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Am J Physiol Heart Circ Physiol 278: H1124-H1133, 2000;
0363-6135/00 $5.00
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Vol. 278, Issue 4, H1124-H1133, April 2000

Dominant role of cAMP in regulation of microvessel permeability

P. He, M. Zeng, and F. E. Curry

Department of Human Physiology, School of Medicine, University of California, Davis, California 95616


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We reported previously that increasing cAMP levels in endothelial cells attenuated ATP-induced increases in hydraulic conductivity (Lp), and that the activation of cGMP-dependent pathways was a necessary step to increase Lp in response to inflammatory mediators. The aim of the present study was to evaluate the role of basal levels of cAMP in microvessel permeability under resting conditions and to evaluate the cross talk between cAMP- and cGMP-dependent signaling mechanisms in regulation of microvessel permeability under stimulated conditions, using individually perfused microvessels from frog and rat mesenteries. We found that reducing cAMP levels by inhibition of adenylate cyclase or inhibiting cAMP-dependent protein kinase through the use of H-89 increased basal Lp in both frog and rat mesenteric venular microvessels. We also found that 8-bromocAMP (8-BrcAMP, 0.2 and 2 mM) was sufficient to attenuate or abolish the increases in Lp due to exposure of frog mesenteric venular microvessels to 8-BrcGMP (2 mM) and ATP (10 µM). Similarly, in rat mesenteric venular microvessels, application of 8-BrcAMP (2 mM) abolished the increases in Lp due to exposure to 8-BrcGMP alone (2 mM) or with the combination of bradykinin (1 nM). In addition, application of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of cGMP-stimulated phosphodiesterase, significantly attenuated both 8-BrcGMP- and bradykinin-induced increases in Lp. These results demonstrate that basal levels of cAMP are critical to maintaining normal permeability under resting conditions, and that increased levels of cAMP are capable of overcoming the activation of cGMP-dependent pathways, therefore preventing increases in microvessel permeability. The balance between endothelial concentrations of these two opposing cyclic nucleotides controls microvessel permeability, and cAMP levels play a dominant role.

hydraulic conductivity; adenosine 3',5'-cyclic monophosphate analog; guanosine 3',5'-cyclic monophosphate analog; erythro-9-(2-hydroxy-3-nonyl)adenine-phosphodiesterase inhibitor; adenylate cyclase inhibitor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MICROVESSEL PERMEABILITY has been shown to be regulated by numerous second messengers through a variety of signaling pathways under different pathological and physiological conditions. In particular, endothelial cell intracellular calcium concentration ([Ca2+]i) and intracellular levels of cAMP and cGMP have been recognized as important second messengers in endothelial cells to regulate microvessel permeability, especially under stimulated states (26). Our previous study (17) in individually perfused intact microvessels demonstrated that changes in endothelial cell [Ca2+]i determined the magnitude of changes in microvessel permeability induced by a variety of inflammatory mediators such as ATP and bradykinin. Most of the in vivo studies from individually perfused or isolated microvessels to intact tissue or organ have shown that these increases in microvessel permeability were attenuated by blocking nitric oxide (NO) synthase or guanylate cyclase (14, 16, 24, 25, 28, 30, 36, 37). Studies in cultured endothelial cells also demonstrated that an increase in endothelial cell [Ca2+]i was associated with increases in NO and cGMP contents in response to inflammatory mediators (11). All of these results indicate that a calcium-activated, NO-cGMP-dependent pathway may be a common pathway to increase venular microvessel permeability in response to acute inflammatory mediators. However, the mechanisms of the action of cGMP in the regulation of microvessel permeability have not been understood. Moreover, the effects of the cGMP-dependent pathway on permeability have not been consistent. One complication is that a cGMP-dependent pathway appears to reduce permeability in some cultured endothelial cells (2, 8, 32, 35). Furthermore, in whole organ studies where leukocytes are present, a cGMP-dependent pathway may regulate leukocyte interaction with the vessel wall (21). We have attempted to overcome some of these complications by studying the mechanisms of the action of cGMP in the regulation of microvessel permeability in individually perfused venular microvessels.

In contrast with the role of cGMP, both in vivo and in vitro studies with a variety of experimental models consistently demonstrated that an elevation of endothelial cAMP levels lowers basal permeability and attenuates the increases in permeability in response to inflammatory agents (1, 3, 15, 26). In addition, results in cultured endothelial monolayers demonstrated that tumor necrosis factor-alpha (TNF-alpha )- and hypoxia-induced changes in endothelial cell barrier function involve a reduction in intracellular cAMP due to increased cyclic nucleotide phosphodiesterase (PDE II) activities (20, 29). If increases in microvessel permeability are cGMP dependent, then it is possible that cGMP-stimulated PDE II is responsible for the accelerated degradation of cAMP, leading to reduced cAMP levels and an increase in microvessel permeability. There have been no data reporting the effect of decreasing endothelial cell cAMP levels on permeability in intact microvessels.

Under our experimental conditions and in some in vivo models described above, cAMP and cGMP appear to play antagonistic roles in the regulation of microvessel permeability (15, 16, 25, 36, 37). Our previous study also demonstrated that the actions of both cAMP and cGMP in the regulation of microvessel permeability occur downstream from calcium entry (15, 16), but the potential site of the cross talk between cAMP- and cGMP-dependent signal transductional pathways in the regulation of microvessel permeability has not been defined. The objective of our current study was to use individually perfused intact microvessels in frog and rat mesentery to investigate the following: 1) the role of basal levels of cAMP and cGMP in maintaining permeability under resting conditions; and 2) the antagonistic relationship and the extent of cross talk between cAMP- and cGMP-dependent pathways in the regulation of microvessel permeability under stimulated conditions.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Preparation

The experiments were carried out in frog and rat mesenteries. Frogs (male Rana pipiens, 2.5-3.0 in.) were supplied by J. M. Hazen, and rats (female Sprague-Dawley, 250-300 g) were supplied by Hilltop Laboratory Animal (Scottdale, PA). The brain of the frog was destroyed by pithing, leaving the spinal cord intact. Preparation of frog mesenteries for measurement of hydraulic conductivity (Lp) has been described in detail (5). For the rat experiments, the tray was extended to fit the size of rat body and mesentery. Rats (age 25-30 days) were anesthetized with pentobarbital sodium (65 mg/kg body wt) given subcutaneously, and kept warm on a heating pad. Anesthesia was maintained by giving additional pentobarbital sodium (3 mg/dose) as needed. After the rat was anesthetized, a midline surgical incision (1.5-2 cm) was made in the abdominal wall, and the mesentery was gently taken out from the abdominal cavity and spread over a pillar for Lp measurements. The upper surface of the rat mesentery was continuously superfused with mammalian Ringer solution during preparation and experimentation. The temperature of the superfusate was maintained at 18-20°C for frogs and 35-37°C for rats. All of the experiments were carried out in venular microvessels, which were classified as segments where there is convergent flow, one or two branches distal to true capillaries. All of the vessels selected for cannulation had brisk blood flow and were free of white cells sticking on the vessel wall.

Measurement of Lp of Microvessel Wall

All measurements were based on the modified Landis technique, which measures the volume flux of water across the walls of microvessels perfused via a glass micropipette after occlusion of the vessel. The assumptions and limitations of the measurement have been evaluated in detail (5). The initial transcapillary water flow per unit area of the capillary wall (Jv/S)0 was measured at a given pressure (20-30 cmH2O for frog microvessels and 30-60 cmH2O for rat microvessels) within each vessel. Microvessel Lp was calculated as the slope of the relation between (Jv/S)0 and pressure. For each experimental design, control and multiple tests were conducted in the same vessel, minimizing the discrepancy of the responses between individual vessels and animals. One vessel was studied per animal.

Solutions

Ringer solutions and albumin-Ringer perfusates. Frog or mammalian Ringer solutions were used for dissecting mesenteries, superfusing tissue, and preparing the perfusion solutions. The frog Ringer solution was prepared as described for all previous experiments (5). The composition of the mammalian Ringer solution was as follows (in mM): 132 NaCl, 4.6 KCl, 2 CaCl2, 1.2 MgSO4, 5.5 glucose, 5.0 NaHCO3, and 20 HEPES and Na-HEPES. The pH of Ringer solution was maintained at 7.40-7.45 by adjusting the ratio of Na-HEPES to HEPES. An albumin-Ringer perfusate was prepared by adding BSA, 1 g/100 ml, to Ringer solution. All perfusates used for control and test perfusion contained BSA (1 g/100 ml).

Test solutions. ATP, bradykinin, 8-bromocAMP (8-BrcAMP), and 8-BrcGMP were purchased from Sigma. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) and 2',5'-dideoxyadenosine (ddA) were purchased from Biomole Research Laboratories (Plymouth Meeting, PA). Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was purchased from BioLog Life Science Institute (La Jolla, CA). All test solutions were freshly prepared in albumin-Ringer solution before each cannulation and applied to the microvessel lumen through perfusates.

Data Analysis and Statistics

All values are means ± SE, except where noted otherwise. Changes in Lp were expressed as the ratio of testing Lp versus control Lp (Lptest/Lpcontrol). For statistical comparisons, data obtained from the same vessel were analyzed by paired t-test and nonparametric Wilcoxon signed-rank test. Fisher's protected least significant differences test was conducted for data comparison among groups. The significance level was P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Roles of Basal Levels of cAMP and cGMP on Microvessel Permeability

Effect of reducing cAMP levels by inhibition of adenylate cyclase on basal Lp. We measured the changes in Lp in both frog and rat mesenteric microvessels after addition of ddA, a cell-permeable inhibitor of adenylate cyclase, to the microvessel perfusate to reduce generation of cAMP. Figure 1A shows the changes in Lp in response to ddA at concentrations of 0.5, 1, and 2 mM in a frog mesenteric microvessel. The mean control Lp of nine frog microvessels was 3.5 ± 0.8 × 10-7 cm · s-1 · cmH2O-1. Exposure to ddA (1 mM) induced an initial peak increase in Lp in 2-3 min, followed by a sustained elevation while ddA was present in the perfusate. Similar results were found in six rat mesenteric microvessels, in which the mean control Lp was 1.1 ± 0.13 × 10-7 cm · s-1 · cmH2O-1. Figure 1B summarizes the results from frog and rat mesenteric microvessels.


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  		Fig. 1.   Effect of inhibition of adenylate cyclase on basal hydraulic conductivity (Lp). A: single experiment to show time course of Lp changes in response to 2',5'-dideoxyadenosine (ddA) in a frog mesenteric microvessel. B: summarized results showing that exposure to ddA (1 mM) induced peak and sustained increases in Lp in both frog (n = 9) and rat (n = 6) mesenteric venular microvessels. *P < 0.05 from control.

The dependence of the effect of ddA on extracellular calcium was tested in four of the nine vessels in frog mesentery. In these four vessels, the mean control Lp was 4.7 ± 1.7 × 10-7 cm · s-1 · cmH2O-1. After extracellular calcium was removed, basal Lp was 4.5 ± 1.5 × 10-7 cm · s-1 · cmH2O-1. When each vessel was exposed to ddA in the absence of extracellular calcium, mean Lp was 5.1 ± 1.6 × 10-7 cm · s-1 · cmH2O-1, which was not significantly different from the control values, with or without extracellular calcium present.

Effect of inhibiting cAMP-dependent protein kinase on basal Lp. To test the downstream signaling pathways of cAMP on microvessel permeability, the effect of H-89, the protein kinase A (PKA) inhibitor, was tested on frog and rat venular microvessels. The results are summarized in Fig. 2.


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  		Fig. 2.   Summarized results showing effect of inhibition of cAMP-dependent protein kinase A on basal Lp. H-89 caused a sustained increase in Lp in both frog and rat microvessels. Left: results of 9 frog microvessels in response to 0.1 µM H-89. Right: results of rat microvessels in response to H-89 at concentrations of 0.1 (n = 3), 1 (n = 5), and 10 (n = 5) µM, respectively. *P < 0.05 from control.

In nine frog mesenteric microvessels, after each vessel was exposed to H-89 (0.1 µM), mean Lp increased from 3.5 ± 0.3 to 10.8 ± 1.6 × 10-7 cm · s-1 · cmH2O-1. The magnitude of this increase in Lp was sustained as long as the inhibitor was present. Albumin-Ringer solution was reperfused in four of nine vessels, and the mean Lp recovered to 1.1 ± 0.04 times the initial control value. Extracellular calcium dependence was tested in three of nine microvessels. Removal of extracellular calcium abolished the H-89-induced increase in Lp. The ratio of Lp measured with H-89 in the absence of extracellular calcium relative to calcium-free control was 0.9 ± 0.1.

The effects of H-89 at concentrations of 0.1, 1, and 10 µM were tested on 13 rat mesenteric microvessels. H-89 at 0.1 µM did not cause a significant increase in Lp, which is 1.4 ± 0.1 times the control (n = 3, P > 0.05). H-89 at concentrations of 1 (n = 5) and 10 µM (n = 5) caused twofold sustained increases in Lp (P < 0.05).

Effect of inhibiting guanylate cyclase on basal Lp. The effect of the guanylate cyclase inhibitor LY-83583 on basal Lp was tested in nine microvessels in frog mesenteries. The mean control Lp was 2.2 × 10-7 cm · s-1 · cmH2O-1. Addition of LY-83583 (10 µM) to the vessel lumen showed no significant effect on basal Lp. The mean ratio of Lp with LY-83583 present relative to control was 1.02 ± 0.11.

Effect of 8-BrcAMP on 8-BrcGMP-Induced Increase in Basal Lp

Experiments in frog mesenteric venular microvessels. We previously reported that the addition of the cGMP analog 8-BrcGMP (2 mM) in the perfusate caused a transient increase in basal Lp in frog mesenteric microvessels (16). To further evaluate the cross talk between cAMP- and cGMP-dependent pathways in the regulation of microvessel permeability, the effect of 8-BrcAMP at concentrations of 2 and 0.2 mM on increases in Lp caused by exposure to 8-BrcGMP (2 mM) were studied in frog mesenteric microvessels.

Exposure of vessels to 8-BrcAMP at 2 mM completely abolished the 8-BrcGMP (2 mM)-induced increases in Lp. The mean control Lp of 13 microvessels was 3.2 ± 0.7 × 10-7 cm · s-1 · cmH2O-1. Each vessel was perfused with 8-BrcAMP (2 mM) for 15 min. Lp in 9 of 13 vessels declined significantly (P < 0.05), and the Lp in the remaining 4 vessels showed no significant changes. The mean value of Lp for all 13 vessels was 2.5 ± 0.7 × 10-7 cm · s-1 · cmH2O-1. When each vessel was then perfused with 8-BrcGMP (2 mM) in the presence of 8-BrcAMP (2 mM), the mean Lp was 2.5 ± 0.6 × 10-7 cm · s-1 · cmH2O-1, which was not significantly different from the mean Lp value when 8-BrcAMP was present alone. After the vessel lumen was perfused with albumin-Ringer perfusate for 30 min, each vessel was exposed to 8-BrcGMP a second time without 8-BrcAMP present. Lp increased transiently to a mean peak value of 5.7 ± 1.4 times the control level. This increase was not significantly different from our previous results in which Lp increased 5.3 ± 0.5 times the control after exposure to 8-BrcGMP (16). The results are shown in Fig. 3.


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  		Fig. 3.   Effect of elevation of intracellular levels of cAMP on 8-BrcGMP-induced increases in basal Lp in frog mesenteric microvessels. Left: 2 mM 8-BrcAMP completely abolished increases in Lp caused by 8-BrcGMP (2 mM, n = 13). Right: addition of a reduced amount of 8-BrcAMP (0.2 mM) significantly attenuated 8-BrcGMP (2 mM) -induced increases in Lp (n = 6). Mean reduction was close to 50%. *P < 0.05 from control; **P < 0.05 from 8-BrcGMP response.

The effect of a lower concentration of 8-BrcAMP (0.2 mM) on 8-BrcGMP (2 mM)-induced increases in Lp was tested in another six microvessels. Results are summarized in Fig. 3, right. These vessels were first exposed to 8-BrcGMP (2 mM), and the mean peak Lp was 5.2 ± 0.8 times the control. Each vessel was then perfused with albumin-Ringer solution for 30 min, and baseline Lp was restored to 3.4 ± 0.3 × 10-7 cm · s-1 · cmH2O-1. When each vessel was exposed to 8-BrcGMP in the presence of 0.2 mM 8-BrcAMP, the transient increase in Lp was significantly reduced to 2.4 ± 0.6 times the control value (P < 0.05). Thus 0.2 mM 8-BrcAMP attenuated the increases in Lp induced by 2 mM of 8-BrcGMP but did not abolish the response as found with 2 mM 8-BrcAMP. A second washout with albumin-Ringer solution for 30 min was performed in three of the six microvessels and restored the baseline Lp to 3.9 × 10-7 cm · s-1 · cmH2O-1. When 8-BrcGMP was reapplied to each of the vessel lumens, Lp increased to a mean peak value of 4.7 ± 1.9 times the control value. In another seven microvessels, we checked that the magnitude of the increases in Lp in response to a second exposure to 8-BrcGMP in the same vessel was similar to the first exposure. The second exposure was given after each vessel was recovered with albumin-Ringer perfusion for 30 min.

Experiments in rat mesenteric microvessels. The effect of 8-BrcAMP on 8-BrcGMP-induced increases in basal Lp was tested in five venular microvessels in rat mesenteries. The mean control Lp was 1.58 ± 0.2 × 10-7 cm · s-1 · cmH2O-1. When each vessel was perfused with 8-BrcAMP (2 mM), the baseline Lp in four of five vessels decreased significantly in 15 min, and in one of the vessels Lp did not change. The mean Lp with 8-BrcAMP perfusion was 0.9 ± 0.1 × 10-7 cm · s-1 · cmH2O-1. Each vessel was then perfused with 8-BrcGMP (2 mM) in the presence of 8-BrcAMP. Mean Lp was 0.8 ± 0.2 × 10-7 cm · s-1 · cmH2O-1, which was not significantly different from the mean Lp value without 8-BrcGMP present. After the testing agents were washed out with albumin-Ringer solution for 30 min, each vessel was exposed to 8-BrcGMP (2 mM) again and mean peak Lp increased significantly to 2.0 ± 0.3 times the control (P < 0.05). Thus 8-BrcAMP (2 mM) completely attenuated the increase in Lp in rat microvessels caused by exposure to 8-BrcGMP (2 mM), a result very similar to that shown in Fig. 3 (left) for frog microvessels.

Effect of 8-BrcAMP on Agonist-Induced Increases in Lp

Effect of 8-BrcAMP on ATP and ATP plus 8-BrcGMP-induced increases in Lp in frog mesenteric microvessels. We carried out a series of investigations to test the effectiveness of cAMP in reducing the increases in permeability caused by exposure to acute inflammatory agonists. Results in nine frog venular microvessels are summarized in Fig. 4. At a concentration of 0.2 mM, 8-BrcAMP reduced the peak increase in Lp due to exposure to ATP (10 µM) alone by ~50%. In seven of the nine microvessels, ATP was given a third time after 8-BrcAMP was washed out with albumin-Ringer solution for another 30 min, and the mean ratio of peak Lp with ATP relative to control was restored. The third exposure to ATP in the same vessel demonstrated that the reduced response to a second exposure to ATP in the presence of 8-BrcAMP was not due to desensitization of ATP receptors (see Fig. 4 legend for Lp values). These data, combined with the results we reported previously that 8-BrcAMP, at a concentration of 2 mM, completely attenuated the ATP-induced increases in Lp (15), demonstrate a competitive interaction between cAMP (in the range of 0.2 to 2 mM) and ATP.


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  		Fig. 4.   Effect of addition of a reduced amount of 8-BrcAMP (0.2 mM) on ATP-induced increases in Lp in frog mesenteric microvessels (n = 9). Summarized results show that 0.2 mM 8-BrcAMP was sufficient to reduce ATP (10 µM)-induced increases in Lp. Mean control Lp was 2.9 ± 0.5 × 10-7 cm · s-1 · cmH2O-1. During the first exposure to ATP, mean ratio of peak value of ATP relative to control was 5.0 ± 0.8. In the presence of 8-BrcAMP (0.2 mM) mean peak value of ATP relative to control was reduced to 2.7 ± 0.8 in the same group of vessels. In 7 of these 9 vessels, ATP was given a third time after washing out the testing agent with albumin-Ringer solution for 30 min, mean peak increase in Lp relative to control was restored to 4.6 ± 0.3 times control. *P < 0.05 from control; **P < 0.05 from ATP response.

Our previous study (16) also demonstrated that 8-BrcGMP (2 mM) significantly potentiated the increase in Lp in vessels exposed to ATP. Figure 5 shows that this effect was completely abolished in the presence of 8-BrcAMP (2 mM). After testing that each of 11 microvessels responded to ATP (10 µM), as shown in Fig. 5, ATP was washed out for 30 min with albumin-Ringer perfusate. When each vessel was then exposed to ATP (10 µM) plus 8-BrcGMP (2 mM) in the presence of 8-BrcAMP (2 mM), the increases in Lp were significantly attenuated. This strong attenuation can be contrasted with the observation that 8-BrcGMP (2 mM) potentiated the peak ATP response from 7.0 ± 0.9 times the control value to 12.4 ± 1.9 times the control (16).


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  		Fig. 5.   Effect of 8-BrcAMP on 8-BrcGMP plus ATP-induced increases in Lp in frog mesenteric microvessels. A: paired measurements of Lp in a single microvessel are shown as a function of time. B: summarized results show that 2 mM 8-BrcAMP was sufficient to reduce 8-BrcGMP (2 mM) plus ATP (10 µM)-induced increases in Lp (n = 9). Mean control Lp was 6.2 × 10-7 cm · s-1 · cmH2O-1. When each vessel was exposed to ATP (10 µM) alone, Lp increased to a mean peak value of 7.0 ± 0.7 times the control. In the presence of 8-BrcAMP, mean peak increase in Lp in response to ATP (10 µM) plus 8-BrcGMP (2 mM) was only 1.3 ± 0.2 times control in the same group of vessels. *P < 0.05 from control; **P < 0.05 from ATP response.

Effect of 8-BrcAMP on bradykinin and bradykinin plus 8-BrcGMP-induced increases in Lp in rat mesenteric venular microvessels. The effect of 8-BrcAMP on bradykinin-induced increases in Lp was studied in five microvessels in rat mesenteries. The mean control Lp was 2.9 ± 0.7 × 10-7 cm · s-1 · cmH2O-1. Perfusion of 8-BrcAMP (2 mM) for 10-15 min decreased basal Lp to 1.8 ± 0.7 × 10-7 cm · s-1 · cmH2O-1. Each vessel was then exposed to bradykinin (1 nM) in the presence of 8-BrcAMP. The mean peak Lp in response to bradykinin was not significantly increased, being 1.1 ± 0.3 times the control (P > 0.05) and 1.7 ± 0.4 times the value of Lp after 8-BrcAMP perfusion (P > 0.05). When each vessel was exposed to bradykinin alone after washing out the 8-BrcAMP with albumin-Ringer solution for 30 min, the mean of peak Lp increased to 5.9 ± 0.8 times the control value within 5 min (P < 0.05) and then declined toward the control level in 10 min. The results are summarized in Fig. 6.


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  		Fig. 6.   Effect of 8-BrcAMP on bradykinin and 8-BrcGMP plus bradykinin-induced increases in Lp in rat mesenteric microvessels. A: paired measurements of Lp in a single microvessel are shown as a function of time. B: summarized results show that 2 mM 8-BrcAMP significantly reduced bradykinin (BK; n = 5)- and bradykinin plus 8-BrcGMP-induced increases in Lp (n = 4). *P < 0.05 from control; **P < 0.05 from BK response.

We demonstrated previously that the bradykinin-induced increases in Lp were potentiated in the presence of 2 mM 8-BrcGMP (16). To test the effect of cAMP (2 mM) on 8-BrcGMP plus bradykinin-induced increases in Lp, experiments were conducted in another four microvessels in rat mesenteries. Again, the 8-BrcGMP plus bradykinin-induced increase in Lp was significantly attenuated by 8-BrcAMP (2 mM), being only 1.3 ± 0.4 times the control value measured with albumin-Ringer perfusate (P > 0.05) and 2.8 ± 1.1 relative to the control measured with the presence of 8-BrcAMP (P > 0.05). The results are shown in Fig. 6B.

Effect of Inhibition of cAMP Degradation on 8-BrcGMP- and Bradykinin-Induced Increases in Lp

We hypothesized that one of the cGMP-dependent enzymes, cGMP-stimulated PDE II, may act to increase venular microvessel permeability by reducing cAMP levels in endothelial cells. To further test this hypothesis, changes in Lp in response to 8-BrcGMP and to bradykinin after pretreatment with EHNA, a specific inhibitor of PDE II, were studied in rat mesenteric microvessels. The effect of EHNA (10 µM) on 2 mM 8-BrcGMP-induced increases in Lp was tested in five microvessels. Mean control Lp was 1.8 ± 0.3 × 10-7 cm · s-1 · cmH2O-1. After perfusing with EHNA for 30 min, the mean Lp was not significantly different from the mean control value. Each vessel was then exposed to 8-BrcGMP. Under these conditions, 8-BrcGMP did not increase microvessel permeability. The mean Lp relative to the original control level was 0.9 ± 0.1 times control. In four of the five vessels, 8-BrcGMP was reapplied after each vessel was perfused with albumin perfusate for 30 min. The mean increase in Lp was 2.0 times the second control value (1.5 ± 0.1 × 10-7 cm · s-1 · cmH2O-1). These results are shown in Fig. 7, left.


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  		Fig. 7.   Effect of inhibition of cAMP degradation on 8-BrcGMP- (left) and bradykinin (right)-induced increases in Lp in rat mesenteric microvessels. Summarized results show that pretreating the vessel with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), a specific inhibitor of phosphodiesterase, prevented 8-BrcGMP (n = 5)- and bradykinin (n = 9)-induced increases in Lp. *P < 0.05 from control; **P < 0.05 from 8-BrcGMP or bradykinin response.

EHNA, an inhibitor of PDE II, also blocked bradykinin-induced increases in Lp in rat venular microvessel. The mean control Lp of nine vessels was 1.8 ± 0.2 × 10-7 cm · s-1 · cmH2O-1. In four of nine vessels, EHNA was applied for 30 min after the initial control measurement, followed by the addition of bradykinin. After EHNA was washed out for a further 30 min, the response to bradykinin alone was measured. In the remaining five vessels, the experimental order was reversed. Data from each individual group are presented in Fig. 7, right. The results were not significantly different between these two groups. Combining data from the two groups, the mean peak increase in Lp in response to bradykinin after pretreatment with EHNA was only 1.4 ± 0.3 times the control value (P > 0.05), whereas with bradykinin alone, Lp increased to 3.5 ± 0.4 times the control (P < 0.05). Thus inhibition of PDE II attenuates the increase in permeability caused by exposure to bradykinin and cGMP analog.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These experiments are an extension of our previous investigation of the individual roles of cAMP and cGMP in the regulation of microvessel permeability. Previously, we and other investigators found that increased levels of cAMP prevented ATP-induced increases in microvessel permeability (15), and that increased levels of cGMP are necessary for inflammatory mediator-induced, NO-dependent increases in microvessel permeability (14, 16, 37). The aim of this study was to link these two processes together and identify the relationship and potential site of the cross talk between cAMP- and cGMP-dependent signal transductional pathways in the regulation of microvessel permeability under resting and activated states. Important new findings are 1) reduction of basal levels of cAMP and inhibition of the downstream pathway of cAMP resulted in an increase in microvessel permeability under resting conditions; 2) activation of cAMP-dependent pathways through the use of the cAMP agonist 8-BrcAMP abolished or significantly attenuated 8-BrcGMP- and inflammatory mediator-induced increases in Lp in both frog and rat mesenteric microvessels; and 3) inhibition of PDE II, the cGMP-stimulated phosphodiesterase, thereby reducing the degradation of cAMP, abolished cGMP- and bradykinin-induced increases in permeability. These results demonstrate that basal levels of cAMP are critical to maintaining the microvessel permeability under resting conditions, and that elevated cGMP levels increased microvessel permeability by a mechanism that may reduce cAMP or compete with the action of cAMP.

Basal Levels of Endothelial Cell cAMP and cAMP-Dependent Pathways are Important for Maintaining Basal Permeability of Microvessels

The effect of increased levels of cellular cAMP, which lowers basal permeability and attenuates inflammatory mediator-induced increases in permeability in both cultured endothelial monolayers and isolated or individually perfused intact microvessels, has been well documented (1, 3, 15, 26). However, the essential role of basal levels of cAMP in maintaining microvessel permeability under resting conditions has not been well defined. The effect of reducing cellular cAMP levels on basal permeability in intact microvessels has not been described previously.

Basal levels of cAMP measured in different types of cultured endothelial cells varied from 1.5 to 26 pmol/mg protein (18, 33, 35). This variation may reflect the differences in cell types, cell conditions, and methodologies. However, basal levels of cGMP, measured by the same methods in the same cell types, had been reported to be much lower than cAMP. In most cases, the cGMP content was 20-27% of cAMP levels (18, 35), although Vigne et al. (33) reported cGMP levels as 60% of cAMP. Those data suggest that, under resting conditions, the content of cAMP in endothelial cells is much greater than that of cGMP. Our results demonstrating that a reduction of basal levels of cAMP caused an increase in resting microvessel permeability, and the use of LY-83583 to inhibit guanylate cyclase showed no effect on basal Lp but significantly attenuated the ATP-induced increases in Lp (17), indicate that the role of cAMP is dominant over cGMP in maintaining basal permeability properties, and that increased levels of cGMP are necessary to increase Lp under stimulated conditions. A previous study by Meyer and Huxley (25) using another guanylate cyclase inhibitor, methylene blue, also showed no effect on baseline microvessel permeability in frog mesenteric microvessels, which was consistent with our results.

In addition, exposure of microvessels to H-89 showed an increase in basal Lp. H-89 has been widely used as an inhibitor of PKA both in vivo and in vitro. It has a more selective inhibition for PKA than for other kinases (4). Its inhibition constant is 10 times higher for protein kinase G, and 600-1,000 times higher for other serine/threonine kinases (4). Our results indicate that basal levels of cAMP exert their effect on microvessel permeability through the activation of PKA-dependent pathways under resting conditions. Another application of H-89 in cultured pulmonary artery endothelial cells (34) demonstrated that H-89 caused translocation of filamin, leading to disassembly of the dense peripheral band in the absence of an inflammatory agent, which was a supportive result for the increases in permeability we observed. Thus those results suggested that basal levels of PKA activity may be needed to maintain cytoskeletal structures essential to maintaining normal permeability.

Previous investigations in our laboratory (15, 17) have demonstrated that, in the presence of an inflammatory mediator, an increase in endothelial cell [Ca2+]i was necessary for an increase in microvessel permeability, although other factors, including cAMP and cGMP, may modulate the changes in permeability downstream from calcium entry. In addition, there are also processes that may bypass the initial calcium mobilization and elicit an increase in permeability without changing endothelial cell [Ca2+]i (13, 14, 16). Our current findings that increased Lp, induced either by inhibition of adenylate cyclase (by ddA) or PKA (by H-89), was abolished by removal of extracellular calcium demonstrate an additional calcium-dependent process, which increases permeability. Specifically, an increase in Lp after reducing cAMP or blocking PKA required the presence of extracellular calcium.

Balance Between Endothelial Concentrations of cAMP and cGMP Controls Microvessel Permeability

We previously demonstrated that increased levels of cAMP and cGMP in endothelial cells play opposite roles in the regulation of microvessel permeability. It has been reported that increasing cAMP levels by the cAMP analog 8-BrcAMP or reducing the degradation of cAMP using an inhibitor of phosphodiesterase IV, reduced basal microvessel permeability and attenuated ATP-induced increases in Lp (1, 10, 15). In addition, an elevation of cGMP levels increased basal Lp and potentiated ATP- and bradykinin-induced increases in permeability (16). Measurement of endothelial cell [Ca2+]i under the same experimental conditions as those in which the effects of cAMP and cGMP were studied demonstrated that both cAMP- and cGMP-dependent signaling pathways to modulate microvessel permeability occur downstream from calcium entry. Neither the action of cAMP nor cGMP involves modification of the receptor-mediated increases in endothelial [Ca2+]i (15, 16). Figure 8 summarizes the results of our previous and current studies in intact venular microvessels, which demonstrate the roles of endothelial cell [Ca2+]i and the two intracellular nucleotides, cAMP and cGMP, in the regulation of microvessel permeability.


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  		Fig. 8.   Potential signal transductional pathways that regulate microvessel permeability. Hatched area has been investigated previously by our laboratory and others. Shaded area represents mechanisms tested in this study. NO, nitric oxide; NOS, nitric oxide synthase; cG, cGMP; LC, light chain; [Ca2+]i intracelluar Ca2+ concentration; PDE II, phosphodiesterase.

Our studies with cAMP conform to consistent in vivo and in vitro observations that experimental conditions that increase intracellular cAMP levels reduce basal permeability and attenuate leakage of plasma proteins induced by a variety of inflammatory mediators. However, the effect of increased levels of cGMP on permeability has been found to vary with experimental conditions and endothelial cell type. In a number of in vitro studies using cultured endothelial cells from large vessels, elevation of cGMP by NO donors, atrial natriuretic peptide, or cGMP analogs was found to decrease agonist-induced increases in endothelial permeability (2, 8, 32, 35), although discrepancies have been reported among different types of endothelial cells and different laboratories. In contrast, in most of the in vivo studies, increases in microvessel permeability have been reported as NO-cGMP dependent (14, 16, 25, 36, 37). For example, inhibitors of NO synthase and guanylate cyclase suppressed both histamine- and vascular endothelial growth factor (VEGF)-induced hyperpermeability in isolated porcine coronary venules (36, 37) and attenuated ATP, bradykinin, and substance P-induced increases in microvessel Lp in intact frog and rat mesenteric venular microvessels (14, 27). Other studies in rat cerebrovasculature and hamster cheek pouch also demonstrated that the production of NO is involved in increases in protein leakage in response to histamine, VEGF, and platelet-activating factor (23, 24, 28). Although cGMP levels have not been measured in vivo, it has been demonstrated in cultured endothelial cells that both ATP and bradykinin increase cGMP production (11). These studies together provide strong evidence to support the idea that a [Ca2+]i-activated NO-cGMP-dependent pathway might be a common pathway leading to an increase in microvessel permeability after exposure to inflammatory agents. The potential downstream targets for increased levels of cAMP and cGMP to regulate microvessel permeability are illustrated in Fig. 8 and are discussed briefly below. We note that in some whole organ studies experimental conditions that increase cGMP protect microvessels against increased permeability (21). The mechanisms responsible for these actions are not well understood, but cGMP may regulate some of the mechanisms favoring the interaction of platelets and leukocytes with the microvessel wall. Neither platelets nor leukocytes were present in our experiments.

Our present results demonstrated that using the same or lesser concentrations of cAMP analog was sufficient to overcome cGMP analog- and/or inflammatory mediator-induced increases in microvessel permeability. One of the mechanisms that may be involved is competition between cAMP and cGMP for a common target to regulate microvessel permeability. The actions of increased cAMP levels in preventing a variety of inflammatory mediator-induced increases in permeability are thought to be a result of the modulation of contractile mechanisms in endothelial cells. For example, phosphorylation of myosin light chain kinase by PKA decreases the force generation (9). Thus one possible site for increased cGMP to compete with the action of cAMP is the cytoskeleton to increase tension. However, at present there is no direct evidence to support this mechanism in venular endothelial cells. The predominant effect of increased cGMP in smooth muscle cells is to reduce tension and cause relaxation by reducing calcium influx and calcium release. The fact that no change in [Ca2+]i was found with an elevation in cGMP in microvessel endothelial cells indicates that the action of cGMP in venular endothelial cells may differ from that in smooth muscle cells.

One of the downstream targets for increased levels of cGMP to regulate microvessel permeability may be via a cGMP-dependent protein kinase (cGK). The existence of cGK has been demonstrated in bovine aortic endothelial cells, in rat pulmonary microvascular endothelial cells, and in human aortic and pulmonary artery endothelial cells, as well as in foreskin microvessel endothelial cells; low expression was found in human umbilical vein endothelial cells (7, 8, 22). Many kinases, including cAMP-dependent protein kinase and cGK, have been shown to be linked to cytoskeletal elements (19), suggesting that cAMP and cGMP might compete with each other and with other kinases (including Rho-dependent kinase, protein kinase N, and protein kinase C) to regulate cytoskeleton in endothelial cells. Studies conducted by Wu et al. (36) showing that an inhibitor of cGK prevented VEGF-induced hyperpermeability in isolated porcine coronary venules directly implicate cGK in increased permeability. However, the direct effect of cGMP on contractile mechanisms in venular endothelial cells requires further study.

A second site of the action of cAMP to reduce permeability is to increase the resistance to water and solute flow in the intercellular cleft between endothelial cells (26). A recent study by Adamson et al. (1) demonstrated that increased cAMP was associated with an increase in the mean number of tight-junction strands between endothelial cells, which may account for a reduction in microvessel permeability. Thus the assembly of junctional complexes might be another potential target of competitive interaction between cGMP and cAMP.

An alternative hypothesis to the competitive interaction of cAMP and cGMP on a common target is that increased cGMP increases microvessel permeability by lowering intracellular levels of cAMP. One possibility is that the interaction of cAMP and cGMP is at the site of cyclic nucleotide degradation by a specific cGMP-dependent cAMP PDE II, which lowers cAMP levels. Thus increased cGMP, by stimulating PDE II, may lower cAMP levels, resulting in cGMP-dependent increases in microvessel permeability. Such mechanisms have been demonstrated in human platelets, which showed that, when cGMP is present, PDE II plays a major role in the hydrolysis of cAMP and restricts any increases in cAMP concentration (6). cGMP (10 µM) will stimulate a three- to sixfold increase in PDE II activity in human brain tissues (31). Studies in cultured endothelial monolayers showed that TNF-alpha -induced increases in permeability were associated with a reduction in intracellular cAMP content, which was partly attributed to increased cyclic nucleotide phosphodiesterase activities. The effect of TNF-alpha was both time and dose dependent and occurred in parallel with a drop in endothelial cell cAMP content. PDE II activity was reportedly increased 150% after TNF-alpha treatment, which was attributed to cGMP-activated hydrolysis of cAMP (20). Another study reported that hypoxia-induced increases in permeability in cultured endothelial monolayers were associated with a progressive decrease in endothelial cAMP levels, which fell from 60 to 15 pmol/mg protein (29). All of these in vitro results suggest that decreased cAMP levels via cGMP-stimulated phosphodiesterase may be the mechanism whereby activation of cGMP-dependent pathways increases microvessel permeability.

Our observations that lowering cAMP levels by inhibition of adenylate cyclase using ddA increased basal Lp, and that using EHNA, a selective inhibitor of PDE II, to prevent decreases in cAMP abolished or attenuated cGMP analog- and bradykinin-induced increases in Lp provide the first in vivo evidence to support the above hypothesis. These results suggest a significant role for a cGMP-dependent phosphodiesterase in the regulation of microvessel permeability. In vitro, the activity of PDE II at a substrate concentration of 10 µM [3H]cGMP was potently inhibited by EHNA (IC50 1.4 µM), whereas the activities of PDE V and PDE III were not significantly affected by EHNA concentrations up to 100 µM. Furthermore, EHNA was more effective as an inhibitor of PDE II enzyme activity in the presence of cGMP rather than in its absence, in which the IC50 values were 3.3 and 14.6 µM, respectively (6). A study using an isolated, blood-perfused rat lung model showed that EHNA reversed the hypoxic pressor response, and anion-exchange chromatography showed that its action was mainly due to the inhibition of PDE II (12). Our observations that EHNA suppressed both 8-BrcGMP- and bradykinin-induced increases in microvessel permeability suggest that when PDE II is inhibited, the cascade of events leading to decreased cAMP is also blocked. The results further support the hypothesis that decreased cAMP levels via cGMP-stimulated PDE II may be the mechanism whereby the activation of cGMP-dependent pathways increase microvessel permeability.

In summary, our observations have shown that basal levels of cAMP are necessary in maintaining basal microvessel permeability, and that increased levels of cAMP inhibit the increases in microvessel permeability brought about by increased endothelial cGMP concentrations. The antagonistic effect between levels of cAMP and cGMP in the regulation of microvessel permeability suggests that the balance between endothelial concentrations of these two opposing cyclic nucleotides controls permeability of the microvessel. cAMP has the dominant role in maintaining basal permeability, and increased levels of cGMP are necessary to increase permeability. If cAMP levels in endothelial cells are not sufficient to inhibit the effect of increased cGMP levels, then an increase in permeability will result. The action of cGMP may be brought about by reducing the level of cAMP due to activation of cGMP-dependent PDE II or by competition with the nucleotides for a common cytoskeletal target to regulate microvessel permeability.


    ACKNOWLEDGEMENTS

This work was supported by the American Heart Association National Center Grant-In-Aid 96011510 (to P. He), and by National Heart, Lung, and Blood Institute Grants HL-56237 (to P. He) and HL-28607 (to F. E. Curry).


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. He, Dept. of Human Physiology, School of Medicine, Univ. of California Davis, One Shields Ave., Davis, CA 95616-8644 (E-mail: pnhe{at}ucdavis.edu).

Received 19 March 1999; accepted in final form 26 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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