Electrophysiological characterization of store-operated and agonist-induced Ca2+ entry pathways in endothelial cells (original) (raw)
Related papers
Cell Calcium, 2008
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 M) 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.
Cell Calcium, 2007
In this study the relationship between the efficiency of endoplasmic reticulum (ER) Ca 2+ refilling and the extent of Ca 2+ entry was investigated in endothelial cells. ER and mitochondrial Ca 2+ concentration were measured using genetically encoded Ca 2+ sensors, while the amount of entering Ca 2+ was controlled by varying either the extracellular Ca 2+ or the electrical driving force for Ca 2+ by changing the plasma membrane potential. In the absence of an agonist, ER Ca 2+ replenishment was fully accomplished even if the Ca 2+ concentration applied was reduced from 2 to 0.5 mM. A similar strong efficiency of ER Ca 2+ refilling was obtained under condition of plasma membrane depolarization. However, in the presence of histamine, ER Ca 2+ refilling depended on mitochondrial Ca 2+ transport and was more susceptible to membrane depolarization. Store-operated Ca 2+ entry (SOCE), was strongly reduced under low Ca 2+ and depolarizing conditions but increased if ER Ca 2+ uptake was blocked or if ER Ca 2+ was released continuously by IP 3. A correlation of the kinetics of ER Ca 2+ refilling with cytosolic Ca 2+ 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 Ca 2+ elevation upon Ca 2+ entry and that this important process is widely achieved even under conditions of diminished Ca 2+ entry.
Inhibition of capacitative Ca2+ entry by a Cl− channel blocker in human endothelial cells
European Journal of Pharmacology: Molecular Pharmacology, 1994
We have used the patch clamp technique in combination with intracellular calcium measurements to measure simultaneously Ca 2+ entry and ionic currents activated by emptying of intracellular Ca 2+ stores (capacitative Ca 2+ entry and Ca 2+ releaseactivated Ca 2+ currents, CRAC) in human endothelial cells from umbilical veins. Intracellular stores were depleted of Ca 2+ by preincubating endothelial cells for 20 minutes with 2 /xM thapsigargin in Ca2+-free solution. Reapplication of 10 mM [Ca2+]~ evoked an increase in [Ca2+] i indicating Ca 2+ influx after the thapsigargin-induced store depletion (capacitative Ca 2+ entry), however no measurable CRAC could be detected. The increase in [CaZ+] i after [Ca2+] e resubmission was substantially reduced in the presence of 50/xM NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid) from 0.77 _+ 0.25/xM to 0.2 _+ 0.06 ~M (n = 6) at a holding potential of -40 mV. Estimates of the capacitative Ca 2+ entry at various membrane potentials from the first time derivative of the Ca 2+ transients showed a highly inwardly rectifying I-V curve with a Ca 2+ inward current amplitude of 1.0 _+ 0.3 pA (membrane capacitance 59 + 9 pF, n = 8) at -80 inV. This current amplitude was decreased to 0.32 + 0.12 pA (n = 6) in the presence of 50 /xM NPPB. This corresponds to a decrease in the Ca 2+ permeability of the endothelial cell membrane from 0.15 • 10 s cm/s (control) to 0.06. l0 s cm/s (50 ~zM NPPB).
The Journal of Physiology, 2000
In a wide variety of non-excitable cell types, release of Ca¥ from intracellular Ca¥ stores initiates a store depletiondependent Ca¥ influx mechanism, termed capacitative Ca¥ entry (for review see Berridge, 1995; Putney, 1997; Parekh & Penner, 1997; Holda et al. 1998). This store-operated or Ca¥ release-dependent capacitative Ca¥ entry (CCE) plays a key role in maintaining the Ca¥ load of intracellular stores (endoplasmic reticulum, ER), but has also been implicated in important cellular functions such as secretion, regulation of adenylate cyclase, gene transcription, cell cycle and proliferation, apoptosis and modulation of [Ca¥]é oscillations and [Ca¥]é waves (for review see e.g. Parekh & Penner, 1997). Furthermore, in endothelial cells CCE has been shown to be a preferential Ca¥ source for the regulation of the Ca¥-calmodulin-dependent NO synthase activity (Xu et al. 1994; Wang et al. 1996). Hoth & Penner (1992) were the first to demonstrate a membrane conductance directly related to Ca¥ store depletion, which subsequently was termed Ca¥ release-activated Ca¥ current (ICRAC). A similar membrane current has been identified in vascular endothelial cells (Fasolato & Nilius, 1998). ICRAC is the best characterized store-operated Ca¥ current, although in recent years an increasing number of store-dependent membrane conductances have been characterized in different cell types that differ from the original ICRAC in their biophysical properties and modes of regulation and modulation (see Clapham, 1995; Parekh & Penner, 1997). Several key questions regarding regulation of CCE have remained unanswered. For example, the signalling mechanism that communicates the degree of store filling to the surface membrane Ca¥-permeable channel remains elusive. Arguments in favour of or against a mechanism based on diffusible signalling messengers or protein-protein interactions between release channel and surface membrane channel are abundant (e.g. Berridge, 1996; Csutora et al.
Amplitude modulation of Ca2+ signals induced by histamine in human endothelial cells
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1994
We have addressed the problem of whether the agonist concentration sensed by endothelial cells is encoded by the sustained rise of the intracellular Ca 2+ concentration ([Ca2+] i) or by the frequency of intracellular Ca 2÷ oscillations. Single or confluent endothelial cells from umbilical veins were stimulated for 15 min with histamine (0.03 to 100 p.mol/i), and the concomitant changes in [Ca2+] i were measured with fura-2/AM. Application of histamine at concentrations above 0.1 /zmol/l resulted always in a fast spike of [Ca2+]i, followed by a slow decline to a sustained plateau level, which depends on the presence of extracellular Ca 2+. At the same time of the development of this plateau phase, quenching of the fura-2/AM signal occurred during agonist stimulation in a Ca2+-free, 1.5 mmol/l Mn 2+ containing solution, indicating influx of divalent cations during this time. From 48 cells in 1.5 mmol/l [Ca2+] e we obtained a close relation between histamine concentration and time integral of [Ca2+] i taken over the 15 min recording of the plateau [Ca2+]i . The half-maximal increase in the integral of [Ca2+]i was at 0.7 /xmol/l for solitary cells, 1.2 p~mol/l for clustered cells and 1.2 /xmol/l for the plateau Ca 2+ level. Repetitive Ca 2+ spikes or Ca 2+ oscillations appeared only in 16 out of 48 cells, but their frequency was not correlated to the agonist concentration. Ca 2+ oscillations were only observed in a concentration window between 0.1 and 1 ~mol/l histamine, both in single and in clustered endothelial cells. Our results indicate that coding of the agonist concentration in endothelial cells is not related to the frequency of Ca 2+ oscillations, but is closely correlated with the plateau level of intracellular Ca 2÷
Ca 2+ Influx Through Ca 2+ Channels in Rabbit Ventricular Myocytes During Action Potential Clamp
Circulation Research, 1999
Ca 2ϩ influx via Ca 2ϩ current (I Ca) during the action potential (AP) was determined at 25°C and 35°C in isolated rabbit ventricular myocytes using AP clamp. Contaminating currents through Na ϩ and K ϩ channels were eliminated by using Na ϩ-and K ϩ-free solutions, respectively. DIDS (0.2 mmol/L) was used to block Ca 2ϩ-activated chloride current (I Cl(Ca)). When the sarcoplasmic reticulum (SR) was depleted of Ca 2ϩ by preexposure to 10 mmol/L caffeine, total Ca 2ϩ entry via I Ca during the AP was Ϸ12 mol/L cytosol (at both 25°C and 35°C). Similar Ca 2ϩ influx at 35°C and 25°C resulted from a combination of higher and faster peak I Ca , offset by more rapid I Ca inactivation at 35°C. During repeated AP clamps, the SR gradually fills with Ca 2ϩ , and consequent SR Ca 2ϩ release accelerates I Ca inactivation during the AP. During APs and contractions in steady state, total Ca 2ϩ influx via I Ca was reduced by Ϸ50% but was again unaltered by temperature (5.6Ϯ0.2 mol/L cytosol at 25°C, 6.0Ϯ0.2 mol/L cytosol at 35°C). Thus, SR Ca 2ϩ release is responsible for sufficient I Ca inactivation to cut total Ca 2ϩ influx in half. However, because of the kinetic differences in I Ca , the amount of Ca 2ϩ influx during the first 10 ms, which presumably triggers SR Ca 2ϩ release, is much greater at 35°C. I Ca during a first pulse, given just after the SR was emptied with caffeine, was subtracted from I Ca during each of 9 subsequent pulses, which loaded the SR. These difference currents reflect I Ca inactivation due to SR Ca 2ϩ release and thus indicate the time course of local [Ca 2ϩ ] in the subsarcolemmal space near Ca 2ϩ channels produced by SR Ca 2ϩ release (eg, maximal at 20 ms after the AP activation at 35°C). Furthermore, the rate of change of this difference current may reflect the rate of SR Ca 2ϩ release as sensed by L-type Ca 2ϩ channels. These results suggest that peak SR Ca 2ϩ release occurs within 2.5 or 5 ms of AP upstroke at 35°C and 25°C, respectively. I Cl(Ca) might also indicate local [Ca 2ϩ ], and at 35°C in the absence of DIDS (when I Cl(Ca) is prominent), peak I Cl(Ca) also occurred at a time comparable to the peak I Ca difference current. We conclude that SR Ca 2ϩ release decreases the Ca 2ϩ influx during the AP by Ϸ50% (at both 25°C and 35°C) and that changes in I Ca (and I Cl(Ca)), which depend on SR Ca 2ϩ release, provide information about local subsarcolemmal [Ca 2ϩ ]. The full text of this article is available at http://www.circresaha.org. (Circ Res. 1999;85:e7-e16.
Calcium entry activated by store depletion in human umbilical vein endothelial cells
Cell Calcium, 1994
We have used the patch clamp technique combined with simultaneous measurement of intracellular Ca*+ to record ionic currents activated by depletion of intracellular Ca*+-stores in endothelial cells from human umbilical veins. Two protocols were used to release Ca*+ from intracellular stores, i.e. loading of the cells via the patch pipette with Ins( 1,4,5)R, and extracellular application of thapsigargin. lns(1,4,5)P3 (10 PM) evoked a transient increase in [Ca2+]i in cells exposed to Ca*+-free extracellular solutions. A subsequent reapplication of extracellular Ca*+ induced an elevation of [Ca*+Ii. These changes in [Ca*+]i were very reproducible. The concomitant membrane currents were neither correlated in time nor in size with the changes in [Ca*']i. Similar changes in [Ca*+]i and membrane currents were observed if the Ca +-stores were depleted with thapsigargin.
The Journal of General Physiology, 2005
Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca 2 ϩ ] i in a Ca 2 ϩ -free medium followed by a secondary [Ca 2 ϩ ] i rise upon Ca 2 ϩ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca 2 ϩ -activated (I Cl(Ca) ) and volume-regulated (I Cl, swell ) chloride currents. NPPB and DIDS more efficiently inhibited I Cl(Ca) and I Cl, swell , respectively. Cell swelling caused by hypotonic solution invariably activated I Cl, swell while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist-and cell swelling-induced I Cl, swell , while its inactive analogue U-73343 had no effect. I Cl(Ca) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca 2 ϩ ] i in the pipette solution at 500 nM. In contrast, I Cl, swell could be directly activated by 1-oleoyl-2-acetyl-sn -glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP 3 infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced I Cl, swell in a nonadditive manner, suggesting their convergence on a common pathway. I Cl, swell and I Cl(Ca) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated I Cl(Ca) , but abolished I Cl, swell , thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that I Cl, swell can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a "selection" switch between closely localized channels.
Journal of Membrane Biology, 2000
We characterized the effects of histamine on intracellular Ca 2+ and activation of ionic currents in human capillary endothelial cells. Histamine produced both a transient and sustained increase in intracellular Ca 2+. The transient response was mediated largely through intracellular Ca 2+ release and the sustained response was due to extracellular Ca 2+ entry. The increase in intracellular Ca 2+ by histamine was not affected by the H2 blocker cimetidine. But was entirely blocked by the H1 antagonist diphenhydramine showing that the histamine response in these cells is mediated through the H1 receptor. A transient ionic current is coactivated with the histamine-induced increase in intracellular Ca 2+ and this current has several properties of a nonselective, Ca 2+ permeable, cation channel (NSC). The magnitude of the NSC current does not strictly correlate with intracellular Ca 2+ levels. A Ca 2+-activated K + current (BKCA) is activated by the increase in intracellular Ca 2+ and this current is blocked by the selective BKCA blocker iberiotoxin.