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Journal of Physiology (2002), 540.1, pp. 209-218
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.012912
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ABSTRACT |
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Inflammatory mediators have a role in the formation of cerebral oedema and there is evidence that cGMP is an important signal in vascular permeability increase. We have investigated the role and the source of cGMP in mediating the permeability response to acutely applied bradykinin and the histamine H2 agonist dimaprit on single cerebral venular capillaries, by using the single vessel occlusion technique. We found that 8-bromo-cGMP applied acutely resulted in a small and reversible permeability increase with a log EC50 -7.2 ± 0.15 M. KT 5823, the inhibitor of cGMP-dependent protein kinase, abolished the permeability responses to both bradykinin and dimaprit, while zaprinast, an inhibitor of type 5 phosphodiesterase, potentiated the response to bradykinin. On the other hand, L-NMMA blocked the response to dimaprit, but not that to bradykinin. Inhibitors of soluble guanylyl cyclase, LY 85353 and methylene blue, also inhibited the permeability response to dimaprit, but not bradykinin. The permeability responses to the natriuretic peptides ANP and CNP were of similar magnitude to that of bradykinin with log EC50 -10.0 ± 0.33 M and -8.7 ± 0.23 M, respectively. The natriuretic peptide receptor antagonist HS-142-1 blocked permeability responses to bradykinin as well as to ANP, and leukotriene D4 blocked the responses to CNP and bradykinin, but not to dimaprit. In conclusion, the histamine H2 receptor appears to signal via cGMP that is generated by a NO and soluble guanylyl cyclase, while bradykinin B2 receptor also signals via cGMP but through particulate guanylyl cyclase.
(Received 27 June 2001; accepted after revision 10 January 2002)
Corresponding author P. A. Fraser: Centre for Cardiovascular Biology and Medicine, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK. Email: paul.fraser{at}kcl.ac.uk
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INTRODUCTION |
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The normally tight blood-brain barrier formed by cerebral endothelium can be modified by inflammatory mediators, and there is evidence that these have a role in the cerebral oedema that can develop following trauma or cerebral ischaemia (Abbott, 2000). Two of these mediators, histamine and bradykinin, have been shown to produce a rapid, small and reversible permeability increase of similar magnitude when briefly applied directly to the pial microvessels. The permeability-increasing effects of histamine are mediated via the H2 receptor (Sarker et al. 1998) and those of bradykinin via the B2 receptor (Sarker et al. 2000). Interestingly, H1 receptor activation results in a decrease in permeability (Sarker et al. 1998), while B1 receptor activation results in an increase in permeability (Sarker et al. 2000), but mediated via histamine release. It appears that different signal transduction mechanisms are used by these two mediators. Thus, the acute response to histamine was inhibited by the calcium-entry blocker SKF 96365, which had no effect on the response to bradykinin. It is possible that calcium entry can occur across the cell membrane through sites of lipid peroxidation (Graier et al. 1998). Although Ca2+ influx has long been associated with an endothelial permeability increase, there is considerable evidence suggesting that cyclic nucleotides have an important role in regulating permeability changes: increased cAMP is associated with decreased permeability whereas raised cGMP increases permeability in vivo (Michel & Curry, 1999).
Both bradykinin and histamine can raise cGMP in endothelial cells in culture via a calcium-calmodulin-dependent activation of endothelial nitric oxide synthase (eNOS) to release nitric oxide, which in turn activates soluble guanylyl cyclase (Furchgott & Vanhoutte, 1989), and this pathway has also been shown to be important for the permeability increase in mesenteric microvessels (He et al. 1998). Cytosolic cGMP also increases when particulate guanylyl cyclase is activated by atrial, brain and C-type natriuretic peptides (ANP, BNP and CNP, respectively), for which cerebral microvessels have receptors (Vigne & Frelin, 1992).
The aim of this study was to investigate the role of the guanylyl cyclases in mediating permeability changes in response to activating the histamine H2 and the bradykinin B2 receptors. Some of the data have been previously presented in a preliminary form (Sarker & Fraser, 1998).
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METHODS |
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The method used in this study, and its theoretical basis, have been described fully (Fraser & Dallas, 1993; Easton & Fraser, 1994). Briefly, the microcirculation of the surface of the brain of rats (aged 20-30 days) was exposed by removing the dura and arachnoid. A low molecular weight fluorescent dye, sulforhodamine B (580 Da), was introduced into the cerebral microcirculation via a bolus injection into the carotid artery, and viewed through a Zeiss ACM fluorescence microscope under 525 to 535 nm illumination. The fluorescent signal, which has been shown to be linearly related to the dye concentration (Fraser & Dallas, 1990), was captured through a microscope, an image-intensifier camera, and analysed through a video-densitometer. Permeability was measured from the rate of loss of dye trapped in a single pial venular capillary by a glass-occluding probe. The fluorescence measurements were made from a small segment 200 to 300 µm from the open end of the occluded vessel, but not so close to the occluding probe that the vessel diameter was distorted. If the vessel was leaky, a transmural hydrostatic pressure gradient would drive fluid across the wall, which would be replaced by fresh dye-free fluid entering the open end of the occluded portion. Dye concentration in the measured portion depends on axial flow and diffusion as well as transmural convection and diffusion. Permeability was calculated from the rate of decrease in fluorescence under the measured portion of the occluded segment. The diameter of the vessels did not change during the occlusion (Easton & Fraser, 1994). As rate of fall of intravascular concentration of a small polar molecule is independent of the hydrostatic pressure (Fraser & Dallas, 1993), the concentration of dye early in the occlusion, before axial volume flux distorts the uniform axial concentration of dye in the region of measurement, will be Ct = C0 e-kt, where k = 4P/d and P is the permeability and d the diameter of the vessel.
Animal preparation
The experiments were performed on Wistar rats of either sex aged between 20 and 30 days and were within guidelines directed by the Animals (Scientific Procedures) Act, 1986. The animals were anaesthetized by an intraperitoneal injection of 60 mg kg-1 body weight sodium pentobarbitone diluted in water (25 % w/v), and maintained by supplementary injection of 10 % of the original dose when necessary. At the end of the experiment the animal was killed by administering an overdose of the anaesthetic. Once anaesthetized, a thermal probe was inserted rectally, and the animal was kept on a heating blanket connected via the probe to a feedback circuit (CFP 8105 Harvard Instruments) to maintain the animal body temperature at 37 ± 1 °C. The trachea and the right common carotid artery were cannulated routinely, the arterial cannula being placed orthograde and filled with heparinized saline (100 U ml-1) to prevent clot formation. A section of the frontoparietal bones on the left side, between coronal and lambdoid sutures, was thinned with a dental drill. A metal ring (internal diameter 7 mm and outside diameter 13 mm) was glued onto the cranium surrounding the thinned bone with cyanoacrylate adhesive. The thinned cranial surface within the ring was constantly superfused by a pump at the rate of 1-2 ml min-1 with artificial cerebrospinal fluid (CSF) warmed to 37 ± 0.3 °C and delivered through a fine plastic tube to ensure that a layer of fluid was present at all times. The thinned bone within the ring was then removed carefully avoiding damage to the meninges and cerebral surface. Pial microvessels were exposed by cutting away the overlying meninges. The rat was then placed on the modified stage of a microscope (ACM, Zeiss Oberkocken) and the exposed cerebral surface illuminated with a 100 W xenon discharge lamp through a 
20 water-immersion objective lens (Cooke, N.A. 0.5).
Artificial cerebrospinal fluid and drug application
The cerebral surface was superfused with an artificial cerebrospinal fluid (CSF) at the rate of 0.5 to 1 ml min-1, warmed so that it arrived at the brain's surface at 37 ± 1 °C and buffered to pH 7.40. The CSF contained (mM): NaCl (110.5), KCl (4.7), CaCl2 (2.5), KH2PO4 (1.1), MgSO4.7H2O (1.25), NaHCO3 (25) and Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid]). All chemicals were obtained from BDH, Poole, except MgSO4.7H2O from Fisons, Loughborough, and Hepes from Sigma. HS-142-1 (Imura et al. 1992) was a kind gift from Dr Y. Matsuda (Kyowa Hakko Kogyo, Tokyo, Japan), leukotriene D4 was purchased from Affinity Research (Exeter, England). KT 5823, the inhibitor of cGMP-dependent protein kinase (PKG; Nazario et al. 1995), the soluble guanylyl cyclase inhibitor LY 85383 (Schmidt et al. 1985) and the type 5 phosphodiesterase (PDE5) inhibitor zaprinast (Frossard et al. 1981) were purchased from CN Biosciences (Nottingham, England). Bradykinin, the histamine H2-specific agonist dimaprit, N-monomethyl-L-arginine (L-NMMA), D-NMMA, super oxide dismutase, catalase, methylene blue, atrial natriuretic peptide and C-type natriuretic peptide were all purchased from Sigma (Poole, England).
A static pool of artificial CSF formed over the exposed pial surface when the delivering pump was switched off. The volume of the static pool was estimated by pipette as being approximately 100 µl. All the drugs were applied abluminally into this pool, and the doses are expressed as the final concentration, unless otherwise specified.
Protocols
Permeability measurements were carried out on venular capillaries between 10 and 18 µm diameter in the following manner. There was sufficient autofluorescence in the fluorescein filter set for the cerebral surface to be viewed directly under the microscope. The long, straight arteries and arterioles were easily distinguished from the venular capillaries, which were shorter and more tortuous. A venular capillary with an unbranching section of at least 200 µm was selected and the glass occluding probe positioned at one end of the vessel. A bolus of fluorescent dye-containing solution was injected through the carotid arterial cannula, and during its transit through the microcirculation the arterioles filled rapidly, followed by a later venular filling. The occluding probe was lowered on to the venular capillary, thereby forming a segment containing dye, while dye was cleared from the rest of the microcirculation, and once a stable level of fluorescence was obtained (after about 30 s) the drug, or mixture of drugs in question, was applied to the brain surface while the occlusion was maintained. Once the videodensitometer trace had stabilized (between 30 and 60 s) the occlusion was released and the drugs washed off the brain surface. The order of application of the drug mixtures was randomized in any series. All the experiments, unless otherwise stated, were completed within 2 h of the first measurement. Dose-response curves were constructed by applying all the different doses of the drug in pseudo-random order to a single vessel, and at least four vessels from four rats were used for each dose-response curve.
Statistics
Unless otherwise stated, the results are expressed as the mean ± S.E.M., and the significance of any changes assessed by using a non-parametric one-way analysis of variance test (Kruskal-Wallis) with Dunn's multiple comparison test, or the Mann-Whitney test where appropriate. Regression lines and sigmoidal dose-response curves were fitted using GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego, CA, USA.
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RESULTS |
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This study was designed to explore the signal transduction pathways linked to acute permeability responses to histamine and bradykinin. Previous experiments have demonstrated that this preparation is stable for the first 2 h following exposure, and acute application of histamine or bradykinin produced reproducible, transiently increased permeability responses without affecting the resting permeability (Sarker et al. 2000).
Permeability response to 8-bromo-cGMP
Previously experiments have shown that raising intraendothelial cGMP resulted in increased permeability of venular microvessels, but often decreased permeability in cultured endothelium (Draijer et al. 1995; He et al. 1998). Which of these responses occurs in pial venular microvessels was assessed by applying the cell permeant analogue of cGMP, 8-bromo-cGMP, from 0.5 nM to 0.5 mM, to the abluminal surface. There was a dose-dependent permeability increase, with the estimated maximal increase being 2.0 ± 0.16 10-6 cm s-1 (mean ± S.E.M.), and the log median effective dose (logEC50) -7.2 ± 0.15 M (Fig. 1).
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Figure 1. Permeability response to applied 8-bromo-cGMP Permeability of single venular capillaries was measured before and during the application of 8-bromo-cGMP to the brain surface, and the change in permeability plotted against the dose. Each of four microvessels (diameters 10 to 16 µm) from four rats received a range of concentrations (from 5 nM to 500 µM). Analysis of variance showed that there was a significant dose dependent permeability increase (P < 0.01). | ||
Requirement of cGMP for acute permeability responses to histamine or bradykinin
The importance of cGMP in the development of a rapid permeability increase was also investigated by using the PKG inhibitor, KT 5823, in conjunction with the histamine H2 and bradykinin B2 receptor agonists, dimaprit and bradykinin. KT 5823 (5 µM) alone had negligible effect on permeability (0.3 ± 0.27 10-6 cm s-1, n = 11), but the permeability response to bradykinin (5 µM bradykinin alone, 1.3 ± 0.15 10-6 cm s-1, n = 6) was very much reduced when co-applied with KT 5823 (0.1 ± 0.18 10-6 cm s-1, n = 6). KT 5823 similarly blocked the response to dimaprit (50 µM dimaprit alone, 1.6 ± 0.44 10-6 cm s-1, n = 5; with KT 5823, 0.0 ± 0.31 10-6 cm s-1, n = 4).
Conversely, a sub-threshold concentration of bradykinin (50 pM) that resulted in no net change in permeability (-0.1 ± 0.16 10-6 cm s-1, n = 10) produced a significant increase when co-applied with the PDE5 inhibitor zaprinast (50 pM + 50 µM zaprinast, 1.4 ± 0.26 10-6 cm s-1, n = 4). Zaprinast alone produced little change in permeability (0.2 ± 0.13 10-6 cm s-1, n = 4). These results are illustrated in Fig. 2.
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Figure 2. The effect of interfering with cGMP metabolism on the permeability response to bradykinin and dimaprit A, the PKG inhibitor KT 5823 (0.5 µM) alone had no effect on permeability (11 vessels, five rats), but when co-applied with bradykinin (5 µM) or dimaprit (50 µM) it blocked the permeability responses (Mann-Whitney; **P < 0.01, *P < 0.05, five vessels from four rats). B, a sub-threshold concentration of bradykinin produced a maximal response when co-applied with the PDE5 inhibitor, zaprinast (50 µM; **P < 0.01; four vessels from four rats). | ||
The role of NO in the permeability response to histamine and bradykinin
The permeability responses to both histamine and bradykinin require the presence of extracellular Ca2+ (Sarker et al. 2000) and a rise in intra-endothelial [Ca2+] can activate endothelial nitric oxide synthase (eNOS) to form nitric oxide (NO), which will, in turn, activate soluble guanylyl cyclase (sGC) to form cGMP from GTP. When the NOS inhibitor L-NMMA (50 µM) was applied to the brain surface a small, significant increase in permeability resulted (Fig. 3; 0.3 ± 0.04 10-6 cm s-1, P < 0.05). L-NMMA blocked the response to 50 µM dimaprit (dimaprit alone 1.2 ± 0.25, with L-NMMA, 0.3 ± 0.20 10-6 cm s-1), but had no effect on the permeability response to 5 µM bradykinin (bradykinin alone, 1.7 ± 0.23; with L-NMMA, 1.4 ± 0.36 10-6 cm s-1). This inhibition was partially reversed by the addition of 50 µM L-arginine (0.8 ± 0.16 10-6 cm s-1). D-NMMA (50 µM), a biologically inactive isomer of L-NMMA, had no effect on the dimaprit-mediated increase in permeability.
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Figure 3. The role of nitric oxide in the permeability response to dimaprit and bradykinin A and B, L-NMMA (50 µM) blocked the response to dimaprit (50 µM; *P < 0.05, Kruskal-Wallis, Dunn's multiple comparison test), but had no effect on the permeability response to bradykinin (5 µM). L-NMMA by itself produced a small permeability increase. D-NMMA (50 µM) had no effect on dimaprit-mediated increase in permeability. These experiments were performed on five venular capillaries (diameter range 10 to 16 µm) from five rats. | ||
The role of soluble guanylyl cyclase
The observation that blocking nitric oxide synthesis has no effect on the permeability response to bradykinin raises the question of the source of the necessary cGMP. This was examined initially by using the inhibitors of sGC, LY 83583 (5 µM) and methylene blue (5 µM). Both inhibitors completely blocked the permeability increase brought about by dimaprit (50 µM dimaprit alone, 1.4 ± 0.52 10-6 cm s-1, dimaprit with LY83583, 0.1 ± 0.13 10-6 cm s-1, and dimaprit with methylene blue, -0.1 ± 0.20 10-6 cm s-1), but had no effect on the response to 5 µM bradykinin (bradykinin alone, 1.3 ± 0.15 10-6 cm s-1; bradykinin with LY83583, 1.5 ± 0.36 10-6 cm s-1, bradykinin with methylene blue, 1.3 ± 0.25 10-6 cm s-1, Fig. 4A).
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Figure 4. The role of soluble guanylyl cyclase in the permeability response to bradykinin and dimaprit A, bradykinin (5 µM) and dimaprit (50 µM) were co-applied to the brain surface with the sGC inhibitors LY 83583 (5 µM) or methylene blue (5 µM), neither of which produced a significant permeability increase). The response to dimaprit was abolished, but neither inhibitor affected the bradykinin-induced permeability increase. B, the zaprinast (50 µM) potentiation of the response to bradykinin (50 pM) was unaffected by either LY 83583 or L-NMMA (50 µM), and did not protect the response to dimaprit from LY 83583. These experiments were performed on 12 venular capillaries (diameter range, 10 to 18 µm) from eight rats (*P < 0.05, Kruskal-Wallis, Dunn's multiple comparison test). | ||
The possibility that the zaprinast-enhanced response to bradykinin (Fig. 2B) was due to potentiation of a low-level activation of the eNOS-sGC pathway was examined by co-applying L-NMMA and LY 83583 (5 µM). Neither of these affected the response (permeability increase 1.7 ± 0.35 10-6 cm s-1 and 1.1 ± 0.18 10-6 cm s-1, respectively, Fig. 4B).
Particulate guanylyl cyclase
Particulate guanylyl cyclase is a possible alternative source for cGMP, but this is usually activated by a natriuretic peptide receptor, and C-type natriuretic peptide (CNP) has been shown to raise cGMP in rat cerebral microvessels (Kobayashi et al. 1994). We found that both atrial natriuretic peptide (ANP) and CNP increased cerebral microvascular permeability in a dose-dependent manner, and the dose-response curves (Fig. 5) give estimated maximal permeability increases for ANP and CNP of 1.2 ± 0.11 10-6 cm s-1 and 1.0 ± 0.1 10-6 cm s-1 with logEC50 of -10.0 ± 0.33 M and -8.7 ± 0.23 M, respectively. The permeability response to CNP (0.5 µM) was unaffected when co-applied with methylene blue (1.2 ± 0.17 10-6 cm s-1 CNP alone, and 1.0 ± 0.25 10-6 cm s-1 with methylene blue, 5 µM).
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Figure 5. Natriuretic peptides increase permeability independently of soluble guanylyl cyclase A, the dose-response curve of particulate guanylyl cyclase (pGC) activators ANP ( | ||
The possibility that bradykinin activates particulate guanylyl cyclase was tested by co-applying bradykinin, natriuretic peptides and dimaprit with leukotriene D4 (LTD4, which has been shown to prevent ANP-dependent production of cGMP; Terada et al. 1989), and the specific natriuretic peptide antagonist HS-142-1 (Imura et al. 1992). Both LTD4 and HS-142-1 blocked the response to bradykinin and the natriuretic peptides. The permeability changes, expressed as the change from the control level (n = 4 to 10 for each group) were: bradykinin (5 µM) alone, 1.2 ± 0.20; co-applied with LTD4 (0.5 µM), 0.1 ± 0.08 10-6 cm s-1; and with HS-142-1(5 µM), -0.1 ± 0.01 10-6 cm s-1; CNP (0.5 µM) alone, 1.0 ± 0.15 10-6 cm s-1; with LTD4, 0.1 ± 0.22 10-6 cm s-1; ANP (5 µM) alone, 0.8 ± 0.08 10-6 cm s-1, and with HS-142-1, -0.3 ± 0.18 10-6 cm s-1. The response to dimaprit was unaffected by the inhibitory action of LTD4 on particulate guanylyl cyclase (dimaprit, 50 µM alone, 1.3 ± 0.18 10-6 cm s-1, with LTD4, 1.1 ± 0.10 10-6 cm s-1).
It is possible to interpret the inhibition of the response to bradykinin by HS-142-1 as evidence that bradykinin causes release of a natriuretic peptide from a nearby tissue (Wennberg et al. 1999) similar to the method by which des-arg9 bradykinin was shown to act via histamine release (Sarker et al. 2000). The question whether cells other than endothelial cells were involved was tested by delivering bradykinin (5 µM), and bradykinin with HS-142-1 (5 µM), to the lumen of venular capillaries via intracarotid bolus injections (Fig. 6). The mean permeability increase with bradykinin alone (compared to a saline bolus immediately preceding) was 3.2 ± 0.62 10-6 cm s-1, which is considerably higher than maximum obtained for the abluminal application. This is possibly due to a difference in the expression of kinin-destroying enzymes (Sarker et al. 2000). The response to bradykinin was reduced in boluses that included HS-142-1 (0.8 ± 0.30 10-6 cm s-1), and applied HS-142-1 alone also resulted in no permeability increase (-0.1 ± 0.59 10-6 cm s-1). These paired experiments were carried out on five vessels in five rats.
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Figure 6. Permeability response to bradykinin was blocked by blocking particulate guanylyl cyclase A, 5 µM bradykinin, 50 µM dimaprit, 0.5 µM CNP and 5 µM ANP all increased permeability significantly. When co-applied with 1 µM LTD4, the response to bradykinin and CNP was blocked (Kruskal-Wallis, Dunn's multiple comparison test, *P < 0.05), but had no effect on the response to dimaprit. Similarly, the natriuretic peptide receptor blocker, HS-142-1, inhibited the responses to 5 µM bradykinin and 5 µM ANP. B, a series of occlusions on a single venular capillary in which bradykinin and the specific natriuretic receptor inhibitor HS-142-1 were applied in the lumen. The permeability coefficients shown were calculated from rate of decrease of fluorescence. C, summary of five paired luminal application experiments. The permeabilities measured with bradykinin alone were significantly greater than those with bradykinin combined with HS-142-1. | ||
We found in a control experiment that there was no effect of HS-142-1 on the relaxation produced by bradykinin in a rat aortic ring preparation, which confirms that the drug does not act by binding to or inactivating bradykinin.
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DISCUSSION |
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The dose-response relationship between 8-bromo-cGMP and permeability is consistent with the association between raised cGMP and increased permeability found in studies in pig coronary, frog mesenteric and rat cerebral microvessels (Yuan et al. 1993; He et al. 1998; Chi et al. 1999). Inhibition of the permeability response to histamine and bradykinin by blocking PKG with KT 5823 suggests that both agonists require cGMP for the permeability response in these microvessels, and thus may involve the activation of vasodilator-stimulated phospho-protein, similar to that found in cultured cerebral endothelium (Sporbert et al. 1999). Potentiation of the sub-threshold concentration of bradykinin by the PDE5 inhibitor zaprinast to a maximal response is consistent with this view. Intriguingly, there is also a suggestion that the lowest 8-bromo-cGMP concentration used here (5 nM) resulted in a permeability decrease (not statistically significant as the baseline permeability was very low), and, if real, some of the processes that lower permeability with raised cGMP in cultured endothelium may operate here (Draijer et al. 1995; Holschermann et al. 1997).
Both histamine and bradykinin are potent mediators of NO release from endothelial cells (Cherry et al. 1982; Lantoine et al. 1998), and a phospholipase C, nitric oxide, soluble guanylyl cyclase pathway was shown to mediate the permeability response to histamine in isolated, perfused porcine coronary venules (Yuan et al. 1993). The permeability response to bradykinin is not related to this pathway so clearly. In a number of in vivo microvascular experiments, it was shown that nitric oxide formation was associated with permeability reduction (e.g. Arnhold et al. 1999), but some do indicate a link between bradykinin-mediated permeability increase and nitric oxide production (e.g. Ramirez et al. 1995), while other experiments showed that there was no link between nitric oxide and the permeability-increasing effects of bradykinin (Cambridge & Brain, 1995; Félétou et al. 1996).
Soluble guanylyl cyclase has been shown to be the source of cGMP following histamine or bradykinin application in the microcirculation of the pig heart (Yuan et al. 1993), brain tumour (Nakano et al. 1996) and rat mesentery (He et al. 1998), and since this enzyme is activated by NO, inhibiting eNOS would be expected to block the permeability response. Such a mechanism appears to operate in this preparation when the histamine H2 receptor was stimulated. Acute bradykinin application, however, produced NO-independent permeability increases. This is surprising, as a rise in [Ca2+]i is normally sufficient to activate eNOS (e.g. Weih et al. 1998), and we have found that bradykinin application results in such a [Ca2+]i increase (Sarker & Fraser, 1999), and that the presence of extracellular Ca2+ is necessary for the bradykinin-stimulated permeability increase (Sarker et al. 2000). In these vessels, however, both the bradykinin-mediated [Ca2+]i and permeability increases depend on free radicals, probably formed from the interaction of arachidonic acid released by B2 receptor activation of the Ca2+-independent phospholipase A2 with either cyclooxygenase or lipoxygenase (Sarker et al. 2000). Similarly, it appears that free radical generation is necessary to induce the acute increase in permeability, independently from any contribution from nitric oxide. This is consistent with the ability of free radicals generated by arachidonic acid to increase permeability (Easton & Fraser, 1998).
Both LY 85385 and methylene blue inhibited dimaprit-mediated responses whilst not affecting those mediated by bradykinin or the natriuretic peptides, but there has been some concern as to the specificity of these compounds for soluble guanylyl cyclase as sometimes they have been shown to reduce responses to natriuretic peptides (Ijioma et al. 1995; Kook et al. 1999). There are, however, a number of instances where it has been found that LY 85385 and methylene blue clearly inhibit the soluble, but not particulate, guanylyl cyclase (Martin et al. 1988; Kawada et al. 1994; Ali et al. 2000). Furthermore, recent work has shown that an alternative and more specific inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Garthwaite et al. 1995), can reduce responses to natriuretic peptides in the coronary circulation (Brunner & Wolkart, 2001), and it is possible that some of the natriuretic peptide signalling involves soluble guanylyl cyclase in some tissues. There are further complications, since it has been shown that LY 85385 and methylene blue are able to inhibit NOS derived from brain tissue directly (Luo et al. 1995), albeit only at higher concentrations than in the present experiments, and it has also been shown that free radicals are generated during the cellular metabolism of LY 85385 and methylene blue, and that these radicals inhibit soluble guanylyl cyclase (Wei et al. 1996). In this preparation, however, free radical generation increases permeability (Easton & Fraser, 1998; Sarker et al. 2000), whereas the soluble guanylyl cyclase inhibitors reduce it.
Particulate guanylyl cyclase
ANP and CNP have been shown to activate the NPR-A and NPR-B receptors (respectively) which are linked across the cell membrane to kinase homology domain and guanylyl cyclase domains, the whole forming particulate guanylyl cyclase (Potter & Hunter, 2001). These natriuretic peptides also have high affinities for the NPR-C clearance receptor, which has been associated with inhibition of adenylyl cyclase (Hempel et al. 1998). Natriuretic peptides have been shown to increase the permeability of cerebral and peripheral microvessels (Huxley et al. 1987; Vigne & Frelin, 1992) and to increase cGMP levels in brain microvessels (Grammas et al. 1991). In the present series of experiments, both ANP and CNP increased permeability in a dose-dependent manner (Fig. 5).
We found that the permeability response to bradykinin was inhibited by the specific natriuretic receptor antagonist HS-142-1, and by LTD4 (Fig. 6). The finding that HS-142-1 was effective in blocking the permeability response when co-applied with bradykinin in the vessel lumen indicates that no other cell type is involved in these responses, in contrast with our previous observations with desArg9-bradykinin (Sarker et al. 2000). There are indications from the literature that bradykinin can increase cGMP formation via particulate guanylyl cyclase directly, in some circumstances (Leibmann et al. 1989), and Wennberg et al. (1999) showed that HS-142-1 application further inhibited the relaxation of coronary arteries to bradykinin following eNOS blockade. The mechanism by which this occurs is not clear, but it is possible to speculate that bradykinin causes some substance to be released from endothelial cells which then activates the natriuretic receptor. For example CNP has been suggested as a candidate for endothelium-dependent hyperpolarizing factor (Wennberg et al. 1999), but Barton et al. (1998) have shown that this is unlikely, and citrulline has been found to cause a HS-142-1-susceptible cGMP formation in endothelial cells (Ruiz & Tejerina, 1998).
There is a possible alternative pathway that is suggested by recent findings. Adrenomedullin has been shown to activate particulate guanylyl cyclase via Gi-protein (Ali et al. 2000), and the bradykinin B2 receptor can signal via Gi-protein in cultured brain endothelium (Fabian et al. 1998). So it is possible that bradykinin in pial venular endothelium stimulates particulate guanylyl cyclase via Gi-protein. Such a mechanism would explain the inhibitory effect of LTD4 on bradykinin, since it can induce the translocation of Gi-protein from the plasma membrane to the cytoskeleton within 15 s (Adolfsson et al. 1996). If this were the mechanism by which bradykinin acts, or if there were any other intracellular activation of particulate guanylyl cyclase, then there are implications for the way in which HS-142-1 inhibits. HS-142-1 is a 3 kDa polysaccharide of bacterial origin, and is unlikely to enter the cell membrane, but may act as a false agonist to keep the particulate guanylyl cyclase complex in an inactive state.
In conclusion, we have found that cGMP is a necessary requirement for permeability increases consequent to acute stimulation of the histamine H2 and the bradykinin B2 receptors, and that in these pial venular capillaries the two receptors activate soluble and particulate guanylyl cyclase, respectively, for its production. A scheme for these signalling pathways, based on cited references and on the work in our laboratory (Easton & Fraser, 1998; Sarker et al. 1998; Sarker et al. 2000), is presented in Fig. 7.
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Figure 7. Signalling pathways for the histamine H2 and the bradykinin B2 receptors on pial venular endothelium The activated H2 receptor probably signals via G | ||
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Acknowledgement
This work was supported by The Wellcome Trust.
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) and CNP (
). ANP and CNP were applied to the brain surface of pial venular capillaries in a range of concentration (5 pmol l-1 to 5 µmol l-1). The full range of concentrations of either ANP or CNP was applied to four microvessels from four rats. Analysis of variance showed that there was a significant dose-dependent permeability increase (P < 0.05). B, methylene blue (MB; 5 µM) produced a small (but insignificant) permeability increase when applied alone, but did not affect the permeability response to CNP (0.5 µM); results from four vessels from four rats (*P < 0.05, Kruskal-Wallis, Dunn's multiple comparison test).
q to phospholipase C (PLC), and the formed IP3 releases Ca2+ from calcium stores. This Ca2+ is able to activate eNOS to produce nitric oxide, which in turn activates soluble guanylyl cyclase (sGC), and the cGMP so formed activates PKG. The depletion of the calcium stores opens a Ca2+ entrance pathway. The B2 receptor on these cells appears to lack a connection with G