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Laboratoire d'Etude de la Microcirculation, Département de Biophysique, Hôpital Fernand Widal, Cedex 10, Paris 75010, France
Submitted 19 September 2002 ; accepted in final form 15 April 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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-nitro-L-arginine (L-NNA) or
cyclooxygenase inhibitor mefenamic acid (MA) alone leads to vasoconstriction.
We found that L-NNA after MA induced an additional constriction,
whereas MA after L-NNA induced a relative dilation. Therefore, an
additional constriction was found when MA was administered after
L-NNA in the presence of the thromboxane A2
synthase-PGH2 (TP) receptor antagonist SQ-29548. We also found a
relative dilation when the TP receptor antagonist was administered after NOS
inhibition by L-NNA. In the presence of superoxide dismutase and
catalase, L-NNA-induced vasoconstriction is reduced, and the
dilation observed after addition of MA in presence of the reactive oxygen
species is no longer present. Taken together, these results showed that NO
inhibition induced a shift in the synthesis or in the effects of
cyclooxygenase products, in favor of constrictor prostanoids. This effect of
NO inhibition disappears when reactive oxygen species are scavenged by
superoxide dismutase and catalase.
prostaglandins; oxidative stress; microcirculation; vascular reactivity
For instance, NO has been shown to interfere in various ways with arachidonic acid pathways because it can stimulate cyclooxygenase (COX) activity (28) and more specifically prostaglandin H synthase (6, 11) or prostaglandin I2 synthase (34). In addition to their possible interactions in the endothelial cells, NO and prostanoids can also interact in the vascular smooth muscle cell by the interactions existing between cAMP and cGMP, mainly via the phosphodiesterase III pathway (8).
Interaction of NO with constrictor prostanoids can also occur via an effect on thromboxane A2 (TxA2) synthase (34) or via an uncoupling effect on the TxA2 receptor via activation of G kinase (35).
Complex interactions also exist between NO and the reactive oxygen species (ROS), which also have many effects on vascular regulation (see Ref. 37 for review). For example, NO is known to quench oxygen-derived free radicals, which can constrict vessels (10), but these radicals are responsible for an impaired NO-mediated dilation in pathological situations such as diabetes (20) or hypertension (31). In different cell types, ROS can also modulate activity of prostacyclin-, PGH2-, or thromboxane synthase (37).
Much less is known about these interactions in microcirculation in vivo. Indeed, it has been shown that many physiological mediators such as acetylcholine or bradykinin, inducing a release of NO, also induce a simultaneous release of dilator prostaglandins (1, 21). This corelease has also been reported for several mediators of inflammation or sepsis (12, 29). Interaction of prostaglandins and NO has also been reported in shear stress or flow-dependent dilation (35, 36). It has also been shown in microcirculation that a synergy can exist between the NO-induced increase in smooth muscle cGMP and the prostacyclin-induced increased in cAMP (7, 13).
The present study was carried out by intravital microscopy to explore more specifically the interactions existing in vivo among NO, dilator and constrictor prostanoids, or ROS at the microvascular level. We showed that in physiological conditions, NO is responsible for permanent negative feedback, which restricts the release of constrictor prostanoids, and that the level of ROS might be a key factor in this interaction.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of cremaster. After anesthesia, the right cremaster muscle was surgically prepared for in vivo visualization by a technique proposed by our group and described in detail elsewhere (33). Briefly, the muscle was detached from the scrotum, and a transverse buttonhole slit about 5-mm long was made in the proximal part of the cremaster pouch. The testicle and epididymis and the cremaster itself were then drawn out through the buttonhole. The small pedicle that attaches the cremaster to the testicle was tied up with two stitches and cut between them to separate the cremaster completely from the testicle. To prepare the cremaster muscle for transillumination microscopy, a flexible extendible ovoid ring was made with metal wire (diameter, 0.1 mm) and placed so that the cremaster acquired a racket shape. The ring had been positioned so that the main cremaster artery was in the center of the racket's upper surface. Throughout these procedures, the muscle was continuously bathed with warm saline solution. The muscle was continuously superfused at 2 ml/min with modified Krebs-Henseleit solution containing (mM/l) 118 NaCl, 5.9 KCl, 1.25 CaCl2 · 2H2O, 0.5 MgSO4 · 7H20, 28 NaHCO3, and 10 glucose. The temperature of the solution was fixed to 34.5°C in the cremaster chamber. By bubbling the solution with a 6% CO2-94% N2 gas mixture, we fixed the pH, PO2, and PCO2 of this solution in the muscle chamber at 7.43 ± 0.03, 25 ± 1.7, and 40 ± 1.0 mmHg, respectively. The chamber was covered with a Plexiglas plate to isolate it from the atmosphere.
To visualize the microcirculation, the chamber was placed on the movable stage of a modified Leitz microscope, and the cremaster muscle was transilluminated using a 100-W tungsten-halogen lamp. The image, magnified by a x20 objective and x10 oculars, was projected into a CCD camera (SONY) connected to a professional videotape recorder (Sony VO 9600 P). Total magnification from the tissue to the video screen was x1,400. Arteriolar diameters were measured by playback analysis of the video record using an electronic caliper.
Experimental protocol. Each set of experiments compared two treatments, and the arterioles were their own controls. Thus the protocols comprised the following steps: The cremaster muscle was allowed to stabilize for 30 min. The diameters of second- or third-order arterioles (see Ref. 38 for classification) were then measured. The first treatment was administered, and diameters were measured after 30 min. The second treatment was then added to the first, and diameters were measured after 30 min. We selected the thirtieth minute for observation because in several other previous studies we found that at this time the effect of the inhibitors used here were maximal. The use of diameter measurements made at earlier times might make the observation dependent on the kinetics of the inhibitors, and the use of measurements made at a later time makes the duration of the experiment unnecessarily longer (thus possibly incurring time-dependent alterations of the preparation). The arterioles studied in each experiment were located in either the same or contiguous microscopic fields. The diameter of each arteriole was recorded over a 10-s period, and then another arteriolar diameter was recorded. Depending on the geometry of the vascular network, between one and five arterioles were studied in each muscle.
Group 1 (n = 5 rats) was given 20 µM mefenamic acid
(MA), a COX inhibitor in the first period, and 200 µM
N-nitro-L-arginine (L-NNA), a
NO synthase (NOS) inhibitor was added in the second period.
In group 2 (n = 5 rats), L-NNA was given first, and MA added in the second period.
Group 3 (n = 6 rats) was given L-NNA first, and 20 µM indomethacin (a structurally different COX inhibitor) was added in the second period.
In group 4 (n = 5 rats) L-NNA was given in the first period, and 1 µM SQ-29548 [an antagonist of PGH2-TxA2 receptors (TP receptors)] was added in the second period.
Group 5 (n = 5 rats) was given L-NNA + SQ-29548 in the first period, and MA added in the second period.
Group 6 (n = 5 rats) was given L-NNA in the first period, and SOD (15,000 U/kg) + catalase (120,000 U/kg) (inhibitors of ROS synthesis) were intravenously added in the second period.
Group 7 (n = 5 rats) rats were pretreated intravenously with SOD + catalase 1 h before the experiment, and during the experiment, MA was given in the first period and L-NNA was added in the second period.
Group 8 (n = 9 rats) rats were pretreated intravenously with SOD + catalase 1 h before the experiment, and during the experiment, L-NNA was given in the first period and MA was added in the second period.
In addition, we checked in a complementary experiment (n = 3 rats) the effect of 1 h SOD + catalase alone.
Drugs. L-NNA, MA, indomethacin, SOD, catalase (all from Sigma-Aldrich; L'Isle d'Abeau, France), and SQ-29548 (RBI, Illkirch, France) were prepared fresh daily. All concentrations referred to final bath concentrations. The doses of NOS inhibitor L-NNA (200 µM) or COX inhibitors MA (20 µM) or indomethacin (20 µM) and of TP receptor antagonist SQ-29548 (1 µM) were those previously used in microcirculation studies (4, 16, 22), respectively. The doses of SOD and catalase were those previously used in our experiments (2).
Statistical analysis. All results are expressed as means ± SE. Mean basal diameter among all groups studied were compared by an ANOVA test. In each group of experiments comparisons between changes in diameter observed in the two different experimental conditions were made by Student's t-test for matched pairs. The significance level was fixed at 5%.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Effect of the order of inhibition of NOS and COX pathways. In group 1, administration of MA in the first period induced significant constriction that reduced the basal diameter by –42 ± 4% of the baseline value, thus indicating that the balance of prostanoids in control conditions was in favor of dilator prostanoids rather than constrictor prostanoids (Fig. 1). Then subsequent administration of L-NNA induced significant additional constriction, which reached –59 ± 4% of the baseline value at the end of the second period (P < 0.001). Even when COX is inhibited, a vasodilator tone, due to NO, still exists.
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In group 2, administration of L-NNA induced significant constriction that reduced the basal diameter by –51 ± 5% of the baseline value. In contrast to what happened in group 1, MA administered during the second period (after 30 min exposure to L-NNA and in its presence) induced a significant dilation from the diameter value observed at the end of the first period. Indeed, at the end of the second period, the diameter was found to be –15 ± 7% of the baseline value (P < 0.001). This result suggested that the balance of prostanoids after NOS inhibition differed from that under control conditions.
We checked whether a significant dilation was also observed when indomethacin was administered in the second period. In this case the constriction varied from –49 ± 5% of the baseline value at the end of the first period (i.e., administration of L-NNA) to –19 ± 6% of the baseline after a 30-min exposure to indomethacin (P < 0.001).
Effect of TP receptor antagonist after NOS inhibition. In group 4, when SQ-29548 (a TP receptor antagonist) was administered on arterioles constricted by L-NNA, we observed a significant dilation, because the diameter varied from –41 ± 4% at the end of the first period to –19 ± 3% of the baseline value after SQ-29548 administration (P < 0.001) (Fig. 2A).
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In contrast, we did not find any significant effect of SQ-29548 on the basal diameter (variation <3%). These results indicate that the level of stimulation of the arterioles of arteriolar TP receptor was largely higher after NOS inhibition than in control conditions.
Effect of COX inhibition after NOS inhibition in the presence of TP receptor antagonist. In group 5, when MA was administered on the arterioles constricted by L-NNA in the presence of SQ-29548, we found a significant additional constriction (Fig. 2B). The diameter varied from –34 ± 6% at the end of the first period to –60 ± 5% of the baseline value after MA administration (P < 0.001).
These results indicate that a production of vasodilator prostanoids still persists after L-NNA administration. When the TxA2-constrictive effects are blocked, the inhibition of COX production induces an additional constriction. This contrasts with what happened in group 2 where the effect of both dilator and constrictor prostanoids was present and where the net effect of COX blockade was a relative dilation.
Effect of SOD ± catalase after NOS inhibition. When SOD + catalase were given after L-NNA, we observed a significant dilation of arterioles because the diameter varied from –32 ± 3% at the end of the first period to –21 ± 4% of the baseline value after SOD + catalase administration (P < 0.001) (Fig. 3). This showed that a part of the vasoconstriction associated with NO inhibition was directly or indirectly due to an increase in the ROS level.
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Effect of the order of inhibition of NOS and COX pathways after pretreatment by SOD ± catalase. When SOD + catalase were administered 1 h before the experiment, MA constriction was lower than without pretreatment (–24.6 ± 5.5%) (Fig. 4). However, L-NNA in the second period induced an additional vasoconstriction (–34.6 ± 4.8%, P < 0.05) as observed in group 1 in the absence of pretreatment (Fig. 4A). When SOD + catalase were administered 1 h before the experiment, L-NNA constriction was also lower than without pretreatment (–9.1 ± 6%) but in contrast with that observed in group 2. The addition of MA in the second period induced a significant additional vasoconstriction (–24.1 ± 3%) (P < 0.01) (Fig. 4B).
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In a complementary experiment, we found that 1 h of SOD + catalase did not induce any change in basal arteriolar diameter (–1.5 ± 5.5%, n = 12).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Moreover, the interactions of NO with the arachidonic acid pathway can take place at different levels and interfere with both dilator or constrictor prostanoid synthesis or effects. The NOS inhibition can lead to a decrease in the synthesis of the dilator prostanoid prostacyclin, because, in the endothelial cell, a synergy exists between the NO level and the rate of prostacyclin synthesis (28, 30). In the smooth muscle cell, a synergy also exists between the second messengers of these mediators cAMP and cGMP (7, 36), thus it can be hypothetized that the absence of NO, which decreases cGMP levels, induces a reduction of the PGI2-dilating effect, which is cAMP dependent.
NOS inhibition can also lead to an increase in the synthesis of constrictor
prostanoids, as observed in long-term treatment with
N-nitro-L-arginine methyl ester
hydrochloride (3). We found a
dilation when the antagonist of TP receptors is administered after the NOS
inhibition, but no effect on the basal diameter was found when TP receptors
were administered in control conditions. This indicates that in our
experimental conditions, there is an increase in PGH2 or
TxA2 when NOS is inhibited. As mentioned before, it is possible
that NO inhibits TxA2 synthase, but it is also possible that
complex interactions exist between NO and PGH synthase
(6) by a mechanism inducing a
S-nitrosation of cysteine residues of the enzyme
(11). In the presence of TP
receptor antagonists, we did not find any relative dilation but, on the
contrary, an additional constriction when COX was inhibited after the NOS
inhibition.
This confirmed that the relative dilation observed could be explained by a TxA2-PGI2 balance of synthesis or effect, which was shifted in favor of TxA2 when NOS was inhibited, whereas it was in favor of PGI2 when NO was present. This also indicates that the major site of the cross talk between the NOS and COX pathway responsible for the present observation is located in the endothelium rather than in the smooth muscle cell.
With regard to the mechanism of interaction between NO and prostaglandins,
the role of ROS has been suggested by several authors
(5,
19,
32). Indeed, administration of
L-NNA also leads to an accumulation of ROS, which could be due to
an increased production of ROS by NOS
(25), but it seems that the
increase in ROS production by NOS occurs with an analog of
L-arginine
(N-monomethyl-L-arginine) different from
the one used in our study (L-NNA), which inhibits production of
both NO and ROS (14).
Moreover, the direct interaction of NO with ROS appears to be an important
aspect of vascular signaling processes, because the rate of reaction of NO
with ROS is three times greater than that with SOD
(37). Indeed, in our
experiments, we found that SOD + catalase after L-NNA induced a
vasodilation, thus showing that the NOS inhibition was associated with an
increase of the ROS level, which was partially responsible for the
vasoconstriction induced by the NOS inhibition. This observation is in
accordance with the findings of Rosenblum and colleagues
(27) in cerebral
microcirculation and was also confirmed by our findings that vasoconstriction
to L-NNA is lower in animals pretreated by SOD + catalase. In
addition, when rats were pretreated by SOD + catalase, we still found an
additional vasoconstriction when NOS was inhibited after COX, but the relative
dilation induced by the COX inhibition after the NOS inhibition was completely
suppressed, and an additional constriction was observed. This strongly
suggested that the level of ROS could be a key factor in the shift of the
TxA2-PGI2 balance of synthesis or effects in favor of
constrictor prostanoids. This hypothesis would be in line with findings in
other vessels, because hydrogen peroxide has been shown to stimulate
thromboxane synthesis in arterial rings
(15,
23,
26).
The mechanisms proposed above stressed the importance of the interactions among the different pathways (NO, COX, and ROS) (Fig. 5). It should be noted that the explanations proposed represent a view that is more complex than the view frequently used where these pathways are considered independently from each other, but we only mentioned direct interactions among the NO, ROS, or COX pathways. However, when COX and/or NO are inhibited, other products from the archidonic acid metabolism may change and affect reactivity of microvessels, thus more complex and indirect interactions, involving these metabolites cannot be ruled out.
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In conclusion, our findings have shown that NO inhibition induced arteriolar constriction is partly due to the vasoconstrictive effect of ROS. This NO inhibition induced a shift in the synthesis or in the effects of COX products, in favor of constrictor prostanoids. This effect disappeared when ROS are scavenged by SOD and catalase.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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on microcirculation in rat. J Appl Physiol
75: 2392–2399,
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