Search

Polycyclic and halogenated aromatic hydrocarbons (PAHs and HAHs) can enhance the generation of reactive oxygen species (ROS) by inducing cytochrome P450 1A (CYP 1A) in vivo and in vitro. While the brain is vulnerable to oxidative injury, whether or not CYP 1A induction in the brain can produce measurable levels of oxidative damage has not been reported. The objective of this study was to investigate the effect of this induction on oxidative damage to the CNS. Time course changes in rat brain CYP 1A activity were determined by measurement of ethoxyresorufin O-deethylase (EROD) activity in whole brain homogenates. Three days after exposure of rats to five daily injections of 3-methylcholanthrene (3-MC) an approximately sevenfold increase in EROD activity was observed. Hepatic levels were increased 60-100 fold. This increase in CYP 1A activity was not accompanied by increased protein or lipid oxidation as measured by tryptophan fluorescence and TBAR formation, or decreased glutamine synthetase (GS) activity. These findings indicate that if increased CYP 1A activity in the brain following 3-MC treatment leads to increased ROS generation, the increase is insufficient to overwhelm the endogenous antioxidant defense system, produce detectable oxidative damage, and alter glutamate homeostasis.

Nucleotide-dependent gating of DeltaF508-CFTR was evaluated in membrane patches excised from HEK 293 and mouse L-cells and compared to observations on wt-CFTR channels recorded in the same expression systems. DeltaF508-CFTR exhibited PKA activated, ATP-dependent channel gating. When compared to wt-CFTR, the Km for ATP was increased by ninefold (260 micron vs. 28 micron) and maximal open probability (Po) was reduced by 49% (0.21 +/- 0.06 vs. 0.41 +/- 0. 02). Additionally, in the absence of PKA, DeltaF508-CFTR inactivated over a 1 to 5 min period whereas wt-CFTR remained active. Time-dependent inactivation could be mimicked in wt-CFTR by the intermittent absence of ATP in the cytosolic solution. The effects of 3-isobutyl-1-methyl xanthine (IBMX), a compound reported to stimulate DeltaF508-CFTR, were evaluated on wt- and DeltaF508-CFTR channels. At concentrations up to 5 mm, IBMX caused a concentration dependent reduction in the observed single channel amplitude (i) of wt-CFTR (maximal observed reduction 35 +/- 3%). However, IBMX failed to significantly alter total patch current because of a concomitant 30% increase in Po. The effects of IBMX on DeltaF508-CFTR were similar to effects on wt-CFTR in that i was reduced and Po was increased by similar magnitudes. Additionally, DeltaF508-CFTR channel inactivation was dramatically slowed by IBMX. These results suggest that IBMX interacts with the ATP-bound open state of CFTR to introduce a short-lived nonconducting state which prolongs burst duration and reduces apparent single channel amplitude. A secondary effect observed in DeltaF508-CFTR, which may result from this interaction, is a prolongation of the activated state. In light of previously proposed linear kinetic models of CFTR gating, these results suggest that IBMX traps CFTR in an ATP-bound state which may preclude inactivation of DeltaF508-CFTR.

CFTR shares structural homology with the ABC transporter superfamily of proteins which hydrolyze ATP to effect the transport of compounds across cell membranes. Some superfamily members are characterized as P-type ATPases because ATP-dependent transport is sensitive to the presence of vanadate. It has been widely postulated that CFTR hydrolyzes ATP to gate its chloride channel. However, direct evidence of CFTR hydrolytic activity in channel gating is lacking and existing circumstantial evidence is contradictory. Therefore, we evaluated CFTR chloride channel activity under conditions known to inhibit the activity of ATPases; i.e., in the absence of divalent cations and in the presence of a variety of ATPase inhibitors. Removal of the cytosolic cofactor, Mg2+, reduced both the opening and closing rates of CFTR suggesting that Mg2+ plays a modulatory role in channel gating. However, channels continued to both open and close showing that Mg2+ is not an absolute requirement for channel activity. The nonselective P-type ATPase inhibitor, vanadate, did not alter the gating of CFTR when used at concentrations which completely inhibit the activity of other ABC transporters (1 mM). Higher concentrations of vanadate (10 mM) blocked the closing of CFTR, but did not affect the opening of the channel. As expected, more selective P-type (Sch28080, ouabain), V-type (bafilomycin A1, SCN-) and F-type (oligomycin) ATPase inhibitors did not affect either the opening or closing of CFTR. Thus, CFTR does not share a pharmacological inhibition profile with other ATPases and channel gating occurs in the apparent absence of hydrolysis, although with altered kinetics. Vanadate inhibition of channel closure might suggest that a hydrolytic step is involved although the requirement for a high concentration raises the possibility of previously uncharacterized effects of this compound. Most conservatively, the requirement for high concentrations of vanadate demonstrates that the binding site for this transition state analogue is considerably different than that of other ABC transporters.

We showed previously that the disulfonic stilbene DNDS (4,4'-dinitrostilben-2,2'-disulfonic acid) was a potent blocker of outwardly rectifying chloride channels (ORCC). The studies reported here were designed to quantify the relationship between electron withdrawal by the 4,4'-substituents and blocker potency. Specifically we compared the blocking effects and molecular properties of the symmetrically substituted 4,4'-diaminostilben-2, 2'-disulfonic acid (DADS) and the hemi-substituted 4-amino, 4'-nitrostilben-2,2'-disulfonic acid (ANDS) with those of DNDS. Blockade was studied using outwardly rectifying colonic chloride channels incorporated into planar lipid bilayers. DADS was 430-fold and ANDS 44-fold less potent than DNDS as blockers of ORCC. Amplitude distribution analysis revealed that all three disulfonic stilbenes act as open channel blockers. Furthermore, this kinetic analysis indicated that the lower potency of DADS and ANDS was due to an increase in off rate. These results support the conclusion that the 4,4'-substituents make an important contribution to blockade by stabilizing the channel-blocker complex. Isopotential electron contour maps illustrated the dramatic shift in charge at the 4,4'-poles of the disulfonic stilbene molecule from electronegative in DNDS to electropositive in DADS as well as the bipolar contour of ANDS. Thus, the greater potency of DNDS results from the symmetric electronegative regions at the 4,4'-poles of the molecule. We hypothesize that the channel protein has two corresponding electropositive areas at the blocker binding site.

Vesicles derived from epithelial cells of the colonic mucosa of the rat were fused to planar phospholipid bilayer membranes, revealing spontaneously switching anion-conducting channels of 50 pS conductance (at -30 mV with 200 mM Cl- each side). The equilibrium selectivity series was I- (1.7)/Br- (1.3)/Cl- (1.0)/F- (0.4)/HCO3- (0.4)/Na (less than 0.11). Only one dominant open-state conductance could be resolved, which responded linearly to Cl- concentrations up to 600 mM. The single-channel current-voltage curve was weakly rectifying with symmetrical solutions. When 50 mV were exceeded at the high-conductance branch of the curve, switching was arrested in the closed state. At more moderate voltages (+/- 40 mV) kinetics were dominated by one open state of about 35-msec lifetime and two closed states of about 2 and 9-msec lifetime. Of these, the more stable closed state occurred less often. At these voltages one additional closed state of significantly longer lifetime (greater than 0.5 sec) was observed.