Diverse mechanisms underlying the regulation of ion channels by carbon monoxide.

Carbon monoxide (CO) is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal-containing proteins, it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, NO and cGMP levels, as well as regulate MAPK signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca(2+) -sensitive K(+) channels. More recent studies have revealed the ability of CO to inhibit T-type Ca(2+) channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

Reversible metabolic depression in hepatocytes of lamprey (Lampetra fluviatilis) during pre-spawning: regulation by substrate availability.

The regulation of oxidative metabolism in hepatocytes of lampreys (Lampetra fluviatilis) during the freshwater pre-spawning period of their life cycle was studied. The energy metabolism in these cells is characterized by a simplified scheme, where glycolytic ATP production is insignificant and fatty acids are the major respiratory substrates. Seasonal changes in aerobic cell metabolism include a considerable reversible depression of metabolic rate in lamprey hepatocytes during the winter months of the pre-spawning period. The depression is characterized by a more than twofold decrease in hepatocyte endogenous respiration rate, a reduction of oxidative phosphorylation and drop in cellular ATP content. The addition of fatty acids to the hepatocyte incubation medium prevents the decrease in the metabolic rate. In spring, before spawning, a marked activation of energy metabolism in lamprey hepatocytes is found. These observations support the conclusion that the regulation of lamprey hepatocyte energy metabolism is realized through the availability of fatty acids for oxidation.

[Oxidative modification of blood proteins in patients with psychiatric disorders (depression, depersonalization)]. / Okislitel'naia modifikatsiia belkov plazmy krovi bol'nykh psikhicheskimi rasstroistvami (depressiia, depersonalizatsiia).

We determined the oxidative modification of proteins (spontaneous and metal-catalysing oxidation, MKO) and the level of corticosteroids in patients with the depersonalization and depression. For detecting oxidative modification of plasma proteins we measured the concentration of protein carbonyl groups formed with 2,4dinitrophenylhydrazine 2,4dinitrophenylhydrazone derivatives; the formation of dityrosine by fluorescence method; protein aggregation and fragmentation. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS)-PAGE in the presence beta-mercaptoethanol was used to determine the aggregation or fragmentation of proteins by oxygen radicals (OH). Acid-soluble peptides were analised as products of the fragmentation oxidative modification proteins. The level of the corticosteroids was determined using HPLS. The increase of the concentration of protein carbonyl groups in blood plasma of patients with mental disorders. In patients with depersonalization we determined the increase of the bityrosyl cross-link, and different degrees of fragmentation compared with depressive patients. The cortisol level was decreased and corticosterone was increased in the blood plasma of patients with depersonalization. In depressive patients the cortisol level was increased and corticosterone was decreased is discussed. We discussed the role oxidative modification proteins in the disturbance of the corticosteroid and opioid receptors functions in the patients with mental disorders.

[High performance liquid chromatography of corticosteroids in patients with depression and depersonalization]. / Vysokoéffektivnaia zhidkostnaia khromatografiia kortikosteroidov u bol'nykh s depressiei i depersonalizatsiei.

Six corticosteroids were measured by high performance microcolumn reverse-phase liquid chromatography in patients with the depressive syndrome and depersonalization. Changes in the blood concentrations of hydrocortisone, cortisone, and corticosterone were revealed. The method is recommended as an additional tool for the differential diagnosis of depressions and depersonalization.

Properties and therapeutic potential of transient receptor potential channels with putative roles in adversity: focus on TRPC5, TRPM2 and TRPA1.

Mammals contain 28 genes encoding Transient Receptor Potential (TRP) proteins. The proteins assemble into cationic channels, often with calcium permeability. Important roles in physiology and disease have emerged and so there is interest in whether the channels might be suitable therapeutic drug targets. Here we review selected members of three subfamilies of mammalian TRP channel (TRPC5, TRPM2 and TRPA1) that show relevance to sensing of adversity by cells and biological systems. Summarized are the cellular and tissue distributions, general properties, endogenous modulators, protein partners, cellular and tissue functions, therapeutic potential, and pharmacology. TRPC5 is stimulated by receptor agonists and other factors that include lipids and metal ions; it heteromultimerises with other TRPC proteins and is involved in cell movement and anxiety control. TRPM2 is activated by hydrogen peroxide; it is implicated in stress-related inflammatory, vascular and neurodegenerative conditions. TRPA1 is stimulated by a wide range of irritants including mustard oil and nicotine but also, controversially, noxious cold and mechanical pressure; it is implicated in pain and inflammatory responses, including in the airways. The channels have in common that they show polymodal stimulation, have activities that are enhanced by redox factors, are permeable to calcium, and are facilitated by elevations of intracellular calcium. Developing inhibitors of the channels could lead to new agents for a variety of conditions: for example, suppressing unwanted tissue remodeling, inflammation, pain and anxiety, and addressing problems relating to asthma and stroke.

Chloride conductance and volume-regulatory nonselective cation conductance in human red blood cell ghosts.

To identify the ion channels involved in erythrocyte volume regulation, whole-cell currents from human red blood cells (RBCs) were recorded in isotonic, hypotonic and hypertonic media. In isotonic NaCl bath solution, whole-cell currents rectified outwardly with reversal potentials (Vrev) between the equilibrium potential of Cl- (ECl) and that of nonselective cations (NSC), ENSC. Replacement of bath Cl by gluconate decreased outward conductance (G outward) by 43 +/- 6% (n = 5) and shifted Vrev with ECl indicating a high fractional Cl- conductance. Hypotonic cell swelling reversibly decreased G outward by 23 +/- 3% (n = 8) while hypertonic cell shrinkage reversibly increased G outward by 27 +/- 8% (n = 5). These shrinkage-activated and swelling-inactivated current fractions rectified outwardly with Vrev at ENSC suggesting that both fractions are generated by the same type of NSC channel. The shrinkage-activated deltaG outward decreased from 4.7 +/- 1.2 nS (n = 3) to 1.4 +/- 0.4 nS (n = 5) and 0.5 +/- 0.4 nS (n = 7) with the increase of pipette [Cl-] from 7 mM to 39 mM and 139 mM, respectively. Similarly, with this increase of pipette [Cl-], G outward under isotonic control conditions decreased from 8.2 +/- 1.4 nS (n = 5) to 7.4 +/- 1.0 nS (n = 20) and 4.1 +/- 0.7 nS (n = 17), due to the differing activity of these NSC channels. In conclusion, human RBCs express, besides a high fractional Cl- conductance, NSC channels that are regulated by cell volume and the cytosolic [Cl-].

Cell volume-sensitive sodium channels upregulated by glucocorticoids in U937 macrophages.

Glucocorticoids exert their anti-inflammatory action in part by influencing macrophages. As regulation of macrophage function involves ion channels, the present study was performed to elucidate the influence of glucocorticoids on macrophage ion channel activity. To this end, the effects of corticosteroids on the sodium conductance in human monocytic cells (U937) was studied using whole-cell and outside-out patch-clamp techniques. Increasing extracellular osmolarity from 310 to 420 mosmol/kg led to cell shrinkage followed by marked activation of inward whole-cell current from -36+/-2 to -72+/-9 pA (n=13; recorded at -150 mV voltage with CsCl intracellular solution, NaCl extracellular solution) while outward current remained unchanged. The increase of inward current was accompanied by a positive shift of reversal potential and was sensitive to amiloride (100 microM). The activation of inward current by shrinkage was not observed when external sodium was replaced by potassium, indicating that the shrinkage-stimulated conductance is sodium selective. Outside-out single-channel measurements revealed a unitary conductance of 6+/-1 pS (n=5) for the sodium-selective amiloride-sensitive current. Pretreating the cells with deoxycorticosterone (100 nM/6 h) markedly upregulated the shrinkage-activated Na+ current. In conclusion, human macrophage-like U937 cells express a sodium-selective shrinkage-activated channel which is upregulated by corticosteroids. Activation of the channel may increase cell volume, an effect of glucocorticoids in other cells.

The heterodimeric amino acid transporter 4F2hc/LAT1 is associated in Xenopus oocytes with a non-selective cation channel that is regulated by the serine/threonine kinase sgk-1.

System L is the major Na(+)-independent amino acid transporter of mammalian cells. It is constituted of the type II membrane protein 4F2hc (CD98) which is covalently linked to the polytopic membrane protein LAT1 via a disulfide bridge. The transporter is known to be regulated by the mineral corticoid aldosterone in Xenopus A6 cells. To understand the regulation of the transporter, the 4F2hc/LAT1 heterodimer was functionally expressed in Xenopus laevis oocytes and its transport properties were analysed using flux measurements and the two-electrode voltage-clamp technique. Expression of 4F2hc/LAT1 resulted in a rapid increase in a Na(+)-independent neutral amino acid antiport activity and simultaneously gave rise to a cation conductance. The cation channel was non-rectifying and non-selective, conducting Li(+) > Cs(+) = Na(+) > K(+). After replacement of Na(+) by NMDG, however, the currents were suppressed almost completely. The cation channel was not inhibited by amiloride, Ba2(+), TEA, Hoe293B, flufenamic acid or substrates of the system L amino acid transporter. Significant inhibition, however, was observed in the presence of La3(+), Gd3(+) and quinidine. Channel activity was upregulated by coexpression of 4F2hc/LAT1 with the aldosterone-regulated protein kinase sgk-1. The cation conductance was sensitive to changes in the redox potential, being inhibited following incubation of the oocytes with DTE for 30 min. Mutation of either of the disulfide bridge-constituting cysteines to serine resulted in a loss of ion channel activity whereas amino acid transport was unaffected. It is concluded that the 4F2hc/LAT1 heterodimer regulates a closely associated cation channel or even constitutes a cation channel itself.

IGF-1 up-regulates K+ channels via PI3-kinase, PDK1 and SGK1.

Involvement of voltage-gated (Kv) potassium channels in IGF-1-induced proliferation of HEK293 cells was studied by patch-clamp, RT-PCR and FACS analysis. IGF-1 up-regulated outwardly rectifying whole-cell K+ current starting after 1 h of incubation and reaching a maximum after 4-6 h. The IGF-1-stimulated current was voltage-gated with an activation threshold of -30 mV to -40 mV, a half-maximal activation at +5.3+/-1.8 mV, and time constants for activation and inactivation of 4.5+/-0.4 ms and 43.5+/-5.6 ms ( n=10), respectively. The current was inhibited by TEA, margatoxin, agitoxin-2 and stichodactyla toxin. PCR amplification of different Kv subunits from HEK293 cDNA demonstrated the expression of Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv3.1 and Kv3.4 mRNA. Quantitative RT-PCR showed up-regulation of Kv1.1, 1.2 and 1.3 mRNA by IGF-1. The effect of IGF-1 on K+ current was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), wortmannin and LY294002, and mimicked by overexpression of human 3-phosphoinositide-dependent protein kinase-1 (hPDK1) or serum- and glucocorticoid-dependent kinase-1 (hSGK1), both sequential downstream targets of PI3-kinase. IGF-1-induced proliferation of HEK293 cells was inhibited by both K+ channel blockers and inhibitors of PI3-kinase. In conclusion, IGF-1 through PI3-kinase, PDK1 and SGK1 up-regulates Kv channels, an effect required for the proliferative action of the growth factor.

Time-dependent regulation of capacitative Ca2+ entry by IGF-1 in human embryonic kidney cells.

In a wide variety of cells, mitogenic factors release Ca(2+) from intracellular stores. The fall of the [Ca(2+)] within the lumen of the Ca(2+)-storing organelles triggers in many cells capacitative Ca(2+) entry (CCE). The present study was performed to elucidate the effect of insulin-like growth factor (IGF-1) on CCE in human embryonic kidney (HEK 293) cells. After depletion of Ca(2+) stores by thapsigargin, CCE was assessed by the increase in cytosolic free [Ca(2+)] (Fura-2 fluorescence imaging) when raising extracellular [Ca(2+)] from 0 to physiological concentrations. IGF-1 exposure (50 ng/ml) for 4 h in serum-free medium markedly enhanced CCE, while a 24-h exposure to IGF-1 depressed CCE profoundly. As some Ca(2+) channels are highly sensitive to the cell membrane potential, and as IGF-1 has been reported to enhance K(+) channel activity, the influence of K(+) channel blockers on the IGF-1-dependent stimulation of CCE was also tested. TEA, charybdotoxin and margatoxin decreased CCE. Similar to the total capacitative calcium entry, the fraction of CCE that was sensitive to K(+) channel blockers was increased after 4 h and decreased after 24 h exposure to IGF-1. Taken together, these data suggest that IGF-1 induces a transient increase followed by a decrease of CCE, and that these effects are at least partly dependent on IGF-1-induced K(+) channel activity.

Cell volume in the regulation of cell proliferation and apoptotic cell death.

Cell proliferation must - at some time point - lead to increase of cell volume and one of the hallmarks of apoptosis is cell shrinkage. At constant extracellular osmolarity those alterations of cell volume must reflect respective changes of cellular osmolarity which are hardly possible without the participation of cell volume regulatory mechanisms. Indeed, as shown for ras oncogene expressing 3T3 fibroblasts, cell proliferation is paralleled by activation of Na(+)/H(+) exchange and Na(+),K(+),2Cl(-) cotransport, the major transport systems accomplishing regulatory cell volume increase. Conversely, as evident from CD95-induced apoptotic cell death, apoptosis is paralleled by inhibition of Na(+)/H(+) exchanger and by activation of Cl(-) channels and release of the organic osmolyte taurine, major components of regulatory cell volume decrease. However, ras oncogene activation leads to activation and CD95 receptor triggering to inhibition of K(+) channels. The effects counteract the respective cell volume changes. Presumably, they serve to regulate cell membrane potential, which is decisive for Ca(++) entry through I(CRAC) and the generation of cytosolic Ca(++) oscillations in proliferating cells. As a matter of fact I(CRAC) is activated in ras oncogene expressing cells and inhibited in CD95-triggered cells. Activation of K(+) channels and Na(+)/H(+) exchanger as well as Ca(++) oscillations have been observed in a wide variety of cells upon exposure to diverse mitogenic factors. Conversely, diverse apoptotic factors have been shown to activate Cl(-) channels and organic osmolyte release. Inhibition of K(+) channels is apparently, however, not a constant phenomenon paralleling apoptosis which in some cells may even require the operation of K(+) channels. Moreover, cell proliferation may at some point require activation of Cl(-) channels. In any case, the alterations of cell volume are obviously important for the outcome, as cell shrinkage impedes cell proliferation and apoptosis can be elicited by increase of extracellular osmolarity. At this stage little is known about the interplay of cell volume regulatory mechanisms and the cellular machinery leading to mitosis or death of the cell. Thus, considerable further experimental effort is required in this exciting area of cell physiology.

K+ channel activation by all three isoforms of serum- and glucocorticoid-dependent protein kinase SGK.

The serum- and glucocorticoid-dependent kinase SGK1 was originally identified as a glucocorticoid-sensitive gene. Subsequently, the two homologous kinases SGK2 and SGK3 have been cloned, being products of distinct genes, which are differentially expressed and share 80% identity in amino acid sequence in their catalytic domains. While SGK1 has been shown to activate ion channels, including K(+) channels, the functions of SGK2 and SGK3 have not been examined. The present study was therefore performed to elucidate the effect of SGK1, SGK2, and SGK3 on electrical properties of renal epithelial cells. To this end human embryonic kidney (HEK293) cells were transfected with the kinases and ion-channel activity determined using the patch-clamp technique. In non-transfected cells and in cells transfected with the empty GFP construct a voltage-gated K(+) current was observed amounting to 303+/-19 pA ( n=13) and 299+/-29 pA ( n=23), respectively. Transfection with SGK1, SGK2 or SGK3 increased the voltage-gated K(+) current to 1056+/-152 pA ( n=17), 555+/-47 pA ( n=17), and 775+/-98 pA ( n=16), respectively. The K(+) current was fully blocked by 3 mM tetraethylammonium chloride and inhibited 45% by the Kv1 channel blocker margatoxin (10 nM). In dual electrode voltage-clamp experiments SGK isoforms up-regulated Kv1 voltage-gated K(+)channels expressed in Xenopus laevis oocytes. The present observations thus reveal a powerful stimulating effect of all three isoforms of SGK on K(+) channels. Those effects may participate in regulation of epithelial transport, cell proliferation, and neuromuscular excitability.

Serum- and glucocorticoid-dependent kinase, cell volume, and the regulation of epithelial transport.

Ample pharmacological evidence points to a role of kinases in the regulation of cell volume. Given the limited selectivity of most inhibitors, however, the specific molecules involved have remained largely elusive. The search for cell volume regulated genes in liver HepG2 cells led to the discovery of the human serum- and glucocorticoid-dependent serine/threonine kinase hsgk1. Transcription and expression of hsgk1 is markedly and rapidly upregulated by osmotic and isotonic cell shrinkage. The effect of osmotic cell shrinkage on hsgk1 is mediated by p38 kinase. Further stimuli of hsgk1 transcription include glucocorticoids, aldosterone, TGF-beta1, serum, increase of intracellular Ca2+ and phorbolesters, whereas cAMP downregulates hsgk1 transcription. The hsgk1 protein is expressed in several epithelial tissues including human pancreas, intestine, kidney, and shark rectal gland. Co-expression of hsgk1 with the renal epithelial Na+-channel ENaC or the Na+/K+/2Cl(-)-cotransporter NKCC2 (BSC1) in Xenopus oocytes, accelerates insertion of the transport proteins into the cell membrane and thus, stimulates channel or transport activity. Thus, hsgk1 participates in the regulation of transport by steroids and secretagogues increasing intracellular Ca2+-activity. The stimulation of hsgk1 transcription by TGF-beta1 may further bear pathophysiological relevance.