The role of BKCa channels on hyperpolarization mediated by hyperosmolarity in human articular chondrocytes

Chondrocytes, the only cell in cartilage, are subjected to hyperosmotic challenges continuously since extracellular osmolarity in articular cartilage increases in response to mechanical loads during joint movement. Hyperosmolarity can affect membrane transport, and it is possible that load modulates matrix synthesis through alterations in intracellular composition. In the present study, the effects of hyperosmotic challenges were evaluated using the whole-cell patch clamp technique, whole cell mode on freshly isolated human and bovine articular chondrocytes. In human chondrocytes, hypertonicity induced the activation of outward Ca(2+)-sensitive K(+) currents, which were inhibited by iberiotoxin and TEA-Cl. The current induced by hypertonic switching (osmolarity from 300 to 400 mOsm/l) caused cell hyperpolarization (from -39 mV to -70 mV) with a reversal potential of -96 ± 7 mV. These results suggest a role for Ca(2+)-activated K(+) channels in human articular chondrocytes, leading to hyperpolarization as a consequence of K(+) efflux through these channels. These channels could have a role in the articular chondrocyte's response to a hyperosmotic challenge and matrix metabolism regulation by load.     

Effects of osmotic challenges on membrane potential in human articular chondrocytes from healthy and osteoarthritic cartilage

Changes in external osmolarity arise from variations in mechanical loads on joints and may affect the homeostasis of chondrocytes, which are the only cell type responsible for matrix turnover. Accordingly, variations in membrane potential may affect cartilage production. The present study assessed the effects of variations in external osmolarity on membrane potential and the possible mechanisms responsible for this response. Membrane potential was measured by the patch clamp whole-cell technique using human articular chondrocytes freshly isolated from healthy and osteoarthritic cartilage. The membrane potential was -39±4 mV in articular human chondrocytes from healthy cartilage and -26±4 mV in those from osteoarthritic cartilage. Increasing the osmolarity produced a reversible hyperpolarization mediated by K+ efflux through BKCa channels in both groups of chondrocytes, but the response in osteoarthritic cells was significantly reduced; no other K+ pathways were involved in this effect. Alternatively, decreasing the osmolarity elicited depolarization in healthy chondrocytes but did not produce any response in chondrocytes from osteoarthritic cartilage. The depolarization was dependent on Na+ influx through Gd3+-sensitive stretch-activated cation channels and was independent of external Ca2+. The differential responses observed in chondrocytes from osteoarthritic cartilage suggest that disregulation on the responses to external osmolarity may be involved in the process that leads to the alterations in the cartilage structure observed in osteoarthritis.     

SEA0400, a novel Na+/Ca2+ exchanger inhibitor, reduces calcium overload induced by ischemia and reperfusion in mouse ventricular myocytes

Given the potential clinical benefit of inhibiting Na+/Ca2+ exchanger (NCX) activity during myocardial ischemia reperfusion (I/R), pharmacological approaches have been pursued to both inhibit and clarify the importance of this exchanger. SEA0400 was reported to have a potent NCX selectivity. Thus, we examined the effect of SEA0400 on NCX currents and I/R induced intracellular Ca2+ overload in mouse ventricular myocytes using patch clamp techniques and fluorescence measurements. Ischemia significantly inhibited inward and outward NCX current (from -0.04+/-0.01 nA to 0 nA at -100 mV; from 0.23+/-0.08 nA to 0.11+/-0.03 nA at +50 mV, n=7), Subsequent reperfusion not only restored the current rapidly but enhanced the current amplitude obviously, especially the outward currents (from 0.23+/-0.08 nA to 0.49+/-0.12 nA at +50 mV, n=7). [Ca2+]i, expressed as the ratio of Fura-2 fluorescence intensity, increased to 138+/-7% (P<0.01) during ischemia and to 210+/-11% (P<0.01) after reperfusion. The change of NCX current and the increase of [Ca2+]i during I/R can be blocked by SEA0400 in a dose-dependent manner with an EC50 value of 31 nM and 28 nM for the inward and outward NCX current, respectively. The results suggested that SEA0400 is a potent NCX inhibitor, which can protect mouse cardiac myocytes from Ca2+ overload during I/R injuries.     

Calcium flux in turtle ventricular myocytes

The relative contribution of the sarcoplasmic reticulum (SR), the L-type Ca(2+) channel and the Na(+)/Ca(2+) exchanger (NCX) were assessed in turtle ventricular myocytes using epifluorescent microscopy and electrophysiology. Confocal microscopy images of turtle myocytes revealed spindle-shaped cells, which lacked T-tubules and had a large surface area-to-volume ratio. Myocytes loaded with the fluorescent Ca(2+)-sensitive dye Fura-2 elicited Ca(2+) transients, which were insensitive to ryanodine and thapsigargin, indicating the SR plays a small role in the regulation of contraction and relaxation in the turtle ventricle. Sarcolemmal Ca(2+) currents were measured using the perforated-patch voltage-clamp technique. Depolarizing voltage steps to 0 mV elicited an inward current that could be blocked by nifedipine, indicating the presence of Ca(2+) currents originating from L-type Ca(2+) channels (I(Ca)). The density of I(Ca) was 3.2 +/- 0.5 pA/pF, which led to an overall total Ca(2+) influx of 64.1 +/- 9.3 microM/l. NCX activity was measured as the Ni(+)-sensitive current at two concentrations of intracellular Na(+) (7 and 14 mM). Total Ca(2+) influx through the NCX during depolarizing voltage steps to 0 mV was 58.5 +/- 7.7 micromol/l and 26.7 +/- 3.2 micromol/l at 14 and 7 mM intracellular Na(+), respectively. In the absence of the SR and L-type Ca(2+) channels, the NCX is able to support myocyte contraction independently. Our results indicate turtle ventricular myocytes are primed for sarcolemmal Ca(2+) transport, and most of the Ca(2+) used for contraction originates from the L-type Ca(2+) channel.     

β-Adrenoceptor/PKA-stimulation, Na(+)-Ca(2+) exchange and PKA-activated Cl(-) currents in rabbit cardiomyocytes: a conundrum

Investigations into the functional modulation of the cardiac Na(+)-Ca(2+) exchanger (NCX) by acute β-adrenoceptor/PKA stimulation have produced conflicting results. Here, we investigated (i) whether or not β-adrenoceptor activation/PKA stimulation activates current in rabbit cardiac myocytes under NCX-'selective' conditions and (ii) if so, whether a PKA-activated Cl(-)-current may contribute to the apparent modulation of NCX current (I(NCX)). Whole-cell voltage-clamp experiments were conducted at 37°C on rabbit ventricular and atrial myocytes. The β-adrenoceptor-activated currents both in NCX-'selective' and Cl(-)-selective recording conditions were found to be sensitive to 10mM Ni(2+). In contrast, the PKA-activated Cl(-) current was not sensitive to Ni(2+), when it was activated downstream to the β-adrenoceptors using 10μM forskolin (an adenylyl cyclase activator). When 10μM forskolin was applied under NCX-selective recording conditions, the Ni(2+)-sensitive current did not differ between control and forskolin. These findings suggest that in rabbit myocytes: (a) a PKA-activated Cl(-) current contributes to the Ni(2+)-sensitive current activated via β-adrenoceptor stimulation under recording conditions previously considered selective for I(NCX); (b) downstream activation of PKA does not augment Ni(2+)-sensitive I(NCX), when this is measured under conditions where the Ni(2+)-sensitive PKA-activated Cl(-) current is not present.     

N-n-Butyl haloperidol iodide inhibits the augmented Na+/Ca2+ exchanger currents and L-type Ca2+ current induced by hypoxia/reoxygenation or H2O2 in cardiomyocytes

N-n-butyl haloperidol iodide (F(2)), a novel quaternary ammonium salt derivative of haloperidol, was reported to antagonize myocardial ischemia/reperfusion injuries. To investigate its mechanisms, we characterized the effects of F(2) on Na(+)/Ca(2+) exchanger currents (I(NCX)) and the L-type Ca(2+) channel current (I(Ca,L)) of cardiomyocytes during either hypoxia/reoxygenation or exposure to H(2)O(2). Using whole-cell patch-clamp techniques, the I(NCX) and I(Ca,L) were recorded from isolated rat ventricular myocytes. Exposure of cardiomyocytes to hypoxia/reoxygenation or H(2)O(2) enhanced the amplitude of the inward and outward of I(NCX) and I(Ca,L). F(2) especially inhibited the outward current of Na(+)/Ca(2+) exchanger, as well as the I(Ca,L), in a concentration-dependent manner. F(2) inhibits cardiomyocyte I(NCX) and I(Ca,L) after exposure to hypoxia/reoxygenation or H(2)O(2) to antagonize myocardial ischemia/reperfusion injury by inhibiting Ca(2+) overload.     

μ-Calpain-mediated deregulation of cardiac, brain, and kidney NCX1 splice variants

μ-Calpain is a Ca(2+)-activated protease abundant in mammalian tissues. Here, we examined the effects of μ-calpain on three alternatively spliced variants of NCX1 using the giant, excised patch technique. Membrane patches from Xenopus oocytes expressing either heart (NCX1.1), kidney (NCX1.3), or brain (NCX1.4) variants of NCX1 were exposed to μ-calpain and their Na(+)-dependent (I(1)) and Ca(2+)-dependent (I(2)) regulatory phenotypes were assessed. For these exchangers, I(1) inactivation is evident as a Na(+)(i)-dependent decay of peak outward currents whereas I(2) regulation manifests as outward current activation by micromolar Ca(2+)(i) concentrations. Notably, with NCX1.1 and NCX1.4 but not in NCX1.3, higher Ca(2+)(i) levels alleviate I(1) inactivation. Our results show that (i) μ-calpain selectively ablates Ca(2+)-dependent (I(2)) regulation leading to a constitutive activation of exchange current, (ii) μ-calpain has much smaller effects on Na(+)-dependent (I(1)) regulation, produced by a slight destabilization of the I(1) state, and (iii) Ca(2+)-dependent regulation (I(2)) and Ca(2+)-mediated alleviation of I(1) appear to be functionally distinct mechanisms, the latter of which is left largely intact after μ-calpain treatment. The ability of μ-calpain to selectively and constitutively activate Na(+)-Ca(2+) exchange currents may have important pathophysiological implications in tissue where these splice variants are expressed.     

Insulin effects on cardiac Na+/Ca2+ exchanger activity: role of the cytoplasmic regulatory loop

Insulin can alter myocardial contractility, in part through an effect on the cardiac sarcolemmal Na(+)/Ca(2+) exchanger (NCX), but little is known about its mechanism of action. The large cytoplasmic domain (f-loop) of NCX is required for regulation by various intracellular factors, and we have shown previously that residues 562-679 are determinants of NCX inhibition by exchanger inhibitory peptide (XIP). Here we show that the same f-loop deletion eliminates the enhancement of NCX current by insulin, and we examine the signal pathways involved in the insulin response. NCX current (I(NCX)) was measured in freshly isolated or cultured (up to 48 h) adult guinea pig myocytes and in myocytes expressing canine NCX1.1 with the 562-679 f-loop deletion (NCX-(Delta562-679)) via adenoviral gene transfer. I(NCX) was recorded by whole-cell patch clamp as the Ni(2+)-sensitive current at 37 degrees C with intracellular Ca(2+) buffered. Insulin (1 microm) increased I(NCX) (at +80 mV) by 110 and 83% in fresh and cultured myocytes, respectively, whereas in myocytes expressing NCX-(Delta562-679) the response was eliminated (with 100 microm XIP included to suppress any native guinea pig I(NCX)). The insulin effect on I(NCX) was not inhibited by wortmannin, a nitric-oxide synthase inhibitor, or disruption of caveolae but was blocked by chelerythrine, implicating protein kinase C, but not phosphatidylinositol-3-kinase, in the mechanism. The insulin effect was also not additive with phosphatidylinositol-4,5-bisphosphate-induced activation of I(NCX). The finding that the 562-670 f-loop domain is implicated in both XIP and receptor-mediated modulation of NCX highlights its important role in acute physiological or pathophysiological regulation of Ca(2+) balance in the heart.     

Mechanisms involved in the increase in intracellular calcium following hypotonic shock in bovine articular chondrocytes

The extracellular osmotic environment of chondrocytes fluctuates during joint loading as fluid is expressed from and reimbibed by the extracellular matrix. Matrix synthesis by chondrocytes is modulated by joint loading, possibly mediated by variations in intracellular composition. The present study has employed the Ca2+-sensitive fluoroprobe Fura-2 to determine the effects of hypotonic shock (HTS) on intracellular Ca2+ concentration ([Ca2+]i) and to characterise the mechanisms involved in the response for isolated bovine articular chondrocytes. In cells subjected to a 50% dilution, [Ca2+]i rapidly increased by approximately 250%, a sustained plateau being achieved within 300 s. The effect was inhibited by thapsigargin or by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i reflects both influx from the extracellular medium and release from intracellular stores. Inhibition of the response by neomycin implicates activation of PLC and IP3 synthesis in the mobilisation of Ca2+ from intracellular stores. The rise was insensitive to inhibitors of L-type voltage-activated Ca2+ channels (LVACC) or reverse mode Na+/Ca2+ exchange (NCE) but could be significantly attenuated by ruthenium red, an inhibitor of transient receptor potential vanilloid (TRPV) channels and by Gd3+, a blocker of stretch-activated cation (SAC) channels. The HTS-induced rise in [Ca2+]i was almost completely absent in cells treated with Ni2+, a non-specific inhibitor of Ca2+ entry pathways. We conclude that in response to HTS the opening of SACC and a member of TRPV channel family leads to Ca2+ influx, simultaneously with the release from intracellular stores.     

钠钙交换在心脏发育早期自主膜电活动发生中的作用

钠钙交换是小鼠心脏发育中最早有功能性表达的通道基因。它的功能主要是通过泵出1个钙,泵入3个钠位置细胞内的钙稳态,此外可能参与兴奋收缩偶联。但是,至今钠钙交换在心脏发育过程中的功能性表达及其在细胞早期兴奋形成中的作用还不是很清楚。采用胚胎干细胞分化的心肌细胞为研究对象,发现在发育极早期,电压钳制在35mV的条件下,10mmol／L咖啡因诱导的内向电流的80％能被灌流液中Na^＋被等浓度的Li^＋取代（n=8）。此为钠钙交换电流。所有钳制的细胞单细胞RT-PCR都检测到了NCX1亚型的mRNA表达。进一步研究了钠钙交换的功能,发现等浓度Li^＋取代灌流液中Na^＋及应用高浓度Ni^2＋阻断了膜电位震荡及与震荡相间的动作电位（早期膜兴奋形式）。因此认为钠钙交换（NCX1亚型）在心脏发育极早期的心肌细胞中已有大量功能性表达,它对于早期自主性兴奋活动的发生起着关键性的作用。     

Regulation of cardiac L-type Ca2+ current in Na+-Ca2+ exchanger knockout mice: functional coupling of the Ca2+ channel and the Na+-Ca2+ exchanger

L-type Ca2+ current (I(Ca)) is reduced in myocytes from cardiac-specific Na+-Ca2+ exchanger (NCX) knockout (KO) mice. This is an important adaptation to prevent Ca2+ overload in the absence of NCX. However, Ca2+ channel expression is unchanged, suggesting that regulatory processes reduce I(Ca). We tested the hypothesis that an elevation in local Ca2+ reduces I(Ca) in KO myocytes. In patch-clamped myocytes from NCX KO mice, peak I(Ca) was reduced by 50%, and inactivation kinetics were accelerated as compared to wild-type (WT) myocytes. To assess the effects of cytosolic Ca2+ concentration on I(Ca), we used Ba2+ instead of Ca2+ as the charge carrier and simultaneously depleted sarcoplasmic reticular Ca2+ with thapsigargin and ryanodine. Under these conditions, we observed no significant difference in Ba2+ current between WT and KO myocytes. Also, dialysis with the fast Ca2+ chelator BAPTA eliminated differences in both I(Ca) amplitude and decay kinetics between KO and WT myocytes. We conclude that, in NCX KO myocytes, Ca2+-dependent inactivation of I(Ca) reduces I(Ca) amplitude and accelerates current decay kinetics. We hypothesize that the elevated subsarcolemmal Ca2+ that results from the absence of NCX activity inactivates some L-type Ca2+ channels. Modulation of subsarcolemmal Ca2+ by the Na+-Ca2+ exchanger may be an important regulator of excitation-contraction coupling.     

Inhibitory effect of n-3 fish oil fatty acids on cardiac Na+/Ca2+ exchange currents in HEK293t cells

Abnormal activity of the cardiac Na+/Ca2+ exchanger (NCX1) can affect intracellular Ca2+ homeostasis and cause arrhythmias. The n-3 polyunsaturated fatty acids (PUFAs), however, may prevent arrhythmias. To test the effect of PUFAs on the cardiac NCX1 current (I(NCX1)), the canine NCX1 cDNA was expressed in human embryonic kidney (HEK293t) cells. The average density of I(NCX1) was 10.9+/-2.6 pA/pF (n=44) in NCX1-transfected cells and eicosapentaenoic acid (EPA, C20:5n-3) significantly inhibited I(NCX1) The suppression of I(NCX1) by EPA was concentration-dependent with an IC50 of 0.82+/-0.27 microM. EPA had a similar effect on outward or inward I(NCX1). Docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (AA, C20:4n-6) also significantly inhibited I(NCX1), whereas the saturated fatty acid, stearic acid (SA, C18:0), did not. Our data demonstrate that the n-3 PUFAs significantly suppress cardiac I(NCX1), which is probably one of their protective effects against lethal arrhythmias.     

Characterization of zebrafish (Danio rerio) NCX4: a novel NCX with distinct electrophysiological properties

Members of the Na+/Ca2+ exchanger (NCX) family are important regulators of cytosolic Ca2+ in myriad tissues and are highly conserved across a wide range of species. Three distinct NCX genes and numerous splice variants exist in mammals, many of which have been characterized in a variety of heterologous expression systems. Recently, however, we discovered a fourth NCX gene (NCX4), which is found exclusively in teleost, amphibian, and reptilian genomes. Zebrafish (Danio rerio) NCX4a encodes for a protein of 939 amino acids and shows a high degree of identity with known NCXs. Although knockdown of NCX4a activity in zebrafish embryos has been shown to alter left-right patterning, it has not been demonstrated that NCX4a functions as a NCX. In this study, we 1) demonstrated, for the first time, that this gene encodes for a novel NCX; 2) characterized the tissue distribution of zebrafish NCX4a; and 3) evaluated its kinetic and transport properties. While ubiquitously expressed, the highest levels of NCX4a expression occurred in the brain and eyes. NCX4a exhibits modest levels of Na+-dependent inactivation and requires much higher levels of regulatory Ca2+ to activate outward exchange currents. NCX4a also exhibited extremely fast recovery from Na+-dependent inactivation of outward currents, faster than any previously characterized wild-type exchanger. While this result suggests that the Na+-dependent inactive state of NCX4a is far less stable than in other NCX family members, this exchanger was still strongly inhibited by 2 microM exchanger inhibitory peptide. We demonstrated that a new putative member of the NCX gene family, NCX4a, encodes for a NCX with unique functional properties. These data will be useful in understanding the role that NCX4a plays in embryological development as well as in the adult, where it is expressed ubiquitously.     

大鼠肥厚心肌细胞钙电流及 Na~ /Ca~(2 )交换电流的研究(英文)

本文观察了心肌肥厚对大鼠心肌细胞Na ／Ca2 交换电流的影响。我们采用Goldblatt两肾一夹方法诱发大鼠心肌肥厚,应用全细胞膜片钳技术记录电流。结果表明：肥厚心肌细胞的Ni2 -敏感Na ／Ca2 交换电流密度大于正常细胞。在钳制电压为＋50mV时,正常细胞的外向交换电流密度为1．53±0．31pA／pF,而肥厚细胞则为2．62±0．53pA／pF（P＜0．01）;钳制电压为-100mV时,正常细胞的内向交换电流密度为0．42±0．14pA／pF,肥厚细胞达1．12±0．33pA／pF（P＜0．001）。这些结果提示,肥厚心肌细胞的Na ／Ca2 交换电流发生了改变,其意义有待进一步探讨。     

Inhibitory effects of purified antibody against α-1 repeat (117-137) on Na(+)-Ca(2+) exchange and L-type Ca(2+) currents in rat cardiomyocytes

Considering that α-1 repeat region may be involved in the ion binding and translocation of Na(+)-Ca(2+) exchanger (NCX), it is possible that the antibodies against NCX α-1 repeat may have a crucial action on NCX activity. The aim of the present study is to investigate the effect of antibody against α-1 repeat (117-137), designated as α-1(117-137), on NCX activity. The antibody against the synthesized α-1(117-137) was prepared and affinity-purified. Whole-cell patch clamp technique was used to study the change of Na(+)-Ca(2+) exchange current (I(Na/Ca)) in adult rat cardiomyocytes. To evaluate the functional specificity of this antibody, its effects on L-type Ca(2+) current (I(Ca,L)), voltage-gated Na(+) current (I(Na)) and delayed rectifier K(+) current (I(K)) were also observed. The amino acid sequences of α-1(117-137) in NCX and residues 1?076-1?096 within L-type Ca(2+) channel were compared using EMBOSS Pairwise Alignment Algorithms. The results showed that outward and inward I(Na/Ca) were decreased by the antibody against α-1(117-137) dose-dependently in the concentration range from 10 to 160 nmol/L, with IC(50) values of 18.9 nmol/L and 22.4 nmol/L, respectively. Meanwhile, the antibody also decreased I(Ca,L) in a concentration-dependent manner with IC(50) of 22.7 nmol/L. No obvious effects of the antibody on I(Na) and I(K) were observed. Moreover, comparison of the amino acid sequences showed there was 23.8% sequence similarity between NCX α-1(117-137) and residues 1?076-1?096 within L-type Ca(2+) channel. These results suggest that antibody against α-1(117-137) is a blocking antibody to NCX and can also decrease I(Ca,L) in a concentration-dependent manner, while it does not have obvious effects on I(Na) and I(K).     

The functional expression of connexin 43 in articular chondrocytes is increased by interleukin 1beta: evidence for a Ca2+-dependent mechanism

Cell-to-cell interactions and gap junctions-dependent communication are crucially involved in chondrogenic differentiation, while in adult articular cartilage direct intercellular communication occurs mainly among chondrocytes facing the outer cartilage layer. Chondrocytes extracted from adult articular cartilage and grown in primary culture express connexin 43 and form functional gap junctions capable of sustaining the propagation of intercellular Ca2+ waves. Degradation of articular cartilage is a characteristic feature of arthritic diseases and is associated to increased levels of interleukin-1 (IL-1) in the synovial fluid. We have examined the effects of IL-1 on gap junctional communication in cultured rabbit articular chondrocytes. Incubation with IL-1 potentiated the transmission of intercellular Ca2+ waves and the intercellular transfer of Lucifer yellow. The stimulatory effect was accompanied by a dose-dependent increase in the expression of connexin 43 and by an enhanced connexin 43 immunostaining at sites of cell-to-cell contact. IL-1 stimulation induced a dose-dependent increase of cytosolic Ca2+ and activates protein tyrosine phosphorylation. IL-1-dependent up-regulation of connexin 43 could be prevented by intracellular Ca2+ chelation, but not by inhibitors of protein tyrosine kinases, suggesting a crucial role of cytosolic Ca2+ in regulating the expression of connexin 43. IL-1 is one of the most potent cytokines that promotes cartilage catabolism: its modulation of intercellular communication represents a novel mechanism by which proinflammatory mediators regulate the activity of cartilage cells.     

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 microM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 microM) or Ni2+ (>100 microM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 microM but did at 30 microM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 microM), Ni2+ (300 microM), or KB-R7943 (30 microM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.     

Changes in intracellular calcium concentration in response to hypertonicity in bovine articular chondrocytes

Intracellular calcium concentration ([Ca2+]i) in articular chondrocytes changes during mechanical challenges associated with joint movements, because of the fluctuation of the extracellular osmotic environment during joint loading. Matrix synthesis by chondrocytes is modulated by loading patterns, possibly mediated by variations in intracellular composition, including [Ca2+]i. The present study has employed the Ca(2+)-sensitive fluoroprobe Fura-2 to determine the effects of hypertonic shock on intracellular Ca2+ concentration ([Ca2+]i) and to characterise the mechanisms involved in the response for isolated bovine articular chondrocytes. In cells subjected to a hypertonic shock, [Ca2+]i rapidly increased by approximately 300%, reaching a maximal value within 50 s following the hypertonic shock with a recovery of more than 90% towards the initial [Ca2+]i within 5 min. The effect was inhibited by removal of extracellular Ca2+ ions, but not by thapsigargin, indicating that the rise in [Ca2+]i is only a result of influx from the extracellular medium. The rise was insensitive to inhibitors of L-type voltage-activated Ca2+ channels, TRPV channels or stretch-activated cation channels. Non-specific inhibitors of Ca2+ channels like CdCl2, NiCl2, LaCl3 and ZnCl2 significantly attenuated the response, although the extent in which CdCl2 and NiCl2 (both of them inhibitors of annexin-mediated Ca2+ fluxes) inhibited the response was significantly greater. The rise was also sensitive to KBR7943, inhibitor of NCE reverse mode and trifluoperazine, inhibitor of the activity of annexins. Hypertonic shock also produced also hyperpolarisation of chondrocytes (Em measured by means of Di-BA-C4(3), a membrane potential sensitive dye), which was inhibited by TEA-Cl and BaCl, but was not affected by changing the extracellular solution to Ca(2+)-free HBS. Inhibition of hyperpolarisation completely abolished the [Ca2+]i rise following hypertonic shock. Treatment with retinoic acid, which can increase the activity of annexins as Ca2+ transport pathways caused a significant increase in [Ca2+]i. The recovery of [Ca2+] was inhibited by benzamil and was dependent on extracellular Na+, but was unaffected by Na-orthovanadate, an inhibitor of plasma Ca(2+)-ATPase. We conclude that in response to hypertonic shock, NCE reverse mode and annexins are the pathways responsible for the [Ca2+]i increase, while forward mode operation of NCE is responsible for the subsequent extrusion of Ca2+ and recovery of [Ca2+]i towards initial values.     

Electrophysiological demonstration of voltage- activated H+ channels in bovine articular chondrocytes.

Matrix synthesis by articular chondrocytes is sensitive to changes in intracellular pH (pH(i)), so characterising the membrane transport pathways that determine pH(i) is important for understanding how chondrocytes regulate the turnover of cartilage matrix. In the present study, the whole-cell patch-clamp technique has been employed to demonstrate the operation of voltage-activated H(+) channels (VAHC) in bovine articular chondrocytes. Using solutions designed to minimise the contribution of ions other than H(+), the application of step voltage-protocols elicited whole-cell currents. These currents were slow activating, observed only in the outward direction, dependent on both extracellular pH (pH(o)) and pH(i), and inhibited by Zn(2+). The reversal potential values, measured by tail current analysis, over a range of different pHo and pHi values, were in good agreement with predicted values for membrane channels having a high selectivity for protons. The results presented here are consistent with the operation of VAHC in articular chondrocytes.