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. 2000 May 1;524(Pt 3):715–724. doi: 10.1111/j.1469-7793.2000.00715.x

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

Maud Frieden 1, Wolfgang F Graier 1
PMCID: PMC2269913  PMID: 10790153

Abstract

  1. 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.

  2. Histamine (10 μM) 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.

  3. 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.

  4. Following the ryanodine-mediated RsCR, the potency of 10 μM histamine to activate KCa channels was reduced by about 60 %. In agreement, an inhibition of RsCR with 25 μM ryanodine diminished KCacurrent in response to histamine by about 70 %.

  5. The effect of 100 μM histamine on KCa channel activity was not reduced by previous RsCR with 200 nM ryanodine, or by an inhibition of RsCR by 25 μM ryanodine.

  6. Histamine (10 μM)-induced Ca2+ elevation was reduced by 30 % following ryanodine-mediated RsCR, whereas no inhibition occurred in the case of 100 μM histamine stimulation.

  7. In cells treated with 10 μM 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 μM histamine-induced KCa current was not obtained in nocodazole-treated cells.

  8. Our data suggest that during moderate cell stimulation (10 μM 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.

Agonist-induced endothelial cell stimulation leads to an increase in intracellular Ca2+ concentration that constitutes a crucial step in the formation of vasoactive compounds. Receptor stimulation results in the activation of phospholipase C (PLC) and the production of inositol 1,4,5-trisphosphate (IP3), which, in turn, releases Ca2+ from the IP3-sensitive Ca2+ stores (Himmel et al. 1993; Graier et al. 1994). The Ca2+ release is accompanied by a Ca2+ influx from extracellular space (Schilling et al. 1992) that is associated with membrane hyperpolarization due to the stimulation of Ca2+-dependent K+ (KCa) channels (Nilius et al. 1997). It is well known that this membrane hyperpolarization plays an important role in Ca2+ signalling and in the production of vasoactive factors, by increasing the driving force for Ca2+ entry (Lückhoff & Busse, 1990).

In addition to that of the IP3 receptor, immunofluorescence staining revealed the presence of ryanodine receptors in endothelial cells (Lesh et al. 1993), but their physiological relevance still remains controversial. However, it was recently shown that during submaximal endothelial cell stimulation localized, subplasmalemmal Ca2+ signalling occurred (Graier et al. 1998). Ca2+ release through ryanodine receptors was detected in this subplasmalemmal region, whereas no increase in the cytosolic Ca2+ concentration was observed. Furthermore it appeared that activation of ryanodine receptors is involved in Ca2+ entry following agonist stimulation, while ryanodine by itself was unable to trigger any Ca2+ influx (Paltauf-Doburzynska et al. 1998). It was therefore proposed that ryanodine-sensitive Ca2+ release (RsCR) may play a role in KCa channel stimulation, thus altering the driving force for Ca2+ entry (Paltauf-Doburzynska et al. 1998). In agreement caffeine, a non-specific activator of Ca2+-induced Ca2+ release (CiCR) stimulated KCa channels and membrane hyperpolarization (Chen & Cheung, 1992; Rusko et al. 1995). However, considering the Ca2+ transient stimulated by caffeine compared with the absence or the slow increase in intracellular Ca2+ concentration due to ryanodine (Corda et al. 1995; Rusko et al. 1995), both agents are likely to have different mechanisms of action.

In this study we used KCa channel activity to monitor subplasmalemmal Ca2+ concentration. The effect of a direct stimulation of RsCR on subplasmalemmal Ca2+ concentration was elucidated. Furthermore, the involvement of RsCR on histamine-mediated activation of KCa channels was assessed, to verify whether KCa channels contribute to the subplasmalemmal Ca2+ control unit (SCCU; Graier et al. 1998). We used the whole-cell configuration of the patch clamp technique in a cell line derived from human umbilical vein (EA.hy926).

METHODS

Materials

Petri dishes were purchased from Corning, Vienna, Austria. The acetoxymethyl ester form of fura-2 (fura-2 AM) was purchased from Molecular Probes Europe BV, Leiden, The Netherlands, and fetal calf serum was obtained from PAA Laboratories, Linz, Austria. Cell culture media and chemicals were obtained from Life Technologies, Vienna, Austria. Ryanodine was purchased from Calbiochem, Vienna, Austria. Iberiotoxin was purchased from RBI, USA. All other materials were from Sigma Chemicals, Vienna, Austria.

Cell culture

Experiments were performed using the endothelial cell line EA.hy926, which was originally derived from a human umbilical vein. This cell line was a gift from Dr Cora-Jean S. Edgell, Pathology Department, University of North Carolina, Chapel Hill, NC, USA. Cells from the EA.hy926 cell line (passage 63 and higher) were grown in Dulbecco's minimum essential medium (DMEM) containing 10 % fetal calf serum, 4.5 mg l−1 D-glucose and 1 % HAT (5 mM hypoxanthine, 20 μM aminopterine, 0.8 mM thymidine). Prior to the experiments, cultured cells were washed twice with Ca2+-free DMEM, incubated with 0.05 % trypsin and 0.02 % EDTA for 1–2 min, centrifuged (5 min at 400 g) and resuspended in storage buffer containing (mM): 130 NaCl, 5.6 KCl, 2 CaCl2, 1 MgCl2, 8 Hepes, 10 glucose (pH 7.45 with NaOH).

Patch clamp recording

We used the whole-cell configuration of the patch clamp (Hamill et al. 1981), and performed experiments in cultured single EA.hy926. Borosilicate glass pipettes were pulled with a Narishige puller (Narishige Co. Ltd, Tokyo, Japan), fire-polished and had a resistance of 3–6 MΩ. Patch clamp recordings were made using a PC-501A amplifier (Warner Instrument Corporation, Dixwell Avenue, Hamden, CT, USA); currents were low-pass filtered at 0.1 kHz, digitized by a PP-50LAB converter (Warner Instrument Corp.) and sampled on a PC running AxoBASIC 2.0 (Axon Instruments, Foster City, CA, USA). Voltage ramps (1.8 s; -80 to +65 mV) were applied repeatedly in order to determine current-voltage relationships. Membrane capacity was measured for each cell tested, by applying a 10 mV voltage step and whole-cell current was expressed in current density (pA pF−1) in order to normalize the results to cell surface. The cell membrane capacitance (Cm) was calculated according to the following equations (de Roos et al. 1996):

graphic file with name tjp0524-0715-mu1.jpg

where Ra is the access resistance, Vp the applied voltage step (10 mV), Io the peak current, Iss the steady-state current, Gm the membrane conductance and τ is the decay constant of the transient.

Experiments were performed at room temperature (20-24°C). A drop of the cell suspension was transferred to the glass-bottomed experimental chamber. After the cells were allowed to reattach for approximately 2 min, the constant superfusion of 2 ml min−1 was switched on. The pipette solution contained (mM): 130 KCl, 1 MgCl2, 5 MgATP, 0.2 Na3GTP, 10 Hepes (pH 7.2 with NaOH). The standard bath solution contained (mM): 130 NaCl, 5.6 KCl, 2 CaCl2, 1 MgCl2, 8 Hepes (pH 7.45 with NaOH). To modify the K+ equilibrium potential, the following bathing solutions were used (mM): 95.6 NaCl, 40 KCl, 2 CaCl2, 1 MgCl2, 8 Hepes (pH 7.45 with NaOH) or 130 KCl, 2 CaCl2, 1 MgCl2, 8 Hepes (pH 7.45 with KOH). TEA and iberiotoxin were applied in the bath solution for at least 10 min preincubation time. Prestimulation with 200 nM ryanodine lasted 4 min. Cells were stimulated with histamine for between 1 and 2 min. Where indicated in experiments, 25 μM ryanodine was added to the pipette solution.

Intracellular Ca2+ measurement

Free bulk Ca2+ concentration ([Ca2+]bulk) was measured using the conventional fura-2 technique for single endothelial cells as described previously (Graier et al. 1995, 1998). Briefly, cells were loaded with 2 μM fura-2 AM for 45 min at room temperature in the dark, centrifuged, washed twice and resuspended in storage buffer (SB: DMEM containing 2 % horse serum, 0.1 % of a vitamin mixture and 0.2 % essential amino acids). After an equilibration period of 10 min, cells were transferred into a glass-bottomed experimental chamber and the cells were allowed to reattach for approximately 2 min before a constant superfusion of 2 ml min−1 was switched on. Cells were perfused in Hepes buffer containing (mM): 130 NaCl, 5.6 KCl, 2 CaCl2, 1 MgCl2, 8 Hepes (pH 7.45 with NaOH). The [Ca2+]bulk concentration was monitored by sampling fluorescence intensity (F) at 510 nm emission alternately at 360 nm (i.e. Ca2+-insensitive wavelength) and 380 nm (i.e. Ca2+-sensitive wavelength) excitation. Fluorescence intensity for each pair of excitation/emission wavelengths was converted to analog by an optical processor and registered by a PC running AxoBASIC 1.0 (Axon Instruments) as previously described (Graier et al. 1995). In view of the reported errors of the [Ca2+]bulk calibration in our system and the general uncertainties of the calibration techniques (Graier et al. 1995, 1998), [Ca2+]bulk is expressed in ratio units (F360/F380). In order to measure Ca2+ concentration using the same procedure as current measurements, we performed simultaneous measurements of ion currents and [Ca2+]bulk as described in more detail in the accompanying manuscript (Paltauf-Doburzynska et al. 2000). The instrumentation combines the microfluorometer and the patch clamp set-up described above. The digitalized signals for ion currents and fluorescence intensity at 510 nm emission at the corresponding excitation at 360 and 380 nm were registered by a PC running AxoBASIC 1.0 (Axon Instruments).

Statistics

Analysis of variance (ANOVA) was performed and statistical significance was evaluated using Scheffé‘s F test. The level of significance was defined as P < 0.05.

RESULTS

K+ current activated by histamine

As shown in Fig. 1, stimulation of endothelial cells with 10 μM histamine resulted in a rapid development of an outward current that gradually declined with time. The current presented a strong outward rectification with a reversal potential of -41.1 ± 5.1 mV (n = 14). The maximal amplitude of the current reached 18.9 ± 5.5 pA pF−1 at +20 mV (n = 15). Due to the small current between -80 and -20 mV, the determination of the reversal potential was problematic and resulted in a large deviation of the values. However, changing the extracellular K+ concentration from 5.6 mM to 40 and 130 mM shifted the reversal potential of the current to -15.5 ± 1.4 mV (n = 8) and +4.0 ± 0.8 mV (n = 7), respectively, strongly suggesting that the main current stimulated by histamine was carried by K+ channels. In agreement with these findings, the application of tetraethylammonium (TEA), a non-specific inhibitor of Ca2+-dependent K+ channels, at 1 mM reduced the current by about 90 % from 18.7 ± 3.2 pA pF−1 (n = 6) to 1.6 ± 0.6 pA pF−1 (n = 6) at +20 mV (P < 0.05). Iberiotoxin (50 nM) a well-known blocker of large-conductance KCa channels (BKCa channels; Galvez et al. 1990) reduced the current by about 80 % from 8.5 ± 1.0 pA pF−1 (n = 6) to 1.4 ± 0.5 pA pF−1 (n = 5) at +20 mV (P < 0.05). Taken together, these results showed that the current triggered by histamine is mainly due to BKCa channel stimulation.

Figure 1. Whole-cell current induced by 10 μM histamine in EA.hy926 cell line.

Figure 1

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A, original recording of the outward current induced by 10 μM histamine. The holding potential was -30 mV, and repetitive voltage ramps (-80 to +65 mV) were applied during the whole recording. Histamine was applied at the time indicated by the horizontal bar. B, the corresponding current-potential curves are represented, with a being obtained before the stimulation, b at the maximum of the stimulation (indicated in A), and b – a corresponding to the stimulated current. The inset highlights the reversal potential of the histamine-stimulated current.

The stimulation of endothelial cells with 100 μM histamine resulted in a large outward current reaching 25.8 ± 6.4 pA pF−1 at +20 mV (n = 5). The reversal potential of the current as well as the sensitivity to 1 mM TEA were not significantly different from what was obtained with a 10 μM histamine stimulation, suggesting that both agonist concentrations stimulated the same type of current (data not shown).

Effect of direct stimulation of ryanodine-sensitive Ca2+ release on K+ current

The application of 200 nM ryanodine, a concentration known to trigger Ca2+ release from ryanodine-sensitive Ca2+ stores in the EA.hy926 (Paltauf-Doburzynska et al. 1998), stimulated an outward current of small amplitude (4.3 ± 0.7 pA pF−1 at +20 mV; n = 20; Fig. 2). This current was observed in about 50 % of the cells. The reversal potential of the current was not significantly different from the one stimulated by histamine (-34.5 ± 4.54 mV; n = 20; n.s. vs. histamine), suggesting that it was due to stimulation of the same channels. In addition, in all cells tested, 50 nM iberiotoxin applied during the response to ryanodine abolished the ryanodine-stimulated K+ current (n = 3). An original recording of the reversible inhibition produced by iberiotoxin is presented in Fig. 3.

Figure 2. Whole-cell current induced by 200 nM ryanodine.

Figure 2

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A, original recording of the outward current induced by 200 nM ryanodine. The holding potential was -10 mV, and repetitive voltage ramps (-80 to +65 mV) were applied during the whole recording. Ryanodine was applied at the time indicated by the horizontal bar. B, the corresponding current-potential curves are represented, with a being obtained before the stimulation, b at the maximum of the stimulation, and b – a corresponding to the stimulated current. Inset: mean values of the current obtained at 0, 20 and 40 mV. Columns represent the means of 21 experiments. Data are means ±s.e.m.

Figure 3. Effect of 50 nM iberiotoxin on the K+ current stimulated by 200 nM ryanodine.

Figure 3

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A, original recording of the outward current induced by 200 nM ryanodine. The holding potential was -20 mV, and repetitive voltage ramps (-80 to +65 mV) were applied during the recording. At the time indicated, 50 nM iberiotoxin was applied for 3 min. B, the corresponding current-potential curves are represented, with a being obtained before the stimulation, b at the maximal of the ryanodine stimulation, c after inhibition by iberiotoxin and d after washout of the inhibitor.

Effect of an inhibition of ryanodine-sensitive Ca2+ release on histamine-induced K+ current

In order to determine the role of RsCR on the activity of KCa channels in response to an agonist, we blocked intracellular ryanodine receptors by applying 25 μM ryanodine inside the patch pipette. After rupturing the patch membrane, ryanodine was allowed to diffuse into the cell for at least 6 min. In these conditions, KCa current stimulated by 10 μM histamine was reduced by about 70 % from 16.7 ± 4.1 pA pF−1 (n = 11) to 4.6 ± 1.2 pA pF−1 (n = 7) at +20 mV (P < 0.05; Fig. 4A). Interestingly, such an inhibition of KCa current was not observed when cells were stimulated with 100 μM histamine (Fig. 4B).

Figure 4. Effect of 25 μM (A and B) or 200 nM (C and D) ryanodine pretreatment on KCa current stimulated with 10 μM (A and C) and 100 μM (B and D) histamine.

Figure 4

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Activated currents recorded at the maximal cell stimulation are represented at 0, 20 and 40 mV. A, 10 μM histamine-induced KCa current is reduced in the presence of 25 μM ryanodine applied inside the patch pipette (▪), compared with control (□). Columns are the means of 7 experiments in the presence of ryanodine and 11 experiments in control conditions. B, in the case of 100 μM histamine stimulation, the current is not affected by ryanodine (▪) compared with control (□). Columns are the means of 8 experiments in the presence of ryanodine and 8 experiments in control conditions. C, 10 μM histamine-induced KCa current is reduced after pretreatment of the cell for 4 min with 200 nM ryanodine (▪), compared with control (□). Columns are the means of 7 experiments in the presence of ryanodine and 12 experiments in control conditions. D, in the case of 100 μM histamine stimulation the current is not affected by ryanodine pretreatment (▪) compared with control (□). Columns are the means of 6 experiments in the presence of ryanodine and 7 experiments in control conditions. Data are means ±s.e.m.*P < 0.05vs. control.

Effect of previous ryanodine-sensitive Ca2+ release on histamine-induced K+ current

Endothelial cells were stimulated with 10 μM histamine after previous stimulation by 200 nM ryanodine. We considered in the further experiments only the cells that previously presented a current stimulated by ryanodine. As shown in Fig. 4C, the histamine-induced K+ current after pretreatment with ryanodine was reduced by about 60 %, from 8.9 ± 1.8 pA pF−1 (n = 12) to 2.9 ± 0.7 pA pF−1 (n = 7) at +20 mV (P < 0.05). In agreement with our data using 25 μM ryanodine, the current stimulated by 100 μM histamine was not affected by a prestimulation with 200 nM ryanodine (Fig. 4D).

Effect of previous ryanodine-sensitive Ca2+ release on histamine-induced Ca2+ elevation

In order to determine whether a prestimulation of the cells with 200 nM ryanodine altered the subsequent histamine-induced Ca2+ increase, the effect of histamine on [Ca2+]bulk with or without prestimulation with 200 nM ryanodine was studied. As previously reported (Paltauf-Doburzynska et al. 1998), 200 nM ryanodine failed to stimulate a bulk Ca2+ increase under normal conditions. However, after ryanodine prestimulation, the response to 10 μM histamine was reduced by 30.3 ± 7.0 % (n = 7; Fig. 5A) compared with control. No reduction of the bulk Ca2+ increase was observed when cells were stimulated with 100 μM histamine (Fig. 5B). Remarkably, the maximal Ca2+ elevation in response to 10 or 100 μM histamine did not statistically differ under control conditions (i.e. without prestimulation with 200 nM ryanodine). However, after ryanodine pretreatment the response to 10 μM histamine was significantly reduced compared with that to 100 μM histamine (Fig. 5C).

Figure 5. Effect of 200 nM ryanodine pretreatment on the Ca2+ elevation stimulated by 10 and 100 μM histamine.

Figure 5

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A and B, original recordings showing the effect of 4 min preincubation time with 200 nM ryanodine on the response to histamine. At the time indicated, the cells were stimulated by histamine following control (^, continuous line) or prestimulation by 200 nM ryanodine (•, dashed line). Each point represents the mean ±s.e.m. (n = 5–11). *P < 0.05vs. control. C, comparison of the effect produced by 10 and 100 μM histamine on the Ca2+ elevation in the absence of (left panel) and following stimulation by (right panel) 200 nM ryanodine. Each column represents the mean ±s.e.m. (n = 5–11). *P < 0.05vs. 100 μM.

Involvement of superficial Ca2+ stores in histamine-induced K+ current

We next investigated whether the integrity of the endoplasmic reticulum (ER) network, and especially the superficial ER (sER), was important for the RsCR-induced KCa channel stimulation. In order to disturb the ER, we used nocodazole. As previously shown (Graier et al. 1998; Paltauf-Doburzynska et al. 1999) preincubation of the endothelial cells with 10 μM nocodazole for 16–20 h results in the collapse of the sER towards the cell nucleus. This has been also shown in EA.hy926 cells (Paltauf-Doburzynsky et al. 2000). In these conditions, prestimulation of endothelial cells with 200 nM ryanodine no longer had an inhibitory effect on the 10 μM histamine-induced KCa current activation (5.8 ± 2.7 pA pF−1 at +20 mV; n = 4; with ryanodine, compared with 6.1 ± 2.2 pA pF−1 at +20 mV; n = 5; without ryanodine; Fig. 6A). In the presence of nocodazole, none of the cells tested developed a current following ryanodine stimulation (5 out of 5 cells).

Figure 6. Effect of pretreatment of the cells with 10 μM nocodazole (A) or the solvent alone (B) on KCa current stimulated by 10 μM histamine.

Figure 6

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Activated currents recorded at the maximal cell stimulation are represented at 0, 20 and 40 mV. A, after the cells were preincubated for 16–20 h in 10 μM nocodazole, the current stimulated by 10 μM histamine was not affected any more by the pretreatment with 200 nM ryanodine for 4 min (▪) compared with control (□). Columns are the means of 5 experiments in the presence or absence of ryanodine. B, cells were preincubated with 0.2 % DMSO (solvent control) for 16–20 h. In these conditions the current stimulated by ryanodine is reduced by pretreatment of the cell with 200 nM ryanodine (▪) compared with control (□). Columns are the means of 5 experiments in the presence of ryanodine and 6 experiments in control conditions. Data are means ±s.e.m.*P < 0.05vs. control.

When cells were preincubated with the solvent alone (0.2 % DMSO), the inhibitory effect of ryanodine on histamine-induced KCa current stimulation occurred, with the current reaching 2.5 ± 0.4 pA pF−1 at +20 mV (n = 5) in the presence of ryanodine, and 10.3 ± 2.4 pA pF−1 at +20 mV (n = 6) in the absence of ryanodine (P < 0.05; Fig. 6B).

DISCUSSION

In this study we have shown that subplasmalemmal ryanodine-sensitive Ca2+ stores contribute to the stimulation of KCa channels in the EA.hy926 cell line. Preventing RsCR either by a previous emptying of subplasmalemmal ryanodine-sensitive Ca2+ stores or by inhibiting CiCR resulted in a reduction of 10 μM histamine-induced KCa channel stimulation. However, this effect was not observed when cells were stimulated with 100 μM histamine. Moreover, collapsing the superficial ER towards the nucleus by treatment with nocodazole prevented ryanodine-induced current. Hence, the inhibitory property of previous RsCR on 10 μM histamine-induced KCa current was not obtained in nocodazole-treated cells. These data indicate that during moderate agonist stimulation, the SCCU contributes to KCa channel activation via RsCR.

In the cell line derived from human umbilical vein (EA.hy926), histamine triggered a transient outward current presenting a strong outward rectification. Changing the extracellular K+ concentration modified the reversal potential of the current that followed the K+ equilibrium potential. Furthermore, 1 mM TEA as well as 50 nM iberiotoxin inhibited the current by about 90 % and 80 %, respectively. Taken together these data strongly suggest that BKCa channels are mainly responsible for the current stimulated by histamine in EA.hy926 cells. The presence of BKCa channels has been shown in many types of endothelial cells (Rusko et al. 1992; Baron et al. 1996; Jow & Numann, 2000; for review see Nilius et al. 1997) as well as in the EA.hy926 cell line used in the present study (Haburcák et al. 1997). Moreover, activation of BKCa channels has been shown to be involved in agonist-induced membrane hyperpolarization (Graier et al. 1993; Wang et al. 1996; Frieden et al. 1999).

In addition to IP3 receptors, Ca2+ release also occurs through ryanodine receptors, but in most cases this release initiates a slow or no increase in bulk Ca2+ concentration instead of the classical Ca2+ transient due to stimulation of IP3 receptors (Ziegelstein et al. 1994; Wang et al. 1995). Recently, it was demonstrated that RsCR preferentially occurred in the subplasmalemmal region, thus most likely explaining the inability to observe a bulk Ca2+ increase (as measured with fura-2) due to ryanodine (Graier et al. 1998; Paltauf-Doburzynska et al. 1998). In the present study the activity of KCa channels was measured in order to monitor increases in the subplasmalemmal Ca2+ concentration, in response to the direct stimulation of RsCR with 200 nM ryanodine, a concentration known to stimulate Ca2+ release by locking the channel in a subconductance open state (Ehrlich et al. 1994; Paltauf-Doburzynska et al. 1998). Our results that ryanodine stimulated an outward current of small amplitude is in line with our previous findings (Paltauf-Doburzynska et al. 1998) and supports the hypothesis that RsCR yields a localized elevation in the subplasmalemmal Ca2+ concentration. Since the ryanodine-evoked current presented a similar outward rectification and reversal potential compared with that stimulated by histamine, and is also sensitive to iberiotoxin, our data suggest that both agonists activated the same channels.

There are reports which show that caffeine, a non-specific activator of CiCR, stimulated a K+ current/membrane hyperpolarization in endothelial cells (Chen & Cheung, 1992; Rusko et al. 1995). Our data presented here are, according to our recent knowledge, the first report showing stimulation of KCa channels by ryanodine in endothelial cells. It should be noted that the effect of ryanodine was observed in only half of the cells tested. Since the RsCR was shown to occur in a restricted subplasmalemmal region (Paltauf-Doburzynska et al. 1998), in some instances the formation of the seal and the subsequent rupture of the patch membrane might alter the organization/integrity of the sER and thus, the vicinity of ryanodine receptors to KCa channels. This hypothesis is further supported by our data obtained in cells with collapsed sER (nocodazole-treated cells), where ryanodine failed to stimulate KCa current, and points to the importance of the close apposition between the sER and the cell membrane. Alternatively, RsCR might be too small or too slow, so that the subplasmalemmal Ca2+ concentration always reached the threshold for BKCa channel stimulation. Indeed, the Ca2+ sensitivity of these channels is relatively weak at resting membrane potential (Rusko et al. 1992; Baron et al. 1996; Haburcák et al. 1997), and RsCR was found to be much slower than the effect of IP3 in intact endothelial cells (Paltauf-Doburzynska et al. 1998).

In order to assess the role of RsCR in KCa channel stimulation, we have only considered cells that previously responded to ryanodine. In these conditions, the current stimulated by 10 μM histamine was reduced by about 70 %. Considering that RsCR takes place in the subplasmalemmal area (Paltauf-Doburzynska et al. 1998), these data suggest that prestimulation of the cell with ryanodine depleted certain subplasmalemmal Ca2+ stores that are responsible for KCa channel activation in response to moderate histamine stimulation. In other words, after ryanodine-induced depletion of the sER Ca2+ pools, subplasmalemmal Ca2+ elevation following histamine might be reduced, resulting in an attenuated activation of KCa channels. In line with this hypothesis, a prestimulation of the cell with 200 nM ryanodine led to a reduced 10 μM histamine-induced Ca2+ increase. This reduction (about 30 %) was smaller than the one obtained on KCa channel activation; this might reflect the fact that we have measured the bulk Ca2+ concentration, and that KCa channel activity reflects the subplasmalemmal Ca2+ concentration. In addition, prevention of CiCR by using 25 μM ryanodine inside the patch pipette also led to a reduced histamine-induced KCa channel activity. Interestingly, when the cells were stimulated with 100 μM histamine, no inhibition of the current by either previous depletion of the ryanodine-sensitive Ca2+ stores or by preventing CiCR was observed. Thus, during stimulation with 100 μM histamine, the high IP3-mediated Ca2+ release and/or Ca2+ entry is sufficient to promote fully the stimulation of KCa channels, even if RsCR does not occur. In line with this suggestion, the prestimulation of the cell with 200 nM ryanodine did not reduce Ca2+ elevation evoked by 100 μM histamine. This difference between the effects triggered by 10 or 100 μM histamine confirmed previous data obtained on SCCU, where it was shown that the localized Ca2+ control is functional in the case of small or moderate cell stimulation, while at supraphysiological stimulation, the tight regulation of this region is overcome (Graier et al. 1998; Paltauf-Doburzynska et al. 1998).

Thus, we suggest that during moderate cell stimulation, RsCR plays a major role in KCa channel stimulation. This is in line with previous observations regarding the role of RsCR in Ca2+ entry and endothelial NO synthase (eNOS) activity. It was reported that prevention of CiCR largely inhibited Ca2+ entry whereas ryanodine by itself did not trigger Ca2+ influx. Furthermore eNOS activity is reduced after ryanodine pretreatment, an effect more pronounced in the case of moderate cell stimulation (Paltauf-Doburzynska et al. 1998). It has been shown that an agonist-evoked stimulation of eNOS strictly depends on Ca2+ entry (Lückhoff et al. 1988). Hence, this Ca2+ entry is controlled by membrane potential (Lückhoff & Busse, 1990). Thus, our results presented here further support our previous hypothesis, that RsCR participates in membrane hyperpolarization and thus provides the driving force for Ca2+ entry and consequently for eNOS activation.

All these data supported the hypothesis that KCa channels contribute to SCCU function. To further confirm this idea, we used nocodazole-treated cells in order to disrupt the sER organization. It was shown that alteration of the sER integrity abolished the SCCU function (Graier et al. 1998; Paltauf-Doburzynska et al. 2000). As mentioned above, in these conditions we never observed KCa current stimulated by ryanodine, which is in agreement with our previous assumption that ryanodine receptors are located in the vicinity of the cell membrane. Furthermore, in cells with collapsed sER, 10 μM histamine-induced KCa current was not reduced after ryanodine pretreatment. Thus, lacking the functionality of the SCCU, the KCa channels are stimulated by cytosolic IP3-sensitive Ca2+ release while CiCR from ryanodine receptors, yet physically removed from the vicinity of the KCa channels, does not contribute any more to the channel stimulation.

In summary we have shown that in the endothelial cell line derived from the human umbilical vein (EA.hy926), histamine induced a K+ current due to stimulation of BKCa channels. This current is activated by subplasmalemmal RsCR under physiological stimulation. In addition, low concentrations of ryanodine triggered a similar current. These findings support previous results showing that in endothelial cells, subplasmalemmal Ca2+ elevation is mainly due to RsCR. In agreement, our data indicate that supraphysiological cell stimulation or disruption of the sER blunted the role played by RsCR in KCa channel stimulation. Hence, the SCCU function can be extended to that of a regulator of KCa channels and thus, to membrane hyperpolarization.

Acknowledgments

We thank Mrs Beatrix Petschar for her excellent technical assistance. This work was supported by the Austrian Funds (P-12341-Med and SFB 714), the Austrian Nationalbank (P7542 and P7902), the Kamillo-Eisner Stiftung (Hergiswil, Switzerland), the Franz Lanyar Foundation and the Swiss National Funds (M.F.).

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