| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Departments of Medicine and Biopharmaceutical Sciences, Colleges of Medicine and Pharmacy, University of Illinois at Chicago, and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois
Submitted 25 August 2006 ; accepted in final form 10 April 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
microcirculation; postcapillary venules; inflammation; plasma exudation; fluorescein isothiocyanate-dextran; adenosine; genistein; tyrphostin 25
Bradykinin has been shown to mediate the increase in microvascular permeability elicited by smokeless (chewing) tobacco and gingipain RgpA, a protease released by Porphyromonas gingivalis, a bacterium thought to underlie periodontal disease in humans, in the intact hamster cheek pouch microcirculation (10, 24). Similarly, substance P mediates grain sorghum dust-induced increase in macromolecular efflux from the hamster cheek pouch microcirculation (11). However, the signal transduction pathway(s) distal to bradykinin and substance P receptor activation in postcapillary venules that mediates this response was not delineated in these studies.
To this end, protein tyrosine kinase is a ubiquitous family of intracellular signaling enzymes that plays an important role in modulating endothelial cell function, including microvascular permeability (1, 3, 16, 29). For instance, Kim and Durán (16) showed that genistein and tyrphostin 25, two structurally unrelated, nonspecific inhibitors of protein tyrosine kinase that affect receptor-linked and cytosolic kinases (13), significantly attenuate platelet-activating factor-induced increase in macromolecular efflux from the intact hamster cheek pouch microcirculation. It is well established that bradykinin modulates endothelial cell function in vitro, in part, by activating the protein tyrosine kinase metabolic pathway (4, 8, 15, 27). However, Félétou et al. (7) showed that genistein had no significant effects on bradykinin-induced increase in macromolecular efflux from the hamster cheek pouch microcirculation. By contrast, Ikezaki et al. (14) showed that both genistein and tyrphostin 25 significantly attenuated bradykinin-induced vasodilation in the same preparation. Taken together, these data suggest that protein tyrosine kinase(s) is involved in bradykinin modulation of arteriolar tone but not microvascular permeability in the hamster cheek pouch. Whether protein tyrosine kinase metabolic pathway also modulates the edemagenic effects of substance P in the hamster cheek pouch microcirculation is uncertain (17). Accordingly, I hypothesized that the protein tyrosine kinase metabolic pathway modulates plasma exudation from postcapillary venules evoked by both bradykinin and substance P in the oral microcirculation.
The purpose of this study was to begin to address these issues by determining whether inhibitors of protein tyrosine kinase attenuate bradykinin- and substance P-induced increase in macromolecular efflux from the intact hamster cheek pouch microcirculation and, if so, whether these effects are specific.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
Preparation of animals. Adult male golden Syrian hamsters weighing 128 ± 2 g were used in these studies as previously described in our laboratory (2, 9–11, 20, 24, 25, 30). Each animal was anesthetized with pentobarbital sodium (6 mg/100 g body wt ip). A tracheostomy was performed to facilitate spontaneous breathing. The left femoral vein was cannulated to inject the intravascular tracer, fluorescein isothiocyanate-labeled dextran (FITC-dextran; molecular mass, 70 kDa) and supplemental anesthesia (2–4 mg·100 g body wt–1·h–1). The left femoral artery was cannulated to obtain arterial blood samples and to monitor systemic arterial pressure and heart rate during the experiment. Body temperature was kept constant (37–38°C) during the experiment using a heating pad.
To visualize the microcirculation of the cheek pouch, we used a method previously described in our laboratory (2, 9–11, 20, 24, 25, 30). Briefly, the left cheek pouch was spread gently over a small plastic baseplate and an incision was made in the skin to expose the cheek pouch membrane. The avascular connective tissue layer was carefully removed, and a plastic chamber was positioned over the baseplate and secured in place by suturing the skin around the upper chamber. This chamber contained the suffusion fluid. This arrangement forms a triple-layered complex: the baseplate, the upper chamber, and the cheek pouch membrane exposed between both plates. The hamster was then transferred to a heated microscope stage. The chamber was connected to a reservoir containing warmed (37–38°C) bicarbonate buffer (composition, in mM: 131.9 NaCl, 2.95 KCl, 1.48 CaCl2, 0.76 MgCl2, and 11.87 NaHCO3), which allowed continuous suffusion of the cheek pouch. The buffer was bubbled continuously with 95% N2-5% CO2 (pH = 7.4). The chamber was also connected via a three-way valve to an infusion pump (Sage Instruments, Cambridge, MA) that allowed for constant administration of drugs into the suffusate.
Determination of clearance of macromolecules. The cheek pouch microcirculation was visualized with an Olympus microscope (Olympus America, Melville, NY) coupled to a 100-W mercury light source at a magnification of x40. Fluorescence microscopy was accomplished with the aid of filters that matched the spectral characteristics of FITC-dextran as previously described in our laboratory (2, 9–11, 20, 24, 25, 30). Macromolecular leakage was determined by extravasation of FITC-dextran, which appeared as fluorescent "spots" or leaky sites around postcapillary venules. The number of leaky sites was determined by counting three random microscopic fields every minute for the first 7 min and then at 5-min intervals for 30–60 min after each intervention (see below). The total number of leaky sites was averaged and expressed as the number of leaky sites per 0.11 cm2 of cheek pouch, which corresponds to an area of one microscopic field (2, 9–11, 20, 24, 25, 30).
In experiments in which clearance of FITC-dextran was calculated, the suffusate was collected at 5-min intervals throughout the experiment by a fraction collector (Microfractionator, Gilson Medical Electronics, Middleton, WI). Samples were collected in glass test tubes and the concentration of FITC-dextran was determined in each tube. Arterial blood samples were collected in heparinized capillary tubes (70-µl volume; Scientific Products, McGaw Park, IL) beginning 5 min before and 5, 30, 60, 120, 180, and 240 min after intravenous injection of FITC-dextran. The concentration of FITC-dextran was determined in all plasma samples.
To quantitate the concentration of FITC-dextran in plasma and suffusate, a standard curve for FITC-dextran concentrations vs. percent emission was generated on a spectrophotofluorometer (Perkin-Elmer, Norwalk, CT). The standard was FITC-dextran prepared on a weight per volume basis. With bicarbonate buffer as background, a standard curve was generated for each experiment and each curve was subjected to linear regression analysis. The percent emission for unknown samples (plasma and suffusate) was determined by the spectrophotofluorometer and the concentration of FITC-dextran was then calculated from the standard curve. In preliminary experiments, minimal fluorescence signal (<2% above background) was detected when drugs were added to the buffer and when plasma and suffusate samples were examined before adding FITC-dextran. Clearance of FITC-dextran was determined by calculating the ratio of suffusate (ng/ml) to plasma (mg/ml) concentration of FITC-dextran and multiplying this ratio by the suffusate flow rate (2 ml/min) (2, 9–11, 20, 24, 25, 30).
Experimental Protocols
Effects of protein tyrosine kinase inhibitors on agonist-induced increase in macromolecular efflux. The purpose of these studies was to determine whether genistein and tyrphostin 25, two structurally unrelated, nonspecific inhibitors of protein tyrosine kinase (1, 3, 7, 8, 13, 16, 29), attenuate bradykinin-, substance P-, and adenosine-induced increase in macromolecular efflux from the intact hamster cheek pouch microcirculation. To this end, adenosine increases macromolecular efflux from the intact hamster cheek pouch microcirculation through a metabolic pathway(s) distinct from that of bradykinin and substance P (2, 25).
The experimental design was similar to that outlined in previous studies from our laboratory (2, 9–11, 14, 20, 24, 25, 30). Briefly, after suffusing buffer for 30 min (equilibration period), FITC-dextran was injected intravenously, and the number of leaky sites and clearance of FITC-dextran were determined for 60 min. Then, bradykinin, substance P (each, 0.5 and 1.0 µM) or adenosine (0.5 µM) were suffused onto the cheek pouch in a random fashion. Each concentration was suffused for 5 min. The number of leaky sites was determined before and every min for 7 min and at 5-min intervals for 45 min thereafter. Clearance of FITC-dextran was determined before and every 5 min thereafter for 45 min. The time interval between subsequent suffusions of each agonist was at least 45 min (2, 9–11, 20, 24, 25, 30). After suffusion of each agonist was stopped and the number of leaky sites returned to baseline within 10–15 min, genistein (1.0 µM) or tyrphostin 25 (10.0 µM) was suffused for 30 min, and suffusion of each agonist was repeated as outlined above (7, 14, 16). The number of leaky sites and clearance of FITC-dextran were determined during each intervention as outlined above.
In preliminary studies, we determined that repeated suffusions of bradykinin, substance P (each, 0.5 and 1.0 µM), and adenosine (0.5 µM) were associated with reproducible results. In addition, suffusion of saline (vehicle) for the entire duration of the experiments was not associated with visible leaky site formation or significant increase in clearance of FITC-dextran. Likewise, suffusion of genistein (1.0 µM) and tyrphostin 25 (10.0 µM) alone for 30 min had no significant effects on macromolecular efflux from the cheek pouch. Lastly, suffusion of DMSO (1.0 µM), the solvent of genistein and tyrphostin 25, for 30 min had no significant effects on bradykinin (0.5 µM)- and substance P (1.0 µM)-induced responses (data not shown; each group, n = 4 animals; P > 0.5). The concentrations of bradykinin, substance P, adenosine, genistein and tyrphostin 25 used in these experiments were based on previous studies in our laboratory and reports in the literature (2, 5–7, 9–11, 14, 16, 20, 22, 24, 25, 30).
Drugs and Chemicals
Fluorescein isothiocyanate-labeled dextran, bradykinin, substance P, genistein, tyrphostin 25, adenosine, and DMSO were purchased from Sigma-Aldrich (St. Louis, MO). Genistein and tyrphostin 25 were dissolved in DMSO as a 1.0 mM stock solution and diluted in saline to the desired concentration. The final concentration of DMSO in the cheek pouch suffusate was 1.0 µM. All other drugs were prepared and diluted in saline to the desired concentrations on the day of the experiment.
Data and Statistical Analyses
Data are expressed as means ± SE except for body weight, which is expressed as means ± SD. Because the number of leaky sites returned to baseline (nil) between successive applications of test compounds, all vehicle (saline) control data are expressed as a single value for each experimental condition. Statistical analysis was performed on actual values using repeated-measures analysis of variance with Neuman-Keuls multiple-range post hoc test to detect values that were different from control values. A P < 0.05 was considered statistically significant; n is given as the number of experiments, with each experiment representing a separate animal.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
Effects of Tyrosine Kinase Inhibitors on Agonist-Induced Increase in Macromolecular Efflux
Suffusion of bradykinin elicited a significant concentration-dependent increase in leaky site formation and clearance of FITC-dextran (Fig. 1; each group, n = 4 animals; P < 0.05). These responses were significantly attenuated by genistein (1.0 µM) and tyrphostin 25 (10.0 µM) (Fig. 1; each group, n = 4 animals; P < 0.05). The number of leaky sites decreased significantly from 12 ± 1/0.11 cm2 during suffusion of bradykinin (1.0 µM) alone to 4 ± 1/0.11 cm2 during suffusion of genistein (1.0 µM) followed by bradykinin (1.0 µM) (Fig. 1, top; each group, n = 4 animals; P < 0.05). Similarly, clearance of FITC-dextran decreased significantly from 43.0 ± 4.4 ml/min x 10–6 during suffusion of bradykinin (1.0 µM) alone to 18.3 ± 2.5 ml/min x 10–6 during suffusion of genistein (1.0 µM) followed by bradykinin (1.0 µM) (Fig. 1, bottom; each group, n = 4 animals; P < 0.05).
|
Suffusion of substance P elicited a significant concentration-dependent increase in leaky site formation and clearance of FITC-dextran (Fig. 2; each group, n = 4 animals; P < 0.05). These responses were significantly attenuated by genistein (1.0 µM) and tyrphostin 25 (10.0 µM) (Fig. 2; each group, n = 4 animals; P < 0.05). The number of leaky sites decreased significantly from 13 ± 1/0.11 cm2 during suffusion of substance P (1.0 µM) alone to 8 ± 1/0.11 cm2 during suffusion of genistein (1.0 µM) followed by substance P (1.0 µM) (Fig. 2, top; each group, n = 4 animals; P < 0.05). Similarly, clearance of FITC-dextran decreased significantly from 39.9 ± 9.3 ml/min x 10–6 during suffusion of substance P (1.0 µM) alone to 23.6 ± 5.0 ml/min x 10–6 during suffusion of genistein (1.0 µM) followed by substance P (1.0 µM) (Fig. 2, bottom; each group, n = 4 animals; P < 0.05). The number of leaky sites decreased significantly from 15 ± 3/0.11 cm2 during suffusion of substance P (1.0 µM) alone to 4 ± 3/0.11 cm2 during suffusion of tyrphostin 25 (10.0 µM) followed by substance P (1.0 µM) (Fig. 2, top; each group, n = 4 animals; P < 0.05). Similarly, clearance of FITC-dextran decreased significantly from 32.9 ± 1.0 ml/min x 10–6 during suffusion of substance P (1.0 µM) alone to 23.2 ± 9.1 ml/min x 10–6 during suffusion of tyrphostin 25 (10.0 µM) followed by substance P (1.0 µM) (Fig. 2, bottom; each group, n = 4 animals; P < 0.05).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
The results of this study extend the observations of Kim and Durán (16), who showed that genistein and tyrphostin 25, at concentrations similar to those used in this study, significantly attenuated platelet-activating factor-induced increase in macromolecular efflux from the intact hamster cheek pouch microcirculation. However, the results of this study contrast with those reported by Félétou et al. (7), who showed that suffusion of genistein (5 and 10 µM) had no significant effects on bradykinin (0.3 µM)-induced increase in macromolecular efflux in the same preparation. They also found that bisindolylmeleimide, a protein kinase C inhibitor, had no significant effects on bradykinin-induced responses in the cheek pouch. By contrast, Murray et al. (22) showed that pharmacological inhibition of protein kinase C significantly attenuated bradykinin-induced increase in macromolecular efflux from the intact hamster microcirculation.
Although the reasons underlying the disparate results reported by Félétou et al. (7) are uncertain, differences in the experimental protocol and scoring technique may have partly accounted for them. For instance, Félétou et al. (6, 7) suffused bradykinin onto the cheek pouch at a flow rate of 6 ml/min, whereas we and other investigators are suffusing bradykinin at 2 ml/min (2, 9–11, 14, 19, 20, 22, 24, 25, 30). They showed that reducing the perfusion rate alters the response of the cheek pouch microcirculation to bradykinin (7). Although these investigators reported a 
10-fold higher number of leaky sites elicited by bradykinin (1.0 µM) in the cheek pouch compared with the number of leaky sites we observed in this study (6), a similar increase in clearance of FITC-dextran from cheek pouch was reported in both studies. The definition of a "leaky site" in the cheek pouch we used in this study, a fluorescent spot with >100-µm diameter, is based on the study of Matsuda et al. (20) and has been used in previous studies in our laboratory as well (2, 9–11, 20, 24, 25, 30). Whether Félétou et al. (6, 7) used a similar definition is uncertain.
The above notwithstanding, the hamster cheek pouch is an established animal model to study the effects of proinflammatory mediators, such as bradykinin and substance P, on macromolecular efflux from postcapillary venules in situ and the mechanisms underlying these phenomenon (2, 3, 5–7, 9–12, 16, 18–20, 22–26, 30). In this model, solute efflux is determined by two reproducible parameters, leaky site formation and clearance of FITC-dextran, thereby providing quantitative appraisal of macromolecular transport across postcapillary venules in the cheek pouch during experimental interventions. Importantly, successive suffusions of test compounds at appropriate time intervals are associated with reproducible formation of leaky sites and increases in clearance of FITC-dextran in the absence of tachyphylaxis. Consequently, changes in macromolecular efflux can be tested repeatedly in the same cheek pouch so that each animal serves as its own control. This experimental approach enabled us to reduce the overall number of animals required to perform the study and facilitates data analysis (2, 3, 5–7, 9–12, 16, 18–20, 22–26, 30).
Conceivably, bradykinin- and substance P-induced increase in macromolecular efflux from the cheek pouch microcirculation and its inhibition by genistein and tyrphostin 25 may have been mediated, in part, by changes in vasomotor tone and/or increase in venular driving pressure in the cheek pouch. However, this possibility seems unlikely because other investigators showed that agonist-induced increase in macromolecular efflux from postcapillary venules in the hamster cheek pouch and other microvascular beds and species are independent of changes in vasomotor tone and increase in venular driving pressure (3, 18, 19, 21–23, 26, 28).
For instance, Rubinstein et al. (25) showed that nitroglycerin elicits vasodilation in the hamster cheek pouch but fails to increase macromolecular efflux evoked by a selective adenosine A1 agonist in the same preparation. Mayhan et al. (18) showed that leukotriene C4 elicits potent vasoconstriction in the hamster cheek pouch but at the same time increases microvascular permeability in a nitric oxide-dependent fashion (19). Kim and Durán (16) showed that both genistein and tyrphostin 25 have no significant effects of platelet-activating factor-induced vasoconstriction in the hamster cheek pouch but they significantly attenuate platelet-activating factor-induced increase in macromolecular efflux in the same preparation. To this end, Ikezaki et al. (14) showed that suffusion of genistein and tyrphostin 25, at concentrations similar to those used in this study, had no significant effects on resting arteriolar diameter in the cheek pouch. In addition, vascular endothelial growth factor elicits vasodilation and increases microvascular permeability in the hamster cheek pouch by different mechanisms (3). Taken together, these data coupled with the results of this study suggest that microvascular smooth muscle tone and postcapillary venular permeability in the hamster cheek pouch are modulated by distinct mechanisms (3, 18, 19, 21–23, 26, 28).
We did not attempt to identify the protein tyrosine kinase(s) that modulates bradykinin- and substance P-induced increase in macromolecular efflux from the cheek pouch microcirculation but rather probed the overall role of the protein tyrosine kinase metabolic pathway in modulating the edemagenic effects of both peptides (8, 15, 17, 27, 29). Our experimental approach was consistent with previous studies that used genistein and tyrphostin 25 to modulate agonist-induced responses in the hamster cheek pouch microcirculation (7, 14, 16). To this end, the results of this study suggest that bradykinin and substance P each activates a distinct protein tyrosine kinase(s) in the cheek pouch microcirculation leading to macromolecular efflux from postcapillary venules because genistein was more effective than tyrphostin 25 in attenuating bradykinin-induced responses, whereas the opposite was observed with substance P. Given that both genistein and tyrphostin 25 are relatively nonspecific protein tyrosine kinase inhibitors that affect both receptor-linked and cytosolic tyrosine kinases (13), further studies using more selective protein tyrosine kinase inhibitors are warranted to support or refute this hypothesis.
In summary, I found that the protein tyrosine kinase metabolic pathway modulates, in part, the edemagenic effects of bradykinin and substance P in the intact hamster cheek pouch microcirculation in a specific fashion. Conceivably, the kinase(s) involved in this process could be agonist specific because genistein was more effective than tyrphostin 25 in attenuating bradykinin-induced responses while the opposite was observed with substance P.
|
GRANTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
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. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
P < 0.05 compared with bradykinin alone.
