INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

VASORELAXATIONS TO AGONISTS such as ACh and bradykinin (BK) are endothelium dependent (11) and are mediated, in part, by endothelium-derived nitric oxide (NO) (21). However, it has become clear that NO does not account for all endothelium-dependent vasorelaxations. The existence of an endothelium-derived hyperpolarizing factor (EDHF), which causes hyperpolarization through activation of K⁺ channels in vascular smooth muscle, has been proposed (6, 26). Although many studies now confirm that EDHF is entirely distinct from NO and cyclooxygenase products (5), there is no consensus view on the identity of this factor. In some vascular preparations, a cytochrome P-450 monooxygenase-derived metabolite of arachidonic acid such as an epoxyeicosatrienoic acid (2) or anandamide (24), a cannabinoid derivative of arachidonic acid, has been proposed to represent EDHF. However, these candidate mediators as EDHF do not seem to extend to every vascular bed (9, 27, 30).

Whatever the chemical nature of EDHF, the EDHF responses are most likely to be associated with an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in endothelial cells (3). This can be supported by the finding that the Ca²⁺ ionophore A-23187 induces endothelium-dependent membrane hyperpolarization of vascular smooth muscle (3, 16). The elevation of [Ca²⁺]_i in endothelial cells induced by agonists such as ACh is the result of Ca²⁺ release from intracellular stores and transmembrane Ca²⁺ influx (18). It has been demonstrated that, when vascular endothelium is stimulated with agonists, Ca²⁺ from intracellular stores and Ca²⁺ from extracellular medium can generate EDHF-mediated membrane hyperpolarization of smooth muscle in rabbit carotid artery (3) and rat mesenteric artery (8, 10). Interestingly, NO formation correlates most closely with transmembrane Ca²⁺ influx rather than Ca²⁺ release from intracellular stores, while prostacyclin formation is almost entirely dependent on Ca²⁺ release from intracellular pools (14). However, the relative contribution of Ca²⁺ release from intracellular pools and transmembrane Ca²⁺ influx in endothelial cells to the EDHF-mediated responses of vascular smooth muscle remains to be fully clarified.

We previously showed that the sustained component of the endothelium-dependent hyperpolarizing response of rat mesenteric artery to ACh requires Ca²⁺ influx via a pathway distinct from the L-type Ca²⁺ channel (10). The possible pathway for Ca²⁺ entry into endothelial cells is thought to be nonselective cation channels (NSCC) (19). Ni²⁺ is known as an NSCC blocker in a variety of cell types including endothelial cells (7, 13). Thus Ni²⁺ may be a useful pharmacological tool to distinguish between roles of endothelial Ca²⁺ sources, Ca²⁺ release from intracellular pools, and transmembrane Ca²⁺ influx, in the EDHF-mediated responses. We therefore investigated the effects of Ni²⁺ on EDHF-mediated relaxations and hyperpolarizations in response to BK and cyclopiazonic acid (CPA), a selective inhibitor of the Ca²⁺-ATPase on the endoplasmic reticulum, in porcine coronary artery to determine the endothelial Ca²⁺ source that plays a predominant role in the EDHF-mediated responses.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mechanical experiments. Porcine hearts were obtained from a slaughterhouse and transported in ice-cold oxygenated physiological salt solution (PSS). The composition of PSS (pH 7.4) was (in mM) 118.2 NaCl, 4.7 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 1.2 KH₂PO₄, 25.0 NaHCO₃, and 10.0 glucose. The left circumflex coronary artery (~2 mm OD) was dissected from the heart in oxygenated PSS. The isolated artery was trimmed of fat and connective tissues under a dissecting microscope and cut into rings 4 mm long. Care was taken to ensure that the endothelium was not damaged during the processing of the tissue preparation. Where indicated, the endothelial cells were removed by gentle rubbing of the intimal surface of the vessel with a moistened cotton ball. The arterial ring was suspended by a pair of stainless steel hooks in a water-jacketed bath filled with 25 ml of PSS. The solution in the bath was gassed with 95% O₂-5% CO₂, and its temperature was maintained at 37°C. The ring was stretched until an optimal tension of 2 g was loaded and then allowed to equilibrate for 60 min before the start of the recordings. Isometric tension was monitored with a transducer and recorded by a pen recorder. The rings were repeatedly challenged with 40 mM K⁺ until the high-K⁺-induced contractions reached a constant value. High-K⁺ PSS was prepared by substitution of KCl for NaCl on an equimolar basis.

2

Electrophysiological experiments. A short piece of coronary artery was prepared by cutting along the longitudinal axis of the rings. The preparation was pinned, intimal side upward, to the bottom of an organ chamber (capacity 3 ml) and superfused at a constant flow rate of 7 ml/min with oxygenated PSS. The temperature of the perfusate was kept at 37°C. After the preparation had been equilibrated for 60 min, glass microelectrodes filled with 3 M KCl (tip resistance 40-80 M) were inserted into the smooth muscle cells from the intimal side. Electrical signals were monitored continuously on an oscilloscope (model VC-10, Nihon Kohden, Tokyo, Japan) and recorded on a chart recorder (model WR3101, Watanabe Sokki, Tokyo, Japan). After membrane potentials were stable for 2 min, the hyperpolarizing responses to BK and CPA were determined by continuous recording of the membrane potential of a single cell. Further details of the experimental procedure have been described elsewhere (8-10).

Measurement of [Ca²⁺]_i in endothelial cells. Porcine aortic endothelial cells were isolated by gentle scraping of the intima of the descending part of porcine aortas. After centrifugation at 250 g for 10 min in medium 199 (M199) solution (Boehringer, Mannheim, Germany), the pellet of endothelial cells was purified from this suspension, resuspended in M199 solution with Earle's salts supplemented with 100 IU/ml penicillin G, 100 µg/ml streptomycin, and 20% newborn calf serum (GIBCO-BRL, New York, NY), and aliquoted into polybiphenyl dishes, fixed on 10 × 10-mm glass coverslips, and incubated at 37°C in 5% CO₂ for 2 days. The medium was renewed every day. Cultured cells were virtually free of contaminating cells as indicated by staining with diiodoacetyl-low-density lipoprotein (LDL); ~99% of the cells took up diiodoacetyl-LDL. The percentage of cells taking up diiodoacetyl-LDL was determined when the nuclei became visible under bisbenzimide staining.

Statistical analysis. Values are means ± SE. The data obtained in cultured endothelial cells are expressed as means ± SD. Statistical assessment of the data was made by Student's t-test or two-way ANOVA, followed by Bonferroni's multiple comparison test when appropriate. P < 0.05 was taken as significant.

Drugs. U-46619 was obtained from Cayman Chemical (Ann Arbor, MI); BK, CPA, L-NNA, indomethacin, apamin, charybdotoxin, and sodium nitroprusside (SNP) were from Sigma Chemical (St. Louis, MO); and pinacidil was from Shionogi (Osaka, Japan). CPA was prepared in DMSO and diluted in ethanol. U-46619 was prepared in ethanol, L-NNA and pinacidil in 0.2 N HCl, and indomethacin in 50 mM Tris. Other compounds were dissolved in distilled water. Further dilutions to the desired concentrations were made with a suitable buffer solution.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Relaxant responses to BK and CPA. Figure 1 shows the time course of changes in the relaxant response to 200 nM BK in the absence and presence of 10 µM indomethacin, 100 µM L-NNA, or 25 mM K⁺ in porcine coronary artery precontracted with 100 nM U-46619. BK caused a sustained relaxation when any of the maneuvers was absent. Removal of the endothelium virtually abolished the relaxant response to BK (data not shown). Indomethacin slightly but significantly enhanced the relaxant response to BK. When the formation of NO was blocked by L-NNA, the relaxant response to BK was markedly depressed. In addition, treatment with L-NNA altered the BK relaxant response to a transient one. Thus, in the presence of L-NNA, the peak relaxation was attained within 2 min after the addition of BK, and then the tone gradually returned toward the contraction level produced by U-46619. In the presence of 25 mM K⁺, where EDHF-associated relaxations can be prevented (10, 16), the relaxant response to BK was also greatly attenuated, but the fade of relaxation was not observed. A similar pattern of relaxation was obtained in the presence of 500 nM apamin and 100 nM charybdotoxin (data not shown). The combination of indomethacin, L-NNA, and 25 mM K⁺ did not allow BK to elicit any effect on the tone (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Influences of indomethacin, NG-nitro-L-arginine (L-NNA), and high K+ on relaxation induced by bradykinin (BK) in pig coronary artery precontracted with U-46619. Time courses of relaxant responses to 200 nM BK in the absence () and presence of 10 µM indomethacin (), 100 µM L-NNA (), and 25 mM K+ () are shown. Indomethacin, L-NNA, and high K+ were given 15 min before application of 100 nM U-46619. Responses are expressed as percent relaxation of U-46619-induced contraction. Values are means ± SE (n = 6). Two-way ANOVA, followed by Bonferroni's multiple comparison test, indicates that the 4 time-response curves are significantly different from each other (P < 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent relaxations induced by BK in the presence of 10 µM indomethacin and 100 µM L-NNA in pig coronary artery precontracted with 100 nM U-46619. Time courses of the relaxant responses to 1 nM (), 10 nM (), 100 nM (), and 1 µM () BK are shown. Responses are expressed as percent relaxation of U-46619-induced contraction. Values are means ± SE (n = 6). Two-way ANOVA, followed by Bonferroni's multiple comparison test, indicates that the 4 time-response curves are significantly different from each other (P < 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Relaxations induced by cyclopiazonic acid (CPA) in pig coronary artery precontracted with U-46619. A: concentration-response curves for CPA-induced relaxation in the absence () and presence of 10 µM indomethacin and 100 µM L-NNA (). Responses are expressed as percent relaxation of 100 nM U-46619-induced contraction. Values are means ± SE (n = 5). B: representative traces showing relaxant response to 10 µM CPA during contraction with U-46619 alone (top trace) and with 100 nM U-46619 and 30 mM K+ (bottom trace). Indomethacin (10 µM) and L-NNA (100 µM) were present throughout the experiment.

Effect of Ni²+ on EDHF-mediated relaxations. At 0.5 and 1 mM, Ni²⁺ had no effect on the resting tension but significantly inhibited the contraction induced by U-46619 in porcine coronary artery. In the presence of 1 mM Ni²⁺, the contraction induced by 100 nM U-46619 was decreased to ~65% of control. Thus, in a series of experiments carried out to examine the effects of this cation on the relaxant responses to BK and CPA, care was taken to match the U-46619-induced contractions in the absence and presence of Ni²⁺. When Ni²⁺ was given, the rings were precontracted by a 10- to 30-fold higher concentration of U-46619.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effect of Ni2+ on relaxations induced by BK and CPA in the presence of 10 µM indomethacin and 100 µM L-NNA in pig coronary artery precontracted with U-46619. Time courses of relaxant responses to 500 nM BK (A) and 10 µM CPA (B) in the absence () and presence of Ni2+ at 0.5 mM () and 1 mM () are shown. Ni2+ was applied 15 min before addition of U-46619. Responses are expressed as percent relaxation of U-46619-induced contraction. Values are means ± SE (n = 5-6).

Hyperpolarizations induced by BK and CPA. The resting membrane potential of vascular smooth muscle cells in porcine coronary artery was 50.3 ± 0.7 mV (n = 10). In tissues with endothelium, 500 nM BK promptly hyperpolarized the membrane potential (Fig. 5A). The peak amplitude of hyperpolarization (22.0 ± 0.4 mV, n = 4) was reached within 30 s. The hyperpolarizing response was then sustained but decayed with a slow time course. In Ca²⁺-free medium, BK produced only a small and transient hyperpolarizing effect (4.6 ± 1.4 mV, n = 4), and sustained hyperpolarization was not generated (Fig. 5B). Treatment with 1 mM Ni²⁺ greatly attenuated the sustained phase of hyperpolarization to BK with marginal change in the resting membrane potential. As a result, the hyperpolarizing response to BK became small and transient (4.8 ± 1.8 mV, n = 4), which was apparently similar to that in the absence of extracellular Ca²⁺ (Fig. 5C).

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Effect of 500 nM BK on cell membrane potential in porcine coronary artery with intact endothelium in normal physiological salt solution (PSS) (A), in Ca2+-free PSS (B), and in the presence of 1 mM Ni2+ (C). BK was applied 5 min after exposure to Ca2+-free PSS or Ni2+.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effect of 10 µM CPA on cell membrane potential in porcine coronary artery with intact endothelium in normal PSS (A), in Ca2+-free PSS (B), and in the presence of 1 mM Ni2+ (C). CPA was applied 5 min after exposure to Ca2+-free PSS or Ni2+.

[Ca²+]_i responses to BK and CPA in cultured endothelial cells. BK (10 nM) induced a rapid increase in [Ca²⁺]_i, as monitored by the ratio of fluorescence at 340 nm to fluorescence at 380 nm, reaching a maximum level within 90 s, followed by a sustained increase in [Ca²⁺]_i that had a tendency to decline slowly toward baseline in cultured porcine aortic endothelial cells loaded with the Ca²⁺-sensitive dye fura 2-AM (Fig. 7A). The rapid increase in [Ca²⁺]_i was dependent on intracellular and extracellular Ca²⁺, while the sustained increase was dependent mainly on the presence of extracellular Ca²⁺, because BK caused only a small and transient increase in [Ca²⁺]_i in the absence of extracellular Ca²⁺ (Fig. 7B). Treatment with 1 mM Ni²⁺ did not affect the basal [Ca²⁺]_i but drastically altered the BK-stimulated increase in [Ca²⁺]_i. Thus the sustained increase in [Ca²⁺]_i was abolished, while the rapid increase was decreased to the same level as in the absence of extracellular Ca²⁺ (Fig. 7A).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effect of 10 nM BK on intracellular Ca2+ concentration ([Ca2+]i) in cultured porcine aortic endothelial cells loaded with fura 2-acetoxymethoxy (AM). A: BK-stimulated [Ca2+]i responses in the absence () and presence of 1 mM Ni2+ (). Cells were pretreated for 10 min with Ni2+ before addition of BK. B: BK-stimulated [Ca2+]i response in Ca2+-free medium containing 1 mM EGTA. A large increase in [Ca2+]i was observed when Ca2+-containing medium was reintroduced. Values (means ± SD) are expressed as ratio of fluorescence at 340 nm to fluorescence at 380 nm (F340/F380); n = 10 cells from 3 separate experiments. Cao2+, extracellular Ca2+.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Effect of 100 µM CPA on [Ca2+]i in cultured porcine aortic endothelial cells loaded with fura 2-AM. A: CPA-stimulated [Ca2+]i responses in the absence () and presence of 1 mM Ni2+ (). Cells were pretreated for 10 min with Ni2+ before addition of BK. B: CPA-stimulated [Ca2+]i response in Ca2+-free medium containing 1 mM EGTA. A large increase in [Ca2+]i was observed when Ca2+-containing medium was reintroduced. Values are means ± SD; n = 10 cells from 3 separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In accordance with the report from another laboratory (16), we clarified that endothelium-dependent relaxation of porcine coronary artery evoked by BK is mediated through two different mechanisms: NO and EDHF that hyperpolarizes vascular smooth muscle cells. This was achieved by using L-NNA, which inhibits the endothelial synthesis of NO, and high K⁺, which prevents membrane hyperpolarization by reducing K⁺ conductance with depolarization. This approach is thought to be the best way to elucidate the contribution of NO and EDHF to endothelium-dependent relaxations (17). The contribution of prostacyclin to the endothelium-dependent relaxant response of porcine coronary artery to BK appeared to be minimal; instead, BK may release vasocontractile prostanoids from the endothelium of this tissue, since indomethacin slightly but significantly enhanced the relaxant response to BK. We found that NO-mediated relaxation in response to BK was well sustained, while EDHF-mediated relaxation was transient. This transient nature of EDHF-mediated relaxation was observed through a wide range of BK concentrations. The BK-stimulated change in [Ca²⁺]_i in porcine aortic endothelial cells exhibited a rapid increase followed by a gradual return to baseline. Thus the time course of EDHF-mediated BK relaxation of porcine coronary artery was apparently the same as that of BK-stimulated increase in [Ca²⁺]_i in aortic endothelial cells. The importance of an elevation of endothelial [Ca²⁺]_i in the EDHF-mediated vascular responses has been well documented (3, 10, 16). Although formation of NO in endothelial cells is known to require an increase in [Ca²⁺]_i (14), we interpret the present results to indicate that EDHF-mediated vascular relaxation is more closely associated with a change in endothelial [Ca²⁺]_i than vasorelaxation related to endothelial NO formation.

In the presence of indomethacin, the endoplasmic reticulum Ca²⁺-ATPase inhibitor CPA produced endothelium-dependent relaxation of porcine coronary artery in a concentration-dependent manner. Although L-NNA greatly suppressed the relaxant responses to the lower concentrations of CPA (3 µM), most of relaxations elicited by the higher concentrations (10 µM) were resistant to L-NNA, reflecting a significant contribution of EDHF to endothelium-dependent relaxations by the higher concentrations of CPA in porcine coronary artery. This can be supported by the observation that the L-NNA-resistant relaxant response to 10 µM CPA was fully suppressed by high K⁺. The potential role of EDHF in CPA-induced relaxations has been demonstrated in our previous work using rat mesenteric artery (8). The EDHF-mediated relaxant response of porcine coronary artery to CPA developed slowly and was long lasting. This temporal aspect was apparently comparable to that of the CPA-stimulated increase in [Ca²⁺]_i in porcine aortic endothelial cells. It is believed that the endoplasmic reticulum Ca²⁺-ATPase inhibitors such as CPA deplete intracellular Ca²⁺ stores, and the emptying of the stores results in Ca²⁺ influx into cells (23). Indeed, we found that CPA failed to increase [Ca²⁺]_i of aortic endothelial cells in Ca²⁺-free medium. The concentration of CPA needed to evoke the increase in [Ca²⁺]_i of aortic endothelial cells was 100 µM, which was higher than that required to produce the maximal endothelium-dependent relaxation of porcine coronary artery. The reason for this is not clear but may be related to less sensitivity of the endoplasmic reticulum Ca²⁺-ATPase to CPA or greater capacity of the endoplasmic reticulum in aortic endothelial cells than in coronary artery endothelial cells. Nevertheless, a significant temporal correlation between the two responses, i.e., CPA-induced EDHF vascular relaxation and endothelial [Ca²⁺]_i elevation, lends further support to the concept that the EDHF-mediated response is crucially dependent on the level of [Ca²⁺]_i in endothelial cells.

In porcine coronary artery with intact endothelium, BK and CPA elicited membrane hyperpolarization of smooth muscle cells. The hyperpolarizing responses to BK and CPA were temporally correlated with the EDHF-mediated relaxant responses to them. Thus EDHF-mediated relaxations of porcine coronary artery are strictly associated with membrane hyperpolarizations. In Ca²⁺-free medium, BK caused only very small and transient hyperpolarizations, and CPA failed to generate hyperpolarization. The same trend of extracellular Ca²⁺ dependency was observed with respect to the BK- and CPA-stimulated [Ca²⁺]_i increase in porcine aortic endothelial cells. The BK-stimulated increase in [Ca²⁺]_i was largely, and the CPA-stimulated increase in [Ca²⁺]_i was entirely, dependent on Ca²⁺ influx into endothelial cells from the extracellular space. These results suggest that Ca²⁺ influx into endothelial cells plays a predominant role in the generation of membrane hyperpolarization due to EDHF.

The present study showed that pretreatment with 1 mM Ni²⁺ had the same effect on the BK- and CPA-stimulated increase in [Ca²⁺]_i of porcine aortic endothelial cells as removal of extracellular Ca²⁺. Thus BK- and CPA-evoked Ca²⁺ influx into endothelial cells was nearly completely eliminated by Ni²⁺. Endothelial cells are known to lack L-type Ca²⁺ channels (1). Furthermore, the agonist-stimulated increase in Ca²⁺ influx into endothelial cells is believed not to be mediated by Na⁺/Ca²⁺ exchange mechanisms (25). Neither verapamil nor replacement of Na⁺ by Li⁺ has an effect on the maximum elevation of [Ca²⁺]_i evoked by the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin in porcine aortic endothelial cells (28). It is thus unlikely that the effect of Ni²⁺ is related to the blocking action on L-type Ca²⁺ channels or Na⁺/Ca²⁺ exchanger. BK has been shown to increase Ca²⁺ influx through activation of NSCC in bovine aortic endothelial cells (15). Also, it has been found that CPA can elicit the NSCC in bovine pulmonary artery endothelial cells (12). In a variety of cell types including endothelial cells, Ni²⁺ is regarded as a blocker of NSCC (7, 13). Therefore, it would be reasonable to conclude that Ni²⁺ prevented BK- and CPA-evoked Ca²⁺ influx into endothelial cells by blocking NSCC.

The inhibitory effect of Ni²⁺ on the EDHF-mediated relaxant responses of porcine coronary artery to BK and CPA was relatively specific, since the endothelium-independent relaxant responses to pinacidil and SNP were unchanged by Ni²⁺. In the presence of 1 mM Ni²⁺, BK-induced transient relaxation was virtually null. Consistent with this is the finding that BK-induced hyperpolarization was greatly diminished by 1 mM Ni²⁺. The hyperpolarizing response to BK in the presence of Ni²⁺ was apparently similar to that obtained in Ca²⁺-free medium, indicating that the remaining response after treatment with Ni²⁺ is due to release of intracellular Ca²⁺ stores in endothelial cells. Since EDHF-mediated relaxation was strictly correlated with membrane hyperpolarization, the tiny relaxant response to BK in the presence of Ni²⁺ suggests that Ca²⁺ released from intracellular stores through inositol 1,4,5-trisphosphate in endothelial cells plays a minor role in EDHF-mediated vascular relaxation when stimulated with BK. However, when a model for Ca²⁺ influx called "capacitative Ca²⁺ entry" (23), where inositol 1,4,5-trisphosphate-induced depletion of intracellular Ca²⁺ stores somehow activates Ca²⁺ influx from the extracellular space, is considered, Ca²⁺ mobilization from intracellular stores in endothelial cells may be important as an initial step leading to the generation of the EDHF-mediated responses. This would account for the mechanism responsible for the action of CPA to cause EDHF-mediated relaxation and hyperpolarization (8). As expected from the data obtained in aortic endothelial cells, the EDHF-mediated relaxant and hyperpolarizing responses of coronary artery to CPA were abolished in the presence of 1 mM Ni²⁺. Taken together, we suggest that EDHF-mediated relaxation and hyperpolarization require primarily an increase in endothelial [Ca²⁺]_i due to Ca²⁺ entry through NSCC.

The chemical identity of EDHF remains controversial (2, 9, 24, 27, 30). However, the existence of humoral EDHF has been suggested by using the bioassay apparatus, where canine coronary artery without endothelium was superfused by the effluent from perfused femoral artery with endothelium (6) and by sandwiched preparations of guinea pig coronary artery (4). Furthermore, the release of EDHF from porcine coronary endothelial cells has been bioassayed by monitoring the membrane potential in vascular smooth muscle cells located downstream (22). If EDHF is a humoral substance, it may be derived from the present study that the increase in endothelial [Ca²⁺]_i that results from Ca²⁺ influx through NSCC is a key step in formation or release of sufficient amounts of EDHF. Recent work has proposed that endothelium-dependent hyperpolarization of vascular smooth muscle cells is electrically conducted from endothelial cells through myoendothelial gap junctions (29). ACh-induced hyperpolarization in endothelial cells appears to involve activation of Ca²⁺-activated K⁺ channels (20). If this is the case, a rise in [Ca²⁺]_i due to Ca²⁺ influx via NSCC may activate the K⁺ channels, thereby producing membrane hyperpolarization in endothelial cells.

In conclusion, the present study demonstrated that EDHF-mediated vascular hyperpolarization and relaxation are closely related to the increase in endothelial [Ca²⁺]_i. Ca²⁺ released from intracellular stores in endothelial cells appears to play a minor role in the development of the EDHF-mediated responses and may be merely a trigger to initiate Ca²⁺ influx from extracellular space for refilling emptied Ca²⁺ stores. We thus suggest that Ca²⁺ entry into endothelial cells through Ni²⁺-sensitive NSCC contributes substantially to the regulation of EDHF.