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We investigated the voltage dependence of nifedipine sensitivity of the ion channels formed by α1 subunits of the cardiac and smooth muscles (CM and SM, respectively) L-type Ca2+ channels stably expressed in Chinese hamster ovary (CHO) cells. Equilibrium inhibition of the α1 subunits, directing Ba2+ current ( I α1), by different concentrations of nifedipine was measured at the holding potentials ( V h ) of −100 mV and −50 mV. At V h =−100 mV, the SM α1 subunit was found to be 6-fold more sensitive for nifedipine than the subunit ( K −100=8.3 and 50.4 nM, respectively). Depolarization to −50 mV resulted in about sevenfold increase in the nifedipine potency for both subunits ( K −50=1.25 and 6.95 nM, respectively). The voltage dependence of steady-state inactivation could be fitted by a sum of two Boltzmann’s equations with slope factors of about 12 and 5 mV. The midpoints of both components in the CM α1 subunit (−75.6 and −42.8 mV) were more negative than those in the SM subunit (−63.7 and −37.7 mV). The relative contribution of the less sloped component in the control was rather low, being less pronounced in the CM (0.15) than in the SM (0.34) subunits. Nifedipine shifted the midpoints of inactivation curves to more negative potentials. The shift was more pronounced for the SM α1 subunit (−24.8 mV compared with −11.8 mV for the CM subunit in the presence of 10 nM nifedipine). Nifedipine differentially affected the two Boltzmann components of inactivation curves, more effectively inhibiting the steeper component. In the presence of 10 nM nifedipine, this component completely disappeared in the SM subunit, while its relative contribution in the CM subunit decreased from 0.85 to 0. 57, resulting in an apparent decrease in the steepness. These results are inconsistent with the receptor modulated hypothesis and suggest the existence of two mechanisms of inactivation characterized by different voltage dependence. [ABSTRACT FROM AUTHOR]

Barium currents through ion channels formed by α1-subunit of L-type Ca2+ channel ( I α1) were recorded from cultured chinese hamster ovary (CHO) cells. The cells were stably transfected with either a cardiac or a smooth muscle (SM) variant of α1-subunit. The I α1 in both cases exhibited similar fast voltage-dependent activation kinetics and slow apparent inactivation kinetics. With 10 mM Ba2+ in the bath solution, I α1 was activated at potentials more positive than −40 mV, peaked between 0 and +10 mV, and reversed at about +50 mV. In addition to slow apparent inactivation of inward current, both subunits provided an extremely slow voltage-dependent inactivation at potentials more positive than −100 mV, with half-maximum inactivation at −43.4 mV for cardiac and −41.4 mV for SM α1-subunits. The onset of inactivation as well as recovery from this process were within a time range of minutes. The voltage dependence of steady-state inactivation could be fitted by the sum of two Boltzmann's equations with slope factors of about 12 mV and 5 mV. A less sloped component has its midpoints at −75.6 and −63.7 mV, and a steeper component has its midpoints at −42.8 and −37.7 mV for cardiac and SM α1-subunits, respectively. Relative contribution of the steeper component was higher in both subunits (0.86 and 0.66 for cardiac and SM subunits, respectively). For comparison, the inactivation curves for 5-sec-long conditioning prepulses could be fitted by single Boltzmann's distribution with a 20 mV more positive midpoint and a slope factor of about 13 mV. In contrast to the steady-state inactivation curves, they showed considerable overlap with the steady-state activation curve. Our results reflect functional consequences of known sequence differences between α1-subunits of the cardiac and SM L-type Ca2+ channels and could be used in structural modeling of Ca2+ channel gating. In addition, they show that depolarization-induced window current has a transient nature and decays with the development of extremely slow inactivation. This is the first demonstration that slow inactivation of the L-type Ca2+ channel is an intrinsic property of its α1-subunits. [ABSTRACT FROM AUTHOR]

4-Aminopyridine is widely used as a Kv channel blocker. However, its mechanism of action is still a matter of debate. Extracellular calcium as well as 4-aminopyridine have been reported to interact with the activation kinetics of particular Kv channels. The objective of the present study was to investigate whether extracellular calcium could modulate the inhibition of Kv current by 4-aminopyridine in vascular myocytes. Kv current was recorded by using whole-cell patch-clamp in freshly isolated smooth muscle cells from rat mesenteric artery. Macroscopic properties of Kv current were not affected by change in extracellular calcium from 0 to 2mM. During a 10s depolarizing pulse, 4-aminopyridine inhibited the peak current without affecting the end-pulse current. The concentration-effect curve of 4-aminopyridine was shifted to the left in the presence of 2mM calcium compared to 0 calcium. After 4-aminopyridine washout, current recovery from block was slower in the presence than in the absence of calcium. Inhibition of Kv current by 4-aminopyridine (0.5mM) and the Kv2 blocker stromatoxin (50nM) was additive and stromatoxin did not alter the potentiation of 4-aminopyridine effect by extracellular calcium. These results showed that extracellular calcium modulated the inhibitory potency of 4-aminopyridine on Kv current in vascular myocytes. The component of Kv current that was inhibited by 4-aminopyridine in a calcium-sensitive manner was distinct from Kv2 current., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The influence of exogenous cytochrome c on reactive oxygen species (ROS) formation and its dependence on mitochondrial permeability transition pore (MPTP) opening is studied in rat liver mitochondria. Fluorescent probe dichlorofluorescein (DCF) was used. It was shown that MPTP activation by increasing concentrations of Ca2+ in the medium results in the increase in mitochondrial ROS production and oxygen consumption, but the decrease in matrix calcium retention, dependent on the amount of added Ca2+. Cytochrome c in the incubation medium does not much influence ROS formation when MPTP opening is blocked by cyclosporine A. However, in the presence of cytochrome c MPTP opening is accompanied by dramatic increase in ROS production. Steep rise in DCF fluorescence because of matrix ROS formation is sensitive to MPTP opening and is not resulted from the direct interaction between the probe and cytochrome c outside the mitochondria. To explain obtained data the hypothesis is put forward that MPTP could serve for ROS exchange between the matrix and the medium where heme iron of cytochrome c would act as a catalytic center to enhance ROS production. We suppose that apart of its conventional function, cytochrome c which is not involved in electron transport, could serve in such way as the amplifier of ROS production which in turn would provide a background for the development of apoptosis due to MPTP opening.

Hypoglossal motoneurones play a major role in maintaining the patency of the upper airways and in determining airways resistance. These neurones receive inputs from many different regions of the neuroaxis including the caudal raphe nuclei. Whilst we have previously shown that glutamate is utilised in projections from one of these caudal raphe nuclei, the raphe pallidus, to hypoglossal motoneurones, these raphe pallidus-hypoglossal projections also contain multiple co-localised neuropeptides, including a population that are immunopositive for enkephalin. The role of enkephalin in the control of hypoglossal motoneurones is unknown. Therefore the aim of these studies was to determine whether enkephalins modulate caudal raphe glutamatergic inputs to hypoglossal motoneurones. Whole cell recordings were made from rat hypoglossal motoneurones in vitro, with glutamate-mediated excitatory postsynaptic currents (EPSCs) evoked in these neurones following electrical stimulation within the raphe pallidus. Superfusion of enkephalin significantly decreased the amplitude of these raphe pallidus evoked EPSCs (56.1+/-29% of control, P<0.001), an action that was mirrored by the tau-opioid receptor agonist, [D-Ala, N-Me-Phe, Gly-ol]-enkephalin acetate (DAMGO;53.8+/-26%, P<0.01), but not by the delta-opioid receptor agonist, [D-Pen]-enkephalin (DPDPE). Enkephalin also increased the amplitude ratio (1.57+/-0.36 vs. 1.14+/-0.27, P<0.01) of pairs of evoked EPSCs (paired pulse ratio), decreased the frequency (P<0.0001) but not the amplitude of miniature EPSCs, whilst having no effect on the inward current evoked by glutamate applied directly to the postsynaptic cell (97.8+/-2.2% of control, P=n.s.). Likewise, DAMGO also increased the paired pulse ratio (1.62+/-0.35 vs. 1.31+/-0.14, P<0.05) and decreased the frequency of miniature EPSCs (P<0.0001). Together, these data suggest that enkephalin acts at tau-opioid receptors located on the presynaptic terminals of raphe pallidus inputs to hypoglossal motoneurones to significantly decrease glutamate release from these projections.

Whole cell recordings were undertaken from vagal motoneurones, including identified gastric vagal motoneurones, located within the medial regions of the dorsal vagal motonucleus of the rat medulla in vitro. The actions of adrenaline on individual channels expressed by these neurones were investigated. Adrenaline directly inhibits Ca2+ currents and delayed rectifier K+ currents and activates a sustained Na+ current. It also inhibits both Ca2+ activated non-selective cationic currents and Ca2+ activated K+ currents, the latter via inhibition of the underlying activating Ca2+ current. Since different sub-populations of vagal motoneurones express different complements of ion channels, this selective modulation of specific conductances by adrenaline may provide a mechanism by which adrenergic inputs, which project non-selectively throughout the DMV, could selectively control different aspects of vagal function.