Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

Abstract

Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence but not in the absence of K ؉ . Studies with purified membranes revealed that CPZ reduced Na ؉ -dependent phosphorylation by interference with Na ؉ binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K ؉ . Low-affinity ATP interaction with the enzyme was strongly reduced after CPZ treatment; in contrast, indirectly measured interaction with ADP was much increased, which suggests that composite regulatory communication with nucleotides takes place during turnover. Studies with lipid vesicles revealed that CPZ reduced ATP-dependent digitoxigenin-sensitive 22 Na ؉ influx into K ؉ -loaded vesicles only at saturating ATP concentrations. The drug apparently abolishes the regulatory effect of ATP on the pump. Drawing on previous homology modeling studies of Na,K-ATPase to atomic models of sarcoplasmic reticulum Ca-ATPase and on kinetic data, we propose that CPZ uncouples an Na ؉ cycle from an Na ؉ /K ؉ cycle in the pump. The Na ؉ cycle possibly involves transport through the recently characterized Na ؉ -specific site. A shift to such an uncoupled mode is believed to produce pumps mediating uncoupled Na ؉ efflux by modifying the transport stoichiometry of single pump units.sodium pump ͉ uncoupling ͉ potassium occlusion N a,K-ATPase (NKA), a P-type cation pump (1), utilizes energy from ATP hydrolysis to exchange intracellular Na ϩ for extracellular K ϩ against their electrochemical potential gradients. The functional pump contains an ␣-subunit that undergoes substrateinduced conformational transitions, coupling ATP hydrolysis to ion transport, and a ␤-subunit important for maturation and targeting of the complex to the membrane (2).According to the Albers-Post scheme, occlusion of three intracellular Na ϩ ions promotes phosphorylation of the ␣-subunit from ATP, whereas occlusion of two extracellular K ϩ ions promotes dephosphorylation (2). Depending on the specific ionic composition on both sides of the membrane, the pump catalyzes several partial reactions (3). In the absence of K ϩ , NKA mediates ATP-driven Na ϩ transport processes, including exchange as well as uncoupled efflux (reviewed in ref. 4). Furthermore, in inside-out vesicles of human red cells, the ratio of net 22 Na ϩ influx/ 86 Rb ϩ efflux was reduced at sub-millimolar cytoplasmic Na ϩ concentrations. This finding indicates that under this particular condition, the pump can function with less than a maximum number of sites filled with Na ϩ ions (5).In sarcoplasmic reticulum (SR) Ca 2ϩ -ATPase (SERCA), the best characterized P-type pump, ATP hydrolysis can take place without Ca 2ϩ transport to allow for thermogenesis (6). This uncoupling is prominent in the presence of a Ca 2ϩ gradient across the SR membrane. Recent studies have shown that sarcolipin, a small SERCA regulator, increases heat production by this pump (7). This finding demonstrates a regulatory inter...

Anxiolytic-like effects induced by blockade of transient receptor potential vanilloid type 1 (TRPV1) channels in the medial prefrontal cortex of rats

“…Although, as pointed out by a recent review, administration of anxiolytic drugs commonly produces biphasic dose responses independent of the animal model employed (Calabrese 2008), this effect could be reflecting a lack of drug specificity at high concentrations. For example, it has been recently showed that at micromolar range, capsazepine can interfere with a Na,K-ATPase pump (Mahmmoud 2008).…”

Capsaicin Stimulates Uncoupled ATP Hydrolysis by the Sarcoplasmic Reticulum Calcium Pump

“…3, showing more than 2-fold activation of ATP hydrolysis after CPS treatment. The ATP activation curves were analyzed by comparing two different fitting procedures, which revealed interesting information (see a similar analysis in the supplemental material provided in Mahmmoud (35). Fitting a Michaelis-Menten equation to the data showed that CPS treatment increases the ATP affinity.…”

Capsaicinoids Regulate Airway Anion Transporters through Rho Kinase– and Cyclic AMP–Dependent Mechanisms

“…Based on the structure of capsaicin, capsazepine was developed as a specific TRPV1 antagonist (7). Although the capsacinoids exert their effects through TRPV1 at submicromolar concentrations, at micro-to millimolar concentrations, they seem to have effects beyond TRPV1-dependent actions, such as inhibitory effects on diverse ion channels and transporters (8)(9)(10)(11). However, little has been documented regarding the effects of the capsaicinoids on the airway ion transport via TRPV1-independent mechanisms.…”