Effects of cocaine and its major metabolites on the HERG-encoded potassium channel.

Cocaine abuse has been reported to result in QT prolongation in humans; however, the mechanisms underlying this effect are still poorly understood. In this study we compared the direct effects of cocaine and its major metabolites in human embryonic kidney 293 cells stably transfected with human ether-a-go-go-related gene (HERG). Cocaine blocked HERG-encoded potassium channels with an IC50 of 4.4 +/- 1.1 microM (22 degrees C). Cocaethylene (a metabolite formed in the presence of ethanol) had a significantly lower IC50 of 1.2 +/- 1.1 microM (P < 0.0001), and cocaine's primary pyrolysis metabolite methylecgonidine blocked HERG with a higher IC50 of 171.7 +/- 1.2 microM. In contrast, 1 mM ecgonine methylester or benzoylecgonine produced only a minimal block (21 +/- 4 and 15 +/- 8%, respectively). Blockade of HERG by cocaine, cocaethylene, and methylecgonidine increased significantly over the voltage range where HERG activates, but became constant at voltages where HERG activation was maximal, indicating that all three drugs block open channels, but by a mechanism that is not highly sensitive to voltage per se. Cocaine and cocaethylene also significantly slowed the time course of deactivation at -60 mV, an effect consistent with open channel block. We conclude that cocaethylene is slightly more potent than cocaine as a blocker of HERG, whereas methylecgonidine has much lower potency, and both benzoylecgonine and ecgonine methyl ester are essentially inactive at clinically relevant concentrations.

Evidence for high molecular weight Na-Ca exchange in cardiac sarcolemmal vesicles.

Cardiac sarcolemma (SL) vesicles were subjected to irradiation inactivation-target sizing analyses and gel permeation high performance liquid chromatography (HPLC) to ascertain the weight range of native Na-Ca exchange. Frozen SL vesicle preparations were irradiated by electron bombardment and assayed for Na-Ca exchange activity. When applied to classical target sizing theory, the results yielded a minimum molecular weight (Mr) of approximately 226,000 +/- 20,000 SD (n = 6). SL vesicle proteins were solubilized in 6% sodium cholate in the presence of exogenous phospholipid and fractionated by size on a TSK 30XL HPLC column. Eluted proteins were mixed 1:1 with mobile phase buffer containing 50 mg/ml soybean phospholipid and reconstituted by detergent dilution. The resulting proteoliposomes were assayed for Na-Ca exchange activity. Na-Ca exchange activity eluted in early fractions containing larger proteins as revealed by SDS-PAGE. Recovery of total protein and Na-Ca exchange activity were 91 +/- 7 and 68 +/- 11%, respectively. In the peak fraction, Na-Ca exchange specific activity increased two- to threefold compared to reconstituted controls. Compared to the elution profile of protein standards under identical column conditions, sodium cholate solubilized exchange activity had a minimum Mr of 224,000 Da. Specific 45Ca2+-binding SL proteins with Mr of 234,000, 112,000, and 90,000 Da were detected by autoradiography of proteins transferred electrophoretically to nitrocellulose. These data suggest that native cardiac Na-Ca exchange is approximately 225,000 Da or larger. The exact identification and purification of cardiac Na-Ca exchange protein(s) remains incomplete.

Hereditary parakeratosis in shorthorn beef calves.

Parakeratosis was diagnosed in 9 Shorthorn beef calves over a 4-year period. When pedigrees of these calves were analyzed, familial associations were strong. Thirty-six coefficients of relationship among all possible combinations of the 9 affected calves ranged from 0.5 to 39.8% and averaged 15.6%. All affected calves were descendants of bull A. Of 9 affected calves, 6 had bull A in their paternal and maternal pedigrees. The 3 remaining affected calves had bull A in their sire's pedigree and were born to 2 full-sib dams. Seemingly, parakeratosis in this Shorthorn herd was hereditary with the mode of inheritance being that of a simple autosomal recessive.