Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry.

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.

Interstitial cells in the vasculature.

Interstitial cells of Cajal are believed to play an important role in gastrointestinal tissues by generating and propagating electrical slow waves to gastrointestinal muscles and/or mediating signals from the enteric nervous system. Recently cells with similar morphological characteristics have been found in the wall of blood vessels such as rabbit portal vein and guinea pig mesenteric artery. These non-contractile cells are characterised by the presence of numerous processes and were easily detected in the wall of the rabbit portal vein by staining with methylene blue or by antibodies to the marker of Interstitial Cells of Cajal c-kit. These vascular cells have been termed "interstitial cells" by analogy with interstitial cells found in the gastrointestinal tract. Freshly dispersed interstitial cells from rabbit portal vein and guinea pig mesenteric artery displayed various Ca2+-release events from endo/sarcoplasmic reticulum including fast localised Ca2+ transients (Ca2+ sparks) and longer and slower Ca2+ events. Single interstitial cells from the rabbit portal vein, which is a spontaneously active vessel, also demonstrated rhythmical Ca2+ oscillations associated with membrane depolarisations, which suggests that in this vessel interstitial cells may act as pacemakers for smooth muscle cells. The function of interstitial cells from the mesenteric arteries is yet unknown. This article reviews some of the recent findings regarding interstitial cells from blood vessels obtained by our laboratory using electron microscopy, immunohistochemistry, tight-seal patch-clamp recording, and fluorescence confocal imaging techniques.

Regulation of muscarinic cationic current in myocytes from guinea-pig ileum by intracellular Ca2+ release: a central role of inositol 1,4,5-trisphosphate receptors.

The dynamics of carbachol (CCh)-induced [Ca(2+)](i) changes was related to the kinetics of muscarinic cationic current (mI(cat)) and the effect of Ca(2+) release through ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP(3)Rs) on mI(cat) was evaluated by fast x-y or line-scan confocal imaging of [Ca(2+)](i) combined with simultaneous recording of mI(cat) under whole-cell voltage clamp. When myocytes freshly isolated from the longitudinal layer of the guinea-pig ileum were loaded with the Ca(2+)-sensitive indicator fluo-3, x-y confocal imaging revealed CCh (10 microM)-induced Ca(2+) waves, which propagated from the cell ends towards the myocyte centre at 45.9 +/- 8.8 microms(-1) (n = 13). Initiation of the Ca(2+) wave preceded the appearance of any measurable mI(cat) by 229 +/- 55 ms (n = 7). Furthermore, CCh-induced [Ca(2+)](i) transients peaked 1.22 +/- 0.11s (n = 17) before mI(cat) reached peak amplitude. At -50 mV, spontaneous release of Ca(2+) through RyRs, resulting in Ca(2+) sparks, had no effect on CCh-induced mI(cat) but activated BK channels leading to spontaneous transient outward currents (STOCs). In addition, Ca(2+) release through RyRs induced by brief application of 5 mM caffeine was initiated at the cell centre but did not augment mI(cat) (n = 14). This was not due to an inhibitory effect of caffeine on muscarinic cationic channels (since application of 5 mM caffeine did not inhibit mI(cat) when [Ca(2+)](i) was strongly buffered with Ca(2+)/BAPTA buffer) nor was it due to an effect of caffeine on other mechanisms possibly involved in the regulation of Ca(2+) sensitivity of muscarinic cationic channels (since in the presence of 5 mM caffeine, photorelease of Ca(2+) upon cell dialysis with 5 mM NP-EGTA/3.8 mM Ca(2+) potentiated mI(cat) in the same way as in control). In contrast, IP(3)R-mediated Ca(2+) release upon flash photolysis of "caged" IP(3) (30 microM in the pipette solution) augmented mI(cat) (n = 15), even though [Ca(2+)](i) did not reach the level required for potentiation of mI(cat) during photorelease of Ca(2+) (n = 10). Intracellular calcium stores were visualised by loading of the myocytes with the low-affinity Ca(2+) indicator fluo-3FF AM and consisted of a superficial sarcoplasmic reticulum (SR) network and some perinuclear formation, which appeared to be continuous with the superficial SR. Immunostaining of the myocytes with antibodies to IP(3)R type 1 and to RyRs revealed that IP(3)Rs are predominant in the superficial SR while RyRs are confined to the central region of the cell. These results suggest that IP(3)R-mediated Ca(2+) release plays a central role in the modulation of mI(cat) in the guinea-pig ileum and that IP(3) may sensitise the regulatory mechanisms of the muscarinic cationic channels gating to Ca(2+).

Smooth muscle cells and interstitial cells of blood vessels.

A rise in intracellular ionised calcium concentration ([Ca(2+)](i)) at sites adjacent to the contractile proteins is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques with special accent on the fluorescence confocal microscopy and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for study of the relationship between the structural organisation of the living smooth muscle myocyte and the features of calcium signalling at subcellular level. Applying fluorescent confocal microscopy and tight-seal recording of transmembrane ion currents to freshly isolated vascular myocytes we have demonstrated that: (1) Ca(2+) sparks originate from clustered opening of ryanodine receptors (RyRs) and build up a cell-wide increase in [Ca(2+)](i) upon myocyte excitation; (2) spontaneous Ca(2+) sparks occurred at the highest rate at certain preferred locations, frequent discharge sites (FDS), which are associated with a prominent portion of the sarcoplasmic reticulum (SR) located close to the cell membrane; (3) Ca(2+)-dependent K(+) and Cl(-) channels sense the local changes in [Ca(2+)](i) during a calcium spark and thereby couple changes in [Ca(2+)](i) within a microdomain to changes in the membrane potential, thus affecting excitability of the cell; (4) an intercommunication between RyRs and inositol trisphosphate receptors (IP(3)Rs) is one of the important determinants of intracellular calcium dynamics that, in turn, can modulate the cell membrane potential through differential targeting of calcium dependent membrane ion channels. Furthermore, using immunohystochemical approaches in combination with confocal imaging we identified non-contractile cells closely resembling interstitial cells (ICs) of Cajal (which are considered to be pacemaker cells in the gut) in the wall of portal vein and mesenteric artery. Using electron microscopy, tight-seal recording and fluorescence confocal imaging we obtained information on the morphology of ICs and their possible coupling to smooth muscle cells (SMCs), calcium signalling in ICs and their electrophysiological properties. The functions of these cells are not yet fully understood; in portal vein they may act as pacemakers driving the spontaneous activity of the muscle; in artery they may have other a yet unsuspected functions.

Identification of interstitial cells of Cajal in the rabbit portal vein.

Two layers of interstitial cells (ICs) of Cajal were detected by c-kit and methylene blue staining in the media of the rabbit portal vein in subendothelial intramuscular and deeper intramuscular positions, displaced radially from each other by about 40-70 microm. Two morphologically distinct types of ICs were found among enzymatically dispersed cells from this vessel: small multipolar cells with stellate-shaped bodies not exceeding 20 microm, and spindle-shaped cells from 40 to 300 microm in length with numerous branching processes. Relaxed smooth muscle cells (SMCs) had a more constant length (90-150 microm). The cell membrane capacitance was 46.5+/-2.2 pF in SMCs, 39.7+/-2.4 pF in spindle-shaped ICs and 27.8+/-0.7 pF in multipolar ICs. Although darker under phase contrast, after loading with fluo-4 AM, single isolated ICs of both types usually had brighter fluorescence than SMCs and displayed various spontaneous calcium events, including Ca(2+) sparks and Ca(2+) waves. Ca(2+) waves were usually followed by contraction of SMCs but no change in shape of ICs. In some ICs spontaneous [Ca(2+)](i) transients (lasting about 2s) which propagated towards the end of the processes were observed. Physical contacts between the processes of ICs and the body of one or more SMCs survived the isolation procedure. Application of noradrenaline (1-10 microM), caffeine (1-10 mM) or high-K(+) solution (60mM) led to a rise of [Ca(2+)](i) in both SMCs and ICs evoking contraction of SMCs but not ICs. No differences in electrophysiological characteristics between single enzymatically isolated IC and SMC were detected; thus, the resting membrane potential estimated under current-clamp conditions was -46.5+/-2.0 mV in spindle-shaped ICs and -45.6+/-2.7 mV in SMCs. Under voltage-clamp, both ICs and SMCs revealed a well-developed voltage-gated nifedipine-sensitive L-type Ca(2+) current, a set of K(+) currents, including spontaneous transient outward currents (STOCs) but no Na(+) current. This study for the first time directly demonstrated the presence in vascular tissue of ICs. Possible roles for ICs including their involvement in spontaneous activity of the vessel were discussed.

Crosstalk between ryanodine receptors and IP(3) receptors as a factor shaping spontaneous Ca(2+)-release events in rabbit portal vein myocytes.

In smooth muscle cells freshly isolated from rabbit portal vein, there was only one site discharging the majority of spontaneous Ca(2+)-release events; the activity of this single site was studied using laser scanning confocal imaging after loading the cells with the fluorescent Ca(2+) indicator fluo-4 acetoxymethyl ester. Localised spontaneous Ca(2+)-release events visualised by line-scan imaging revealed two predominant spatiotemporal patterns: (i) small-amplitude, fast events similar to Ca(2+) sparks in cardiomyocytes and (ii) larger and slower events. The sum of two Gaussian profiles was well fitted to the amplitude histogram (peak frequencies at 1.8 and 3.2 F/F(0)) and spatial spread (full width at half-maximal amplitude) histogram (peak frequencies at 2 and 3.8 microm) for the 230 localised Ca(2+)-release events analysed. The existence of two populations of Ca(2+)-release events was also supported by the histograms of the rise times and half-decay times, which revealed modes at 38 and 65 ms, respectively. Shifting the scan line along the z-axis during imaging from a single discharge site suggested that the appearance of two populations of Ca(2+)-release events is not due to out-of-focus imaging. Both small and large events persisted upon 3-5 min exposure to 1-5 microM nicardipine, but were abolished after 10-15 min exposure to 50-100 microM ryanodine, 0.1 microM thapsigargin or 10 microM cyclopiazonic acid. Only small-amplitude, fast events persisted in the presence of inhibitors of inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, 10 microM xestospongin C or 30 microM 2-aminoethoxy-diphenylborate (2-APB), or in the presence of 2.5 microM U-73122 (a phospholipase C (PLC) inhibitor). Coupling between neighbouring Ca(2+)-release domains giving rise to spontaneous [Ca(2+)](i) waves was abolished in the presence of 2-APB. Examination of the saltatory propagation of the waves suggested that the critical factor that determines propagation between domains is a time-dependent change in the sensitivity of ryanodine receptors and/or IP(3) receptors to Ca(2+), which can give rise to 'loose coupling' between release sites. These results suggest that activation of IP(3) receptors (due to the tonic activity of PLC and ongoing production of IP(3)) recruits neighbouring domains of ryanodine receptors, leading to larger Ca(2+) releases and saltatory propagation of [Ca(2+)](i) waves in portal vein myocytes.

Calcium release events in excitation-contraction coupling in smooth muscle.

Although smooth muscle cells are not organized in sarcomeres, as are striated muscles, nevertheless Ca2+ for contraction is released from the sarcoplasmic reticulum (SR) at certain preferred sites. These sites commonly discharge packets of Ca2+ spontaneously and have been called frequent discharge sites (FDSs). Each spontaneous release of a Ca2+ packet usually leads to a burst of openings of Ca2+-activated K+ channels in the cell membrane which produces a spontaneous transient outward current (STOC) in smooth muscle cells under voltage clamp. When fluorescent Ca2+ indicators such as Fluo-3 became available, the spontaneous transient increases in [Ca2+]i produced by Ca2+ packets released from the SR were also detected in cardiac muscle as flashes of fluorescence or 'sparks'. Sparks in smooth muscle consist of smaller Ca2+ packets that can give rise to 'microsparks'. In some smooth muscles which have Ca2+-activated Cl- channels, STICs (spontaneous transient inward currents) are also found to be associated with sparks. FDSs have been found to be important initiating sites for a Ca2+ wave in response to an action potential or in response to receptor activation and possibly other stimuli, such as stretch. In both cases Ca2+-induced Ca2+ release seems to be crucially involved.

[Monoamine, GABA, and glutamergic mechanisms of the anterior hypothalamus in anti-aversive effects of sedative and selective drugs in various models of anxiety]. / Issledovanie monoamin-, GAMK- i glutamatergicheskikh mekhanizmov perednego gipotalamusa v antiaversivnykh éffektakh anksiosedativnykh i anksioselektivnykh sredstv na razlichnykh modeliakh trevogi.

The experiments using "illuminated site" and "threatening situation" avoidance tests on rats microinjected with GABA, glutamic acid, monoamines and their agonists and antagonists, as well as anxiosedative and anxioselective agents into the anterior hypothalamus revealed functional ambiguity in the neurochemical profile of this limbic brain formation in the anxiety states of various aversive genesis. Preliminary intraperitoneal injection of the monoamine and GABA antagonists, followed by the local administration of the antianxiety drugs studied, showed that the antiaversive action of chlordiazepoxide, fenibut, and indoter is manifested in both anxiety models via a GABAergic mechanism in the anterior hypothalamus. The anxiolytic effect of campiron is manifested only under negative-stressor zoosocial conditions and is mediated by the serotoninergic systems of this limbic brain formation.

The role of neurochemical mechanisms of ventromedial hypothalamus in various models of anxiety in rats.

Microinjections of glutamic acid, serotonin, and sulpiride in the ventromedial hypothalamus reduced anxiety in an illuminated platform avoidance task in rats, while dopamine, apomorphine, picrotoxin, and memantine increased it. Similar injections of phenylephrine and yohimbine reduced anxiety in threatening situation task only, while GABA reduced it in both tasks. It is suggested that various emotional and stress phenotypes are realized through functionally different neurochemical mechanisms of ventromedial hypothalamus.

Direct visualization of sarcoplasmic reticulum regions discharging Ca(2+)sparks in vascular myocytes.

Localized Ca(2+)-release events, Ca(2+)sparks, have been suggested to be the 'elementary building blocks' of the calcium signalling system in all types of muscles. In striated muscles these occur at regular intervals along the fibre corresponding to the sarcomeric structures which do not exist in smooth muscle. We showed previously that in visceral and vascular myocytes Ca(2+)sparks occurred much more frequently at certain sites (frequent discharge sites [FDSs]). In this paper, we have related the position of FDSs to the distribution of the sarcoplasmic reticulum in the same living myocyte. The three-dimensional distribution of the SR in freshly isolated rabbit portal vein myocytes was visualized by means of high-resolution confocal imaging after staining with DiOC(6)and/or BODIPY TR-X ryanodine. Both fluorochromes revealed a similar staining pattern indicating a helical arrangement of well-developed superficial SR which occupied about 6% of the cell volume. Computing the frequency of spontaneous Ca(2+)sparks detected by means of fluo-4 fluorescence revealed that in about 70% of myocytes there was only one major FDS located on a prominent portion of superficial SR network usually within 1-2 microm of the nuclear envelope, although a few sparks occurred at other sites scattered generally in superficial locations throughout the cell. Polarized mitochondria were readily identified by accumulation of tetramethylrhodamine ethyl ester (TMRE). These were closely associated with the SR network in extra-nuclear regions. TMRE staining, however, failed to reveal any mitochondria near the FDS-related SR element. When observed, propagating [Ca(2+)](i)waves and associated myocyte contractions were initiated at FDSs. This study provide first insight into the three-dimensional arrangement of the SR in living smooth muscle cells and relates the peculiarity of the structural organization of the myocyte to the features of Ca(2+)signalling at subcellular level.

[The neurochemical profile of the caudate nucleus in the anxiolytic action of benzodiazepine and nonbenzodiazepine tranquilizers on different models of anxiety]. / Neirokhimicheskii profil' khvostatogo iadra v anksioliticheskom deistvii benzodiazepinovykh i nebenzodiazepinovykh trankvilizatorov na razlichnykh modeliakh trevogi.

In the experiments on rats in avoidance response to "eliminated area" and "threatening situation" it is found that the microinjection of monoamines and amino-acids of mediator action, adreno- and dophaminemimetics, their antagonists, and blocker of GABAA receptors picrotoxine into dorsal and ventral sections of caudate nucleus elucidates different functional value and neurochemical special features of examined neostriatum specimens in realization of anxiety states of different origin. Local injection of chlordiazepoxide, phenibutum, indoterum, campirone and campironine into aforementioned sections of the caudate nucleus reduces the state of anxiety in "eliminated area" and/or "threatening situation" response tests thus revealing the similarity with well-known effects of GABA and serotonin and substantial difference in the effects of catecholamines and glutamine acid. It is found that anti-anxious effect of benzodiazepine and nonbenzodiazepine anxiolytics can be mediated through participation of neuron matrices of dorsal and ventral sections of the caudate nucleus having functionally ambiguous neurochemical profile.

Membrane ion channels as physiological targets for local Ca2+ signalling.

Ionized calcium plays a central role as a second messenger in a number of physiologically important processes determining smooth muscle function. To regulate a wide range of cellular activities the mechanisms of subcellular calcium signalling should be very diverse. Recent progress in development of visible light-excitable fluorescent dyes with high affinity for Ca2+ (such as oregon green 488 BAPTA indicators, fluo-3 and fura red) and confocal laser scanning microscopy provides an opportunity for direct visualization of subcellular Ca2+ signalling and reveals that many cell function are regulated by the microenvironment within small regions of the cytoplasm ('local control' concept). Here confocal imaging is used to measure and locate changes in [Ca2+]i on a subcellular level in response to receptor stimulation in visceral myocytes. We show that stimulation of muscarinic receptors in ileal myocytes with carbachol leading to activation of inositol 1,4,5-trisphosphate receptors (IP3Rs) accelerates the frequency of spontaneous calcium sparks (discharged via ryanodine receptors, RyRs) and gives rise to periodic propagating Ca2+ waves oscillating with a frequency similar to that of carbachol-activated cationic current oscillations. Furthermore, by combining the whole-cell patch clamp technique with simultaneous confocal imaging of [Ca2+]i in voltage-clamped vascular myocytes we demonstrate that calcium sparks may lead to the opening of either Ca2+-activated Cl- channels or Ca2+-activated K+ channels, and the discharge of a spontaneous transient inward current (STIC) or a spontaneous transient outward current (STOC), respectively.

Excitation-contraction coupling in gastrointestinal and other smooth muscles.

The main contributors to increases in [Ca2+]i and tension are the entry of Ca2+ through voltage-dependent channels opened by depolarization or during action potential (AP) or slow-wave discharge, and Ca2+ release from store sites in the cell by the action of IP3 or by Ca(2+)-induced Ca(2+)-release (CICR). The entry of Ca2+ during an AP triggers CICR from up to 20 or more subplasmalemmal store sites (seen as hot spots, using fluorescent indicators); Ca2+ waves then spread from these hot spots, which results in a rise in [Ca2+]i throughout the cell. Spontaneous transient releases of store Ca2+, previously detected as spontaneous transient outward currents (STOCs), are seen as sparks when fluorescent indicators are used. Sparks occur at certain preferred locations--frequent discharge sites (FDSs)--and these and hot spots may represent aggregations of sarcoplasmic reticulum scattered throughout the cytoplasm. Activation of receptors for excitatory signal molecules generally depolarizes the cell while it increases the production of IP3 (causing calcium store release) and diacylglycerols (which activate protein kinases). Activation of receptors for inhibitory signal molecules increases the activity of protein kinases through increases in cAMP or cGMP and often hyperpolarizes the cell. Other receptors link to tyrosine kinases, which trigger signal cascades interacting with trimeric G-protein systems.

[Study of monoamino- and amino acidergic mechanisms of the rat caudate nucleus in antiaversive effects of tranquilizers in various anxiety models]. / Issledovanie monoamin- i aminokislotergicheskikh mekhanizmov khvostatogo iadra krys v relizatsii antiaversivnykh éffektov trankvilizatorov na razlichnykh modeliakh trevogi.

Administration of anxio-sedative drugs into the rat caudate nucleus revealed that antiaversive effects of chlordiazepide, phemibut, and indoter only occur under dominating fear motivation, whereas antiaversive effects of campirone and campironine occur under the influence of negative or stressful zoo-social actions and are realised via the GABA- and serotoninergic type of synaptic switching in the dorsal part of the caudate nucleus.

Variability in spontaneous subcellular calcium release in guinea-pig ileum smooth muscle cells.

1. Spontaneous, localized transient increases in [Ca2+]i ('Ca2+ sparks') were observed in about 40 % of fluo-3-loaded myocytes examined using laser scanning confocal microscopy. Ca2+ sparks persisted after application of Cd2+ (200 microM), but were abolished by ryanodine (30 microM) or thapsigargin (0.1 microM), suggesting that they arise from the spontaneous activation of ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR). 2. Ca2+ sparks occurred much more frequently at certain sites (or 'frequent discharge sites', FDSs) within any confocal plane of the cell and line-scan imaging revealed a wide variation in their spatial size, amplitude and time course. Some spontaneous local transients were very similar to 'Ca2+ sparks' observed in heart, i.e. lasting approximately 200 ms with a peak fluorescence ratio of 1.75 +/- 0.23 (mean +/- s.d., n = 33). Other events were faster and smaller, lasting only approximately 40 ms with a peak normalized fluorescence of 1.36 +/- 0.09 (mean +/- s.d., n = 28). 3. Spontaneous Ca2+ waves with a wide range of propagation velocities (between 30 and 260 micron s-1) were also observed. In about 60 % of records (n = 33), Ca2+ sparks could be detected at the sites of wave initiation. Waves of elevated [Ca2+]i propagated with non-constant velocity and in some cases terminated. These observations could be explained by heterogeneity in the distribution of subcellular release sites as well as variability in the contribution of each release site to the wave. 4. Spontaneous [Ca2+]i transients in single dispersed visceral smooth muscle cells have a wide spectrum of behaviour that is likely to be the result of spatio-temporal recruitment of smaller local events, probably via a calcium-induced calcium release (CICR) mechanism. The spatial non-uniformity of SR and RyR distribution within the cell may account for the existence of 'frequent discharge sites' firing the majority of the smooth muscle Ca2+ sparks and the wide variation in the Ca2+ wave propagation velocities observed.

Inwardly rectifying whole cell potassium current in human blood eosinophils.

1. Membrane currents were studied in single human blood eosinophils using the whole cell voltage clamp technique. The whole cell current-voltage relationship exhibited rectification about the membrane potential which followed the potassium equilibrium potential when [K+]o was raised. Elevation of [K+]o considerably potentiated inward current amplitude, and in some cells channel activity was discernible in the whole cell membrane current recordings. The single channel conductance was 24 +/- 1 pS ([K+]o, 100 mM; [K+]i, 140 mM), and eosinophils were found to have as few as three, and on average twenty, inward rectifier channels each. 2. The inward current was inhibited in a voltage-dependent manner by extracellular cations in order of potency Ba2+ > Cs+ > Na+. Intracellular acidification inhibited while alkalization augmented the inward current. Mg2+ contributed to rectification as dialysis with nominally Mg(2+)-free pipette solution was associated with an increase in the outward current during membrane polarization. 3. By reverse transcription-polymerase chain reaction (RT-PCR) using suitable primers on human eosinophils mRNA, an inward rectifier channel, Kir2.1, was identified, which is known from expression studies to have very similar properties to those found in this study. 4. Superoxide anion production or its stimulation by phorbol 12-myristate 13-acetate (PMA) was not significantly affected by depolarization with 140 mM [K+]o, or by 1 mM BaCl2. 5. It is concluded that the single channel currents and the whole cell current rectification observed in human blood eosinophils resulted from the presence of an inwardly rectifying potassium channel, probably Kir2.1.

Confocal imaging of calcium release events in single smooth muscle cells.

Localized [Ca2+]i transients ('sparks') first directly detected in cardiac myocytes were considered to represent 'elementary' Ca(2+)-release events playing a key role during excitation-contraction coupling (Cheng et al. 1993). In this study we employed confocal [Ca2+]i imaging to characterize subcellular calcium signalling in fluo-3 loaded visceral and vascular smooth muscle cells. In some experiments membrane potential of the myocyte was controlled using whole-cell patch clamp technique and changes in membrane current were recorded simultaneously with [Ca2+]i imaging. Some local [Ca2+]i transients were very similar to 'Ca2+ sparks' observed in heart, i.e. lasting approximately 200 ms with a peak fluorescence ratio of 1.75 +/- 0.23 (mean +/- SD, n = 33). Ca2+ sparks were found to occur in certain preferred locations in the cell, termed frequent discharge sites. Other events were faster and smaller, lasting only approximately 40 ms with a peak normalized fluorescence of 1.36 +/- 0.09 (mean +/- SD, n = 28). A high correlation between spontaneous transient outward currents and spark occurrence was observed. Proliferating waves of elevated [Ca2+]i initiated during membrane depolarization seem to arise from spatio-temporal recruitment of local Ca(2+)-release events. The spatial non-uniformity of sarcoplasmic reticulum and ryanodine receptor distribution within the cell may account for the existence of 'frequent discharge sites' and the wide variation in the Ca2+ wave propagation velocities observed.

Voltage-activated proton current in eosinophils from human blood.

1. The resting membrane potential of freshly purified normodense human eosinophils bathed in and dialysed with quasi-physiological solutions was -63 +/- 2 mV (n = 100). 2. In voltage-clamp mode with quasi-physiological internal and external solutions, voltage steps from the holding potential of -60 mV to levels positive to +20 mV resulted in development of a quasi-instantaneous outward current and a slowly developing outward current. The instantaneous current was absent when the cells were bathed in and dialysed with K(+)-free solution. 3. The slow outward current persisted following simultaneous replacement of K+, Na+ and most of the Cl- with largely impermeant ions (tetraethylammonium, N-methyl-D-glucamine and methanesulphonate) and was augmented when the cell was dialysed with a solution of increased buffering capacity for protons. The observed reversal potential of the current closely followed the hydrogen equilibrium potential over a wide range of internal-external pH combinations, indicating that the conductance underlying the slow outward current was highly selective for H+ ions. 4. Acidification of the pipette solution (increasing [H+]i) augmented the outward H+ current and shifted its activation range negatively, whilst acidification of the external solution had the opposite effect. The voltage dependence of the current is modulated by the transmembrane pH gradient so the only outward current could be activated. However, when the outward current was activated by a voltage step, rapid acidification of external solution produced an inward H+ current which rapidly deactivated. 5. The proton current was reversibly inhibited in a voltage-dependent manner by extracellular application of Zn2+. The apparent dissociation constants were 8 nM (at +40 mV), 36 nM (at +70 mV) and 200 nM (at +100 mV). 6. The proton current was augmented by exposure to 10 microM arachidonic acid. This augmentation consisted of a shift of the voltage dependence of activation to more negative potentials and enhancement of maximum conductance (gH,max). The proton current recorded in eosinophils was significantly augmented under conditions of elevated cytosolic free calcium concentration ([Ca2+]i). The threshold level of [Ca2+]i associated with this effect lay between 0.1 and 1 microM and was not measurably affected by cytosolic acidification. 7. Eosinophils from human blood possess a voltage-dependent H+ conductance (gH) which normally allows protons to move outwards only; raising [Ca2+]i was associated with augmentation of gH and intracellular acidification or arachidonate shifted its activation range negatively towards physiological potentials.

Tetrodotoxin-blockable depolarization-activated Na+ currents in a cultured endothelial cell line derived from rat interlobar arter and human umbilical vein.

Voltage-dependent Na+ current (INa) was identified in cultured endothelial cells derived from rat interlobar artery (RIAE cells) and human umbilical vein (HUVE cells). Tetrodotoxin (TTX) reduced INa in a dose-dependent manner with the apparent dissociation constant (Kd) of 1.4 microM. Low sensitivity of INa to TTX as well as its kinetics and voltage-dependent properties indicates that voltage-gated Na channels expressed in vascular endothelial cells belong to the so-called TTX-resistant type.