Modulation of the prostaglandin-endoperoxide synthase 2 gene expression by variant haplotypes: influence of the 3'-untranslated region

The inducible inflammatory enzyme cycloxigenase-2 is up-regulated in cancer, and favors tumor progression. Cycloxigenase-2 is encoded by the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which presents sequence variations in the promoter region (PR) and in the 3′-untranslated region (3′-UTR). Different PR (rs689465, rs689466, rs20417) and 3′-UTR (rs5275) variants were generated by site-directed mutagenesis, and combined in haplotypes to access expression levels using a reporter system (luciferase) in human cells (MCF-7 and HEK293FT). Luciferase activity did not differ significantly among PTGS2 PR constructs, except for pAAC (containing variant allele rs20417 C), with 40% less activity than pAAG (wild-type sequence) in MCF-7 cells (P<0.01). Despite the lack of individual significant differences, PTGS2 PR constructs enclosing rs689466 G (pAGG and pAGC) showed an approximate two-fold increase in luciferase activity when compared to those containing rs689466 A (pAAG, pGAC, pAAC and pGAG) in both cell lines (P<0.001 for MCF-7 and P=0.03 for HEK293FT). The effect of PTGS2 3′-UTR sequences varied between MCF-7 and HEK293FT: MCF-7 cells showed significant reduction (40-60%) in luciferase activity (at least P<0.01), whereas HEK293FT cells showed more diverse results, with an average 2-fold increase when combined constructs (PR and 3′-UTR) were compared to respective parental PR sequences. The contribution of 3′-UTR variant (rs5275) was not consistent in either cell line. Despite the modulation of the 3′-UTR, with variable effects of rs5275, the enhancing transcriptional effect of rs689466 G was still detectable (P<0.0001 in MCF-7 or P=0.03 in HEK293FT cells).

Modulation of the prostaglandin-endoperoxide synthase 2 gene expression by variant haplotypes: influence of the 3'-untranslated region.

The inducible inflammatory enzyme cycloxigenase-2 is up-regulated in cancer, and favors tumor progression. Cycloxigenase-2 is encoded by the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which presents sequence variations in the promoter region (PR) and in the 3'-untranslated region (3'-UTR). Different PR (rs689465, rs689466, rs20417) and 3'-UTR (rs5275) variants were generated by site-directed mutagenesis, and combined in haplotypes to access expression levels using a reporter system (luciferase) in human cells (MCF-7 and HEK293FT). Luciferase activity did not differ significantly among PTGS2 PR constructs, except for pAAC (containing variant allele rs20417 C), with 40% less activity than pAAG (wild-type sequence) in MCF-7 cells (P<0.01). Despite the lack of individual significant differences, PTGS2 PR constructs enclosing rs689466 G (pAGG and pAGC) showed an approximate two-fold increase in luciferase activity when compared to those containing rs689466 A (pAAG, pGAC, pAAC and pGAG) in both cell lines (P<0.001 for MCF-7 and P=0.03 for HEK293FT). The effect of PTGS2 3'-UTR sequences varied between MCF-7 and HEK293FT: MCF-7 cells showed significant reduction (40-60%) in luciferase activity (at least P<0.01), whereas HEK293FT cells showed more diverse results, with an average 2-fold increase when combined constructs (PR and 3'-UTR) were compared to respective parental PR sequences. The contribution of 3'-UTR variant (rs5275) was not consistent in either cell line. Despite the modulation of the 3'-UTR, with variable effects of rs5275, the enhancing transcriptional effect of rs689466 G was still detectable (P<0.0001 in MCF-7 or P=0.03 in HEK293FT cells).

CYP2C9 genotypes and the quality of anticoagulation control with warfarin therapy among Brazilian patients.

OBJECTIVE: To evaluate the impact of the two most common CYP2C9 variant alleles (*2 and *3) on the maintenance dose of warfarin and on the quality of anticoagulation control in Brazilians. METHODS: Patients (n = 103) initiated warfarin therapy with 5 mg/day (or 2.5 mg/day when over 80 years old). The international normalized ratio (INR) was targeted between 2 and 3, monitored every week until four consecutive adequate measures had been obtained, and then monthly. Serious hemorrhagic events were defined by the need for inpatient hospitalization. CYP2C9 genotyping was obtained by PCR-RFLP. RESULTS: The frequencies of CYP2C9*2 and CYP2C9*3 were 0.097 and 0.073, respectively, with genotypic distribution fitting Hardy-Weinberg equilibrium. CYP2C9 genotype was the only clinical feature associated with the risk of severe bleeding (one-sided P = 0.019, Fisher exact method), with an odds ratio of 4.8 (95% confidence interval of 1.4-16.6) for any variant genotype as compared to CYP2C9*1*1. Patients with either CYP2C9*2 or CYP2C9*3 were equally difficult to maintain in the INR target range, showing significantly (one-sided P = 0.038, Mann-Whitney U-test) reduced ratio of adequate INR measures (0.54 +/- 0.2), when compared to CYP2C9*1*1 patients (0.63 +/- 0.2). Patients with CYP2C9*3, but not CYP2C9*2, required significantly (one-sided P = 0.001, Mann-Whitney U-test) lower warfarin maintenance doses (3.1 +/- 1.8 mg) than CYP2C9*1*1 patients (5.3 +/- 2.1 mg). CONCLUSION: Patients with either CYP2C9*2 or CYP2C9*3 show higher risk of over-anticoagulation compared to CYP2C9*1*1 subjects and could benefit from a reduction in the initial warfarin standard dose (e.g., to 2.5 mg/day).

WIN 17317-3 blocks Ca2+-activated K+ channels and enhances motility of guinea-pig detrusor muscle.

Patch-clamp experiments in both clonal GH3 cells and guinea-pig bladder myocytes reveal that 1-benzyl-7-chloro-4-(n-pentylimino)-1,4-dihydroquinoline hydrochloride (WIN 17317-3), a potent blocker of Kv1.3 channels and potential immunomodulator, reduces, in a reversible manner and at low micromolar concentrations, K+ currents through Ca2+-activated high conductance K+ channels (BK channels). Blockade of BK channels is thought to account for the stimulatory effect of WIN 17317-3 on the contractility of guinea-pig bladder. This effect is not modified by tetrodotoxin (1 microM), but is abolished by nifedipine (0.1 microM). In conclusion. WIN 17317-3 lacks selectivity for the Kv1.3 channels, its postulated target for immunosuppression.

Correolide, a nor-triterpenoid blocker of Shaker-type Kv1 channels elicits twitches in guinea-pig ileum by stimulating the enteric nervous system and enhancing neurotransmitter release.

Correolide (1 - 10 microM), a nortriterpene purified from Spachea correae and a selective blocker of Kv1 potassium channels, elicits repetitive twitching in guinea-pig ileum. This effect is not seen in guinea-pig duodenum, portal vein, urinary bladder or uterine strips, nor in rat or mouse ileum. The time course and amplitude of the correolide-induced twitches in guinea-pig ileum are similar to those elicited by electrical stimulation of the enteric nervous system. The correolide-induced twitching is not affected by pre-treatment with capsaicin (1 microM), but is facilitated by the NO synthase inhibitor, N(G)-nitro-L-arginine methyl esther (L-NAME, 200 microM). The correolide-induced twitching is abolished by tetrodotoxin (1 microM) or hexamethonium (100 microM), and is markedly inhibited by nifedipine (0.3 microM) or atropine (0.2 microM). The atropine-resistant component is inhibited by selective antagonists of NK1 and NK2 tachykinin receptors, namely GR 82334 and GR 94800 (1 microM each). The former compound is more effective in inhibiting the correolide-induced, atropine-resistant activity. Correolide intensified the twitching of ileum segments exposed to saturating concentrations of margatoxin (MgTX), which suggests that Kv1 sub-types other than Kv1.1 (Kv1.4 or Kv1.5) are involved in the relatively greater degree of stimulation of the enteric nervous system by correolide, as compared to MgTX. We propose that blockade of Kv1 channels by correolide increases the excitability of intramural nerve plexuses promoting release of acetylcholine and tachykinins from excitatory motor neurons. This, in turn, leads to Ca(2+)-dependent action potentials and twitching of the muscle fibres.

Peptidyl inhibitors of shaker-type Kv1 channels elicit twitches in guinea pig ileum by blocking kv1.1 at enteric nervous system and enhancing acetylcholine release.

Potent and selective peptidyl blockers of the Shaker-type (Kv1) voltage-gated potassium channels were used to determine the role of these channels in regulating the spontaneous motility of smooth muscle preparations. Margatoxin (MgTX), kaliotoxin, and agitoxin-2 at 1 to 10 nM and agitoxin-1 at 50 to 100 nM induce twitches in guinea pig ileum strips. These twitches are abolished by tetrodotoxin (TTX, 0.5 microM), atropine (1 microM), hexamethonium (10 microM), or nifedipine (0.1 microM). It is proposed that blockade of Kv1 channels by MgTX, kaliotoxin, or the agitoxins increases excitability of intramural nerve plexuses in the ileum, promoting release of acetylcholine from excitatory motor nerve terminals. This, in turn, leads to Ca2+-dependent action potentials and twitching of the muscle fibers. MgTX does not induce twitches in several other guinea pig and/or rat vascular, genitourinary, or gastrointestinal smooth muscles, although small increases in spontaneous myogenic activity may be seen in detrusor muscle exposed to >30 nM MgTX. This effect is not reversed by TTX or atropine. The TTX- and atropine-sensitive twitches of guinea pig ileum are also induced by nanomolar concentrations of alpha-dendrotoxin, a selective blocker of Shaker Kv1.1 and 1.2 subtypes, or stichodactylatoxin, a peptide isolated from sea anemone that displays high affinity for Kv1.1 and 1.3, but not by charybdotoxin, which blocks Kv1.2 and 1.3 but not 1.1. The data taken together suggest that high-affinity blockade of Kv1.1 underlies the ability of MgTX, kaliotoxin, agitoxin-1, agitoxin-2, alpha-dendrotoxin, and stichodactylatoxin to elicit TTX-sensitive twitches in guinea pig ileum.

Mechanisms of the UTP-induced tension in mammalian skeletal muscles.

The mechanisms of UTP-induced tension in human and rat skinned fibers were investigated using isometric tension recordings, electrophysiological techniques and biochemical methods. In fast-type fibers from rat extensor digitorum longus (EDL) the UTP-induced tension: a) required previous loading of Ca2+ into the sarcoplasmic reticulum (SR); b) was inhibited by previous exposure to caffeine; c) was abolished by functional disruption of the SR; d) was not affected by blockade of the SR Ca(2+)-release channels by ruthenium red or heparin; e) was prevented by spermidine. These data point to the SR as the target of UTP action and suggest a pathway of UTP-induced Ca(2+)-release independent of the ryanodine- or the IP3-sensitive Ca(2+)-release channels. Accordingly, UTP failed to stimulate the electrophysiological activity of ryanodine-sensitive channels, incorporated into lipid bilayers. We suggest that UTP-induced Ca(2+)-release might occur via the channel form of the SR Ca(2+)-ATPase. The UTP-induced tension in human slow-type fibers was not affected by the SR Ca2+ content or by disruption of the SR, but was accompanied by changes in the tension-pCa relationship, namely increase in maximum Ca(2+)-activated tension, and in apparent Ca(2+)-affinity of troponin. The UTP-induced tension in slow-type fibers from rat soleus was partially inhibited by Ca(2+)-depletion from, or by disruption of the SR, and was accompanied by changes in tension/pCa relationship, similar to those observed in human fibers. Both in skinned fibers and in isolated SR vesicles, UTP was less effective than ATP as a substrate for the SR Ca(2+)-ATPase. This effect might contribute to the UTP-induced tension.

Mechanisms of the UTP-induced tension in mammalian skeletal muscles.

The mechanisms of UTP-induced tension in human and rat skinned fibers were investigated using isometric tension recordings, electrophysiological techniques and biochemical methods. In fast-type fibers from rat extensor digitorum longus (EDL) the UTP-induced tension: a) required previous loading of Ca2+ into the sarcoplasmic reticulum (SR); b) was inhibited by previous exposure to caffeine; c) was abolished by functional disruption of the SR; d) was not affected by blockade of the SR Ca(2+)-release channels by ruthenium red or heparin; e) was prevented by spermidine. These data point to the SR as the target of UTP action and suggest a pathway of UTP-induced Ca(2+)-release independent of the ryanodine- or the IP3-sensitive Ca(2+)-release channels. Accordingly, UTP failed to stimulate the electrophysiological activity of ryanodine-sensitive channels, incorporated into lipid bilayers. We suggest that UTP-induced Ca(2+)-release might occur via the channel form of the SR Ca(2+)-ATPase. The UTP-induced tension in human slow-type fibers was not affected by the SR Ca2+ content or by disruption of the SR, but was accompanied by changes in the tension-pCa relationship, namely increase in maximum Ca(2+)-activated tension, and in apparent Ca(2+)-affinity of troponin. The UTP-induced tension in slow-type fibers from rat soleus was partially inhibited by Ca(2+)-depletion from, or by disruption of the SR, and was accompanied by changes in tension/pCa relationship, similar to those observed in human fibers. Both in skinned fibers and in isolated SR vesicles, UTP was less effective than ATP as a substrate for the SR Ca(2+)-ATPase. This effect might contribute to the UTP-induced tension.

The beta subunit of the high conductance calcium-activated potassium channel. Identification of residues involved in charybdotoxin binding.

Coexpression of alpha and beta subunits of the high conductance Ca2+-activated K+ (maxi-K) channel leads to a 50-fold increase in the affinity for 125I-charybdotoxin (125I-ChTX) as compared with when the alpha subunit is expressed alone (Hanner, M., Schmalhofer, W. A., Munujos, P., Knaus, H.-G., Kaczorowski, G. J., and Garcia, M. L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2853-2858). To identify those residues in the beta subunit that are responsible for this change in binding affinity, Ala scanning mutagenesis was carried out along the extracellular loop of beta, and the resulting effects on 125I-ChTX binding were determined after coexpression with the alpha subunit. Mutagenesis of each of the four Cys residues present in the loop causes a large reduction in toxin binding affinity, suggesting that these residues could be forming disulfide bridges. The existence of two disulfide bridges in the extracellular loop of beta was demonstrated after comparison of reactivities of native beta and single-Cys-mutated subunits to N-biotin-maleimide. Negatively charged residues in the loop of beta, when mutated individually or in combinations, had no effect on toxin binding with the exception of Glu94, whose alteration modifies kinetics of ligand association and dissociation. Further mutagenesis studies targeting individual residues between Cys76 and Cys103 indicate that four positions, Leu90, Tyr91, Thr93, and Glu94 are critical in conferring high affinity 125I-ChTX binding to the alpha.beta subunit complex. Mutations at these positions cause large effects on the kinetics of ligand association and dissociation, but they do not alter the physical interaction of beta with the alpha subunit. All these data, taken together, suggest that the large extracellular loop of the maxi-K channel beta subunit has a restricted conformation. Moreover, they are consistent with the view that four residues appear to be important for inducing an appropriate conformation within the alpha subunit that allows high affinity ChTX binding.

Functional effects of uridine triphosphate on human skinned skeletal muscle fibers.

Chemically skinned human skeletal muscle fibers were used to study the effects of uridine triphosphate (UTP) on the tension-pCa relationship and on Ca2+ uptake and release by the sarcoplasmic reticulum (SR). Total replacement (2.5 mM) of adenosine triphosphate (ATP) with UTP (i) displaced the tension-pCa relationship to the left along the abcissae and increased maximum Ca(2+)-activated tension, both effects being larger in slow- than in fast-type fibers; (ii) markedly reduced Ca2+ uptake by the SR (evaluated by the caffeine-evoked tension) in both fiber types; (iii) had no effect on the rate of depletion of caffeine-sensitive Ca2+ stores during soaking in relaxing solutions; (iv) induced tension in slow- but not in fast-type fibers. The effects on the SR functional properties are consistent with the notion that UTP is a poor substitute for ATP as a substrate for the Ca ATPase pump and as an agonist of the ryanodine-sensitive Ca(2+)-release channel. The UTP-induced tension in human slow-type fibers is attributed to effect(s) of the nucleotide on the tension-pCa relationship of the contractile machinery. The present data reveal important differences between the effects of UTP on human versus rat muscle fibers.

Effects of uridine triphosphate on skinned skeletal muscle fibers of the rat.

Chemically skinned muscle fibers from rat extensor digitorum longus muscle were used to study the effects of uridine triphosphate (UTP) on Ca2+ uptake and release by the sarcoplasmic reticulum (SR) and on Ca2+-activated tensions. Total replacement (2.5 mM) of adenosine triphosphate (ATP) with UTP (i) increased submaximal Ca2+-induced tension (pCa 6.2-5.8) but diminished Po, the maximum tension elicited by pCa 4.2, by ca. 15%, (ii) markedly reduced Ca2+ uptake by the SR (evaluated by caffeine-elicited tension); and (iii) induced tension in Ca2+-loaded fibers. The UTP-induced tension averaged 55% of Po and its rates of development and decay were considerably slower than those of caffeine-evoked tension. The UTP-induced tension (i) depended on the Ca2+-loading conditions; (ii) was reversibly blocked by brief (15 s) exposures of Ca2+-loaded fibers to 5 mM EGTA or by pretreatment with caffeine; (iii) was abolished by functional disruption of the SR with the nonionic detergent Brij-58; and (iv) persisted after blockade of the SR Ca2+ release channels with ruthenium red. Exposure of Ca2+-loaded fibers to UTP depressed the tension elicited subsequently by caffeine, and enhanced the rate of depletion of caffeine-sensitive Ca2+ stores during soaking in relaxing solutions containing 5 mM EGTA. The UTP-induced tension is attributed to increased release of Ca2+ from the SR, via a ruthenium red insensitive pathway(s), combined with reduced Ca2+ uptake by the SR and increased Ca2+ affinity of the contractile proteins.

Comparative pharmacokinetics of different oral nifedipine preparations in healthy Brazilian volunteers.

1. The pharmacokinetics of different pharmaceutical preparations of oral nifedipine--Adalat (capsule), Oxcord and Cardalin (tablets)--was determined after administration of single oral doses of 10 mg to nine healthy young Brazilian volunteers (7 men). 2. There were no significant changes in heart rate or systolic and diastolic blood pressure measured in the sitting position within 8 h of nifedipine administration to these normotensive volunteers. No side effects were reported by the volunteers or observed by the attending physicians during the study. 3. No significant differences were observed among the three preparations in relation to the following pharmacokinetic parameters obtained from the plasma concentration-time curves: area under the curve (AUC), slope (beta) and half-life (T1/2) of the elimination phase, volume of distribution (Vd/F) and total body clearance (CL/F), both expressed as functions of the oral bioavailability (F) of nifedipine. 4. The peak plasma concentration of nifedipine (Cmax) and the time to reach Cmax (Tmax) were not different for the two tablet preparations. However, Cmax was significantly higher, and Tmax was significantly shorter for the capsule. These data indicate that the capsule and the tablet preparations are not bioequivalent.

Comparative pharmacokinetics of different oral nifedipine preparations in healthy brazilian volunteers

The pharmacokinetics of different pharmaceutical preparations of oral nifedipine-Adalat (capsule), Oxcord and Cardalin (tablets)-was determined after administration of single oral doses of 10 mg to nine healthy young Brazilian volunteers (7 men). There no significant changes in heart rate or systolic and diastolic blood pressure measured in the sitting position within 8 h of nifedipine administration to these normotensive volunteers. No side effects were reported by the volunteers or observed by the attending physicians during the study. No significant differences were observed among the three preparations in relation to the following pharmacokinetic parameters obtained from the plasma concentration-time curves: area under the curve (AUC), slope (beta) and half-life (T½) of the elimination phase, volume of distribution (Vd/F) and total body clearance (CL/F), both expre4ssed as functions of the oral bioavailability (F) of nifedipine. The peak, plasma concentration of nifedipine (C max) and the time to reach C max (T max) were not different for the rwo tablet preparations. However, C max was significantly higher, and T max was significantly shorter for the capsule. These data indicate that the capsule and the tablet preparations are not bioequivalent