ORAI1 channel gating and selectivity is differentially altered by natural mutations in the first or third transmembrane domain.

KEY POINTS: Gain-of-function mutations in the highly selective Ca2+ channel ORAI1 cause tubular aggregate myopathy (TAM) characterized by muscular pain, weakness and cramping. TAM-associated mutations in ORAI1 first and third transmembrane domain facilitate channel opening by STIM1, causing constitutive Ca2+ influx and increasing the currents evoked by Ca2+ store depletion. Mutation V107M additionally decreases the channel selectivity for Ca2+ ions and its inhibition by acidic pH, while mutation T184M does not alter the channel sensitivity to pH or to reactive oxygen species. The ORAI blocker GSK-7975A prevents the constitutive activity of TAM-associated channels and might be used in therapy for patients suffering from TAM. ABSTRACT: Skeletal muscle differentiation relies on store-operated Ca2+ entry (SOCE) mediated by STIM proteins linking the depletion of endoplasmic/sarcoplasmic reticulum Ca2+ stores to the activation of membrane Ca2+ -permeable ORAI channels. Gain-of-function mutations in STIM1 or ORAI1 isoforms cause tubular aggregate myopathy (TAM), a skeletal muscle disorder with muscular pain, weakness and cramping. Here, we characterize two overactive ORAI1 mutants from patients with TAM: V107M and T184M, located in the first and third transmembrane domain of the channel. When ectopically expressed in HEK-293T cells or human primary myoblasts, the mutated channels increased basal and store-operated Ca2+ entry. The constitutive activity of V107M, L138F, T184M and P245L mutants was prevented by low concentrations of GSK-7975A while the G98S mutant was resistant to inhibition. Electrophysiological recordings confirmed ORAI1-V107M constitutive activity and revealed larger STIM1-gated V107M- and T184M-mediated currents with conserved fast and slow Ca2+ -dependent inactivation. Mutation V107M altered the channel selectivity for Ca2+ ions and conferred resistance to acidic inhibition. Ca2+ imaging and molecular dynamics simulations showed a preserved sensitivity of T184M to the negative regulation by reactive oxygen species. Both mutants were able to mediate SOCE in Stim1-/- /Stim2-/- mouse embryonic fibroblasts expressing the binding-deficient STIM1-F394H mutant, indicating a higher sensitivity for STIM1-mediated gating, with ORAI1-T184M gain-of-function being strictly dependent on STIM1. These findings provide new insights into the permeation and regulatory properties of ORAI1 mutants that might translate into therapies against diseases with gain-of-function mutations in ORAI1.

Evidence for a receptor-activated Ca2+ entry pathway independent from Ca2) store depletion in endothelial cells.

Ca(2+) entry in endothelial cells is a key signaling event as it prolongs the Ca(2+) signal activated by a receptor agonist, and thus allows an adequate production of a variety of compounds. The possible routes that lead to Ca(2+) entry in non-excitable cells include the receptor-activated Ca(2+) entry (RACE), which requires the presence of an agonist to be activated, and the store-operated Ca(2+) entry (SOCE) pathway, whose activation requires the depletion of the ER Ca(2+) store. However, the relative importance of these two influx pathways during physiological stimulation is not known. In the present study we experimentally differentiated these two types of influxes and determined under which circumstances they are activated. We show that La(3+) (at 10 microM) is a discriminating compound that efficiently blocks SOCE but is almost without effect on histamine-induced Ca(2+) entry (RACE). In line with this, histamine does not induce massive store depletion when performed in the presence of extracellular Ca(2+). In addition, inhibition of mitochondrial respiration significantly reduces SOCE but modestly affects RACE. Thus, agonist-induced Ca(2+) entry is insensitive to La(3+), and only modestly affected by mitochondrial depolarization. These data shows that agonist relies almost exclusively on RACE for sustained Ca(2+) signaling in endothelial cells.

Ca2+ refilling of the endoplasmic reticulum is largely preserved albeit reduced Ca2+ entry in endothelial cells.

In this study the relationship between the efficiency of endoplasmic reticulum (ER) Ca2+ refilling and the extent of Ca2+ entry was investigated in endothelial cells. ER and mitochondrial Ca2+ concentration were measured using genetically encoded Ca2+ sensors, while the amount of entering Ca2+ was controlled by varying either the extracellular Ca2+ or the electrical driving force for Ca2+ by changing the plasma membrane potential. In the absence of an agonist, ER Ca2+ replenishment was fully accomplished even if the Ca2+ concentration applied was reduced from 2 to 0.5mM. A similar strong efficiency of ER Ca2+ refilling was obtained under condition of plasma membrane depolarization. However, in the presence of histamine, ER Ca2+ refilling depended on mitochondrial Ca2+ transport and was more susceptible to membrane depolarization. Store-operated Ca2+ entry (SOCE), was strongly reduced under low Ca2+ and depolarizing conditions but increased if ER Ca2+ uptake was blocked or if ER Ca2+ was released continuously by IP(3). A correlation of the kinetics of ER Ca2+refilling with cytosolic Ca2+ signals revealed that termination of SOCE is a rapid event that is not delayed compared to ER refilling. Our data indicate that ER refilling occurs in priority to, and independently from the cytosolic Ca2+ elevation upon Ca2+ entry and that this important process is widely achieved even under conditions of diminished Ca2+entry.

Dopamine modulates von Willebrand factor secretion in endothelial cells via D2-D4 receptors.

OBJECTIVE: von Willebrand factor (VWF) is acutely released from endothelial cells in response to numerous calcium-raising agents (e.g. thrombin, histamine) and cAMP-raising agents (e.g. epinephrine, adenosine, vasopressin). In contrast, very few inhibitors of endothelial VWF secretion have been described. The neurotransmitter dopamine is a modulator of exocytosis in several endocrine cells, and is possibly involved in the regulation of several endothelial cell functions. We therefore investigated the effect of dopamine on endothelial VWF secretion. RESULTS: Dopamine, D2/D3- and D4-specific agonists inhibited histamine- but not thrombin-induced VWF secretion. Expression of dopamine D2, D3 and D4 receptors was demonstrated by reverse transcription polymerase chain reaction (RT-PCR) in both human aortic (HAEC) and umbilical vein (HUVEC) endothelial cells. D2-D4 agonists did not inhibit histamine-induced rise in [Ca(2+)](i): they inhibited histamine-induced secretion even in the absence of extracellular calcium. Thus, the dopamine effects are not mediated by [Ca(2+)](i)-dependent signalling. D2/D3- and D4-specific agonists inhibited neither the rise in cAMP nor VWF secretion in response to epinephrine and adenosine, arguing against an effect on cAMP-mediated signalling. D1 and D5 receptors were not detected in HAEC or HUVEC by RT-PCR, and the D1/D5-specific agonist SKF 38 393 failed to modulate VWF secretion, arguing against a role for these receptors in endothelial exocytosis. CONCLUSIONS: Dopamine inhibits histamine-induced endothelial exocytosis by activating D2-D4 receptor, via a mechanism distinct from [Ca(2+)](i)-or cAMP-mediated signaling. In contrast, D1 and D5 receptors are not functionally expressed in cultured endothelial cells. Dopamine agonists may be useful as inhibitors of endothelial activation in inflammation and cardiovascular disease.

Mechanisms of Ca2+ store depletion in single endothelial cells in a Ca(2+)-free environment.

Depletion of agonist-sensitive Ca2+ stores results in activation of capacitative Ca2+ entry (CCE) in endothelial cells. The proportion of Ca2+ stores contributing to the regulation of CCE is unknown. In fura-2/am loaded single endothelial cells freshly isolated from bovine left circumflex coronary arteries, we investigated whether a resting period in a Ca(2+)-free environment results in emptying of bradykinin-sensitive Ca2+ stores (BsS) and activation of CCE. In a Ca(2+)-free environment, depletion of BsS occurred in a time-dependent manner (59% after 10 min in Ca(2+)-free solution). This effect was prevented by inhibition of the Na(+)-Ca2+ exchange but not by a blockade of ryanodine-sensitive Ca2+ release (RsCR). In contrast to BsS, mitochondrial Ca2+ content remained unchanged in the Ca(2+)-free environment. Remarkably, activity of CCE (monitored as Mn2+ influx) did not increase after depletion of BsS in the Ca(2+)-free environment. In contrast to Mn2+ influx, the effect of re-addition of Ca2+ to elevate bulk Ca2+ concentration ([Ca2+]b) decreased with the time the cells rested in Ca(2+)-free buffer. This decrease was prevented by an inhibition of RsCR. In low Na+ conditions the effect of Ca2+ on [Ca2+]b was reduced while it did not change the time the cells rested in Ca(2+)-free solution. After a 2 min period in low Na+ conditions, ryanodine-induced Ca2+ extrusion was markedly diminished. Inhibition of RsCR re-established the effect of Ca2+ on [Ca2+]b in low Na+ conditions. Collapsing subplasmalemmal Ca2+ stores with nocodazole, increased the effect of Ca2+ on [Ca2+]b. In nocodazole-treated cells, the effect of Ca2+ on [Ca2+]b was not reduced in Ca(2+)-free environment. These data indicate that activation of CCE is not associated with the agonist-sensitive Ca2+ pools that deplete rapidly in a Ca(2+)-free environment. Subplasmalemmal ryanodine-sensitive Ca2+ stores (RsS) are emptied in Ca(2+)-free/low Na+ solution and re-sequester Ca2+ which enters the cells prior an increase in [Ca2+]b occurs. Thus, in endothelial cells there are differences in the functions of various subplasmalemmal Ca2+ stores (i.e. BsS and RsS), which include either activation of CCE or regulation of subplasmalemmal Ca2+.

Effect of 5-hydroxytryptamine on the membrane potential of endothelial and smooth muscle cells in the pig coronary artery.

1. Many endothelium-dependent vasodilators hyperpolarize the endothelial cells in blood vessels. It is not known whether these hyperpolarizations are linked to nitric oxide synthesis or to an endothelium-derived hyperpolarizing phenomenon, since most of the vasodilators release both factors. In this context, we first verified that the endothelium-dependent relaxations induced by 5-hydroxytryptamine (5-HT) on pig coronary arteries are due only to the activation of the nitric oxide pathway. Then we studied the effects of 5-HT on membrane potential of endothelial and smooth muscle cells. 2. In the absence of endothelium, 5-HT caused a concentration-dependent contraction of coronary artery strips. No change of the smooth muscle cell membrane potential was observed during contraction to 1 microM 5-HT. 3. In the presence of 1 microM ketanserin to suppress the contractile effect of 5-HT, 5-HT induced concentration-dependent relaxation of endothelium-intact strips precontracted by 10 microM prostaglandin F2 alpha (PGF2 alpha). These relaxations were suppressed by 1 microM NG-nitro-L-arginine, an inhibitor of nitric oxide synthesis, showing that they were produced predominantly by nitric oxide. 4. In the presence of 1 microM ketanserin, 1 microM 5-HT did not change the smooth muscle cell membrane potential of strips precontracted by either 10 microM PGF2 alpha or by 10 microM acetylcholine (ACh). In the same conditions, 1 microM 5-HT caused a weak 2.6 +/- 0.4 mV hyperpolarization, of the endothelial cells. 5. In conclusion, the fact that 5-HT did not change the membrane potential of smooth muscle cells and only weakly hyperpolarized the endothelial cells during relaxations, suggests that in both cell types no electrical events accompany activation of the nitric oxide pathway. This is in contrast to the hyperpolarizations observed in endothelial and smooth muscle cells when the endothelium-derived hyperpolarization factor (EDHF) pathway is activated.

Charybdotoxin-sensitive small conductance K(Ca) channel activated by bradykinin and substance P in endothelial cells.

1 In cultured porcine coronary artery endothelial cells, we have recently shown that substance P and bradykinin stimulated different types of Ca(2+)-dependent K(+) (K(Ca)) current. A large part of this current was insensitive to iberiotoxin and apamin. The aim of the present study was to characterize the K(Ca) channel responsible for this current. 2 In cell-attached configuration and asymmetrical K(+) concentration, 100 nM bradykinin or substance P activated a 10 pS K(+) channel. In inside-out configuration, the channel was half-maximally activated by 795 nM free Ca(2+). 3 Apamin (1 micro M) added to the pipette solution failed to inhibit the channel activity while charybdotoxin (50 nM), completely blocked it. Perfusion at the intracellular face of the cell, of an opener of intermediate conductance K(Ca) channel, 500 micro M 1-ethyl-benzimidazolinone (1-EBIO) increased the channel activity by about 4.5 fold. 4 In whole-cell mode, bradykinin and substance P stimulated an outward K(+) current of similar amplitude. Charybdotoxin inhibited by 75% the bradykinin-induced current and by 80% the substance P-induced current. Charybdotoxin plus iberiotoxin (50 nM each) inhibited by 97% the bradykinin-response. Charybdotoxin plus apamin did not increase the inhibition of the substance P-response obtained in the presence of charybdotoxin alone. 5 1-EBIO activated a transient outward K(+) current and hyperpolarized the membrane potential by about 13 mV. Charybdotoxin reduced the hyperpolarization to about 3 mV. 6 Taken together these results show that bradykinin and substance P activate a 10 pS K(Ca) channel, which largely contributes to the total K(+) current activated by these agonists. Despite its small conductance, this channel shares pharmacological characteristics with intermediate conductance K(Ca) channels.

How can detection of infectious tuberculosis be improved? Experience in the Somali region of Ethiopia.

In early 1999, 48% of pulmonary tuberculosis (PTB) cases detected in the Somali region of Ethiopia were smear-positive. Actions at the laboratory level and peer-review of smear-negative PTB diagnoses were proposed. Clinicians knew, but did not adhere to, the algorithm recommended by the National Tuberculosis Programme for these diagnoses, partly due to the costs involved to patients. Challenging clinicians, in a non-threatening way, to become more clinically rigorous proved successful, and the proportion of smear-positive PTB increased to 65%. Operational research is needed to assess the feasibility of these widely-recommended smear-negative PTB diagnosis guidelines.

Properties of triple helices formed by oligonucleotides containing 8-aminopurines.

The synthesis of parallel hairpins carrying 8-aminopurines is described. These hairpins have a high affinity for specific polypyrimidine sequences resulting in the formation of very stable triplexes.

Tightening the Belt on Polymerases: Evaluating the Physical Constraints on Enzyme Substrate Size.

To test the limits of polymerase enzyme activity on geometrically constrained DNAs, four very small synthetic circular DNAs were constructed by using newly developed methods. Surprisingly, even a 13-nucleotide circular DNA (1) can be copied successfully by both DNA and RNA polymerases, despite the very small diameter and large degree of distortion in this synthetic DNA. The picture shows models to indicate the relative sizes of 1 and the Klenow fragment of the DNA polymerase I from E. coli.

Protein kinase C-delta mediates von Willebrand factor secretion from endothelial cells in response to vascular endothelial growth factor (VEGF) but not histamine.

BACKGROUND: Vascular endothelial growth factor (VEGF) and histamine induce von Willebrand factor (VWF) release from vascular endothelial cells. Protein kinase C (PKC) is involved in the control of exocytosis in many secretory cell types. OBJECTIVES: We investigated the role of PKC and the interactions between PKC and Ca2+ signaling in both VEGF-induced and histamine-induced VWF secretion from human umbilical vein endothelial cells (HUVECs). RESULTS: Several PKC inhibitors (staurosporine, Ro31-8220, myristoylated PKC peptide inhibitor and Go6983) block VEGF-induced but not histamine-induced VWF secretion. PKC-alpha and novel PKCs (PKC-delta, PKC-epsilon, and PKC-eta), but not PKC-beta, are expressed in HUVECs. Both VEGF and histamine activate PKC-delta. However, gene inactivation experiments using small interfering RNA indicate that PKC-delta (but not PKC-alpha) is involved in the regulation of VEGF-induced but not histamine-induced secretion. Both VEGF and histamine induce a rise in cytosolic free Ca2+ ([Ca2+]c), but the response to VEGF is weaker and even absent in a significant subset of cells. Furthermore, VEGF-induced secretion is largely preserved when the rise in [Ca2+]c is prevented by BAPTA-AM. CONCLUSIONS: Our study identifies striking agonist specificities in signal-secretion coupling. Histamine-induced secretion is dependent on [Ca2+]c but not PKC, whereas VEGF-induced secretion is largely dependent on PKC-delta and significantly less on [Ca2+]c. Our data firmly establish the key role of PKC-delta in VEGF-induced VWF release, but suggest that a third, VEGF-specific, signaling intermediate is required as a PKC-delta coactivator.

Subplasmalemmal ryanodine-sensitive Ca2+ release contributes to Ca2+-dependent K+ channel activation in a human umbilical vein endothelial cell line.

The whole-cell configuration of the patch clamp technique was used to assess the involvement of ryanodine-sensitive Ca2+ release (RsCR) in histamine-activated Ca2+-dependent K+ (KCa) channels in the human umbilical vein endothelial cell line EA.hy926. Histamine (10 microM) induced a transient outward current that reached 18.9 +/- 5.5 pA pF-1 at +20 mV. This current was diminished by 1 mM tetraethylammonium or 50 nM iberiotoxin, by 90 % and 80 %, respectively, suggesting that this current results from the stimulation of large-conductance KCa (BKCa) channels. In about 50 % of the cells tested, stimulation of RsCR with 200 nM ryanodine initiated a small outward current that was also sensitive to iberiotoxin. Following the ryanodine-mediated RsCR, the potency of 10 microM histamine to activate KCa channels was reduced by about 60 %. In agreement, an inhibition of RsCR with 25 microM ryanodine diminished KCacurrent in response to histamine by about 70 %. The effect of 100 microM histamine on KCa channel activity was not reduced by previous RsCR with 200 nM ryanodine, or by an inhibition of RsCR by 25 microM ryanodine. Histamine (10 microM)-induced Ca2+ elevation was reduced by 30 % following ryanodine-mediated RsCR, whereas no inhibition occurred in the case of 100 microM histamine stimulation. In cells treated with 10 microM nocodazole for 16 h to collapse the superficial endoplasmic reticulum, 200 nM ryanodine failed to initiate any KCa current. Furthermore, the inhibitory effect of previous RsCR on 10 microM histamine-induced KCa current was not obtained in nocodazole-treated cells. Our data suggest that during moderate cell stimulation (10 microM histamine), subplasmalemmal RsCR greatly contributes to the activation of KCa channels in endothelial cells. Thus, the function of the subplasmalemmal Ca2+ control unit (SCCU) described previously must be extended as a regulator for KCa channels.

Substance P and bradykinin activate different types of KCa currents to hyperpolarize cultured porcine coronary artery endothelial cells.

1. Substance P and bradykinin, endothelium-dependent vasodilators of pig coronary artery, trigger in endothelial cells a rise in cytosolic Ca2+ concentration ([Ca2+]i) and membrane hyperpolarization. The aim of the present study was to determine the type of Ca2+-dependent K+ (KCa) currents underlying the endothelial cell hyperpolarization. 2. The substance P-induced increase in [Ca2+]i was 30 % smaller than that induced by bradykinin, although the two peptides triggered a membrane hyperpolarization of the same amplitude. The two agonists evoked a large outward K+ current of the same conductance at maximal stimulation. Agonists applied together produced the same maximal current amplitude as either one applied alone. 3. Iberiotoxin (50 nM) reduced by about 40 % the K+ current activated by bradykinin without modifying the substance P response. Conversely, apamin (1 microM) inhibited the substance P-induced K+ current by about 65 %, without affecting the bradykinin response. Similar results were obtained on peptide-induced membrane hyperpolarization. 4. Bradykinin-induced, but not substance P-induced, endothelium-dependent relaxation resistant to NG-nitro-L-arginine and indomethacin was partly inhibited by 3 microM 17-octadecynoic acid (17-ODYA), an inhibitor of cytochrome P450 epoxygenase. Similarly, the bradykinin-induced K+ current was reduced by 17-ODYA. 5. Our results show that responses to substance P and bradykinin result in a hyperpolarization due to activation of different KCa currents. A current consistent with the activation of large conductance (BKCa) channels was activated only by bradykinin, whereas a current consistent with the activation of small conductance (SKCa) channels was stimulated only by substance P. The observation that a similar electrical response is produced by different pools of channels implies distinct intracellular pathways leading to KCa current activation.

Ca(2+)-dependent non-selective cation and potassium channels activated by bradykinin in pig coronary artery endothelial cells.

1. Using the cell-attached and inside-out modes of the patch-clamp technique, we studied the Ca(2+)-dependent ionic channels activated by bradykinin in cultured pig coronary artery endothelial cells to further understand electrophysiological events underlying cellular activation. 2. In the cell-attached mode, bradykinin (94 nM) activated two types of Ca(2+)-dependent channels: a high conductance K+ channel (285 pS in high symmetrical K+), whose open state probability was increased by depolarization, and a lower conductance inwardly rectifying non-selective cation channel (44 pS in high symmetrical K+). 3. The 285 pS K+ channel was half-maximally activated by cytosolic Ca2+ levels of 1.6 and 4.5 microM at +10 and -30 mV, respectively. Such local concentrations should be reached in the presence of bradykinin, which induces a mean maximal cytosolic Ca2+ rise of 1.3 microM. 4. The 285 pS K+ channel was inhibited by d-tubocurarine, which acted by reducing the mean open time duration (flickering pattern), finally reducing the channel conductance. 5. Divalent cations such as Ca2+ could flow through the 44 pS non-selective cation channel, with nearly the same permeability (P) as monovalent cations (PK: PNa: PCa = 1:1:0.7). 6. The cation channel appeared to be more sensitive to Ca2+ than the K+ channel, with a half-maximal open probability induced by 0.7 microM Ca2+ on the intracellular side of the membrane. 7. In contrast to the K+ channel, the cation channel mean open time was clearly increased by bradykinin. This effect was delayed compared with the increase in the channel open state probability and was rapidly lost in the inside-out configuration. Caffeine also activated the cation channel but more transiently than bradykinin and without any effect on the open duration. 8. In the absence of extracellular Ca2+, the bradykinin-induced increase in cytosolic free Ca2+ was shortened temporally by 52% and reduced in amplitude by 88%, whereas the bradykinin-induced hyperpolarization was not significantly reduced in amplitude but was shortened by 70%, thus illustrating the major role of Ca2+ influx in endothelial cell activation by bradykinin. 9. We conclude that bradykinin activates two types of Ca(2+)-dependent channels in coronary endothelial cells: a high conductance K+ channel regulated by membrane potential, and an inwardly rectifying cation channel allowing Ca2+ entry, the cation channel being about 6 times more sensitive to Ca2+ than the K+ channel. The increase in cation channel open state probability involves an increase in open number, like the K+ channel, but also involves a rise in channel open duration. Ca2+ entry via cation channels could contribute to increase the cytoplasmic Ca2+ level, activate Ca(2+)-dependent K+ channels, thus triggering membrane hyperpolarization when the endothelial cell is stimulated by a vasoactive agonist such as bradykinin.

Epoxyeicosatrienoic acids activate a high-conductance, Ca(2+)-dependent K + channel on pig coronary artery endothelial cells.

1. Epoxyeicosatrienoic acids (EETs) have been described as endothelium-derived hyperpolarizing factors (EDHFs), based on their stimulatory effects on smooth muscle K+ channels. In order to reveal a putative autocrine effect of EETs on endothelial channels, we have studied the effects of the four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET) on the high-conductance, Ca(2+)-dependent K+ (BKCa) channel recorded in inside-out patches of primary cultured pig coronary artery endothelial cells. Currents were recorded in the presence of either 500 nm or 1 microM free Ca2+ on the cytosolic side of the membrane. 2. In 81% of experiments, EETs at < 156 nM, applied on the cytosolic side of the membrane, transiently increased BKCa channel open state probability (PO) without affecting its unitary conductance, thus providing evidence for direct action of EETs, without involvement of a cytosolic transduction pathway. 3. The four EET regioisomers appeared to be equally active, multiplying the BKCa channel PO by a mean factor of 4.3 +/- 0.6 (n = 15), and involving an increase in the number and duration of openings. 4. The EET-induced increase in BKCa channel activity was more pronounced with low initial PO. When the BKCa channel was activated by 500 nM Ca2+, application of EETs increased the initial PO value of below 0.1 by a factor of 5. When the channel was activated by 1 microM Ca2+, application of EETs increased the initial PO value by a factor of 3. 5. Our results show that EETs potentiate endothelial BKCa channel activation by Ca2+. The autocrine action of EETs on endothelial cells, which occurs in the same concentration range as their action on muscle cells, should therefore fully participate in the vasoactive effects of EETs, and thus be taken into account when considering their putative EDHF function.

Histamine-induced Ca2+ oscillations in a human endothelial cell line depend on transmembrane ion flux, ryanodine receptors and endoplasmic reticulum Ca2+-ATPase.

Using single cell microfluorometry to monitor changes in bulk Ca2+ concentration ([Ca2+]bulk) and the whole-cell configuration of the patch clamp technique to measure K+ currents (voltage clamp) and membrane potential (current clamp), the mechanisms of histamine-induced Ca2+ oscillations in the umbilical vein endothelial cell-derived cell line EA.hy926 were studied. In single cells, histamine (10 microM) evoked sinusoidal Ca2+ oscillations in low extracellular Ca2+ concentrations ([Ca2+]o = 10-30 microM). In contrast, histamine did not initiate Ca2+ oscillations either in the absence of extracellular Ca2+ (10 microM EGTA) or in the presence of 2.5 mM extracellular Ca2+. Ca2+ oscillations were accompanied by rhythmic activation of Ca2+-activated K+ (KCa) channels and membrane hyperpolarization of 18.1 +/- 3.9 mV. Hence, cell depolarization with 70 mM extracellular K+ or the inhibition of non-selective cation channels (NSCCs) and KCa channels by 10 microM Loe 908 and 10 mM tetrabutylammonium prevented histamine-evoked Ca2+ oscillations. Preventing Na+-Ca2+ exchange (NCX) by 10 microM 2', 4'-dichlorobenzamil, or removal of extracellular Na+, abolished histamine-induced Ca2+ oscillations. Lowering the extracellular Na+ concentration and thus promoting the reversed mode of NCX (3Na+ out and 1Ca2+ in) increased the amplitude and frequency of histamine-induced Ca2+ oscillations by 25 and 13 %, respectively. Hence, in the absence of extracellular Ca2+, 10 microM histamine induced an elevation of intracellular Na+ concentration in certain subplasmalemmal domains. The inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2,5-di-tert-butyl-1, 4-benzo-hydroquinone (15 microM) prevented histamine-induced Ca2+ oscillations. In addition, blockage of ryanodine-sensitive Ca2+ release (RsCR) by 25 microM ryanodine blunted Ca2+ oscillations. In endothelial cells that were treated for 16 h with 10 microM nocodazole to collapse the superficial endoplasmic reticulum (sER), no histamine-induced Ca2+ oscillations were found. We conclude that in low [Ca2+]o conditions histamine-induced Ca2+ oscillations depend on transmembrane Na+ loading through NSCCs that leads to Ca2+ entry via NCX. Cation influx is controlled by KCa channel activity that triggers membrane hyperpolarization and, thus, provides the driving force for cation influx. Hence, the Ca2+ entering needs to be sequestrated via SERCA into sER to become released by RsCR to evoke Ca2+ spiking. These data further support our previous work on localized Ca2+ signalling as a key phenomenon in endothelial Ca2+ homeostasis.

An intercellular regenerative calcium wave in porcine coronary artery endothelial cells in primary culture.

1. A regenerative calcium wave is an increase in cytosolic free calcium concentration ([Ca2+]i) which extends beyond the stimulated cells without decrement of amplitude, kinetics of [Ca2+]i increase and speed of propagation. 2. The aim of the present study was to test the hypothesis that such a wave could be evoked by bradykinin stimulation and by scraping cultured endothelial cells from porcine coronary arteries. 3. Calcium imaging was performed using the calcium-sensitive dye fura-2. A wound or a delivery of bradykinin to two to three cells on growing clusters of approximately 300 cells caused an increase in [Ca2+]i which was propagated throughout the cluster in a regenerative manner over distances up to 400 micrometer. This wave spread through gap junctions since it was inhibited by the cell uncoupler palmitoleic acid. 4. The same experiments performed in confluent cultures caused a rise in [Ca2+]i which failed to propagate in a regenerative way. The wave propagation probably failed because the confluent cells were less dye coupled than the growing cells. This was confirmed by immunohistology which detected a dramatic decrease in the number of connexin 40 gap junctions in the confluent cultures. 5. The regenerative propagation of the wave was blocked by inhibitors of calcium-induced calcium release (CICR) and phospholipase C (PLC), and by suppression of extracellular calcium, but not by clamping the membrane potential with high-potassium solution. 6. We conclude that regenerative intercellular calcium waves exist in cultured islets but not in confluent cultures of endothelial cells. An increase in [Ca2+]i is not sufficient to trigger a regenerative propagation. The PLC pathway, CICR and extracellular calcium are all necessary for a fully regenerated propagation.

Parallel-stranded hairpins containing 8-aminopurines. Novel efficient probes for triple-helix formation.

We describe novel oligomers with a greater propensity to form triplexes than oligomers containing only natural bases. They consist of a polypyrimidine sequence linked head-to-head with a polypurine sequence carrying one or several 8-aminoadenine or 8-aminoguanines. The presence of 8-aminopurines also stabilised the parallel-stranded duplex structure.