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(Circulation Research. 1995;76:396-404.)
© 1995 American Heart Association, Inc.

Articles

Na⁺-Ca²⁺ Exchange in Intact Endothelium of Rabbit Cardiac Valve

Li Li, Cornelis van Breemen

From the Department of Pharmacology and Therapeutics, The University of British Columbia, Vancouver, Canada.

Correspondence to Cornelis van Breemen, Department of Pharmacology and Therapeutics, Faculty of Medicine, 2176 Health Sciences Mall, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

	Abstract

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Abstract A new method of measuring cytoplasmic free Ca²⁺ ([Ca²⁺]_i) of individual intact cardiovascular endothelial cells by using imaging fluorescence microscopy was designed. Application of agonist to the aortic or pulmonary valve of the rabbit triggered an increase in [Ca²⁺]_i, which depended on the existence of endothelium on the surface of the valve. Under resting conditions, sudden reversal of the Na⁺ gradient by substituting external Na⁺ with N-methyl D-glucamine (NMDG) resulted in a [Ca²⁺]_i spike, which then returned toward the resting level. Increasing intracellular Na⁺ concentration ([Na⁺]_i) by application of ouabain or monensin induced a sustained [Ca²⁺]_i increase. Na⁺ substitution by NMDG during the agonist- or monensin-induced [Ca²⁺]_i increase gave rise to a further [Ca²⁺]_i spike, which subsequently declined to a level higher than that before removal of external Na⁺. A selective inhibitor of Na⁺-Ca²⁺ exchange, 3',4'-dichlorobenzamyl (DCB), abolished the transient [Ca²⁺]_i increase induced by Na⁺ substitution, and Mg²⁺, an inorganic inhibitor of Na⁺-Ca²⁺ exchanger, markedly reduced this transient [Ca²⁺]_i increase. On the other hand, the selective Na⁺-H⁺ exchanger blocker 5-(N,N-hexamethylene)amiloride (HMA) did not abolish the transient [Ca²⁺]_i increase caused by Na⁺ substitution. In summary, decreasing the Na⁺ gradient of the endothelial cells through either receptor stimulation (agonist), Na⁺-K⁺ pump inhibition (ouabain), pretreatment with Na⁺ ionophore (monensin), or reversing the Na⁺ gradient through Na⁺ substitution (NMDG) all increased [Ca²⁺]_i. This raised [Ca²⁺]_i was antagonized by agents such as DCB or Mg²⁺, which are thought to inhibit Na⁺-Ca²⁺ exchange, but not by HMA, an inhibitor of Na⁺-H⁺ exchange. Taken together, these results strongly imply the presence of Na⁺-Ca²⁺ exchange as a viable mechanism for Ca²⁺ transport in intact cardiovascular endothelium and that the Ca²⁺ entry component is enhanced when [Na⁺]_i is elevated.

Key Words: endothelium • Na⁺-Ca²⁺ exchange • cytoplasmic Ca²⁺ • Na⁺ • cardiac valves

	Introduction

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The cardiovascular endothelial cell is a source of various vasoactive factors. It has the capability of secreting both antiaggregatory and aggregatory, antithrombotic and thrombotic, and vasorelaxant and vasoconstrictive factors as well as growth inhibitors and stimulators.¹ The role of the endothelial cell in the relaxation response of vascular smooth muscle to acetylcholine (ACh) was proposed,² and a nonprostanoid substance, termed endothelium-derived relaxing factor (EDRF), was shown to be released from the endothelium.³ In addition to relaxing vascular smooth muscle, EDRF released locally may inhibit platelet aggregation^{4 5 6} and adhesion.⁷ Currently, one EDRF is identified as nitric oxide (NO).^{8 9} Endothelial responses also appear to be altered in various cardiovascular pathologies, including atherosclerosis, diabetes, hypertension, septic shock, and dementia.^{10 11 12 13 14}

In regulating the release and/or the production of EDRF, cytoplasmic Ca²⁺ plays an important role. It was demonstrated that relaxation of the rabbit aorta with a functional endothelium was initiated by the Ca²⁺ ionophore A23187^{2 15 16 17 18 19} and suppressed by the deletion of Ca²⁺ from the incubation medium or by adding Ca²⁺ entry blockers.^{2 15 17 19} Subsequent studies showed that NO in mammalian endothelial cells is derived from L-arginine and that a cytoplasmic enzyme, NO synthase, the activity of which is regulated by Ca²⁺,^{20 21} is involved in the formation of NO. Therefore, it is crucial to characterize the Ca²⁺ transport pathways that control cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) to understand the physiology of the endothelium as well as its role in atherosclerotic and thrombotic vascular diseases.

One ubiquitous Ca²⁺ transport process is Na⁺-Ca²⁺ exchange in the plasmalemma, first identified in cardiac muscle²² and nerve membrane.²³ The exchanger functions reversibly so that Ca²⁺ can be transported in either direction (inwardly or outwardly) across the plasmalemma in exchange for Na⁺, depending on the electrochemical gradients of Na⁺ and Ca²⁺ across the membrane.²⁴ Variation of intracellular or extracellular Na⁺ concentration ([Na⁺]_i or [Na⁺]_o, respectively) thus affects the level of [Ca²⁺]_i.

The existence of Na⁺-Ca²⁺ exchange in endothelial cells has been a controversial issue. The voltage dependence of resting [Ca²⁺]_i, which was consistent with a simple pump-leak model, provided no evidence for the existence of Na⁺-Ca²⁺ exchange in the surface membrane of endothelium.²⁵ A similar conclusion was reached from the observation in cultured endothelium that isotonic substitution of extracellular Na⁺ with K⁺ (which would be expected to depolarize the cell) decreased [Ca²⁺]_i²⁶ and that application of the Na⁺-K⁺ pump inhibitor ouabain was without effect on [Ca²⁺]_i.²⁷ Since the activity of the exchanger is expected to be steeply dependent on [Na⁺]_i, it is possible that changes in [Ca²⁺]_i due to the exchange will not become apparent until [Na⁺]_i is sufficiently elevated. Agonist application may increase Na⁺ influx via nonselective surface membrane channels,^{28 29} thereby increasing [Na⁺]_i. It has been suggested that such an increase in [Na⁺]_i, coupled to Na⁺-Ca²⁺ exchange, may contribute to the plateau phase of the [Ca²⁺]_i response to agonist.²⁵ However, it was reported that isotonic Li⁺ substitution for Na⁺ in cultured endothelium had no effect on [Ca²⁺]_i regardless of whether the cells were at rest or activated by the agonist bradykinin.³⁰ Similarly, bradykinin-stimulated changes in [Ca²⁺]_i were unaffected by isotonic substitution of external Na⁺ with N-methyl D-glucamine (NMDG).³¹ These results suggest that Na⁺-Ca²⁺ exchange does not significantly contribute either to Ca²⁺ extrusion or entry in the cultured endothelium.

However, there are other observations suggesting the existence of the exchanger in the endothelium. More recently, it was reported that if cultured endothelial cells were first Na⁺-loaded with the Na⁺ ionophore monensin and then exposed to physiological saline solution (PSS) with the external Na⁺ substituted by Li⁺, a large transient increase in [Ca²⁺]_i ensued; it was further reported that combined pretreatment with ouabain and monensin doubled this [Ca²⁺]_i transient.²⁷ This observation clearly established the presence of Na⁺-Ca²⁺ exchange in cultured endothelial cells, even though it was not shown to regulate [Ca²⁺]_i either at rest or during activation. A similar conclusion was obtained from the observation that the rate of ⁴⁵Ca influx was increased by application of ouabain in bovine aortic cultured endothelium and was increased further when the ouabain-treated endothelium was transferred to a solution in which external Na⁺ was substituted by choline.³² Data obtained from intact endothelium also support a role for Na⁺-Ca²⁺ exchange. It has been reported that putative blockers of Na⁺-Ca²⁺ exchanger, such as dichlorobenzamyl (DCB), blocked the relaxation responses to ACh and A23187 in rat aorta, which was interpreted in terms of the blockers' decreasing [Ca²⁺]_i by reducing Ca²⁺ influx via the exchanger.³³

These somewhat conflicting data discussed above cast some doubt on the physiological role of Na⁺-Ca²⁺ exchange in intact endothelium. In this study, experiments were conducted to investigate the role of Na⁺-Ca²⁺ exchange in regulating [Ca²⁺]_i in intact endothelium of rabbit cardiac valve by using digital imaging microscopy. The valve was chosen for studying intact endothelium because the complete endothelial covering of this thin structure allows for selective loading with Ca²⁺-sensitive dyes, as was first reported by Aoki et al.³⁴ Digital imaging fluorescence microscopy allows recording of fura 2 fluorescence ratio changes in individual as well as in groups of endothelial cells on the surface of the valve in their native intercellular interactions. The method has recently been elaborated in our previous study.³⁵ It circumvents the problems of studies in cultured endothelial cells, where cell surface receptor, ion channel expression, and intercellular coupling might be changed by enzymatic or mechanical treatments used to separate the endothelial cells from surrounding smooth muscle and subsequent culture procedures.²¹ Furthermore, it may be used for comparative studies of endothelium-related pathophysiologies, and immunohistochemical and other fluorescent indicators are readily adapted for use in studies of other facets of intracellular signaling pathways and intercellular communication. Thus, the ability to record changes in [Ca²⁺]_i in intact endothelial monolayer provides a more realistic assessment of excitation-response coupling within individual cells and communication between cells.

	Materials and Methods

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Tissue Preparation
Adult New Zealand White rabbits weighing 2.0 to 2.5 kg were killed by CO₂ asphyxiation and exsanguinated from the carotid artery. The heart was quickly excised and placed in normal physiological saline solution (N-PSS). The composition of different solutions used is shown in the Table. N-PSS was adjusted to pH 7.4 at 37°C by using NaOH. The solution referred to as 0 Ca²⁺-PSS is similar to N-PSS, except CaCl₂ is omitted from the solution. Sodium substitution was achieved by isotonic substitution of extracellular NaCl with NMDG, and the pH was adjusted to 7.4 by using HCl. The solutions appearing on the Table as 2 Mg²⁺-NMDG-PSS and 4 Mg²⁺-NMDG-PSS contain 2 mmol/L and 4 mmol/L MgCl₂, respectively.

View this table: [in this window] [in a new window]   Table 1. Compositions of Representative Solutions

The apex of the ventricle was then cut away to facilitate removal of coagulated blood. Both the aortic and pulmonary arteries were opened carefully with a longitudinal incision at their respective attachments to the left and right ventricles. The pulmonary and aortic valves (each composed of three fibrous cusps suspended from the root of aortic and pulmonary arteries) were dissected free with fine tools, placed in N-PSS in separate Petri dishes, and kept in the refrigerator until fura 2-AM loading. After loading, the valves were then mounted in a specially designed chamber.

Fig 1 illustrates the specially designed tissue chamber. The bottom of the chamber was made of a glass coverslip coated with Sylguard (Dow Corning Corp) with a fine polytetrafluoroethylene (Teflon) tube (internal diameter, 0.28 mm) secured in it. Through one end of this tube, experimental solutions passed continuously to a channel (width, 1.5 mm; length, 4 mm), which was made by cutting off the Sylguard at the center of the chamber bottom. The temperature of the chamber and solutions flowing through the chamber were kept at 37°C by a bipolar temperature controller (Medical System Corp). The fura 2–loaded valve was then pinned over this channel. Test solutions were restricted to the channel bathing the lower surface of the valve so that various solution changes could be made. The upper surface of the valve was continuously bathed in N-PSS by a second inflow-and-outflow system established above the tissue (total volume of bath, 1.5 mL) to maintain the temperature at 37°C and prevent overflow. On the other end of this Teflon tube, an adjustable vacuum pump continuously removed the perfusion solution from the channel. The flow was maintained by matching the inflow and outflow rates of 800 µL/min.

View larger version (25K): [in this window] [in a new window]   Figure 1. Schematic diagram of the experimental chamber designed for visualization of fura 2–loaded intact valvular endothelium. The valve leaflet is pinned flat over a narrow channel cut in Sylguard (width, 1.5 mm; length, 4 mm; Dow Corning Corp). Only the lower surface of the valve is exposed to solution changes while the upper surface is continuously bathed in normal physiological saline solution. Polytetrafluoroethylene (Teflon) tubing (internal diameter, 0.28 mm) is secured by the Sylguard (see method).

Measurement of [Ca²⁺]_i
A 1-mmol/L stock solution of the acetoxymethyl ester of fura 2 (fura 2-AM) in dimethyl sulfoxide (DMSO) was dissolved in N-PSS solution to a final concentration of 1 µmol/L (final concentration of DMSO, 0.1%). The valves were incubated in the loading solution at room temperature for 45 to 60 minutes in a gently shaking water bath. The valves were subsequently transferred to fresh N-PSS for a further 15 minutes before use. This procedure yielded cells that were evenly loaded with fura 2.³⁶

The fura 2–loaded valve where no obvious disruption of the cell monolayer occurred was then pinned flat over the channel in the chamber with the perfusion system (illustrated in Fig 1). The chamber was placed on the stage of an inverted fluorescence microscope so that a x20 phase/fluor objective (numerical aperture, 0.75; Nikon Diaphot) was focused on the lower surface of endothelial cells forming the ceiling of the narrow perfusion channel in the chamber, and individual cells were visualized. The recordings were from the lower surface endothelial cell monolayer, since the endothelial cell monolayer of the valve leaflet facing the upper chamber did not respond to the addition of agonist to the underlying channel in control experiments, in which the upper surface was brought into focus.

The lower surface of the valve was exposed to alternating 340- and 380-nm (bandwidth, 10 nm) UV light (1/s), and emission light was passed through a 510-nm (bandwidth, 40 nm) cutoff filter before acquisition by the ICCD camera (an intensified charge-coupled device, model ER 4093G; Cohu, 4810 series), which was connected to a Trinitron color video monitor (Sony Corp). Autofluorescence of the unloaded valve was minimal, and background images (340 and 380 nm) of the fura 2–loaded valve were obtained from a focal plane 1 mm away from the endothelial plane. The fluorescence ratio (F340/F380, the ratio of excitation at 340 nm to that at 380 nm) was determined as background-subtracted ratio, and [Ca²⁺]_i was calculated by using the following equation³⁷ :

where K_d is the dissociation constant for the fura 2–Ca²⁺ complex; R is the above-mentioned fluorescence ratio (F340/F380); R_max and R_min are the maximal and minimal fluorescence ratios measured by the addition of 10 µmol/L of Ca²⁺ ionophore ionomycin to Ca²⁺-replete (2 mmol/L CaCl₂) solution and Ca²⁺-free (10 mmol/L EGTA) solution, respectively; and b is the fluorescence ratio at 380-nm excitation determined at R_min and R_max, respectively. The average resting [Ca²⁺]_i was calculated to be 90±8 nmol/L (SEM, n=400 cells from six experiments). However, responses were routinely recorded and expressed as changes of signal ratio of individual cells, indicating relative [Ca²⁺]_i because cellular conditions are not readily duplicated in calibration solutions.

Regions containing single or groups of cells loaded evenly with fura 2 were selected for recording. Up to 64 regions of interest could be simultaneously monitored for changes in fluorescence ratio signal in any preparation. Pairs of the fluorescence signal ratio, which are recorded as digital image data by using a Sun Sparc1+ workstation and INOVISION acquisition and analysis software (Inovision Corp) controlled by a Data Translation frame grabber (DT3861) housed in a PC 80286 computer, were plotted as background-subtracted ratios (F340/F380) on-line collected every 10 seconds at 340- and 380-nm excitation wavelengths during the experimental procedure. Consecutive images were averaged over 32 frames (1 s) and stored for subsequent analysis.

The single tracing of ratio data shown in each figure was an average ratio measured simultaneously with individual cells of interest chosen from the same valve; where applicable, ratio values were expressed in the text as mean±SEM of these cells in the same preparation (n=number of cells). All cells in the same valve preparation responded similarly. Chemicals and/or drugs were applied, as indicated by the horizontal bars or arrows in each figure. Each tracing was obtained from a different preparation and is representative of those responses obtained in at least six experiments, each of which had similar results.

Chemicals
Ouabain, monensin, NMDG, 5-(N,N-hexamethylene)amiloride (HMA), ACh, carbamylcholine chloride (carbachol [CCh]), DMSO, ionomycin, and analytical grade reagents for PSS were purchased from Sigma Chemical Co. DCB was kindly provided by Merck Research Laboratories. Fura 2-AM was from Molecular Probes. All drugs were dissolved in N-PSS from their respective stock solutions. The stock solutions of ouabain and ACh were made in N-PSS; monensin, in ethanol; and DCB and HMA, in DMSO.

	Results

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Bioassay experiments have shown that the ability to respond to ACh is a characteristic of endothelium in vivo. To establish that the fura 2 fluorescence signals in the present study were originating from the endothelial layer of the valve, we compared the ACh-induced fura 2 signal obtained from the intact valve with a signal obtained from a valve whose endothelium was gently scraped off after fura 2 loading. Fig 2 shows that intact valvular endothelium responded to ACh (10 µmol/L) with an initially rapid increase of fluorescence ratio to a peak of 1.09±0.05 and then declined slowly to a level of 0.84±0.03 (SEM, n=64), almost equal to that before activation (tracing a), whereas removal of endothelium on the side of the valve from which the signal was recorded greatly reduced the fluorescence intensity and abolished the ACh-induced transient ratio increase (tracing b). These results demonstrated that the preparation used was physiologically well preserved and that the fura 2 fluorescence was derived from the cells facing the experimental solutions.

View larger version (13K): [in this window] [in a new window]   Figure 2. The average fluorescence ratio signal (F340/F380) in response to continuous perfusion of acetylcholine (ACh, 10 µmol/L). Signal was observed in intact endothelial cells (n=64) from rabbit valve in normal physiological saline solution (tracing a, solid line) and from rabbit valve after the removal of endothelium by gently scraping the surface of the valve (tracing b, dashed line). Each tracing was obtained from a different preparation.

The objective of the present study was to evaluate the contribution of Na⁺-Ca²⁺ exchange to the regulation of [Ca²⁺]_i. Since the activity of the Na⁺-Ca²⁺ exchange is critically dependent on the electrochemical Na⁺ gradient across the cell membrane, external Na⁺ removal would first reverse the Na⁺ gradient and, after loss of intracellular Na⁺, abolish it; correspondingly, it would first cause Ca²⁺ entry in exchange for Na⁺ efflux and then slow down after loss of intracellular Na⁺. In the present study, isotonic substitution of external Na⁺ with Li⁺ in N-PSS yielded a transient fluorescence ratio increase in intact endothelium; however, a fluorescence ratio transient of variable size was often seen when Na⁺ was substituted with Li⁺ in 0 Ca²⁺ PSS (data not shown), which cannot be interpreted as an effect due to Na⁺-Ca²⁺ exchange. Since Li⁺ has an inhibitory effect on the hydrolysis of inositol trisphosphate (IP₃), a substance that causes Ca²⁺ release from endoplasmic reticulum (ER), this transient ratio increase induced by Li⁺ in the absence of extracellular Ca²⁺ could be due to IP₃ accumulation.^{38 39} Therefore, Li⁺ substitution for Na⁺ cannot be used to investigate the Na⁺-dependent transport process in intact endothelial cells. An alternative substitute, NMDG, was thus used. Fig 3 shows that NMDG-PSS induced a fluorescence ratio peak of 2.09±0.05 from 1.35±0.03 and then declined almost to the previous level of 1.37±0.06 (SEM, n=50) (tracing a). This is consistent with the internal Na⁺-dependent Ca²⁺ entry pathway. The decline in [Ca²⁺]_i during the period of exposure to Na⁺-free solution had also been observed in cardiac muscle.^{40 41} It might be due to the loss of Na⁺ from the cell during the exposure to Na⁺-free solution, thus abolishing the Na⁺ gradient and leading to diminution of Ca²⁺ entry via the exchange process. This peak response was not seen in the control experiment using NMDG in the absence of extracellular Ca²⁺ (NMDG, 0 Ca²⁺-PSS) (tracing b).

View larger version (13K): [in this window] [in a new window]   Figure 3. Effect of external Na+ substitution by N-methyl D-glucamine (NMDG) on the average fluorescence ratio signal (F340/F380) of intact endothelial cells (n=50) in the field of view of rabbit valve. External Na+ was isotonically substituted by NMDG in normal physiological saline solution (tracing a, solid line) and in 0 Ca2+ physiological saline solution (tracing b, dashed line). Each tracing was obtained from a different preparation.

Alternatively, the transmembrane Na⁺ gradient may be reduced by inhibition of the Na⁺-K⁺ pump with cardiotonic steroids. In cardiac muscle, squid axon, and barnacle muscle, inhibition of the Na⁺-K⁺ pump causes an increase in [Na⁺]_i, which promotes Na⁺ extrusion coupled to Ca²⁺ entry, an effect believed to be mediated by Na⁺-Ca²⁺ exchange.⁴² Fig 4 shows that application of 100 µmol/L ouabain, an inhibitor of the Na⁺-K⁺ pump, increased the resting fluorescence ratio from a basal level of 1.34±0.04 to a new steady state level of 1.59±0.06 (SEM, n=30) in N-PSS, a result that is consistent with the presence of an internal Na⁺-dependent Ca²⁺ entry.

View larger version (11K): [in this window] [in a new window]   Figure 4. Tracing showing the effect of ouabain (100 µmol/L) on the average fluorescence ratio signal (F340/F380) of intact endothelial cells (n=30) from rabbit valve.

A Na⁺ ionophore, monensin, which increases [Na⁺]_i, represents yet another way of reducing the transmembrane Na⁺ gradient. Fig 5 shows that application of 50 µmol/L monensin in N-PSS promoted a fluorescence ratio increase to a value of 1.23±0.07 from 1.09±0.06 (SEM, n=35). Monensin causes an increase in [Na⁺]_i that couples to Ca²⁺ entry via Na⁺-Ca²⁺ exchange. The result is consistent with that observed in cultured endothelial cells.²⁷ The elevated ratio was maintained because high [Na⁺]_i was sustained by the continued presence of monensin and external Na⁺. Removal of external Na⁺ after the [Na⁺]_i has first been elevated should prolong the period of Na⁺ efflux mediated by Ca²⁺ entry. Fig 6, top, shows that when external Na⁺ was substituted by NMDG in the presence of monensin (monensin first increased the fluorescence ratio from 1.23±0.07 to 1.39±0.08), a peak of 1.84±0.05 was observed, which declined to an elevated plateau level of 1.51±0.06 (SEM, n=40). Fig 6, bottom, shows what might be the physiological correlate of this experiment with the application of an agonist. The agonist may increase Na⁺ influx via nonselective plasma membrane channels. Na⁺ replacement after stimulation with the agonist CCh, an analogue of ACh, induced a further broad fluorescence ratio peak of 1.61±0.05 from 1.37±0.07, followed by an elevated level of the ratio of 1.45±0.08 (SEM, n=70).

View larger version (12K): [in this window] [in a new window]   Figure 5. Tracing showing the effect of monensin (50 µmol/L) on the average fluorescence ratio signal (F340/F380) of intact endothelial cells (n=35) from rabbit valve.

View larger version (14K): [in this window] [in a new window]   Figure 6. Effect of external Na+ replacement by N-methyl D-glucamine (NMDG) on the elevated fluorescence ratio signal (F340/F380) of intact endothelial cells (n=40) from rabbit valve. Fluorescence ratio signal was elevated by application of 50 µmol/L monensin (n=40) (top) and agonist carbamylcholine chloride (carbachol [CCh], 10 µmol/L) (n=70) (bottom).

However, it must be considered that reversal of the Na⁺ gradient alters two possible Na⁺-dependent processes: (1) the reversal of the putative Na⁺-H⁺ exchange, leading to a lowering of pH_i, and (2) the reversal of Na⁺-Ca²⁺ exchange, thereby increasing [Ca²⁺]_i.

The presence of Na⁺-H⁺ exchange has been demonstrated in rat brain capillary endothelial cells in vivo⁴³ and in cultured bovine aortic endothelial cells.⁴⁴ It is important for the regulation of pH_i. Changes in pH_i might affect [Ca²⁺]_i and Ca²⁺-dependent intracellular events. Therefore, the contribution of Na⁺-Ca²⁺ or Na⁺-H⁺ exchanger to [Ca²⁺]_i should be verified by using their selective blockers.

To confirm the contribution of Na⁺-Ca²⁺ exchange to [Ca²⁺]_i, DCB, a selective Na⁺-Ca²⁺ exchanger inhibitor (IC₅₀, 17 µmol/L), was thus applied. As one of a number of amiloride analogues, DCB inhibits Na⁺-Ca²⁺ exchange. Fig 7, top, shows that the transient ratio increase in response to external Na⁺ removal by NMDG was abolished after the valve was incubated in N-PSS with 25 µmol/L DCB for 8 minutes (n=66 cells). At this concentration of DCB (25 µmol/L), Na⁺-Ca²⁺ exchange can be effectively inhibited while the osmotic integrity of the membrane, Na⁺,K⁺-ATPase enzymatic activities, Na⁺-H⁺exchange, mitochondrial oxygen consumption, and Na⁺, Ca²⁺, or K⁺ conductance are not changed.⁴⁵

View larger version (12K): [in this window] [in a new window]   Figure 7. Effect of inhibitors of Na+-Ca2+ exchange on the average fluorescence ratio signal (F340/F380) of intact endothelial cells from rabbit valve. After recording a control response (upper tracing) to N-methyl D-glucamine physiological saline solution (NMDG-PSS), the fura 2–loaded valve was exposed to normal physiological saline solution (N-PSS) containing dichlorobenzamyl (DCB, 25 µmol/L) for 8 minutes. After a brief wash with N-PSS, the valve was again exposed to NMDG-PSS (middle tracing) (n=65). These two tracings were obtained from the same preparation. The bottom tracing shows the fura 2–loaded valve exposed to 2 Mg2+ NMDG-PSS and 4 Mg2+ NMDG-PSS. The control response was subsequently recorded in NMDG-PSS (n=60).

Another inhibitor of the Na⁺-Ca²⁺ exchange is the Mg²⁺ ion.^{46 47 48} Mg²⁺ is thought to compete with Ca²⁺ at the divalent binding site of the carrier protein on the plasmalemma without being transported.⁴⁹ Fig 7, bottom, shows the effect of Mg²⁺ present in the NMDG-PSS solution on the fluorescence ratio. The response to 2 Mg²⁺-NMDG-PSS was from 0.80±0.03 to 1.11±0.05, faster and larger than that in 4 Mg²⁺-NMDG-PSS, which was from 0.73±0.04 to 0.86±0.06, whereas an NMDG response from 0.75±0.05 to 1.12±0.07 in NMDG-PSS was faster and larger than that in 2 Mg²⁺-NMDG-PSS (SEM, n=58). The response was completely abolished in 8 Mg²⁺ NMDG-PSS (data not shown). In contrast to DCB, the inhibitory effect of Mg²⁺ was reversed after washing. Since Mg²⁺ acts from the outside, these results strongly support the idea that the Na⁺-Ca²⁺ exchange is involved in the [Ca²⁺]_i change induced by external Na⁺ removal. In control experiments without addition of DCB or raised extracellular Mg²⁺, the intact endothelium responded to consecutive applications of NMDG with similar amplitudes of ratio increases (data not shown).

Conversely, the selective blocker of the Na⁺-H⁺ exchanger (IC₅₀, 0.3 µmol/L), HMA, which is also an analogue of amiloride, did not affect the NMDG-stimulated transient ratio increase. NMDG induced a ratio increase from a basal level of 0.78±0.02 to a peak of 1.95±0.03 in the presence of 10 µmol/L HMA, similar to that obtained in the absence of HMA, which is from 0.74±0.02 to 2.25±0.03 (SEM, n=75) (Fig 8). At this concentration of HMA (10 µmol/L), pH_i changes mediated by Na⁺-H⁺exchange could be effectively blocked.⁴⁴

View larger version (15K): [in this window] [in a new window]   Figure 8. Tracing showing the effect of inhibitor of Na+-H+ exchanger on the average fluorescence ratio signal (F340/F380) of intact endothelial cells (n=60) from rabbit valve. After recording a control response to N-methyl D-glucamine physiological saline solution (NMDG-PSS), the fura 2–loaded valve was exposed to 10 µmol/L 5-(N,N-hexamethylene)amiloride (HMA) and subsequent NMDG-PSS in the presence of HMA.

Combined with the results due to changes in Na⁺ gradient, these data clearly indicate the presence of Na⁺-Ca²⁺ exchange in this preparation. The transient [Ca²⁺]_i peak caused by external Na⁺ removal may be due to Ca²⁺ influx via the Na⁺-Ca²⁺ exchange driven by the outward Na⁺ movement, which, however, does not appear to influence the steady state [Ca²⁺]_i. The increased resting level of [Ca²⁺]_i by ouabain and monensin and the greater amplitude of [Ca²⁺]_i transient resulting from external Na⁺ removal after [Na⁺]_i was first elevated by monensin or agonist can be explained by stimulation of the Ca²⁺ entry via the Na⁺-Ca²⁺ exchange due to elevated [Na⁺]_i.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intracellular Ca²⁺ signaling controls the secretory function (eg, EDRF release) of endothelial cells. Characterization of mechanisms regulating [Ca²⁺]_i in endothelial cells has been mostly carried out by the use of subcultured cells. However, treatment of isolated tissues with enzymes for the purpose of dislodging endothelial cells from the extracellular matrix and adjoining cells will modify the influence of surrounding tissue on the function of the endothelial monolayer. In addition, proliferation of endothelial cells in artificial media will alter the expression of cell-surface receptors, transport processes, and intercellular coupling.²¹ For example, ACh-induced [Ca²⁺]_i responses^{50 51} and EDRF release^{52 21} are generally not observed in passaged cultured cells, whereas endothelium-dependent relaxation of arteries by ACh is diagnostic for healthy intact endothelium.^{53 54} Furthermore, the physiological importance of Na⁺-Ca²⁺ exchange in endothelial cells is questionable, since evidence for Na⁺-Ca²⁺ exchange was obtained only in cultured endothelium under extreme experimental conditions,²⁷ whereas studies on intact arteries showed that several Na⁺-Ca²⁺ exchange inhibitors altered vessel contraction in an endothelium-dependent manner.³³ Collectively, these findings suggest that there are fundamental differences in Ca²⁺ regulation between cultured and intact endothelial cells.

Recent experiments using the cardiac valve³⁴ demonstrate that it is possible to obtain new insight into [Ca²⁺]_i regulation in intact endothelium.³⁵ The semilunar valves are delicate tricuspid projections (valve leaflets) covered entirely with endothelium and attached to the wall of the endocardium by a ring of dense fibrous connective tissue. Thus, valvular endothelium is continuous with endocardium. Endocardial endothelium possesses secretory mechanisms similar to those observed in vascular endothelium, despite several morphological differences due to the direction of blood flow.^{55 56} Endocardial endothelium also contains the enzyme NO synthase and releases EDRF in response to vasoactive agents^{57 58} and has been shown to modulate inotropic responses of subjacent myocardium.⁵⁹ The study of valvular endothelium, therefore, has significance for both vascular and cardiac research.

Precautions have been taken in studying intact endothelium to ensure that all recordings reflect responses from endothelial cells only. Regions near the apex of the valve leaflet where individual cells could be visualized were chosen, since focusing through the thickness of this valve section (100 µm) revealed that the endothelial cells were uniformly loaded and that the fura 2 fluorescence was observed only on the surface endothelial cell layer. Electron micrographs showed endothelial cell monolayers, with a continuous basal lamina as well as gap junctions between cells, sandwiching a ground matrix of collagen with varying amounts of elastic elements and sparsely populated fibroblast-like cells; there were no underlying smooth muscle cells that might influence endothelial cell responses and contribute to [Ca²⁺]_i signaling.³⁵ There was also no additional fluorescence from the central tissue when focusing through the valve leaflet, and gently rubbing the surface greatly reduced the raw fura 2 fluorescence.

Although ACh responses have been reported for primary endothelial cell cultures⁵¹ and freshly dissociated endothelial cells,⁶⁰ the intercellular communications are disrupted by the isolation process. In our preparation, individual cells with intact intercellular connections and functional muscarinic ACh receptors were observed. The intact valvular endothelium shares the ability of its arterial counterpart to increase [Ca²⁺]_i in response to vasoactive ACh or CCh, which relates to endothelium-dependent relaxation of smooth muscle with an intact endothelium after stimulation of endothelial muscarinic receptors with ACh.^{2 56 57} The increase in [Ca²⁺]_i is thought to be due to Ca²⁺ influx across the plasmalemma and Ca²⁺ release from ER after receptor activation. Recent investigations have shown that freshly isolated bovine aortic endothelial cells express m1, m2, and m3 muscarinic receptor subtypes,⁶¹ whereas only the m2 muscarinic receptor subtype was identified in cultured endothelial cells.⁶² It is known that m1 and m3 receptor subtypes are coupled to stimulation of phospholipase C (PLC).⁶³ PLC stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces two physiologically active substances, diacylglycerol and IP₃.⁶⁴ Diacylglycerol stimulates protein kinase C, but inhibitors of protein kinase C do not block ACh-evoked responses.⁶⁵ Therefore, ACh exerts its effect mainly through the synthesis of IP₃. IP₃ elevates [Ca²⁺]_i by acting on specific receptors to release Ca²⁺ from ER and in many cells, including endothelium, by evoking a subsequent store-regulated Ca²⁺ entry by an unknown mechanism.⁶⁶ We found that Li⁺ cannot be used as a Na⁺ substitute in studying the role of Na⁺-Ca²⁺ exchange in an intact endothelial preparation because of its effect on IP₃ hydrolysis. The inactivation process of IP₃ is the hydrolysis of IP₃ to inositol 1,4-bisphosphate (IP₂) and inositol 1-phosphate (IP) and then to inositol and inorganic phosphate. Li⁺ directly inhibits IP monoesterase, an enzyme present in the cytoplasm of the cell that hydrolyzes IP to inositol and inorganic phosphate. So inhibition of IP monoesterase by Li⁺ finally causes IP₃ accumulation and [Ca²⁺]_i increases.⁶⁷ It was observed that acute administration of Li⁺ to rats resulted in increased endogenous IP levels especially in brain and that the small increase of IP that followed the administration of centrally acting agonists in vivo was greatly enhanced in the presence of Li⁺.⁶⁸ Our data suggest that IP₃ accumulation in Li⁺-PSS was sufficient to release Ca² from the ER. In cultured endothelial cells, this effect of Li⁺ was not observed.³⁰

Instead of Li⁺, NMDG was used to replace external Na⁺. NMDG induced a transient [Ca²⁺]_i increase that was not observed in the absence of extracellular Ca²⁺. Since the removal of external Na⁺ did not cause a [Ca²⁺]_i increase because of mobilization from intracellular stores,⁶⁹ this transient [Ca²⁺]_i increase is thought to be due to an internal Na⁺-dependent Ca²⁺ entry pathway, such as provided by the Na⁺-Ca²⁺ exchange in the plasmalemma. The net Ca²⁺ movement [J_Ca(Na/Ca)] mediated by exchange is determined by the difference between the membrane potential (E_m) and the reversal potential of the exchange (E_Na/Ca), as well as by the kinetic parameters (k) that control the rate of exchange: J_Ca(Na/Ca)=k(E_m-E_Na/Ca). It has been shown that the Na⁺-Ca²⁺ exchange stoichiometry is 3Na⁺:1Ca²⁺.⁷⁰ Thus, the reversal potential for the Na⁺-Ca²⁺ exchange is E_Na/Ca =3E_Na-2E_Ca, where the equilibrium potential for Na⁺ is E_Na=(RT/F)ln([Na]_o/[Na]_i) and the equilibrium potential for Ca²⁺ is E_Ca=(RT/2F)ln([Ca]_o/[Ca]_i). R, T, and F are the gas constant, absolute temperature, and Faraday's number, respectively. In freshly dissociated endothelial cells from rabbit aorta, E_m is -50 mV⁶⁰ ; in cultured endothelial cells, the resting [Ca²⁺]_i ranges between 30 and 120 nmol/L,²¹ and the [Na⁺]_i is 24 mmol/L.³² If this holds true in intact endothelium, when [Na⁺]_o is 140 mmol/L and [Ca²⁺]_o is 1 mmol/L, the reversal potential for the exchanger (E_Ca/Na) is calculated to be -110 mV, which is considerably more negative than E_m. Thus, the Na⁺-Ca²⁺ exchange in vivo most likely operates in a net Ca²⁺-influx mode at rest. However, activation of Ca²⁺ entry mode of the exchange requires internal Ca²⁺. This portion of internal Ca²⁺ is not transported by the exchanger and allosterically activates exchanger in the Ca²⁺ influx mode of operation.⁷¹ We found that Na⁺ substitution does not decrease [Ca²⁺]_i, which implies that the Na⁺-Ca²⁺ exchanger might be relatively dormant in the resting condition unless the [Ca²⁺]_i is elevated to a certain level. This is different from the Na⁺-K⁺ pump, which normally operates at about half-maximal velocity in resting cells.⁷² The threshold of [Ca²⁺]_i for activation of the Na⁺-Ca²⁺ exchanger is different in different types of cells. When the cells are activated and [Ca²⁺]_i rises to a certain level, the exchanger is activated; the direction of net Ca²⁺ movement is then determined by whether V_m is more positive (Ca²⁺ influx) or more negative (Ca²⁺ efflux) than E_Na/Ca.⁴² We have demonstrated in intact endothelial cells a transient [Ca²⁺]_i increase evoked by merely substituting external Na⁺ with NMDG, which is different from that in cultured endothelial cells, where external Na⁺ removal did not increase [Ca²⁺]_i³⁰ unless the [Na⁺]_i was first elevated by monensin and/or ouabain.²⁷ The Na⁺-Ca²⁺exchange mediates Ca²⁺ flux, which showed a transient [Ca²⁺] increase when the Na⁺ gradient was reversed by sudden removal of Na⁺. The decline of [Ca²⁺]_i during the period of Na⁺ replacement could be due to the fact that Na⁺ leaves the cell during the exposure to zero external Na⁺ solution, which will lead to a reduction of Ca²⁺ entry via the exchange, and the level to which it declined was equal to that before the maneuver. The observation that the removal of external Na⁺ did not change the steady state [Ca²⁺]_i after the transient [Ca²⁺]_i increase supports the view that at rest Na⁺-Ca²⁺ exchange is dormant; moreover, Ca²⁺ is likely to be extruded via some Na⁺-independent mechanism (eg, Ca²⁺, Mg²⁺-ATPase).⁷³

Since the activity of the Na⁺-Ca²⁺ exchanger is critically dependent on both [Ca²⁺]_i and [Na⁺]_i, the effect of Na⁺-Ca²⁺ exchange was tested under various levels of [Na⁺]_i and [Ca²⁺]_i. To increase [Na⁺]_i, the Na⁺-K⁺ pump inhibitor ouabain was applied. Both electrophysiological measurements⁷⁴ and influx measurements⁷⁵ have shown the existence of ouabain-sensitive Na⁺,K⁺-ATPase in endothelial cells. Inhibition of the Na⁺ pump will cause depolarization,⁷⁴ which could decrease [Ca²⁺]_i by reducing the electrochemical gradient for Ca²⁺ entry (E_m-E_Ca).²⁷ However, depolarization would favor Ca²⁺ entry via Na⁺-Ca²⁺ exchange, especially when [Na⁺]_i is increased.^{40 41 76} In cultured endothelium, ouabain was without effect on [Ca²⁺]_i, which was interpreted to mean that the increase in [Na⁺]_i that is induced by ouabain possibly promotes Ca²⁺ entry (or inhibits Ca²⁺ extrusion) via the Na⁺-Ca²⁺ exchange, but that is offset by the decrease in Ca²⁺ entry through a leak pathway resulting from the depolarization after Na⁺ pump current inhibition. A Na⁺-K⁺-Cl^- cotransport mechanism in endothelial cells⁷⁷ might also help limit the increase in [Na⁺]_i in the presence of ouabain. Without a sufficient increase in [Na⁺]_i, the Na⁺-Ca²⁺ exchange may be unable to transport Ca²⁺ into the cell at a high enough rate to overcome other Ca²⁺ buffering systems. We have found that in intact endothelium, ouabain caused an increase in fluorescence ratio at rest. Combined with the finding that removal of external Na⁺induced a transient elevation in [Ca²⁺]_i in intact but not cultured endothelial cells, these data indicate that Na⁺-Ca²⁺ exchange has a more prominent role in the intact endothelium than in cultured endothelial cells.

An alternative method for inducing an increase in [Na⁺]_i is application of the Na⁺ ionophore monensin. It resulted in an elevated basal ratio signal. Under this condition, the Na⁺ substitution resulted in a further increase in fluorescence ratio that relaxed to a level higher than that before Na⁺ removal. Na⁺ substitution during agonist stimulation also led to a prolonged elevation in the fluorescence ratio. The reason that [Ca²⁺]_i remained high for a longer period of time may be that after application of monensin and agonist, [Na⁺]_i was dissipated more slowly. The agonist application in itself may increase Na⁺ influx via nonselective surface membrane channels and thereby increase [Na⁺]_i, change the Na⁺ gradient, and finally increase [Ca²⁺]_i via Na⁺-Ca²⁺ exchange. Na⁺-Ca²⁺ exchange may thus play a physiological role when [Ca²⁺]_i is elevated.

Endothelial cells have also been shown to possess Na⁺-H⁺exchange, which could affect pH_i when cells were exposed to NMDG or monensin and indirectly affect [Ca²⁺]_i. Our findings that the Na⁺-H⁺ exchanger blocker HMA was ineffective but that the blockade of Na⁺-Ca²⁺ exchange with DCB and Mg²⁺ was highly effective in blocking the transient increase of fluorescence ratio caused by Na⁺ substitution with NMDG confirm the involvement of the Na⁺-Ca²⁺ exchanger. In conclusion, the Na⁺-Ca²⁺ exchanger was shown to be present in the intact endothelium and appears to play a physiological role related to agonist-induced [Ca²⁺]_i signaling.

	Acknowledgments

 
This study was supported by a grant from Medical Research Council of Canada (5-91189-3220).

Received April 8, 1994; accepted November 28, 1994.

	References

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