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Specificity for the Na K 2Cl cotransport pathway. Kinase Inhibitors Block the cpt-CAMP-mediated Stimulation of pH]Bumetanide Binding to Isolated Red Cell Membranes-we previously found an excellent correlation between the activation state of Na K 2Cl cotransport in intact cells and [3H]bumetanide binding to isolated membranes from those cells Pewitt et al., 1990 ; . To examine whether this relationship held in cells treated with kinase inhibitors, measurements of both parameters were made in cells treated with K-252a. cpt-CAMP caused a 25-fold increase in 86Rb influx in these cells and an II-fold increase in specific [3H]bumetanide binding in the isolated membranes, similar to our previous observations made with NE-stimulated cells Pewitt et al., 1990 ; . Preincubation of cells with K-252a 40 ELM ; reduced the cpt-CAMP stimulation of s6Rb uptake by 90% and correspondingly reduced the stimulation of specific [3H]bumetanide binding in isolated membranes by 83% Fig. 3 ; . To assess whether the effect of K-252a was at the level of cptCAMP activation of Na K 2Cl cotransport rather than the function of the transporter after activation, we tested the ability of the inhibitor to directly modulate ["Hlbumetanide binding in isolated membranes from stimulated cells. No effect of the compound on this parameter was observed at concentrations up to 40 pM, suggesting that K-252a does not directly interact with the diuretic-binding component of the transport system. Kinase Inhibitors ALSO Block Na K 2Cl Cotransport Stimulation by CAMP-independent Stimuli-The effect of kinase inhibitors on two CAMP-independent stimuli of Na K 2Cl cotransport was tested. In a direct comparison, cotransport was activated by three different agents: cpt-CAMP 0.5 mM ; , hypertonicity 398 mOsm ; , and NaF 10 mM ; in the presence of increasing concentrations of K-252a 0.3-50 ; . The doseresponse curves for K-252a inhibition of fluoride and hypertonic stimulation were clearly right-shifted from that obtained with cpt-CAMP Fig. 4 ; . The effect of the kinase inhibitor on either CAMP-independent stimulus was half-maximal at 2030 and virtually complete at 100 jrM. A similar differential effect was noted with H-9; at 0.5 mM this compound completely inhibited cpt-CAMP stimulation while fluoride and hypertonic stimulation were only slightly affected data not shown ; . However, at this concentration of H-9, nonspecific effects i.e. agglutination and discoloration of the erythroT f 125 1.4. CYP2C9 and CYP2D6 in this biotransformation by human liver microsomes was further confirmed. Finally, kinetic assessments of the formation of R-norfluoxetine by expressed CYP2D6, CYP2C9, and CYP2C19 determined apparent Km values of 1.8, 9.7, and 8.5 M, respectively. Interestingly, the kinetic analyses with expressed CYP2D6 and CYP2C9 exhibited product inhibition at high R-fluoxetine concentrations, which was similar to that observed in three microsomal liver samples. Expressed CYP2C9 also exhibited substrate activation at low concentrations. Taken together, the data presented indicate that in microsomal samples containing a full complement of CYPs, the contribution of CYP2C9 and CYP2D6 to the formation of R-norfluoxetine at low R-fluoxetine concentrations is similar. Although present in relatively small amounts 2%, Shimada et al., 1994 ; in the human liver, CYP2D6 has a low Km value, which indicates a high affinity for R-fluoxetine. This coupled with the antibody inhibition data suggests that CYP2D6 plays an important role 40% ; in the formation of R-norfluoxetine. Although CYP2C9 appears to have a 5-fold higher Km value for R-fluoxetine, results with inhibitory antibodies suggest it also plays a primary role in R-fluoxetine metabolism 55% ; , which is likely due to CYP2C9 levels in the liver that are about 10-fold greater than those of CYP2D6 Shimada et al., 1994 ; . Expressed CYP2C19 and CYP2C9 have a similar affinity for R-fluoxetine; however, CYP2C19 represents only 1% of the CYPs in the liver Inoue et al., 1997 ; , therefore it would not be expected to play a major role in this biotransformation. Furthermore, the correlation studies indicated that CYP2C19 levels were not related to the formation of R-norfluoxetine, suggesting that CYP2C19 does not play a significant role in this biotransformation. Similar studies were performed examining the conversion of S-fluoxetine to S-norfluoxetine. Enzyme kinetic studies in two liver samples containing a full complement of enzymes were consistent with two enzymes being involved in this biotransformation, with the low Km enzyme exhibiting an apparent Km value of about 0.2 M. Interestingly, a microsomal sample deficient in CYP2D6 HLK ; apparently contained only the high Km enzyme Km 109 M ; able to form S-norfluoxetine. In the correlation studies, the only activity that correlated with S-norfluoxetine formation following incubation with 2.5 M S-fluoxetine was that for CYP2D6. Only expressed CYP2D6 was able to form this metabolite apparent Km value of 0.58 M ; at a low S-fluoxetine concentration. These results confirm the apparently exclusive role of CYP2D6 in this biotransformation at low, pharmacological S-fluoxetine concentrations. In the current study, multiple CYPs were found to be capable of forming both R- and S-norfluoxetine following incubation with high concentrations of R- and S-fluoxetine. As reported herein, at high concentrations of substrate, CYP2D6 is only one of many CYPs that may participate in this biotransformation. This may explain the conclusions of von Moltke et al. 1997 ; and Margolis et al. 2000 ; who suggested that in addition to CYP2D6 and CYP2C9 playing a role in norfluoxetine formation, that CYP2C19 and CYP3A may also be involved. These conclusions were confirmed in the current studies where CYP2C19 and CYP3A along with other CYPs ; were able to form R- and S-norfluoxetine at high substrate concentrations. The involvement of CYP2D6 and CYP2C9 in the metabo.

Metaphase II oocytes were obtained from consenting patients from our IVF-ICSI programme only when an adequate number of oocytes was retrieved. Ovarian stimulation was conducted in all patients as described previously Ubaldi et al., 1999 ; using a combination of GnRH agonist subcutaneous buserelin acetate 0.2 mg twice daily; Suprefact; Hoechst, Marion Roussel, Milan, Italy ; started in late luteal phase of the previous cycle, recombinant FSH Gonal-F, 75 IU, Serono, Rome Italy or Puregon 100 IU, Organon, Oss, The Netherlands ; and HCG Profasi; Serono ; . Oocyte decumulation has been described extensively elsewhere Rienzi et al., 1998 ; . Briey, the cumulus and corona radiata were removed immediately after ovum pick-up by a brief exposure to HEPES-buffered medium Gamete, Vitrolife, Gothenburg, Sweden ; containing 20 IU ml hyaluronidase fraction VIII Hyase-10X; Vitrolife ; and by gentle aspiration in and out of a plastic pipette Flexipet, 130 mm i.d., COOK, Australia ; . The denuded oocytes were then evaluated to assess their nuclear maturation stage. Only oocytes that had released the rst polar body metaphase II ; were included in this study. Immediately after decumulation, the supernumerary metaphase II oocytes obtained were used for meiotic spindle observation and the freezing procedure. Freezing and thawing procedures The freezing solution consisted of phosphate-buffer saline PBS ; with 25 mg ml human serum albumin HSA; Cryo-PBS, Vitrolife ; , freezing solution 1 containing 1.5 M 1, 2-propanediol PrOH ; in Cryo-PBS FS1, Vitrolife ; and freezing solution 2 containing 1.5 M PrOH + 0.1 M sucrose in Cryo-PBS FS2, Vitrolife ; . A maximum of three oocytes were frozen per straw. Equilibration with the freezing solutions was carried out at room temperature. Oocytes were rst incubated for 2 min in Cryo-PBS, then for 10 min in freezing solution 1 and nally in freezing solution 2, and immediately loaded into straws paillette cristal, Cryo Bio System, IMV Technologies, France ; . The straws were placed into a freezing chamber CL-863, Cryologic, Australia ; at room temperature. The temperature was decreased to 7C at rate of 2C min. Seeding was done manually by touching the straw close to the cotton plug with a nitrogen-cooled forceps. After holding for 10 min at 7C, the temperature was lowered slowly at a rate of 0.3C min to 30C, followed by rapid cooling 10C min ; to 110C. The straws were then plunged into liquid nitrogen LN2 ; , and stored submerged in LN2 until thawing. For thawing, the straws were removed from LN2 and rapidly warmed to room temperature. The retrieved oocytes were then transferred to a plastic 4-well dish where they were washed free from the cryoprotectant at room temperature by stepwise dilutions in 1.0, 0.5 and 0.0 M PROH in the presence of 0.1 M sucrose and Cryo-PBS Thaw Kit 1, Vitrolife ; . The exposure times for thawing solutions were 5 min solution 1 ; , 10 min solution 2 ; , 10 min solution 3 ; and 10 min PBS ; . All surviving oocytes with an intact membrane and clear cytoplasm ; were cultured further in IVF-medium Vitrolife ; at 37C, 5% CO2 for 3 h. Spindle visualization by Polscope Meiotic spindles were imaged at sequential steps during the freezing thawing procedure. Briey, the oocytes were placed in a 5 drop of the corresponding medium covered with mineral oil ovoil, Vitrolife, Gothemburg, Sweeden ; in a glass-bottomed culture dish Willco Wells, Amsterdam, The Netherlands ; . The meiotic spindle visualization was performed at 20Q magnication with LC Polscope optics and controller SpindleView, CRI, Woburn, MA ; , combined with a computerized image analysis system SpindleView software, CRI ; . For this purpose, each oocyte was rotated with the use of the injection and focalin.

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