Mechanosensitive Ca2+ transients in endothelial cells from human umbilical vein

Transcription

1 Proc. Natd. cad. Sci. US Vol. 91, pp , pril 1994 Cell iology Mechanosensitive Ca2+ transients in endothelial cells from human umbilical vein MSHIRO OIKE, GuY DROOGMNS, ND ERND NILIUS* KU Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, -3 Leuven, elgium Communicated by Erwin Neher, December 27, 1993 (received for review October 14, 1993) STRCT We have investigated the changes in intracellular calcium concentration ([Ca2+]) in human endothelal cells induced by mechanial stretch due to osmotic cell swelling. Hypotonic solutions also activate a Cl- conductance that has been described elsewhere and mainly serves to clamp the membrane potential at negative values to provide a driving force for Ca2+ influx. The Increase in [Ca2W+ caused by hypotonic solutions is due to release from inouitol-1,4,5- t tive Ca2+ pools and a subsequent Ca2+ influx, apparently activated by store depletion. These changes in [Ca2e+ are completely abolished if the phospholipase 2 (PL2) activity is inhibited by either 4-bromophenacyl bromide or cyclosporin. rachidonic acid, applied either extracellularly or intracellularly via the patch pipette, mimics the mechanosensitive response even in cells with blocked PL2. Metabolites of the ipo- and cyclooxygenase pathways can be excluded. Phospholipase C activation and the protein kinase pathway are not involved in this mechanical response. lthough no specific ph cal tools for probing the role of PL2 are available, our evidence suggests that mecansensitivity in endothelial cells may be modulated by ar onic acid. The biological responses of vascular endothelial cells to mechanical forces, such as shear stress and mechanical stretch, are very diverse. Some of them develop fast, while others occur within the range of several hours (1, 2). One of the earliest responses to mechanical activation is an increase in intracellular [Ca2W] ([Ca2W+, which, for instance, activates the production of nitric oxide (3). Until now, there is no clear indication by which mechanism endothelial cells act as mechanosensors. We have shown recently (4) that mechanical stimulation of endothelial cells activates a Cl- conductance that is not related to the changes in [Ca2~+i. ctivation of this conductance may clamp the resting potential of the endothelial cells at negative values to provide a sufficiently high driving force for Ca2+ influx (4). In the present report, we have studied the changes in [Ca2eli that occur upon mechanical stimulation and the various signal transduction pathways that might be involved in the mechanosensitivity of endothelial cells. MTERILS ND METHODS Endothelial cells were isolated, as described in more detail previously (5), from human umbilical cord veins by a collagenase digestion method and grown in medium 199 containing 1% human serum, 2 mm L-glutamine, 1 units of penicillin per ml, and 1 pg of streptomycin per ml. We have used only nonconfluent voltage-clamped cells in our experiments that were responsive to stimulation with histamine (1,pM). For intracellular Ca2+ measurements, cells were incubated with 2,LM fura-2/m (acetoxymethylester) (Molecular The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. 294 Probes) dissolved in normal Krebs' solution for 2 min at room temperature and thereafter for another 2 min at 37TC. The extracellular solution was a modified Krebs' solution, containing mm NaCl, 5.9 mm KCl, 1.2 mm MgC2, 1.5 mm CaCl2, 11.5 mm glucose, 11.5 mm Hepes-NaOH (ph 7.3). The pipette solutions in all experiments contained 1 mm potassium aspartate, 4 mm KCl, 5 mm NaCl, 1 mm MgCl2,.5 mm Na2TP, 1 mm Hepes,.1 mm EGT (ph 7.2). Cell swelling was induced by a hypotonic solution (), which was obtained by diluting the normal Krebs' solution with an osmolarity of 29 mosm to an osmolarity of 185 mosm. The ionic concentrations in this solution were 94. mm NaCl, 4.2 mm KC1,.9 mm MgCl2, 1.1 mm CaCl2, 8.2 mm glucose, 8.2 mm Hepes. Ca2+-free contained.7 mm EGT. To exclude an effect of changes in ionic concentrations, a series of experiments were performed in which the described above was compared to an isotonic solution with the same ionic composition to which 81.7 mm D-mannitol was added. The system for Ca2+ measurements and its calibration are based on a previously described method (). Single cells were excited alternately with light of 3 and 39 nm via a rotating filter wheel (speed between 2 and 3 per s) and fluorescence was measured at 51 nm. pparent concentration of free calcium was calculated from the fluorescence ratio of the background corrected fluorescence signals. The patch-clamp technique was applied in the standard whole-cell mode or in the perforated patch configuration using a List EPC-9 patchclamp amplifier. Membrane currents were filtered at 1 khz with an eight-pole essel filter and digitized on line at sample intervals of 25 s. In the experiments with heparin (1 MM; 5 kda; Sigma H-54), neomycin (1 mm; Sigma), protein kinase (PK) inhibitory peptide [3 p;m; amino acid residues 5-24 ofthe heat stable PK inhibitor (7); kindly provided by M. ollen, Department of iochemistry, KU Leuven, elgium], and for some patches with arachidonic acid () (1 p;m; Sigma), which were all added to the pipette solution, we have used ruptured patches. In all other experiments we used nystatinperforated patches (8) (a stock solution of nystatin at 5 mg/ml dissolved in dimethyl sulfoxide was diluted 1 times in the pipette solution immediately before use). The following compounds were added to the bath: (Sigma), nordihydroguaiaretic acid {4,4'-(2,3-dimethyl-1,4-butanediyl)bis[1,2- benzenediol]; NDG} (4,uM; Sigma), 4-bromophenacyl bromide (pp; Sigma), cyclosporin (1,uM; Sandoz), indomethacin (4,uM; Sigma). ll experiments were performed at room temperature (2 C-22 C). bbreviations:, arachidonic acid; [Ca2+]i, intracellular free calcium concentration; PL2, phospholipase 2; PLC, phospholipase C; PK, protein kinase ; Ins(1,4,5)P3, inositol 1,4,5- trisphosphate; NDG, nordihydroguaiaretic acid; pp, 4-bromophenacyl bromide. *To whom reprint requests should be addressed.

2 RESULTS Superfusion of voltage-clamped endothelial cells with Ca2+free induces cell swelling and membrane stretch, which are accompanied by a transient increase in [Ca2+]i (Fig. 1). Internal application of hypertonic solution via the patch pipette induces a similar Ca2+ transient in cells superfused with normotonic Ca2+-free solutions, suggesting that these Ca2+ signals are due to changes in osmolarity rather than to changes in extracellular ionic strength. These Ca2+ transients developed within 2-1 s and reached their maximum within 4- min, after which [Ca2+]i decayed to its basal value. To quantify our results, we have determined the amplitude ofthe maximum increase of [Ca2+]i above its resting level. These values showed a large variability, especially between various batches of cells. Values ranged between.1 and.4 jum, with a mean value of.23 ±.3 jum (n = 5 cells). t the same time, an inward current was activated at a holding potential of -4 mv. This current has been described in detail elsewhere (4) and has been identified as a current through a small conductance, Ca2+-independent Cl- channel. Cell iology: Oike et al. Proc. Nadl. cad. Sci. US 91 (1994) 2941 This current was completely blocked by 1,9-dideoxyforskolin, but this compound did not affect the increase in [Ca2+] (n = 9 cells). Fig. 1 shows a similar Ca2+ transient obtained by omitting mannitol from a normotonic Ca2+-free extracellular solution, keeping all other ionic concentrations constant. subsequent stimulation with a supramaximal concentration of histamine (1 pm), which has been shown previously (9, 1) to induce a Ca2+ transient by depleting intracellular inositol 1,4,5-trisphosphate [Ins(1,4,5)P3d-sensitive Ca2+ pools, did not induce any significant increase in [Ca2e]i ( pm in five cells). It can therefore be concluded that cell swelling induces Ca2+ release and depletion of intracellular agonistsensitive Ca2+ stores. Depletion of these Ins(1,4,5)P3- sensitive stores by pretreatment with thapsigargin (Fig. 1C) in Ca2+-free solution completely abolished the Ca2+ response induced by. similar result was obtained if the stores were depleted by exposure to histamine in Ca2+-free solution before application of. Other mechanical stimuli, such as shear stress induced by directing a stream ofnormotonic solution along the cell surface 15a 1Ca L. 4 C5 =L a C14. C Thapsigargin 2pM Nc 2 \ I = (3) (8)... 2 D Shear E Stretch.5 - FIG. 1. Mechanical activation of a membrane current and Ca2+ release in endothelial cells. () Exposure of endothelial cells to hypotonic Ca2+-free bath solutions activates an inward current (upper trace) and induces a transient increase in [Ca2+]i (lower trace). The holding potential was -4 mv. () ctivation of a Ca2+ signal by hypotonic stress at constant extracellular ion concentrations by omitting mannitol from the superfusion solution () in Ca2+-free solutions. subsequent application ofa supramaximal concentration of histamine did not cause any substantial increase in [Ca2e]j, indicating that the Ins(1,4,5)P3-sensitive Ca2+ stores are depleted. (C), applied after depletion of intracellular Ca2+ stores with 2,uM thapsigargin, does not evoke a Ca2+ transient. (D and E) Shear stress and direct mechanical stretch also induce a transient Ca2+ increase in Ca2+-free solutions. ll these mechanical stimuli were applied to cells voltage clamped at -4 mv. 8) (4) (4) (4) (4) 5 1 [Ca21e [mm] FIG. 2. Changes in [Ca2+]i after a short period of cell swelling are modulated by extracellular [Ca2W] ([Ca2W+. () Swelling of endothelial cells by evoked an increase in [Ca2+]i together with a C1- current. The recovery of [Ca2+]1 in normotonic solution is accelerated in Ca2+-free -solution, but [Ca2+h] increases again after resubmission of extracellular Ca2+. pparently a Ca2+-entry pathway is activated by the exposure to, but it is not accompanied by a significant change in transmembrane current. () Dependence of [Ca2+]i on the extracellular [Ca2+] in cells that had been exposed previously to (solid symbols) or not (open symbols). It is obvious that [Ca2+]j strongly depends on extraceliular [Ca2+J after exposing them to but not under control conditions. This dependence on extracellular Ca2+ may also explain the increases in [Ca2+]i after switching back to the normotonic solution () that has a higher extracellular [Ca2+1 than the hypotonic solution (1.1 M in compared to 1.5 M in the normal physiological solution). Number of cells is indicated in parentheses.

3 2942 Cell iology: Oike et al. or direct mechanical stretch induced by moving a patch pipette in close contact with the cell surface, also induced transient changes in intracellular Ca2+ (Fig. 1 D and E). In the presence of extracellular Ca2+, the -induced increase in [Ca2+]i was sustained, suggesting a contribution of Ca2+ influx to the mechanically evoked Ca2+ signal. fter reperfusion with normotonic solution, [Ca2e]i slowly decayed to its resting level with t I2 = 328 ± 122 s (n = 5 cells). It is well established that store depletion by agonists activates a Ca2+-entry pathway in many nonexcitable cells (11, 12). Fig. 2 provides some evidence for activation of a similar mechanism by store depletion induced by cell swelling. In the presence of extracellular Ca2+, cell swelling induced a sustained increase in [Ca2e]i and activated a Cl- current. subsequent superfusion with normotonic solution was accompanied by a slow decline of [Ca2+]i, which was accelerated after removal of extracellular Ca2+. subsequent readmission of extracellular Ca2+ caused a pronounced increase in [Ca2+]i. In contrast, similar changes in extracellular Ca2t, if applied to the same cell but before induction of cell swelling, did not affect the level of intracellular calcium. The dependence of the level of intracellular calcium on the extracellular Ca2+ concentration is shown in Fig. 2 and strongly suggests that store depletion induced by cell swelling may activate a Ca2+-entry mechanism. We were unable, however, to detect any changes in transmembrane current during these changes in [Ca2+]j. This current is either too small and beyond the resolution of our technique, or these changes might occur through some electroneutral mechanism. ctivation of phospholipase C (PLC) was not involved in the mechanically induced release of intracellular Ca2+. The elevation of [Ca2+]i due to was not affected if the endothelial cells were loaded via the patch pipette with either 1 mm neomycin, a blocker ofplc, or with the low molecular weight heparin that blocks Ins(1,4,5)P3 receptors. In contrast, both substances completely abolished the histamine (1 um)-evoked Ca2+ signals in the same cell. modulatory role of PK is also unlikely, because the [Ca2+1j responses to mechanical stimulation were not affected if endothelial cells were loaded via the patch pipette with a peptide consisting of C-.4-1-T 1 7? (7): i :: ::.::...:....:...:... :. : Proc. Natl. cad. Sci. US 91 (1994) amino acid residues 5-24 of the heat stable PK inhibitor (7) that specifically blocks the catalytic subunit of PK. The quantified effects of these various putative mediators are summarized in Fig. 3, which shows the -induced increase in [Ca2eJi for different experimental conditions; neither of these conditions significantly altered the response to. The variability of the -induced responses between different batches of cells can be judged from the scatter ofthe control values in this figure. It has been reported that, but not its metabolites, is able to release Ca2+ from internal stores (13-15). Fig. 4 shows that (1,uM) induces a transient increase in intracellular Ca2+ in endothelial cells exposed to Ca2+-free solutions. The dependence of these changes in [Ca2 I on the concentration of is shown in Fig. 4. The increase in [Ca2+]i by extracellularly applied still occurred in cells loaded with heparin (Fig. 4C). Histamine, however, was unable to evoke a release of intracellular Ca2+ in heparin-loaded cells (data not shown), indicating that costimulation of Ins(1,4,5)P3 receptors by is unlikely. This similarity between the and the mechanically induced changes in [Ca2J+] may point to a stimulation of phospholipase 2 (PL2) by. Inhibition of PL2 by pretreating the cells with pp (1 lim) or cyclosporin (1) (1,uM) completely abolished the Ca2+ response induced by (Fig. 5 and ). In contrast, the release of intracellular Ca2+ induced by was not significantly affected in pppretreated cells (Fig. 4C), indicating that the inhibitory effects on the responses cannot be entirely explained by nonspecific effects of these compounds. The concentration dependence of the inhibitory effect of pp is shown in Fig. 5C. lso, the Ca2+ transients induced by the other mechanical stimuli-i.e., shear stress and mechanical stretch of the cell-were completely abolished in the presence of 1 um pp. The mechanically induced increase of [Ca2+]i still occurred in the presence of blockers of cyclooxygenase (indomethacin) or lipoxygenase (NDG). It is therefore likely that the increase in [Ca2i]i is caused by rather than by some of its metabolites. The quantified effects of these substances are included in Fig (4 L.. r_---. ly 1) /I.4 v.x;. FiG c _ > E Co o a o Z * C Evaluation of various possible pathways of signal transduction in mechanically stimulated endothelial cells. Effects of inhibitors of the PLC and PK pathways on changes in [Ca2+]i induced by cell swelling. Pooled data from endothelial cells clamped at a potential of -4 mv. Neither ofthese procedures significantly affected the swelling-induced increase in [Ca2+hi. On the right are shown the effects oflipoxygenase and cyclooxygenase inhibitors. These substances also did not significantly alter the Ca2+ responses, suggesting that metabolites of are not M) were loaded into the cell via the patch involved in the sigial transduction. Neomycin (1 mm), heparin (5 NM), and PK inhibitor (PKI; 3 pipette, and measurements were done after 15-2 min. Indomethacin (Indo; cyclooxygenase blocker; 4,uM) and NDG (lipoxygenase blocker; 4 PM) were applied to the bath throughout the experiment. (Inset) How [Ca2]-ii.e., the increase in [Ca2e]i above the resting level-has been determined is shown. Large differences between amplitudes of the control responses reflect the large variability between various batches of cells. Control and test values were always obtained from the same batch of cells. Number of cells is indicated in parentheses.

4 Cell iology: Oike et Proc. Natl. cad. Sci. US 91 (1994) 2943 rachidonic cid L *=i I L;4m.4 - *hk control +Heparin +pp (1) C.3-4 mn control pp cyclosporin _;q cli C.2- i () CD <.1 - I. - FiG [rachidonic cid] in KIM 1 1 Ca2+ transients induced by in Ca2+-free solutions. () Typical Ca2+ response induced by 1 M in Ca2+-free bathing solutions. () Concentration dependence of these Ca2+ transients. Solid line represents hyperbolic fit through data points. Concentration of evoking a half-maximal effect was.7 jum. The Ca2+ transient evoked by 1.M (C) also occurred in cells loaded with heparin via the patch pipette and was not affected by 1 pm PL2 inhibitor pp. n.s., Not significantly different. Number of cells is indicated in parentheses. DISCUSSION It is well established that endothelial cells function as a mechanosensitive signal transduction interface between blood and the vessel wall (2, 3, 17). However, until now it was not clear how these mechanical events were linked to their biological effects. In this report, we have shown that various mechanical stimuli evoke a release of Ca2W from Ins(1,4,5)P3- sensitive stores in endothelial cells. This can be inferred from the observation that histamine, applied after mechanical stimulation in Ca2+ solution, does not evoke a release of Ca2+. The ensuing store depletion may trigger Ca2+ entry via the plasmalemma, but this pathway has until now not been well characterized in endothelial cells. We were able to show an increase in [Ca2J+] that depends on extracellular Ca2+, but we were unable to record a change in transmembrane current during these changes in [Ca2+]i. The absence of any current during Ca2+ influx could be explained by a tiny influx through a Ca2+-selective channel, but an electroneutral mechanism cannot be excluded. This Ca2+ release is accompanied by activation of a Ca2+-independent Cl- conductance that may contribute to stabilization of the driving force for Ca2+ entry and has been described in detail elsewhere (4). The Ca2+ release induced by mechanical stimulation could be mimicked by. This observation and inhibition of these mechanical responses by PL2 blockers indicate that they may be mediated by that is generated by mechanical stimulation of PL2. It is unlikely that the effects of the inhibitors are entirely due to nonspecific actions, because they do not affect the response to...1 'g I... t.i I I....,.., [pp] in pm FiG. 5. Effects of PL2 inhibitors on changes in (Ca2+1i induced by mechanical stimulation. () Ca2+ transient induced by is completely abolished after pretreating the cell with 1 M pp. () PL2 inhibitors pp (1 M) and cyclosporin (1 jtm) significantly reduced the Ca2+ transient induced by. *, P <.1. (C) Concentration dependence of inhibition of the -induced Ca2+ transient by pp. Data were fitted with a hyperbolic equation with a concentration for half-maximal inhibition of 1.1 M. Number of cells is indicated in parentheses. Increased synthesis of has been observed in endothelial cells from human umbilical vein during stimulation by laminar shear stress (18) and is consistent with our observations. Our data about a direct mobilization of intracellular Ca2+ by are in agreement with a mechanical activation of an [Ca2+]j-independent PL2 by. However, we cannot exclude the possibility that a mechanically activated release of intracellular Ca2+ activates a Ca2+-dependent PL2 and that the ensuing increase in plays a permissive role for further Ca2+ release rather than being the trigger for release. Ca2+ release from intracellular Ca2+ stores by has been demonstrated in human neutrophils, T and lymphocytes, and human platelets (13-15). ctivation of a plasmalemmal Ca2+ entry by store depletion induced by has been described in platelets (19). Our results show that but not its metabolites, as described previously (2), modulate the mechanoeffects on endothelial cells. Our experiments also show that neither the PLC pathway nor a sensitization of Ins(1,4,5)P3 receptors by is involved in the signal transduction (18, 21, 22). It can be concluded that the mechanically induced changes of intracellular calcium in endothelial cells may be modulated via. This mechanism, in addition to the well-known agonist-dependent activation via Ins(1,4,5)P3 receptors, could explain the synergistic effects of shear stress on agonist-induced changes in intracellular Ca2+. We thank G. Schwarz for his initial help in some of the shear stress

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