Cardiovascular Research

Cardiovascular Research 1997 36(3):437-444; doi:10.1016/S0008-6363(97)00197-1
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Indomethacin enhances endothelial NO release — evidence for a role of PGI₂ in the autocrine control of calcium-dependent autacoid production

Steffen-Sebastian Bolz^* and Ulrich Pohl

Institute of Physiology and Pathophysiology, Johannes-Gutenberg-University Mainz, Duesbergweg 6, D-55099 Mainz, Germany

^* Corresponding author. Tel. (+49-6131) 395212; Fax (+49-6131) 395644; E-mail: bolz@mzdmza.zdv.uni-mainz.de

Received 28 January 1997; accepted 26 June 1997

	Abstract

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Objective: We studied whether NO or prostacyclin (PGI₂), which are continuously released by endothelial cells, have autocrine/paracrine effects on the calcium-dependent autacoid production by modulating the intracellular Ca²⁺ concentration ([Ca²⁺]_i). Methods: Histamine(His)-induced [Ca²⁺]_i increases (Fura 2-method) and NO-dependent cGMP increase were measured in human umbilical vein endothelial cells (HUVECs) before and after cyclooxygenase inhibition or application of cAMP- and cGMP-elevating drugs. Results: 0.3 µM His increased endothelial [Ca²⁺]_i from 77±2 nM to 418±59 nM. The His-induced [Ca²⁺]_i increases were significantly attenuated following treatment with PGI₂ (by 23%) and forskolin (by 33%), both increasing the cAMP release from HUVECs (by 49% and 66%). The His-induced [Ca²⁺]_i increases were inhibited by the protein kinase A-activator cBIMPS (by 61%) which also abolished the His-induced PGI₂ release. Conversely, inhibition of the PGI₂ production with indomethacin significantly augmented the His-induced [Ca²⁺]_i increases (by 32%), resulting in a significantly augmented NO production as indicated by an enhanced LNNA-sensitive cGMP increase in HUVECs. In contrast, neither increases of cGMP (basal 0.4±0.1 pmol/mg) elicited by 10 µM SNP (21±2 pmol/mg) or 10 µM C-type natriuretic peptide (CNP, 4.6±1.6 pmol/mg) nor its reduction by 30 µM LNNA had any effect on the His-induced [Ca²⁺]_i increases. Conclusion: PGI₂ attenuates agonist-induced [Ca²⁺]_i increases by a cAMP-dependent mechanism, thereby modulating not only its own synthesis via a negative feedback but also that of NO. Consequently, reduced PGI₂ levels result in an increased NO production. NO which does not cause a negative feedback control by cGMP might therefore compensate for the lack of PGI₂.

KEYWORDS HUVECs; Histamine; cAMP; cGMP; Feedback

	1 Introduction

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Nitric oxide (NO) and prostacyclin (PGI₂) are continuously released by endothelial cells, particularly when exposed to the physical stimuli stretch [1]and shear stress [2, 3]. The tonic release of NO plays an important role in the control of basal vascular tone. Consequently, the inhibition of nitric oxide synthesis with N-nitro-L-arginine (LNNA) leads to general vasoconstriction and subsequent blood pressure increases [4]. In contrast, with a few exceptions, systemic blood pressure remains unaffected if the synthesis of the vasodilator PGI₂ [6, 7]is blocked by cyclooxygenase inhibitors [5]although it is also continuously released as indicated by its tonic effects on platelet cAMP [8]. One possible explanation for the lack of a pressure increase following cyclooxygenase inhibition would be that the attenuation of PGI₂ production is compensated for by the enhanced release of another vasodilator, e.g. NO. Such a compensatory mechanism would require that PGI₂ normally attenuates the activity of nitric oxide synthase (NOS) or modulates the signal transduction cascade leading to the activation of NOS. While there is no evidence for the former, experimental data (mostly obtained in smooth muscle and in platelets [9–11]) suggest that PGI₂ is involved in the regulation of endothelial cytosolic free calcium concentration ([Ca²⁺]_i) by altering the cellular levels of cAMP [12–14]. This autocrine or paracrine control by cAMP could be part of a negative feedback mechanism controlling the NO synthesis, since the key enzyme, the endothelial NO synthase [eNOS], is mainly activated in a calcium-calmodulin-dependent manner [15].

Autocrine effects of PGI₂ on endothelial cAMP levels and related effects on endothelial [Ca²⁺]_i have been shown previously [13, 16]. However, in these studies high concentrations of stable autacoid analogues and additional inhibition of the phosphodiesterases were required to modulate [Ca²⁺]_i. Thus the question remains, whether cAMP still plays a significant role for the regulation of [Ca²⁺]_i in a more physiologic situation. Furthermore, the possible autocrine effects of PGI₂ on the [Ca²⁺]_i-dependent synthesis of NO might be modified by simultaneous feedback effects of NO and cGMP on endothelial cells. This potential mutual interaction of both autacoids at the endothelial level is still poorly understood.

The aim of the study was to analyze the role of PGI₂ and cAMP in the control of endothelial NO production. Furthermore it was asked whether NO, via cGMP-dependent steps, could influence the agonist-induced changes of [Ca²⁺]_i in endothelial cells. This enables to analyze a potential mechanism at endothelial cell level, which could increase NO production to compensate for the lack of PGI₂ but fail to compensate for a reduced or lacking NO production. We therefore compared the effects of both cyclic nucleotides on [Ca²⁺]_i in HUVECs and studied the agonist-induced [Ca²⁺]_i changes as well as the release of NO in the presence and absence of PGI₂ and other agents involved in the cAMP pathway.

	2 Materials and methods

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
2.1 Cell culture
Endothelial cells from human umbilical veins were prepared following a protocol described previously [17]with slight modifications. Briefly, fresh human umbilical veins were filled with Dispase II (2.4 U/ml, 37°C, Boehringer, Germany) and diluted in Mg²⁺ and Ca²⁺-free phosphate buffered saline (PBS). After an incubation time of 30 min the veins were perfused with PBS to wash out the removed cells. These were separated from the perfusate by centrifugation at 100 rpm for 5 min. After resuspension in culture medium they were seeded onto Fibronectin-coated (5 µg/cm², Boehringer] 24-well culture plates (NUNCLON). Medium 199 (pH 7.4, Biochrom, Germany) containing 25% fetal calf serum (FCS, Boehringer), 50 U/ml penicillin (Sigma, Deisenhofen) and 50 µg/l streptomycin (Sigma, Deisenhofen) was used. Confluent monolayers showed a cobblestone pattern, which is typical for endothelial cells, within 3 days. In addition, the endothelial cells were characterized by their ability to uptake acLDL [18]. For all experiments only cells from primary cultures were used.

2.2 Determination of intracellular free calcium ([Ca²⁺]_ii)
The concentration of [Ca²⁺]_i was determined using the Fura 2 method [19]on a microscope-based photometer (Photomed, Wedel, Germany). The culture plates with endothelial cells were transferred to the stage of an inverted microscope (IMT-2, Olympus, Japan) and incubated for 1 h at 37°C with HEPES-buffer (see Section 2.4) containing Fura 2-AM (stock solution 1 mg in 1 ml dimethyl sulfoxide, Molecular Probes, Oregon, USA) in a final concentration of 2 µM and 0.5% bovine serum albumin (BSA). Before starting the measurements the cells were rinsed twice with HEPES buffer (37°C) and given 15 minutes for further hydrolysis of the remaining Fura 2-AM. Measurements were performed in the wells of the culture plate with a supernatant of 500 µl buffer. All substances for incubation or stimulation of the cells were added in 250 µl aliquots by exchanging 250 µl of the supernatant. The supernatant's volume was kept constant to ensure artefact-free fluorescence measurements. The excitation wavelength alternated between 340 nm and 380 nm every 0.25 s during recording of the emission wavelength at 510 nm. The photomultiplier (PTI) signal represented the light emission (510 nm) of about 50 cells. All measurements were corrected for autofluorescence of the cells and the carrier medium (culture plate) obtained by quenching the Fura 2 fluorescence with 8 mM MnCl₂ at the end of every experiment. The corrected fluorescence ratio F_{340 nm}/F_{380 nm} was converted to concentrations of [Ca²⁺]_i using a computer-stored calibration curve (PTI calibration software). As determined in randomly selected experiments, the computer-generated values for [Ca²⁺]_i did not differ significantly from values calculated using the equation published by Grynkiewicz et al. [19](nM):

R represents the ratio of the (corrected) fluorescence values at 340 nm and 380 nm. R_min and R_max are the minimal and maximal ratio values determined by sequential addition of ionomycin (10 µM) and EGTA (5 mM). β represents the fluorescence ratio at 380 nm of free Fura 2 and calcium-saturated Fura 2. According to Grynkiewicz et al. the dissociation constant of the Fura 2-calcium complex, K_d, was assumed to be 224 nM at 37°C.

2.3 Determination of the cyclic nucleotides cGMP and cAMP
The basal and stimulated cyclic nucleotide release was determined in the same cells by measuring the cAMP or cGMP concentration in the supernatant, which was freshly added with or without the respective agonist and removed after 2 min. The release was calculated from the absolute nucleotide content divided by the exposure time (2 min).

Since the determination of intracellular cyclic nucleotide concentrations in resting and stimulated cells required their lysis, these concentrations had to be measured in different cells. After 2 min incubation without treatment (basal) or after 2 or 5 min exposure to agonists, the cells were lysed by incubating them with trichloroacetic acid (TCA, Sigma, 6% in HEPES buffer solution). The cell lysate was kept on ice for 30 min. After extraction of TCA with water saturated ether (4x) the samples were kept frozen (–20°C) until they were analyzed. Cyclic AMP and cGMP were determined using a commercially available radioimmunoassay (New England Nuclear, Dreieich, FRG). Cyclic GMP was acetylated before assaying.

Values of cAMP and cGMP were normalized to total cell protein per well as determined by the Lowry method [20].

2.4 Drugs
The HEPES-buffered salt solution was composed as follows (concentrations in mmol/l): HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethansulfonic acid) 10, NaCl 144, KCl 4, CaCl₂ 1.6, MgCl₂ 1, NaH₂PO₄ 0.4 and glucose 10 (pH adjusted to 7.4 by titration with 1 M NaOH).

Bradykinin, histamine, sodium nitroprusside (SNP), C-type natriuretic peptide (CNP), forskolin, isobutylmethylxanthine (IBMX) and EGTA were purchased from Sigma (Deisenhofen, Germany), ionomycin from Calbiochem (La Jolla, CA, USA), 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-3'-5'-cyclo-mono-phosphorothioate (cBIMPS) from Biolog (Bremen, Germany), N-nitro-L-arginine (LNNA) from Serva (Heidelberg, Germany), bovine serum albumin from Biomol (Hamburg, Germany), 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethansulfonic acid (HEPES), MnCl₂ and trichloroacetic acid (TCA) from Merck (Darmstadt, Germany). PGI₂ (Flolan^®) was a generous gift from Wellcome (London, UK).

All buffers and solutions were freshly prepared.

SNP (stock solution 10 mM in Na acetate) and PGI₂ (stock solution 1 mM in glycine buffer) were diluted in HEPES buffer solution to their final concentration immediately before use. All control groups received treatment with the solvent alone.

All concentrations given in the text refer to final concentrations in the cell supernatant.

2.5 Statistical analysis
All data are presented as means±SEM (n denotes the number of experiments). The intracellular concentrations of the cyclic nucleotides were compared by Student's t-test for non-paired data since they were obtained from different cells. The data about cyclic nucleotide release, which were obtained from the same cells before and after stimulation (see protocols) were compared by the t-test for paired data.

For comparing changes of [Ca²⁺]_i over time under different treatments, a non-linear regression analysis was employed. Briefly, the goodness of the fit to a Gompertz function was calculated first for every individual curve and then after pooling the two data sets. The individual curves were considered to be significantly different if the F-test indicated a significantly smaller sum of squares for the deviations in each individual fit as compared to the deviation in the fit to the pooled data [21]. Single values at the different times were subsequently compared using the Student's t-test for paired data.

Differences were considered to be significant at error probabilities less than 0.05 (p<0.05).

	3 Results

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
3.1 Agonist-induced increases of [Ca²⁺]_i
Histamine and bradykinin increased [Ca²⁺]_i in HUVECs dose-dependently (data not shown). Within the same cells histamine (n = 9, 0.3 µM) increased [Ca²⁺]_i from basal 77±2 nM to 418±59.5 nM and bradykinin (n = 9, 3 nM) from basal 74±4 nM to 391±57 nM. For all experiments regarding the influence of cyclic nucleotides on agonist-induced [Ca²⁺]_i changes, those concentrations of histamine and bradykinin were used that increased [Ca²⁺]_i to a comparable extent. The intracellular calcium changes in response to histamine as shown in Fig. 1 had a characteristic kinetic, with an initial peak reached within seconds followed by a sustained plateau phase.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 The histamine-induced [Ca2+]i increase under control conditions (, n = 8) was significantly reduced (by 61%) after activation of protein kinase A with cBIMPS (500 µM, , n = 8). Furthermore, cBIMPS significantly prolonged the time period required to reach the maximal [Ca2+]i concentration (initial peak). (*=p<0.05 control vs. cBIMPS treatment).

 
3.2 Influence of PGI₂, forskolin and cBIMPS on agonist-induced [Ca²⁺]_i increase
Preincubation with PGI₂ (0.1 µM, 2 min) did not influence resting calcium levels but did reduce the bradykinin-induced [Ca²⁺]_i peak in HUVECs by 23% from 386±62 nM to 296±48 nM (p<0.005, n = 19). Virtually the same results were obtained in three experiments using 0.3 µM histamine as stimulus (data not shown). A 2 min preincubation of the cells (n = 7) with 0.1 µM PGI₂ did not significantly increase the intracellular cAMP (from 55±3 to 56±1 pmol/mg_protein). The cAMP release from HUVECs, however, increased significantly following a 2 min stimulation period with PGI₂ (from 3.9±0.4 to 5.8±0.4 pmol/min/mg_protein, p<0.005, n = 14).

To study further a potential role of cAMP and cAMP-dependent protein kinase (cAMP-Pk) in the control of agonist-induced increases of [Ca²⁺]_i, the adenylyl cyclase stimulator forskolin and the cAMP-Pk activator cBIMPS were applied. Incubation of the cells with 10 µM forskolin for 2 min elevated intracellular cAMP from 53.6±7.2 to 85.5±11.3 pmol/mg_protein (p<0.05, n = 12) and the cAMP release from 4.2±0.4 to 7±0.7 pmol/min/mg_protein (p<0.005, n = 12). Forskolin also reduced histamine-induced [Ca²⁺]_i increases by 33% (p<0.05) but it did not affect resting [Ca²⁺]_i levels. Preincubation with cBIMPS (500 µM, 1 min) reduced the histamine-induced [Ca²⁺]_i increases significantly from 313±13 nM to 120±16 nM (i.e. by 61%, p<0.005, n = 8) and significantly prolonged the time period needed to reach maximal [Ca²⁺]_i (Fig. 1). The histamine-induced increase in PGI₂ release (by 40±3% under control conditions, p<0.005, n = 16) was completely abolished in cBIMPS-pretreated cells.

3.3 Effects of cyclooxygenase inhibition on agonist-induced [Ca²⁺]_i increase and NO production
After preincubation of HUVECs with the cyclooxygenase inhibitor indomethacin (30 µM, 30 min) the PGI₂ concentration in the supernatant was significantly reduced from 1928±300 pmol/ml to 113±23 pmol/ml (p<0.005, n = 7). Indomethacin also completely abolished the increase of extracellular PGI₂ by histamine (control increase from 1928±300 pg/ml to 3462±561 pg/ml, p<0.005, n = 7).

Pretreatment of the cells with indomethacin enhanced the histamine-induced initial [Ca²⁺]_i increases significantly (p<0.05, Fig. 2). [Ca²⁺]_i-levels during the plateau phase were not affected by indomethacin treatment. The indomethacin effects on bradykinin-induced [Ca²⁺]_i rises were similar (data not shown).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 The increase of [Ca2+]i by histamine (0.3 µM, , n = 19) was significantly enhanced after inhibition of PGI2 production with indomethacin (, n = 19). (*=p<0.05 control vs. indomethacin treatment).

 
Since arachidonic acid (AA) and AA-metabolites (13-HODE; 14,15 EET; 15-HETE), which reportedly increase [Ca²⁺]_i in smooth muscle cells [22, 23], could accumulate during cyclooxygenase inhibition, we studied the effects of AA on agonist-induced [Ca²⁺]_i increases. Increasing concentrations of AA (0.1–10 µM) were applied for 0.5, 2 and 30 min. After a 30 min incubation period using the highest AA concentration (10 µM) neither resting [Ca²⁺]_i (75±3 nM, n = 7) nor histamine-induced [Ca²⁺]_i increases (398±58 nM, n = 7) were significantly different from control (basal: 75±2 nM; peak: 402±63 nM, n = 7).

In order to study the effects of cyclooxygenase inhibition on the synthesis of NO, we measured the LNNA-sensitive increases of intracellular cGMP. Stimulation with 0.3 µM histamine in the presence of the phosphodiesterase inhibitor IBMX increased cGMP 12-fold after 2 min and 22-fold after 5 min. The increase after 5 min was significantly higher in indomethacin-treated cells (16.9±0.8 pmol/mg_protein) than in control cells (9.7±0.8 pmol/mg_protein). The cGMP increase after histamine was completely blocked by LNNA. (Fig. 3)

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 The histamine(0.3 µM)-induced cGMP increase in HUVECs (Con, n = 8) was significantly enhanced after inhibition of endogenous PGI2 production with indomethacin (Indo, n = 8). The increase was completely inhibited after preincubation of the cells with LNNA (LNNA, n = 8), indicating that it was mediated by NO. (*=p<0.05 control vs. indomethacin treatment).

 
3.4 Effects of histamine, SNP and CNP on intracellular cGMP and [Ca²⁺]_i
Stimulation of human umbilical endothelial cells (HUVECs) with increasing concentrations of histamine (3–300 nM, n = 9) elevated the intracellular levels of cGMP dose-dependently (Fig. 4). Additional unselective inhibition of cellular phosphodiesterases with IBMX (10 µM, 30 min) enhanced the increases in cGMP in response to 0.3 µM histamine further (9.4±1.1 pmol/mg_protein vs. 2.9±0.5 pmol/mg_protein, n = 9, p<0.01). LNNA (30 µM, 30 min) treatment completely abolished the histamine-induced elevations of cGMP.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Agonist-induced increase of cGMP in endothelial cells. The top panel depicts increases of intracellular cGMP following stimulation with increasing concentrations of histamine (3–300 nM, n = 9). Pretreatment with 30 µM N-nitro-L-arginine (LNNA) completely abolished the response to 300 nM histamine (n = 9). Bottom panel: increases of intracellular cGMP following stimulation with C-type natriuretic peptide (CNP, 0.01–10 µM, n = 5). Pretreatment with 30 µM N-nitro-L-arginine did not alter the cGMP.

 
C-type natriuretic peptide (1 nM–10 µM for 120 s) increased cGMP in HUVECs dose-dependently. In contrast to histamine stimulations, LNNA did not reduce the cGMP increase (Fig. 4). Similarly, LNNA did not affect the increases of cGMP elicited by stimulation with 10 µM SNP for 120 s (control: from 0.42±0.07 pmol/mg_protein to 21.9±2.1 pmol/mg_protein, LNNA: 22±2.1 pmol/mg_protein, n = 8).

None of these treatments to modulate cGMP reduced basal [Ca²⁺]_i or histamine-induced [Ca²⁺]_i peaks (Table 1). To exclude the possibility that already the basal levels of cGMP could inhibit increases of [Ca²⁺]_i, cells were pretreated with LNNA for 30 min before being exposed to histamine. Again, no significant effects on histamine-induced [Ca²⁺]_i increases could be detected (Table 1).

View this table: [in this window] [in a new window]   Table 1 Intracellular cGMP levels in HUVECs as modulated by LNNA, SNP and CNP (treatment) neither affected basal [Ca2+]i levels nor histamine (0.3 µM)-induced [Ca2+]i peaks

 
In contrast, SNP treatment of VSMCs using the same protocol reduced the histamine-induced calcium increases by 30% (n = 6, p<0.05). Furthermore the time required to reach peak [Ca²⁺]_i was significantly longer in SNP-treated than in control cells (66±4.7 vs. 33±1.6 s).

	4 Discussion

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
The study shows that the autacoids PGI₂ and NO exert autocrine or paracrine effects on cultured human umbilical vein endothelial cells (HUVECs) by increasing the second messengers cAMP and cGMP. However, increases in cGMP were found not to have any measurable effects either on the intracellular calcium or on the calcium-dependent autacoid synthesis. In contrast, cAMP elevations effectively reduced agonist-induced intracellular calcium increases and subsequently the calcium-dependent synthesis of PGI₂ and NO. An inhibition of PGI₂ production by the cyclooxygenase blocker indomethacin therefore led to augmented agonist-induced [Ca²⁺]_i levels and enhanced NO release. This might provide a cellular pathway to compensate for the lack of the vasodilator PGI₂ in the control of smooth muscle tone (Fig. 5). PGI₂- or NO-dependent increases of cAMP and cGMP in endothelial cells have been demonstrated before in the presence of phosphodiesterase (PDE) inhibitors [24, 25], whereas this study now reveals that the autocrine effects of NO or PGI₂ are sufficient to increase the cyclic nucleotides without concomitant PDE-inhibition. The increase of cAMP in response to PGI₂ was consistently detectable only as an increase in the release of cAMP, which would require cAMP to be increased at least locally within the cells. In fact, following stimulation of the adenylyl cyclase with forskolin, which increased intracellular cAMP to much higher levels than PGI₂, a parallel increase of cAMP release was also detectable. Highly localized cAMP elevations following stimulation with forskolin, PGE₂ and isoproterenol have been demonstrated before in fibroblasts and LLC-PK1 cells [26]. Depending on the ‘background level’ of cAMP, which was high in the endothelial cells studied here, these localized cAMP elevations might not have been detectable any more after cell lysis. However, such local increases in cAMP apparently play an important role in the feedback control of the endothelial autacoid synthesis as discussed below.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Proposed model for the augmented endothelial NO production after inhibition of the cyclooxygenase pathway. Grey arrows depict signal transduction pathways related to the synthesis of NO and PGI2. Black arrows mark the compensatory pathway leading to an enhanced NO/cGMP production after inhibition of the cyclooxygenase.

 
In contrast to the effects of increased cAMP, neither decreases of cGMP below resting levels due to LNNA treatment nor increases elicited by different stimuli (endothelium-derived NO, NO donors and CNP [27, 28]) influenced basal [Ca²⁺]_i or stimulated [Ca²⁺]_i increases in HUVECs. This corresponds to the previously described lack of cGMP effects on thrombin-induced increases of [Ca²⁺]_i in these cells [29]. The same authors reported that cGMP-dependent protein kinase (cGMP-Pk) is not found in freshly isolated or cultured HUVECs [29], which could explain the ineffectiveness of cGMP on [Ca²⁺]_i. It should be noted, however, that endothelial cells of other origin do contain small amounts of cGMP-Pk [30, 31], which is mainly associated with the cytoskeleton. Cyclic GMP-PK may not be involved in the control of [Ca²⁺]_i in this instance either, due to the fact that NO in bovine aortic endothelial cells —in spite of the presence of the enzyme in these cells [30]— had no effect on the calcium-dependent NO release (Bolz and Pohl, unpublished data) [24]. Only recently, it has been described that the reaction product of NO and O₂^–, peroxynitrite, rather than NO itself reduced the agonist-induced increase of [Ca²⁺]_i in calf pulmonary endothelial cells [32]. However, peroxynitrite exerts part of its physiologic effects also by a thiol-mediated activation of soluble guanylyl cyclase [33]and might therefore have been inefficient in our setting. The lack of cGMP effects on the control of [Ca²⁺]_i does not mean that there were no effects of cGMP at all. It is well conceivable that cGMP was effective on other cellular processes such as control of macromolecule permeability by cGMP-Pk-independent pathways [34].

In contrast to cGMP, elevations of intracellular cAMP, induced by the adenylyl cyclase activator forskolin, reduced agonist-induced [Ca²⁺]_i increases in endothelial cells. The [Ca²⁺]_i increases were also reduced by cBIMPS, an activator of the cAMP-dependent protein kinase (cAMP-PK) [35]. This suggests that cAMP-Pk is functionally integrated into the control of [Ca²⁺]_i by mediating the effects of cAMP on target proteins. In the same way as forskolin and cBIMPS, the physiological vasodilator PGI₂ reduced [Ca²⁺]_i, although the effects were smaller in magnitude. Conversely, inhibition of PGI₂ synthesis with indomethacin resulted in an augmentation of agonist induced increases of [Ca²⁺]_i. All PGI₂ effects on [Ca²⁺]_i ran in parallel to changes of cAMP release from the cells, reflecting localized changes of intracellular cAMP as previously discussed. It is of note that exogenous PGI₂ had to be applied in 10-fold higher concentrations than those measured in the supernatant of HUVECs following stimulation with histamine or bradykinin in order to elicit similar effects on [Ca²⁺]_i and cAMP. Similar observations have been reported by other authors [36, 37]. The requirement for higher concentrations most likely reflects an auxiliary role of prostaglandins other than PGI₂, which may be released from stimulated HUVECs and may contribute to the control of cellular cAMP.

The cAMP-mediated modulation of agonist-induced [Ca²⁺]_i increases by PGI₂ had in fact distinct effects on the calcium-dependent PGI₂ and NO synthesis. For technical reasons we were unable to study the effects of exogenous PGI₂ on the PGI₂ release from endothelial cells. However, the cBIMPS-induced reduction of [Ca²⁺]_i increases was associated with a complete abolition of the agonist-induced PGI₂ release, suggesting that the PLA₂ activity is regulated via cAMP-dependent control of [Ca²⁺]_i. Our results are consistent with the view that PGI₂ controls its own synthesis via a cAMP-dependent negative feedback mechanism. Therefore the inhibition of the PGI₂ synthesis with indomethacin resulted in augmented increases of [Ca²⁺]_i after stimulation with histamine, which led to an enhanced increase of the intracellular cGMP concentration. Since the latter could be completely blocked by LNNA, it reflects an enhanced production of NO. This enhancement suggests that PGI₂ affects not only its own synthesis but also the [Ca²⁺]_i-dependent NO production.

Similar results were obtained in preliminary experiments where we measured NO directly by an amperometric technique and found an enhanced NO release from indomethacin-treated cells following stimulation with the calcium ionophore ionomycin (Bolz and Pohl, unpublished data). It is of note that the inhibition of PGI₂-synthesis augmented only the initial [Ca²⁺]_i increase, which most likely represents the [Ca²⁺]_i release from intracellular stores. This resulted in a significant enhancement of NO release as derived from an increase of LNNA-sensitive cGMP levels after 5 min. Based on this, we conclude that modulations of the initial [Ca²⁺]_i increase have significant impact on the long term NO output. This corresponds to the [Ca²⁺] concentration–time profiles published by Blatter et al. [38]. These profiles suggest that the initial [Ca²⁺]_i increase is sufficient to activate NOS and that there is no need for the continued presence of increased [Ca²⁺]_i for the surge of NO production. This is in accordance with a recently published study which demonstrated that the eNOS activity is highly sensitive to even small [Ca²⁺]_i changes and that the initial [Ca²⁺]_i increase following stimulation with an agonist participates considerably in eNOS activation [39]. It is, however, surprising that the differences in increases in cGMP between indomethacin-treated and control cells were significant only after 5 min. This finding suggests that under our experimental conditions the translation of [Ca²⁺]_i increase to the cGMP increase was delayed.

To summarise, by attenuating agonist-induced [Ca²⁺]_i increases, cAMP not only modulates the production of PGI₂ but also of NO. Consequently, at low levels of PGI₂, e.g. following inhibition of cyclooxygenase, an increased endothelial NO production occurs. Since this does not underlie a feedback inhibition by cGMP, NO might compensate for the lack of PGI₂ in the control of vascular tone under these circumstances. Further studies in intact organs will have to prove whether the lack of an effect of cyclooxygenase inhibition on vascular resistance is due to this compensatory mechanism.

Time for primary review 21 days.

	Acknowledgements

 
This work was supported by the Deutsche Forschungsgemeinschaft (DFG Po 307/1-3).

	References

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 

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