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Aim: Gold nanoparticles are widely used for biomedical applications, but the precise molecular mechanism of their interaction with cellular structures is still unclear. Assuming that intracellular calcium fluctuations associated with surface plasmon‐induced calcium entry could modulate the activity of potassium channels, we studied the effect of 5 nm gold nanoparticles on calcium‐dependent potassium channels and associated calcium signaling in freshly isolated rat pulmonary artery smooth muscle cells and cultured hippocampal neurons. Methods: Outward potassium currents were recorded using patch‐clamp techniques. Changes in intracellular calcium concentration were measured using the high affinity Ca2+ fluorescent indicator fluo‐3 and laser confocal microscope. Results: In pulmonary artery smooth muscle cells, plasmonic gold nanoparticles increased the amplitude of currents via large‐conductance Ca2+‐activated potassium channels, which was potentiated by green laser irradiation near plasmon resonance wavelength (532 nm). Buffering of intracellular free calcium with ethylene glycol‐bis‐N,N,N′,N′‐tetraacetic acid (EGTA) abolished these effects. Furthermore, using confocal laser microscopy it was found that application of gold nanoparticles caused oscillations of intracellular calcium concentration that were decreasing in amplitude with time. In cultured hippocampal neurons gold nanoparticles inhibited the effect of EGTA slowing down the decline of the BKCa current while partially restoring the amplitude of the slow after hyperpolarizing currents. Conclusion: We conclude that fluctuations in intracellular calcium can modulate plasmonic gold nanoparticles‐induced gating of BKCa channels in smooth muscle cells and neurons through an indirect mechanism, probably involving the interaction of plasmon resonance with calcium‐permeable ion channels, which leads to a change in intracellular calcium level. [ABSTRACT FROM AUTHOR]

There is little information about the mechanisms by which G-protein-coupled receptors gate ion channels although many ionotropic receptors are well studied. We have investigated gating of the muscarinic cationic channel, which mediates the excitatory effect of acetylcholine in smooth muscles, and proposed a scheme consisting of four pairs of closed and open states. Channel kinetics appeared to be the same in cell-attached or outside-out patches whether the channel was activated by carbachol application or by intracellular dialysis with GTP[gamma]S. Since in the latter case G-proteins are permanently active, it is concluded that the cationic channel is the major determinant of its own gating, similarly to the [K.sub.Ach] channel (Ivanova-Nikolova, T.T., and G.E. Breitwieser. 1997. J. Gen. Physiol. 109:245-253). Analysis of adjacent-state dwell times revealed connections between the states that showed features conserved among many other ligand-gated ion channels (e.g., nAChR, B[K.sub.Ca] channel). Open probability ([P.sub.O]) of the cationic channel was increased by membrane depolarization consistent with the prominent U-shaped I-V relationship of the muscarinic whole-cell current at negative potentials. Membrane potential affected transitions within each closed-open state pair but had little effect on transitions between pairs; thus, the latter are likely to be caused by interactions of the channel with its ligands, e.g., [Ca.sup.2+] and G[alpha]o-GTP. Channel activity was highly heterogeneous, as was evident from the prominent cycling behavior when [P.sub.o] was measured over 5-s intervals. This was related to the variable frequency of openings (as in the [K.sub.Ach] channel) and, especially, to the number of long openings between consecutive long shuttings. Analysis of the underlying Markov chain in terms of probabilities allowed us to evaluate the contribution of each open state to the integral current (from shortest to longest open state: 0.1, 3, 24, and 73%) as [P.sub.O] increased 525-fold in three stages. KEY WORDS: Markov chain * channel kinetics * G-protein * carbachol * smooth muscle

The transient receptor potential melastatin 8 (TRPM8) channel has been characterized as a cold and menthol receptor expressed in a subpopulation of sensory neurons but was recently identified in other tissues, including the respiratory tract, urinary system, and vasculature. Thus TRPM8 may play multiple functional roles, likely to be in a tissue-and activation state-dependent manner. We examined the TRPM8 channel presence in large arteries from rats and the functional consequences of their activation. We also aimed to examine whether these channels contribute to control of conscious human skin blood flow. TRPM8 mRNA and protein were detected in rat tail, femoral and mesenteric arteries, and thoracic aorta. This was confirmed in single isolated vascular myocytes by immunocytochemistry. Isometric contraction studies on endothelium-denuded relaxed rat vessels found small contractions on application of the TRPM8-specific agonist menthol (300 [micro]M). However, both menthol and another agonist icilin (50 [micro]M) caused relaxation of vessels precontracted with KC1 (60 mM) or the [alpha]-adrenoceptor agonist phenylephrine (2 [micro]M) and a reduction in sympathetic nerve-mediated contraction. These effects were antagonized by bromoenol lactone treatment, suggesting the involvement of [Ca.sup.2+]-independent phospholipase [A.sub.2] activation in TRPMS-mediated vasodilatation. In thoracic aorta with intact endothelium, menthol-induced inhibition of KCl-induced contraction was enhanced. This was unaltered by preincubation with either [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME; 100 nM), a nitric oxide synthase inhibitor, or the ACh receptor antagonist atropine (1 [micro]M). Application of menthol (3% solution, topical application) to skin caused increased blood flow in conscious humans, as measured by laser Doppler fluximetry. Vasodilatation was markedly reduced or abolished by prior application of L-NAME (passive application, 10 mM) or atropine (iontophoretic application, 100 nM, 30 s at 70 [micro]A). We conclude that TRPM8 channels are present in rat artery vascular smooth muscle and on activation cause vasoconstriction or vasodilatation, dependent on previous vasomotor tone. TRPM8 channels may also contribute to human cutaneous vasculature control, likely with the involvement of additional neuronal mechanisms. human; rat; artery

In recent years, the transient receptor potential melastatin member 8 (TRPM8) channel has emerged as a promising prognostic marker and putative therapeutic target in prostate cancer (PCa). However, the mechanisms [...]

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [[Ca.sup.+]] in a [Ca.sup.2+]-free medium followed by a secondary [[Ca.sup.2+]] rise upon [Ca.sup.2+] readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as [Ca.sup.2+]-activated ([I.sub.Cl(Ca)]) and volume-regulated ([[I.sub.Cl, swell]) chloride currents. NPPB and DIDS more efficiently inhibited [I.sub.Cl(Ca)] and [[I.sub.Cl, swell] respectively. Cell swelling caused by hypotonic solution invariably activated [I.sub.Cl, swell] while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling-induced [I.sub.Cl, swell], while its inactive analogue U-73343 had no effect. [I.sub.Cl(Ca)] could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [[[Ca.sup.2+]].sub.i] in the pipette solution at 500 nM. In contrast, [I.sub.Cl, swell] could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by Ins[P.sub.3] infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced [I.sub.Cl, swell] in a nonadditive manner, suggesting their convergence on a common pathway. [I.sub.Cl, swell] and [I.sub.Cl(ca)] showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated [I.sub.Cl(Ca), but abolished [I.sub.Cl, swell], thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that [I.sub.Cl, swell] can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a 'selection' switch between closely localized channels. KEY WORDS: human keratinocytes * histamine receptors * cell swelling * chloride channels * store-operated [Ca.sup2+] entry

Excitation-contraction coupling is discussed relative to gastrointestinal and other smooth muscles. A change in calcium-ion concentration is most important among the control mechanisms of tension in smooth muscles, but other G protein- and kinase-mediated mechanisms can modulate or even bring on tension or give rise to relaxation. Some effects may come from vectorial transport of calcium by the sarcoplasmic reticulum, but there are some evidence gives rise to arguments against that viewpoint.

Functional biomechanical parameters of muscle soleus contraction in rats as well as selected blood biochemical parameters were studied during the first 3 days of post-traumatic syndrome progression caused by the destruction of muscle cells by compression. Single administration of the antioxidant C60 fullerene and the selective agonist of TRPM8 channels menthol were used as therapeutic agents. Injection of C60 fullerene at a concentration of 1 mg/kg into the damaged muscle improved its contractile function by 25–28%. The use of combined injections of C60 fullerene and menthol (at the concentration 1 mg/kg) improved this index by additional 27–39% and simultaneously stabilized the decrease in muscle strength observed throughout the experiment. A tendency towards a decrease in the indexes of the above described biochemical parameters by 10–15% were found with the therapeutic administration of C60 fullerene. With combined injections of C60 fullerene and menthol, the above described biochemical parameters decreased by an additional 17–24%. The synergism between the action of menthol and C60 fullerene on the post-traumatic recovery of skeletal muscle function opens up new perspectives for the clinical application of this combination therapy. [ABSTRACT FROM AUTHOR]