non-steroidal antiinflammatory drugs (NSAIDs) during MTX therapy led us to investigate whether these compounds could reduce the binding of 7-OH-MTX in vitro. Equilibrium dialysis experiments demonstrated that naproxen and indomethacin concentration dependently reduced the binding of 1 microM 7-OH-MTX. After ingestion of 1000 mg naproxen, per cent unbound 7-OH-MTX in sera from volunteers increased 2-3-fold in vitro, positively correlated to naproxen concentrations (P less than 0.00015). In addition, etacrynic acid, bilirubin, sulphamethizole and acetylsalicylic acid displaced 7-OH-MTX from its binding protein(s) in a competitive manner. The data suggest that 7-OH-MTX interacts with several exogenous and endogenous substances associated with HSA in human serum. Displacement of 7-OH-MTX from HSA may contribute to the interaction between NSAIDs and MTX.

Reduced β‐adrenergic sensitivity in healthy volunteers induced by hypoglycemia

Summary— A single causative mechanism for development of hypoglycemia unawareness in insulin‐dependent diabetes mellitus (IDDM) is not yet apparent. Reduced adrenergic sensitivity may be part of the explanation. This study was carried out to investigate the effect of hypoglycemia on β‐adrenergic sensitivity. Ten healthy male subjects (age 19–23 years) gave informed consent to take part in the study. They were hospitalized overnight at the University Hospital of Tromsø, Department of Clinical Research, on two occasions. Isoprenaline and metoprolol sensitivity tests were performed the morning after hospitalization: once after an intravenous (iv) injection of placebo (0.9% NaCl), and once after an iv injection of insulin (0.15 IU insulin/kg body weight) to induce hypoglycemia. The dose of isoprenaline needed to increase heart rate (HR) by 25 beats per minute (bpm) (I25), and the dose of metoprolol (M–12.5) needed to inhibit I25 with 50% or 12.5 bpm, when injected simultaneously, were used as determinants of isoprenaline and metoprolol sensitivity. In this study, there was a significant (p < 0.05) increase both in I25 and M–12.5 after hypoglycemia. The dose‐response curve of isoprenaline/HR was significantly shifted to the right after hypoglycemia. This study shows that acute hypoglycemia induces a reduction in β‐adrenegic sensitivity, and it supports the hypothesis of reduced β‐adrenergic sensitivity as an important pathophysiological mechanism in hypoglycemia unawareness in IDDM.

Interactions of vinblastine and vincristine with methotrexate transport in isolated rat hepatocytes

The accumulation of methotrexate (MTX) in the presence of vinblastine (VBL) and vincristine (VCR) was studied in isolated rat hepatocytes. In accordance with our recent study on vindesine (VDS), we found VBL and VCR to reduce net MTX accumulation significantly at 15 min after MTX addition. Drug concentrations of 100 μM VBL and 500 μM VCR led to 67% and 82% reductions in intracellular MTX, respectively. Since there was only a slight inhibition of MTX efflux by 100 μM VBL, the accumulation data demonstrate that the major effect of VBL is on MTX influx. Dixon-plot analyses are suggestive of competitive inhibition of the MTX influx, yielding inhibition constants (Ki values) of 55 μM for VBL and 110 μM for VCR. Since theKi values correspond grossly to plasma levels obtained in humans shortly after the infusion of therapeutic doses of the vinca alkaloids studied herein, the interaction with MTX uptake could serve to diminish the toxicity of MTX to nonmalignant cells.