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(Circulation Research. 1995;76:922-924.)
© 1995 American Heart Association, Inc.

Articles

Simultaneous Measurements of Ca²⁺ and Nitric Oxide in Bradykinin-Stimulated Vascular Endothelial Cells

Lothar A. Blatter, Ziad Taha, Stefan Mesaros, Philip S. Shacklock, Withrow G. Wier, Tadeusz Malinski

From the Department of Physiology (L.A.B.), Loyola University Chicago, Maywood, Ill; the Department of Chemistry and Institute of Biotechnology (Z.T., S.M., T.M.), Oakland University, Rochester, Mich; and the Department of Physiology (P.S.S., W.G.W.), University of Maryland, Baltimore, Md.

Correspondence to Tadeusz Malinski, PhD, Department of Chemistry, Institute of Biotechnology, Oakland University, Rochester, MI 48309-4401.

	Abstract

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Abstract The production of endothelium-derived relaxing factor (EDRF), known to be nitric oxide (NO), is triggered by a rise in the cytoplasmic calcium concentration ([Ca²⁺]_i) subsequent to receptor binding of vasoactive agonists. In vascular endothelial cells, NO is synthesized from L-arginine by the Ca²⁺/calmodulin–dependent NO synthase. In this study, we report the first simultaneous measurements of [Ca²⁺]_i and [NO] at the level of single endothelial cells. In cultured bovine aortic endothelial cells, extracellular application of bradykinin (BK, 10 to 20 µmol/L) caused transient (sometimes oscillatory) increase in [Ca²⁺]_i, which was measured with the fluorescent Ca²⁺ indicator fura 2 and fluorescence imaging microscopy. BK caused an increase in [Ca²⁺]_i, primarily through release from intracellular stores. Under identical experimental conditions, BK caused a transient increase in [NO], which was measured by application of a porphyrinic NO microsensor. [NO] peaked at 0.5 µmol/L. Simultaneous measurements of [Ca²⁺]_i and [NO] in BK-stimulated endothelial cells revealed that a transient increase in [Ca²⁺]_i was rapidly followed by an increase in [NO] that outlasted the [Ca²⁺]_i transient.

Key Words: endothelium-derived relaxation factor/nitric oxide • bradykinin • cytoplasmic calcium concentration • porphyrinic NO microsensor • fura 2

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Endothelial cells modulate the contraction of vascular smooth muscle cells by releasing factors that cause relaxation or contraction.^{1 2} The endothelial cell layer has been referred to as a "transducing surface."³ It transduces shear stress (flow) and signals the presence of many substances in the blood, and it is known to be intimately involved in the pathophysiology of atherosclerosis, coronary vasospasm, and coronary thrombosis.^{4 5}

Endothelium-derived relaxation factor (EDRF) was discovered more than a decade ago⁶ and recently has been confirmed to be identical to nitric oxide (NO). NO has been determined to be a unique, ubiquitous messenger of cellular signals. NO is not only involved in the regulation of blood pressure but also has been characterized as a neurotransmitter and plays an important role in the immune system.⁷ Its chemical nature makes NO an excellent candidate for short-term and short-range signaling: NO is very lipophilic (and therefore diffuses readily through cellular membranes), it is synthesized rapidly on demand, and its short life ensures a localized response.⁸

NO is synthesized in vascular endothelial cells from L-arginine by the Ca²⁺/calmodulin–dependent enzyme NO synthase (NOS). NOS is fully activated by [Ca²⁺]_i that is also encountered as a result of exogenous stimulation with various agonists such as the vasoactive peptide bradykinin (BK).⁹ Although there is substantial indirect evidence that synthesis and release of NO by endothelial cells is [Ca²⁺]_i dependent, experimental limitations have heretofore precluded the direct simultaneous in situ measurements of [NO] and [Ca²⁺]_i. Recently, a porphyrinic microsensor has been developed¹⁰ with characteristics, namely dimensions, response time, sensitivity, and selectivity, for NO, and it has been applied to the in situ monitoring of NO release from single cells.

In this study we report for the first time the simultaneous measurements of [NO] and [Ca²⁺]_i obtained from individual vascular endothelial cells. [NO] was measured either intracellularly or directly from the cell surface by placing a flexible NO microsensor onto the cell membrane. BK caused transient increases of [Ca²⁺]_i that were followed by longer-lasting transient increases of [NO]. A 20 µmol/L [BK] caused [NO] transients that peaked at approximately 0.5 µmol/L.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Cultured endothelial cells were derived from fetal bovine aorta. The cells were cultured in minimal essential medium (MEM) with 10% fetal bovine serum and 0.004% gentamicin. During the experiments, the cells were kept at 37°C in an atmosphere of 5% CO₂/95% air and were passaged twice a week by enzymatic (trypsin) procedure. For experimentation the cells were plated on glass coverslips and grown to confluence. The cells were bathed in a physiological salt solution of the following composition (mmol/L): NaCl 135, KCl 4, MgCl₂ 1, CaCl₂ 2, dextrose 10, L-arginine 1, HEPES 10, titrated to pH 7.3 with NaOH (3.9 mmol/L). In the nominally Ca²⁺-free solution, the addition of CaCl₂ was omitted. BK and L-arginine were obtained from Sigma.

[Ca²⁺]_i was measured with the fluorescent calcium indicator fura 2, using the membrane-permeant fura 2 acetoxymethyl ester (fura 2-AM). The cells were loaded with the indicator by exposure to a physiological salt solution containing 0.5 µmol/L fura 2-AM for 30 minutes. Dye loading and experiments were carried out at room temperature (20°C). For the [Ca²⁺]_i measurements, fura 2 fluorescence was excited at 360 and 380 nm. Although no intracellular calibration was obtained, the fura 2 ratio signal was translated into a calcium concentration term, using calibration solutions with a typical intracellular ionic background and containing fura 2 pentapotassium salt, in order to provide an approximate quantification of the observed changes in [Ca²⁺]_i. The experimental setup for fluorescence microscopy and digital imaging of [Ca²⁺]_i has been described in detail previously.^{11 12} Its main components are a charge- coupled device (CCD) camera (TM-745E, PULNiX America Inc) fiber-optically coupled to a microchannel plate intensifier (Philips Electronic Instruments Inc) and a real-time image processor (series 151, Imaging Technology, Inc) under the control of a microcomputer. Typically [Ca²⁺]_i images were obtained every 10 s, calculated from an average of eight successive background-subtracted video frames recorded at both excitation wavelengths.

Several microsensors were prepared as described previously.¹⁰ Briefly, a single sharpened carbon fiber (0.5 to 7.0 µm diameter) encapsulated in a glass capillary with 1 mm protruding (for the extracellular measurements) was modified by coating with nickel(II) tetrakis(3-methoxy-4-hydroxyphenyl)porphyrin by use of cyclic scanning at a potential between -0.2 and 1.0 V. The polymeric porphyrin was subsequently coated by dipping for 10 s in 1% Nafion solution and left to dry in air. Prior to the electrochemical measurements of NO, the electrode was placed on the surface of a cell and a constant potential of 0.65 V (versus saturated calomel electrode) was applied using an EC 225 IBM voltametric analyzer and current-sensitive preamplifier. The amperograms were recorded on an Omniscribe strip chart recorder. Standard solutions of NO were prepared as decribed previously.¹³ Data are mean±SEM of n separate experiments and were analyzed by ANOVA. Post hoc tests of the significance of difference among individual means were performed using Tukey's procedure. Significance was established at P<.05.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
BK-Evoked [Ca²⁺]_i Transients in Fetal Bovine Aortic Endothelial Cells
The vasodilator BK was used to stimulate [Ca²⁺]_i production, which was measured with fluorescence imaging microscopy in cultured fetal bovine aortic endothelial cells. BK has been shown to increase [Ca²⁺] in endothelial cells originating from various vascular beds.¹⁴ In the presence of extracellular calcium ([Ca²⁺]_o=2 mmol/L), repetitive stimulation with BK (10 µmol/L) at short intervals caused [Ca²⁺]_i transients of decreasing amplitude (Fig 1). After removal of extracellular calcium, the [Ca²⁺]_i transient evoked by BK initially remained the same. However, repetitive stimulation under Ca²⁺-free conditions resulted in [Ca²⁺]_i transients that were attenuated and eventually abolished. From these experiments, it was concluded that BK-evoked [Ca²⁺]_i transients were due primarily to release of Ca²⁺ from intracellular stores. The decreasing amplitude of the [Ca²⁺]_i transients in the presence of extracellular Ca²⁺ may be related to incomplete refilling of the stores during repetitive stimulations at short intervals and/or partial desensitization of the surface membrane receptor for BK.

View larger version (14K): [in this window] [in a new window]   Figure 1. Bradykinin-evoked [Ca2+]i transients in vascular endothelial cells. Cultured vascular endothelial cells were repetitively stimulated with 10 µmol/L bradykinin in the presence (2 mmol/L) and absence of extracellular Ca2+.

Simultaneous Measurements of [NO] and [Ca²⁺]_i
Fig 2 shows a group of confluent endothelial cells loaded with fura 2. Fig 2A shows the fura 2 fluorescence excited at 380 nm. Fig 2B shows the same group of cells in bright-field illumination and the position of the NO sensor on a cell's surface. The simultaneous measurements of [NO] and [Ca²⁺]_i were done with a porphyrinic sensor and fluorescence imaging microscopy, respectively. BK-stimulated endothelial cells revealed a transient increase of [NO] that outlasted the [Ca²⁺]_i transient. A typical concentration-time profile obtained during simultaneous measurements of [NO] and [Ca²⁺]_i is shown in Fig 3. Addition of BK (20 µmol/L) initiated an increase in [Ca²⁺]_i within 3.0±0.4 s (n=5) (Fig 3A). A maximum concentration of 1.0±0.1 µmol/L was reached over 10±1 s. The peak [Ca²⁺]_i increase was followed by a small decrease, with formation of a semiplateau at a level of 0.9 µmol/L. The semiplateau was observed for about 50±5 s, after which [Ca²⁺]_i declined to near basal levels after about 350±20 s.

View larger version (74K): [in this window] [in a new window]   Figure 2. Fluorescence imaging microscopy (A) and bright-field (B) images of the same group of fetal bovine aortic endothelial cells. A, Image of fura 2 fluorescence recorded from the group of confluent cultured endothelial cells. Fura 2 fluorescence was excited at 380 nm, and the emitted signal was recorded at 510 nm. B, Bright-field image of the same group of cells showing the NO microsensor in place.

View larger version (17K): [in this window] [in a new window]   Figure 3. Graph shows concentration-time profile obtained during simultaneous measurements of [NO] and [Ca2+]i in a group of endothelial cells stimulated with bradykinin (20 µmol/L). [NO] was measured from the surface of the single cell.

NO was detected by the porphyrinic sensor after 8.0±0.5 s (n=5) following the addition of BK (Fig 3B). This was a delay of 5 s from the release of detectable amounts of Ca²⁺. The initial rate of NO release was 8.0±0.7 nmol/L per second. After 15 s, the rate of NO release decreased to 0.5±0.1 nmol/L per second, and a plateau was established after 175±20 s that lasted for about 200±50 s. After that time, [NO] decayed slowly with a rate of 0.8±0.2 nmol/L per second and reached an undetectable level after 14 minutes. The time of appearance of a detectable signal of Ca²⁺ or NO depends on several factors. The major factor is mass transport of BK (diffusion/convection) from the point of its injection to the cell membrane. Other factors are interaction with receptors, signal transduction, release and diffusion of Ca²⁺, and synthesis and diffusion of NO. The mass transport of BK depends on its gradient of concentration between the point of injection and the cell surface. Therefore, the mass transport of BK results in a lag time between the time of injection and the time at which the analytical signal is recorded at the cell surface. There was an observed decrease in time between injection and measurement of the analytical signal as the applied concentration of BK increased (15±2 s and 8.0±0.7 s for BK concentrations of 0.1 µmol/L and 20 µmol/L, respectively). However, the maximum [NO] remains approximately the same in the concentration range of 0.1 to 20 µmol/L of BK. The contribution of the response time of the sensor (10 ms for [NO] of 10 nmol/L and 1 ms for [NO] of 1 µmol/L) is negligible in comparison with the observed time of the appearance of detectable NO signal. The concentration-time profile of [Ca²⁺]_i suggests that a rapid and transient increase is sufficient to activate NOS. However, there is apparently no need for the continuous presence of increased [Ca²⁺]_i for the surge in NO production.

[NO], when monitored on the cell membrane after stimulation with BK, can be observed for a time interval of several minutes, which coincides with previous observations for porcine aorta endothelial cells.¹⁰ NO synthesis and release from cultured aortic endothelial cells was measured with the porphyrinic microsensor either from the surface of the cells or intracellularly after impalement of the microelectrode into a single cell. A difference between surface [NO] and intracellular [NO], with a lower concentration in the cytoplasm, was observed only in the first few seconds of the NO release process. After that, the surface and intracellular concentrations reached similar levels of 0.50±0.05 µmol/L. As previously reported,⁸ the cell membrane does not present a barrier to the diffusion of NO, and NO tends to be preconcentrated in the hydrophobic environment of the membrane. The concentration in the membrane creates a high concentration gradient on both sides of the membrane. This gradient facilitates an efficient diffusion-controlled supply of NO to adjacent cells. Detection of NO on the surface of the cell membrane, the location with the highest level of [NO], has been found to be an efficient, accurate, and reproducible method of measuring NO release. Therefore, this method was used in the studies being presented. The profile of the NO concentration-time curve depends on the kinetics of NO release (supply) and the dynamics of NO mass transport, which is controlled by depletion or dilution of NO and its consumption due to chemical reactions. The time of duration of the plateau depends on these same three factors, and it can also be affected by NO production by neighboring cells. Generally, a maximum concentration measured on the membrane of a single isolated cell or a single cell within a group of cells will be identical within an experimental error. However, the time of duration of the plateau is shorter when the measurements are done on a single isolated cell. The depletion process (diffusion/convection), which is due to a higher gradient of [NO], will be faster for the isolated cell than for the same cell located within a group of cells.

	Acknowledgments

 
Financial support was provided by grants from the National Institutes of Health; the American Heart Association, Maryland Affiliate, Inc; Cassella AG; and the Biotechnology Research Program, Oakland University. We wish to thank Dr Robert Koos, Department of Physiology, University of Maryland, Baltimore, Md, who provided the cell cultures for these studies.

Received September 9, 1994; accepted December 29, 1994.

	References

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Blatter, L. A. Articles by Malinski, T. Search for Related Content PubMed PubMed Citation Articles by Blatter, L. A. Articles by Malinski, T. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL NITRIC OXIDE