Exposure of the acetylcholine receptor to acetylcholine, or its stable analogue carbamylcholine, inactivates (desensitizes) the receptor. Inactivation of receptor-controlled ion (86Rb+) flux in the presence of different concentrations of carbamylcholine (12.5 microM to 28 mM) was measured in the millisecond to minute time region, using a quench flow technique and membrane vesicles prepared from the electric organ of Electrophorus electricus. Three different kinetic measurements were made to establish the relationship between carbamylcholine concentration and the ion translocation process: (i) the rate of inactivation of the ion translocation process; (ii) the rate of recovery of the inactivated receptor upon removal of carbamylcholine; and (iii) the rate of the ion flux mediated by equilibrium mixtures of active and inactive receptor forms. The kinetics of these three processes follow single-exponential rate laws, and simple analytical expressions for their ligand concentration dependence could be used. Therefore, it was possible to determine the value of the rate constants in a scheme relating the ligand binding steps to ion translocation, and to predict the dependence of these rate constants on carbamylcholine concentration over the 200-fold range investigated.
We examined the effects of various types of oxidative stress on cell survival and on mitochondrial membrane potential (delta psi m) in PC12 cells transfected with BCL-2. Several types of oxidative stress such as exposure to hydrogen peroxide, 13-L-hydroperoxylinoleic acid, and xanthine + xanthine oxidase triggered apoptotic nuclear condensation and DNA fragmentation in normal PC12 cells. These types of oxidative stress induced significant increases in level of reactive oxygen species (ROS) before cell death. By contrast, BCL-2 prevented the apoptosis induced by these oxidative stresses. However, BCL-2 did not reduce ROS levels, indicating that it functions downstream of ROS generation. We measured delta psi m as a potential target of ROS during oxidative stress-induced cell death. Hydrogen peroxide, 13-L-hydroperoxylinoleic acid, and xanthine + xanthine oxidase induced a significant loss of delta psi m simultaneously with cell death. BCL-2 prevented the decrease in delta psi m as well as apoptosis induced by oxidative stress. These observations suggest that the oxidative stress triggers apoptosis associated with both increased generation of ROS and decreases in level of delta psi m and that BCL-2 prevents cell death as well as delta psi m but not ROS production.
To study the effects of tea components on ionotropic gamma-aminobutyric acid (GABA) receptor response, ionotropic GABA receptors (GABA(A) receptors) were expressed in Xenopus oocytes by injecting cRNAs synthesized from cloned cDNAs of the alpha(1) and beta(1) subunits of the bovine receptors, and their electrical responses were measured by a voltage clamping method. Extracts of green tea, black tea, and oolong tea in an aqueous solution induced the GABA-elicited response, which showed that these teas contain GABA, whereas coffee does not. Caffeine weakly inhibited the response in a competitive manner (K(i) = 15 mM), and (+)-catechin inhibited it in a noncompetitive one (K(i) = 1.7 mM). Especially, two catechin derivatives, (-)-epicatechin gallate and (-)-epigallocatechin gallate, inhibited the response strongly. Alcohols such as leaf alcohol or linalool potentiated the response, possibly because their binding to the potentiation site enhances the GABA-binding affinity to GABA(A) receptors when they bind. Extracts of green tea made with ethyl ether, which must contain lipophilic components of green tea, inhibited the response elicited by GABA, possibly because the amounts of caffeine and catechin derivatives were much larger than fragrant alcohols in such extracts of tea.
To study the effects of polyphenols on the Na(+)/glucose cotransporter (SGLT1) response, SGLT1 was expressed in Xenopus oocytes by injecting cRNA synthesized from the cloned cDNA of the small intestine cotransporter of rats, and the electrical response elicited by glucose or galactose was measured by a voltage clamping method. Most phenol derivatives had no effect on the response. However, the polyphenols (+)-catechin, (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg), which are components of green tea, caused an inhibition of the response, which was almost independent of glucose concentration. The inhibition constants were estimated to be 2.3 mM for (+)-catechin and 0.45 mM for both ECg and EGCg, assuming the noncompetitive inhibition mechanism. Saponin prepared from tea seeds also inhibited the response significantly. Tannic acid and aqueous extracts of teas induced nonspecific electrical responses in both cRNA-injected and noninjected oocytes at lower concentrations than those that caused an inhibition of the SGLT1 response when their dose-dependent effects were examined. These results are possibly helpful in the development of a dietary supplement for diabetic patients.
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