TRPV1 activation power can switch an action mode for its polypeptide ligands.

TRPV1 (vanilloid) receptors are activated by different types of stimuli including capsaicin, acidification and heat. Various ligands demonstrate stimulus-dependent action on TRPV1. In the present work we studied the action of polypeptides isolated from sea anemone Heteractis crispa (APHC1, APHC2 and APHC3) on rat TRPV1 receptors stably expressed in CHO cells using electrophysiological recordings, fluorescent Ca2+ measurements and molecular modeling. The APHCs potentiated TRPV1 responses to low (3-300 nM) concentrations of capsaicin but inhibited responses to high (>3.0 µM) concentrations. The activity-dependent action was also found for TRPV1 responses to 2APB and acidification. Thus the action mode of APHCs is bimodal and depended on the activation stimuli strength-potentiation of low-amplitude responses and no effect/inhibition of high-amplitude responses. The double-gate model of TRPV1 activation suggests that APHC-polypeptides may stabilize an intermediate state during the receptor activation. Molecular modeling revealed putative binding site at the outer loops of TRPV1. Binding to this site can directly affect activation by protons and can be allosterically coupled with capsaicin site. The results are important for further investigations of both TRPV1 and its ligands for potential therapeutic use.

Muscarinic M1 modulation of acid-sensing ion channels.

Acid-sensing ion channels (ASICs) are ligand-gated cation channels that are highly expressed in nervous system. Little is known about the regulation of these channels. Therefore, we tested whether muscarinic M1 receptors can modulate ASICs. The muscarinic agonist oxotremorine methiodide applied to the bath solution strongly inhibited the whole-cell current in Chinese hamster ovary cells heterologously expressing ASIC1a and M1 receptors. Maximal current was inhibited 30% during muscarinic receptor stimulation. These effects were fast, fully reversible and subunit specific. The acid-sensing current in population of isolated rat hippocampus CA1 and striatum interneurons, thought to be carried primarily by ASIC1a, was similarly inhibited by oxotremorine methiodide. Thus, the current study identifies ASIC1a as a novel target for muscarinic signaling.

Mechanisms of non-steroid anti-inflammatory drugs action on ASICs expressed in hippocampal interneurons.

The inhibitory action of non-steroid anti-inflammatory drugs was investigated on acid-sensing ionic channels (ASIC) in isolated hippocampal interneurons and on recombinant ASICs expressed in Chinese hamster ovary (CHO) cells. Diclofenac and ibuprofen inhibited proton-induced currents in hippocampal interneurons (IC(50) were 622 +/- 34 muM and 3.42 +/- 0.50 mM, respectively). This non-competitive effect was fast and fully reversible for both drugs. Aspirin and salicylic acid at 500 muM were ineffective. Diclofenac and ibuprofen decreased the amplitude of proton-evoked currents and slowed the rates of current decay with a good correlation between these effects. Simultaneous application of acid solution and diclofenac was required for its inhibitory effect. Unlike amiloride, the action of diclofenac was voltage-independent and no competition between two drugs was found. Analysis of the action of diclofenac and ibuprofen on activation and desensitization of ASICs showed that diclofenac but not ibuprofen shifted the steady-state desensitization curve to more alkaline pH values. The reason for this shift was slowing down the recovery from desensitization of ASICs. Thus, diclofenac may serve as a neuroprotective agent during pathological conditions associated with acidification.

Subunit-dependent cadmium and nickel inhibition of acid-sensing ion channels.

Acid-sensing ion channels (ASIC) are ligand-gated cation channels that are highly expressed in peripheral sensory and central neurons. ASIC are transiently activated by decreases in extracellular pH and are thought to play important roles in sensory perception, neuronal transmission, and excitability, and in the pathology of neurological conditions, such as brain ischemia. We demonstrate here that the heavy metals Ni(2+) and Cd(2+) dose-dependently inhibit ASIC currents in hippocampus CA1 neurons and in Chinese hamster ovary (CHO) cells heterologously expressing these channels. The effects of both Ni(2+) and Cd(2+) were voltage-independent, fast, and reversible. Neither metal affected activation and desensitization kinetics but rather decreased pH-sensitivity. Moreover, distinct ASIC isoforms were differentially inhibited by Ni(2+) and Cd(2+). External application of 1 mM Ni(2+) rapidly inhibited homomeric ASIC1a and heteromeric ASIC1a/2a channels without affecting ASIC1b, 2a, and ASIC3 homomeric channels and ASIC1a/3 and 2a/3 heteromeric channels. In contrast, external Cd(+) (1 mM) inhibited ASIC2a and ASIC3 homomeric channels and ASIC1a/2a, 1a/3, and 2a/3 heteromeric channels but not ASIC1a homomeric channels. The acid-sensing current in isolated rat hippocampus CA1 neurons, thought to be carried primarily by ASIC1a and 1a/2a, was inhibited by 1 mM Ni(2+). The current study identifies ASIC as a novel target for the neurotoxic heavy metals Cd(2+) and Ni(2+).

Phorbol diacetate potentiates na(+)-k(+) ATPase inhibition by a putative endogenous ligand, marinobufagenin.

Several vasoconstrictor agents can regulate the phosphorylation status of the Na(+)-K(+) ATPase (NKA). We have recently demonstrated that mammalian tissues contain an endogenous bufadienolide, digitalis-like alpha(1)-NKA-selective ligand, marinobufagenin (MBG). Protein kinase C induces phosphorylation of the alpha(1)-NKA isoform, the major isoform in vascular smooth muscle, kidney, and heart cells. We hypothesized that protein kinase C-induced phosphorylation of NKA can potentiate the effect of endogenous digitalis-like ligands, and that such potentiation can occur in an NKA isoform-specific fashion. A protein kinase C activator, phorbol 12,13-diacetate (PDA, 50 nmol/L), induced phosphorylation of the alpha1-NKA from human mesenteric artery (HMA) sarcolemma and rat kidney but not that of the alpha(3)-NKA from rat fetal brain. In HMA sarcolemma, which predominantly contains alpha(1)-NKA, PDA (50 nmol/L) potentiated the NKA-inhibitory effect of MBG at the level of high-affinity binding sites (0.05 +/- 0.03 nmol/L versus 4.0 +/- 1.7 nmol/L, P<0.05). In contrast, PDA did not affect the NKA inhibition by ouabain, an alpha(3)-NKA ligand. In isolated endothelium-denuded HMA artery rings, 50 nmol/L PDA potentiated the MBG-induced vasoconstriction (EC(50), 17 +/- 6 nmol/L versus 150 +/- 40 nmol/L; P<0.01). Our results suggest that alpha(1)-isoform-specific NKA inhibition by the endogenous digitalis-like ligand, MBG, is substantially enhanced via NKA phosphorylation by protein kinase C. Thus, an interaction of protein kinase C-dependent phosphorylation and MBG on NKA activity may underlie the synergistic vasoactive effects of MBG and other endogenous vasoconstrictors in hypertension.