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The role of endogenous prostanoids in the response of the rat gastric microcirculation to vasoactive agents
MICROVASCULARRESEARCH23, 336--346 (1982)
The Role of Endogenous Prostanoids in the Response of the Rat Gastric Microcirculation to Vasoactive Agents PAUL H . GUTH AND TERRY L . MOLER
Medical and Research Services and Center for Ulcer Research and Education, VA Wadsworth Hospital Center, Los Angeles, California 90073, and UCLA School of Medicine, Los Angeles, California 90024 Received April 13, 1981 The effect of prostaglandin E 2 on the gastric microcirculation and the role of endogenous prostanoids in the response of the gastric microcirculation to vasoactive agents were studied using an in vivo microscopy technique in the rat. Changes in diameter of gastric submucosal arterioles (the vessels controlling mucosal blood flow) in response to superfusion with different agents were measured by an image-splitting technique. Prostaglandin E2 produced dose-related dilatation of gastric submucosal arterioles. Administration of indomethacin to inhibit endogenous prostanoid synthesis significantly reduced basal arteriolar diameter but did not alter the magnitude of the change in vessel diameter in response to norepinephrine or histamine. Although the final diameter achieved with these agents was smaller after indomethacin than before, this difference was entirely accounted for by the decrease in basal arteriolar diameter following inhibition of prostanoid synthesis. There was no evidence of any direct modifying effect of endogenous prostanoids on norepinephrine vasoconstriction or histamine vasodilatation. The present study also demonstrated that neither endogenous prostanoids nor endogenous histamine are responsible for vascular escape from adrenergic vasoconstriction.
INTRODUCTION
Prostaglandins are potent, naturally occurring vasoactive hormones. They are synthesized in tissues throughout the body including the stomach (5,18,19). Microcirculatory studies involving various species and circulatory beds have demonstrated that prostaglandins are able to affect vascular smooth muscle either directly or through interactions with other vasoactive substances (3,6,16,17). Gastric mucosal blood flow experiments have shown that prostaglandins increase flow in this vascular bed (13,14). Furthermore, inhibition of prostanoid synthesis reduces basal mucosal blood flow (12,15). These studies suggest an important local circulatory role for prostaglandins in the stomach. Previous in vivo microscopy studies of the rat gastric mucosal microcirculation have shown that local control of blood flow to the gastric mucosa is exerted via constriction or dilatation of submucosal arterioles (8). The present experiment was performed to study the role of prostaglandins in this control mechanism. The direct effect of prostaglandin E2 (PGE2) on rat gastric submucosal arterioles was observed. Inhibition of prostanoid (prostaglandins, including prostacyclin, 336 0026-2862/82/030336-I 1$02.00/0 Copyright © 1982by AcademicPress, Inc. All rights of reproductionin any form reserved. Printed in U.S.A.
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and thromboxane) synthesis by indomethacin was studied for its effect on basal arteriolar diameter and arteriolar response to vasoconstrictor and vasodilator agents. Additionally, the role of endogenous vasodilator substances, including prostanoids and histamine, in microvascular escape from norepinephrine-induced vasoconstriction was studied. METHODS Male Sprague-Dawley rats weighing 150-200 g were fasted for 24 hr but allowed water ad libitum prior to the experiment. The rats were anesthetized with sodium pentobarbital, 45 mg/kg intraperitoneally, and supplementary doses were given as needed. To ensure a free airway, the trachea was exposed and cannulated with a 3-cm length of polyethylene tubing (Intermedic-260). For animals in which inhibition of prostanoid synthesis was desired, the internal jugular vein was cannulated with a short length of polyethylene tubing (Intermedic-50) for injection of indomethacin. The in vivo microscopy technique described by Rosenberg and Guth (20) and Guth et al. (I0) was utilized to study gastric submucosal arteriolar responses. Briefly, exteriorization of the stomach through a ventral midline abdominal incision was followed by the placement of a fiber-optic light rod in the stomach lumen via a small incision in the forestomach. The stomach wall was then transilluminated by passing cool light from a high-intensity source through the rod. After careful removal of the serosal and muscle layers from a small area in the corpus, a shallow aluminum disk with a hole in the center was placed over the exposed submucosa and sealed fluid tight to the surrounding intact serosa with a silicone plastic adherent. This preparation permitted direct visualization of the gastric submucosal vascular networks. The exposed submucosa was continuously superfused with a Krebs solution at 37.5 ° and body temperature (rectal) was maintained at 37.5 ° by a heating pad. Following the surgical preparation the animal was undisturbed for 15 min to assure vessel diameter stability. A microscope equipped with long-working-distance objectives permitted excellent visualization of blood flow through the submucosal arteriolar network down to the deep layer of the mucosa and back through mucosal collecting venules to the submucosal venular plexus. The response of the small branches of the submucosal arteriolar plexus to vasoactive agents was studied. The vasoactive substances used were prostaglandin EE (Upjohn), norepinephrine bitartrate (Levophed bitratrate, Winthrop), and histamine dihydrochloride (Sigma). Other compounds studied which were not vasoactive by themselves, but influenced the synthesis or activity of vasoactive compounds included mepyramine (ICN), an Hi-histamine antagonist, metiamide (Smith, Kline and French), an H2histamine antagonist, and indomethacin (Sigma), an inhibitor of prostanoid synthesis. Prostaglandin stock solution was prepared by dissolving 10 mg in 0.1 ml of absolute ethanol and then adding 0.9 ml of 0.2 mg/ml Na2CO3. Aliquots of the prostaglandin stock solution were stored in a freezer for not more than 3 weeks. On the day of the experiment, aliquots of the stock solution were thawed and then diluted with Krebs solution to the appropriate concentration. Solutions of indomethacin were prepared daily by dissolving 20 mg of indomethacin in 1
338
GUTH AND MOLER
ml of 5% NaHCO3 and then diluting with 3 ml of distilled water. Norepinephrine, histamine, mepyramine, and metiamide were prepared in Krebs solution daily. Changes in diameter of a submucosal arteriole were measured with an image splitter (Vickers) connected to a recorder (2). This system permitted multiple measurements of vessels before, during, and after the topical application of solutions of known molarity of vasoactive substances. One-half milliliter of each vasoactive agent to be studied, was placed in the aluminum disk for 3 min, during which time the Krebs solution superfusion was discontinued. Test treatment order for the various concentrations of each drug studied was random. After each test the drug was aspirated and the exposed submucosal bed was continuously superfused for a period of 8 min with Krebs solution. To inhibit prostanoid synthesis, indomethacin, 10 mg/kg, was injected as a bolus into the jugular vein and the in vivo microscopic studies were performed 15 min later. For the indomethacin experiments, each rat was studied with each dose of a selected vasoactive agent before indomethacin administration and the full study was repeated after indomethacin. Six studies, each consisting of the results from six rats, were performed. Each rat in each study received all doses of the agent being studied. Study 1: Arteriolar diameter changes were recorded in response to the local application of 2.8 x 10 -5 to 2.8 x 10 -1° M prostaglandin E2. Study 2: Norepinephrine-induced vasoconstriction of arterioles was studied before and after inhibition of prostanoid synthesis. Concentrations of norepinephrine used were 1, 2.5, and 5 x 10 -6 M. Study 3: The effect of inhibition of prostanoid synthesis on arteriolar response to histamine was studied. Arteriolar diameter changes in response to 10 -3, 104, and 10 -5 M histamine were recorded in each rat before and after injection of indomethacin. Studies 4, 5, and 6: The phenomenon of escape from norepinephrine-induced vasoconstriction was studied prior to and after inhibition of prostanoid synthesis and histamine receptor blockade, separately and in combination. Histamine receptor blockade was accomplished by adding 10 -5 M mepyramine and 10 -5 M metiamide (final concentrations) to the norepinephrine solution (10). Quantification of escape from norepinephrine constriction was accomplished by measuring arteriolar diameter at the end of the 3-min exposure to norepinephrine (just before aspirating the norepinephrine solution). Postconstriction dilatation was measured 1 min after removing the norepinephrine solution. Vessel diameter was measured in micrometers. Changes in vessel caliber were expressed either as percentage of control diameter or micrometer change from control diameter. Statistical significance was assessed by the sign test, Wilcoxon sum of ranks, and t test for correlated means. RESULTS Gastric Submucosal Arteriolar Response to Prostaglandin Ez Significant dose-related dilatation of gastric submucosal arterioles occurred with 2.8 x 10 -9 to 2.8 x 10 -6 M PGE2 (P < 0.05, sign test) (Fig. 1). The vehicle in which the PGE2 was dissolved had no effect by itself on arteriolar diameter. Arterioles selected for measurement in this part of the investigation had a mean
PROSTANOIDS AND GASTRIC MICROCIRCULATION
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FIG. 1. Log dose-response curve of maximum response of gastric submucosal arterioles to increasing doses of prostaglandin Ez. Maximum dilatation occurred within 15-30 sec of prostaglandin applications. The control diameter averaged 25.3 _+ 2.4 Ixm (n = 6). Results in this and subsequent figures are expressed as mean _+ standard error.
luminal diameter of 25.3 + 2.4 ~xm. Dilatation of the arterioles was prompt, maximal dilatation occurring within 15 to 30 sec of prostaglandin application.
Effect of lndomethacin on Arteriolar Constrictor Response to Norepinephrine The maximum constriction of arterioles to topically applied norepinephrine before and after injection of indomethacin is presented in Fig. 2. Arterioles selected for study had a mean luminal diameter of 38.8 -+ 0.9 p~m. Maximum norepinephrine vasoconstriction occurred within the same time period both before and after indomethacin, i.e., 15 to 30 sec. Before inhibition of prostanoid synthesis there was a dose-related vasoconstriction (P < 0.05) of the arterioles in response to norepinephrine. Following indomethacin injection alone, the mean arteriolar diameter was significantly reduced (P < 0.05, Wilcoxon sum of ranks test) to 20.1 ___ 0.8 p~m. Dose-related vasoconstriction (P < 0.05) to norepinephrine was still observed after indomethacin. The vasoconstrictor effect, when expressed as percentage of control diameter, appeared much greater for postindomethacin arterioles and the log dose-response curve was shifted to the left. The data suggested that endogenous prostanoids attenuated the constrictor response to norepinephrine. However, when the same data were analyzed with vessel diameter expressed in micrometers instead of as percentage of control another explanation became apparent (Fig. 3). Although there was still a log dose response for norepinephrine constriction, the pre- and postindomethacin regression lines were parallel and the difference in response of the vessels after indomethacin could be entirely accounted for by the decrease in control diameter when prostanoid synthesis was inhibited. If the log dose-response curves were
340
GUTH AND MOLER
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FIG. 2. Log dose response of gastric submucosal arterioles to topically applied norepinephrine before and after inhibition of prostanoid synthesis by 10 mg kg -I indomethacin iv. Vessel diameter is expressed as percentage of control. The control diameter averaged 38.8 ___ 0.9 ixm before, and 20.1 - 0.8 Ixm after, indomethacin (n = 6).
plotted as micrometer change from the respective control diameters, the two lines would be identical.
Effect of lndomethacin on Arteriolar Dilator Response to Histamine Prior to indomethacin administration there was a dose-related vasodilatation (P < 0.06) in response to histamine (Fig. 4). As was the case with the other vasoactive substances, arterioles reached peak diltation within 15 to 30 sec after stimulation with histamine. Following indomethacin alone mean basal vessel 40. :3 I
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FIG. 3. Log dose response of gastric submucosal arterioles to topically applied norepinephrine before and after injection of indomethacin. Same data as in Fig. 2 except vessel diameter is expressed in micrometers. ** = significantly different from preindomethacin value at P < 0.01, t test for correlated means.
341
PROSTANOIDS AND GASTRIC MICROCIRCULATION
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FIG. 4. Log dose response of gastric submucosalarterioles to topicallyapplied histaminebefore and after injection of indomethacinin six rats. *,** = significantlydifferentfrom preindomethacin value at P < 0.05 and 0.01, t test for correlated means.
diameter decreased significantly (P < 0.05) from 20.8 ___ 0.9 to 14 ___ 0.6 ~m. The arterioles still demonstrated prompt dose-related dilatation (P < 0.05) in response to histamine. The pre- and postindomethacin regression lines for 105-10-4 M histamine were parallel. The significantly smaller final diameters for the postindomethacin regression could be entirely accounted for by the decrease in control diameter when prostanoid synthesis was inhibited. If the responses were plotted as micrometer change from the respective control diameters, the two lines would be identical. The loss of the dilating effect of naturally occurring prostanoid on basal diameter was overcome with 10 -3 M histamine which caused near-maximum vasodilatation and there was no significant difference between pre- and postindomethacin vessel size.
Effect of Indomethacin and Histamine Receptor Blockade on Escape from Norepinephrine-Induced Vasoconstriction The effect of indomethacin administration on escape from adrenergic vasoconstriction is shown in Fig. 5. The arterioles selected had an average luminal diameter of 41.5 - 2.9 ~m. Because of the importance of expressing results as micrometer change from control demonstrated in the first part of this investigation, all results are expressed in this manner. Before inhibition of prostanoid synthesis, 2.5 × 10-6 M norepinephrine produced a constriction with the arteriolar diameters decreasing an average of 12.9 ~m. Despite the continued presence of norepinephrine, the arterioles began to dilate and after 3 min the average vessel diameter was 5.8 txm less than control values. A diameter measurement taken 1 min following removal of the norepinephrine indicated that the vessels had dilated slightly above control values--a postconstriction dilatation. Following injection of indomethacin, the basal vessel size decreased to 25.5 --- 1.3 txm; however, inhibition of prostanoid synthesis by indomethacin had no effect on the escape phenomenon. After indomethacin the arterioles constricted
342
GUTH AND MOLER
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FIG. 5. Effect of inhibition of prostanoid synthesis by indomethacin on escape from norepinephrine-induced vasoconstriction in six rats. In this figure and in Figs. 6 and 7 results are expressed as change in micrometers from control diameter. Maximum constriction occurred 15-30 sec after the application of norepinephrine. The escape diameter was measured at the end of the 3-min application of norepinephrine. The postnorepinephfine diameter was measured 1 min after removal of the norepinephrine solution.
in response to norepinephrine, but still escaped and dilated significantly (P < 0.5, t test for correlated means) toward control values in spite of the continued presence of norepinephrine. After removal of norepinephrine there again was a slight dilatation above control values. The same response was seen with both doses of norepinephrine. There were no significant differences (t test for correlated means) in the magnitude of the constriction, escape, or postnorepinephrine dilatation responses before and after indomethacin. The effect of histamine receptor blockade on escape from norepinephrine vasoconstriction is shown in Fig. 6. The arterioles selected had an average control luminal diameter of 42.4 ___ 4.4 Ixm in the preblockade phase and 40.2 ___ 4.9 p,m in the blockade phase of this study. There was no significant difference in norepinephrine-induced vasoconstriction, escape from vasoconstriction, or postnorepinephrine dilatation between norepinephrine alone and with H1 + Hz histamine receptor antagonists. The effect of indomethacin administration plus histamine receptor blockade is shown in Fig. 7. The arterioles selected had an average luminal diameter of 39.7 ___ 1.7 t~m before and 25.6 ___ 1.8 txm after indomethacin. The pattern of response was similar to that observed in the previous two studies. In spite of inhibition of prostanoid synthesis plus histamine receptor blockade, the vessels still escaped from norepinephrine-induced vasoconstriction and showed a slight postnorepinephrine dilatation. Norepinephrine-induced vasoconstriction, however, was significantly less after indomethacin plus histamine blockade (P < 0.05), although escape and postnorepinephrine dilatation were not.
343
PROSTANOIDS AND GASTRIC MICROCIRCULATION
+10-
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Constriction 2.5x10-6M Norepinephrine CNE)
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Escape
Post NE
Constriction 5 x 10-6 M Norepinephrine
F]6. 6. Effect of H1- and H2-histamine receptor blockade on escape from norepinephrine-induced vasoconstriction in six rats. H~A = H r h i s t a m i n e receptor antagonist, m e p y r a m i n e 10 5 M, and HzA = Hz-histamine receptor antagonist, metiamide 10 -5 M. The times for taking diameter m e a s u r e m e n t s were the same as in Fig. 5.
DISCUSSION Prostaglandins have been shown to play an important role in the regulation of microcirculatory blood flow (3,16,17). Our previous in vivo microscopic studies of the gastric microcirculation have demonstrated that local control of blood flow to the gastric mucosa is exerted via constriction or dilatation of submucosal [ ] Pre-lndomethacin ,lO
[ ] Post- Indornethacin.HIA+H2A
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'
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wl
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Escape
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2~5x10-6M Norepinephrine ~NE)
Maximum Escape Constriction
Post NE
5x110-6MNorepinephrine
FIG. 7. Effect of indomethacin plus histamine receptor blockade on escape from norepinephrineinduced vasoconstriction. The times for taking diameter m e a s u r e m e n t s were the same as in Fig. 5. * ** = significantly different from postindomethacin + H~A + H2A value at P < 0.05 and 0.01, t test.
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GUTH AND MOLER
5. DEBIAK, D. M., MILLER, E. R., HUSLIG, R. L., AND SMITH, W. L. (1975). Distribution of prostaglandin-forming cyclooxygenase in the porcine stomach. Fed. Proc. 38, 884. 6. FERREIRA,S. H., AND MONCADA,S. (1971). Inhibition of prostaglandin synthesis augments the effects of sympathetic nerve stimulation of the cat spleen. Br. J. Pharmacol. 43, 419-420. 7. GERKINS,J. F., STRAND,D. G., FLEXNER, C., NIES, A., OATES, J., AND DATA, J. L. (1977). Effect of indomethacin and aspirin on gastric blood flow and acid secretion. J. Pharmacol. Exp. Ther. 203, 646-652. 8. GUTH, P. H., AND SMITH, E. (1975). Neural control of gastric mucosal blood flow in the rat. Gastroenterology 69, 935-940. 9. GUTH, P. H., AND SMITH, E. (1975). Escape from vasoconstriction in the gastric microcirculation. Amer. J. Physiol. 228, 1893-1895. 10. GUTH, P. H., MOLER, T. L., AND SMITH, E. (1980). H~ and H2 histamine receptors in rat gastric submucosal arterioles. Microvasc. Res. 19, 320-328. ll. HEDQVlST,P. (1977). Basic mechanism of prostaglandin action on autonomic neurotransmission. Annu, Rev. Pharmacol. Toxicol. 17, 259-279. 12. KAUFFMAN,G. L., JR., AURES, D., AND GROSSMAN,M. I. (1980). Intravenous indomethacin and aspirin reduce basal gastric mucosal blood flow in dogs. Amer. J. Physiol. 238, G131-G134. 13. KONTUREK,S. J., LANCASTER,C., HANCHAK,A. J., NEZAMIS,J. E., AND ROBERT, A. (1979). The influence of prostacyclin on gastric mucosal blood flow in resting and stimulated canine stomach. Gastroenterology 76, 1173. 14. MAIN, I. H. M., AND WHITTLE, B. J. R. (1973). The effects of E and A prostaglandins on gastric mucosal blood flow and acid secretion in the rat. Br. J. Pharmacol. 49, 428-436. 15. MAIN, I. H. M., AND WHITTLE, B. J. R. (1975). Investigation of the vasodilator and antisecretory role of prostaglandins in the rat gastric mucosa by use of non-steroidal anti-inflammatory drugs. Br. J. Pharmacol. 53, 217-224. 16. MESSINA, E. J., WEINER, R., AND KALEV, G. (1974). Microcirculatory effects of prostaglandins E , E2, and A~ in the rat mesentery and cremaster muscle. Microvasc. Res. 8, 77-89. 17. MESSlNA, E. J., WEINER, R., AND KALEY, G. (1975). Inhibition of bradykinin vasodilation and potentiation of norepinephrine and angiotensin vasoconstriction by inhibitors of prostaglandin synthesis in skeletal muscle of the rat. Circ. Res. 37, 430-437. 18. MONCADA,S., SALMON,J. A., VANE, J. R., AND WHITTLE, B. J. R. (1977). Formation of prostacyclin (PGI2) and its product, 6-oxo-PGFla, by the gastric mucosa of several species. J. Physiol. 275, 4-5P. 19. PESKAR,B. M. (1978). Regional distribution of prostaglandin-metabolizing enzymes in the mucosa of the human upper gastrointestinal tract. Acta Hepato-Gastroenterol. 25, 49-51. 20. ROSENBER6,A., AND GUTH, P. H. (1970). A method for the in vivo study of the gastric microcirculation. Microvasc. Res. 2, 111-112. 21. Ross, G. (1971). Escape of mesenteric Vessels from adrenergic and nonadrenergic vasoconstriction. Amer. J. Physiol. 221, 1217-1222. 22. SELVE, H. (1965). "The Mast Cells," p. 385. Butterworths, London. 23. ZIMMERMAN,B. G., RYAN, M. I., GOMER, S., AND KRAFT, E. (1973). Effect of the prostaglandin synthesis inhibitors indomethacin and eieosa-5-8-11-14-tetraynoic acid on adrenergic responses in dog cutaneous vasculature. J. Pharmaeol. Exp. Ther. 187, 315-323.
The Role of Endogenous Prostanoids in the Response of the Rat Gastric Microcirculation to Vasoactive Agents PAUL H . GUTH AND TERRY L . MOLER
Medical and Research Services and Center for Ulcer Research and Education, VA Wadsworth Hospital Center, Los Angeles, California 90073, and UCLA School of Medicine, Los Angeles, California 90024 Received April 13, 1981 The effect of prostaglandin E 2 on the gastric microcirculation and the role of endogenous prostanoids in the response of the gastric microcirculation to vasoactive agents were studied using an in vivo microscopy technique in the rat. Changes in diameter of gastric submucosal arterioles (the vessels controlling mucosal blood flow) in response to superfusion with different agents were measured by an image-splitting technique. Prostaglandin E2 produced dose-related dilatation of gastric submucosal arterioles. Administration of indomethacin to inhibit endogenous prostanoid synthesis significantly reduced basal arteriolar diameter but did not alter the magnitude of the change in vessel diameter in response to norepinephrine or histamine. Although the final diameter achieved with these agents was smaller after indomethacin than before, this difference was entirely accounted for by the decrease in basal arteriolar diameter following inhibition of prostanoid synthesis. There was no evidence of any direct modifying effect of endogenous prostanoids on norepinephrine vasoconstriction or histamine vasodilatation. The present study also demonstrated that neither endogenous prostanoids nor endogenous histamine are responsible for vascular escape from adrenergic vasoconstriction.
INTRODUCTION
Prostaglandins are potent, naturally occurring vasoactive hormones. They are synthesized in tissues throughout the body including the stomach (5,18,19). Microcirculatory studies involving various species and circulatory beds have demonstrated that prostaglandins are able to affect vascular smooth muscle either directly or through interactions with other vasoactive substances (3,6,16,17). Gastric mucosal blood flow experiments have shown that prostaglandins increase flow in this vascular bed (13,14). Furthermore, inhibition of prostanoid synthesis reduces basal mucosal blood flow (12,15). These studies suggest an important local circulatory role for prostaglandins in the stomach. Previous in vivo microscopy studies of the rat gastric mucosal microcirculation have shown that local control of blood flow to the gastric mucosa is exerted via constriction or dilatation of submucosal arterioles (8). The present experiment was performed to study the role of prostaglandins in this control mechanism. The direct effect of prostaglandin E2 (PGE2) on rat gastric submucosal arterioles was observed. Inhibition of prostanoid (prostaglandins, including prostacyclin, 336 0026-2862/82/030336-I 1$02.00/0 Copyright © 1982by AcademicPress, Inc. All rights of reproductionin any form reserved. Printed in U.S.A.
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and thromboxane) synthesis by indomethacin was studied for its effect on basal arteriolar diameter and arteriolar response to vasoconstrictor and vasodilator agents. Additionally, the role of endogenous vasodilator substances, including prostanoids and histamine, in microvascular escape from norepinephrine-induced vasoconstriction was studied. METHODS Male Sprague-Dawley rats weighing 150-200 g were fasted for 24 hr but allowed water ad libitum prior to the experiment. The rats were anesthetized with sodium pentobarbital, 45 mg/kg intraperitoneally, and supplementary doses were given as needed. To ensure a free airway, the trachea was exposed and cannulated with a 3-cm length of polyethylene tubing (Intermedic-260). For animals in which inhibition of prostanoid synthesis was desired, the internal jugular vein was cannulated with a short length of polyethylene tubing (Intermedic-50) for injection of indomethacin. The in vivo microscopy technique described by Rosenberg and Guth (20) and Guth et al. (I0) was utilized to study gastric submucosal arteriolar responses. Briefly, exteriorization of the stomach through a ventral midline abdominal incision was followed by the placement of a fiber-optic light rod in the stomach lumen via a small incision in the forestomach. The stomach wall was then transilluminated by passing cool light from a high-intensity source through the rod. After careful removal of the serosal and muscle layers from a small area in the corpus, a shallow aluminum disk with a hole in the center was placed over the exposed submucosa and sealed fluid tight to the surrounding intact serosa with a silicone plastic adherent. This preparation permitted direct visualization of the gastric submucosal vascular networks. The exposed submucosa was continuously superfused with a Krebs solution at 37.5 ° and body temperature (rectal) was maintained at 37.5 ° by a heating pad. Following the surgical preparation the animal was undisturbed for 15 min to assure vessel diameter stability. A microscope equipped with long-working-distance objectives permitted excellent visualization of blood flow through the submucosal arteriolar network down to the deep layer of the mucosa and back through mucosal collecting venules to the submucosal venular plexus. The response of the small branches of the submucosal arteriolar plexus to vasoactive agents was studied. The vasoactive substances used were prostaglandin EE (Upjohn), norepinephrine bitartrate (Levophed bitratrate, Winthrop), and histamine dihydrochloride (Sigma). Other compounds studied which were not vasoactive by themselves, but influenced the synthesis or activity of vasoactive compounds included mepyramine (ICN), an Hi-histamine antagonist, metiamide (Smith, Kline and French), an H2histamine antagonist, and indomethacin (Sigma), an inhibitor of prostanoid synthesis. Prostaglandin stock solution was prepared by dissolving 10 mg in 0.1 ml of absolute ethanol and then adding 0.9 ml of 0.2 mg/ml Na2CO3. Aliquots of the prostaglandin stock solution were stored in a freezer for not more than 3 weeks. On the day of the experiment, aliquots of the stock solution were thawed and then diluted with Krebs solution to the appropriate concentration. Solutions of indomethacin were prepared daily by dissolving 20 mg of indomethacin in 1
338
GUTH AND MOLER
ml of 5% NaHCO3 and then diluting with 3 ml of distilled water. Norepinephrine, histamine, mepyramine, and metiamide were prepared in Krebs solution daily. Changes in diameter of a submucosal arteriole were measured with an image splitter (Vickers) connected to a recorder (2). This system permitted multiple measurements of vessels before, during, and after the topical application of solutions of known molarity of vasoactive substances. One-half milliliter of each vasoactive agent to be studied, was placed in the aluminum disk for 3 min, during which time the Krebs solution superfusion was discontinued. Test treatment order for the various concentrations of each drug studied was random. After each test the drug was aspirated and the exposed submucosal bed was continuously superfused for a period of 8 min with Krebs solution. To inhibit prostanoid synthesis, indomethacin, 10 mg/kg, was injected as a bolus into the jugular vein and the in vivo microscopic studies were performed 15 min later. For the indomethacin experiments, each rat was studied with each dose of a selected vasoactive agent before indomethacin administration and the full study was repeated after indomethacin. Six studies, each consisting of the results from six rats, were performed. Each rat in each study received all doses of the agent being studied. Study 1: Arteriolar diameter changes were recorded in response to the local application of 2.8 x 10 -5 to 2.8 x 10 -1° M prostaglandin E2. Study 2: Norepinephrine-induced vasoconstriction of arterioles was studied before and after inhibition of prostanoid synthesis. Concentrations of norepinephrine used were 1, 2.5, and 5 x 10 -6 M. Study 3: The effect of inhibition of prostanoid synthesis on arteriolar response to histamine was studied. Arteriolar diameter changes in response to 10 -3, 104, and 10 -5 M histamine were recorded in each rat before and after injection of indomethacin. Studies 4, 5, and 6: The phenomenon of escape from norepinephrine-induced vasoconstriction was studied prior to and after inhibition of prostanoid synthesis and histamine receptor blockade, separately and in combination. Histamine receptor blockade was accomplished by adding 10 -5 M mepyramine and 10 -5 M metiamide (final concentrations) to the norepinephrine solution (10). Quantification of escape from norepinephrine constriction was accomplished by measuring arteriolar diameter at the end of the 3-min exposure to norepinephrine (just before aspirating the norepinephrine solution). Postconstriction dilatation was measured 1 min after removing the norepinephrine solution. Vessel diameter was measured in micrometers. Changes in vessel caliber were expressed either as percentage of control diameter or micrometer change from control diameter. Statistical significance was assessed by the sign test, Wilcoxon sum of ranks, and t test for correlated means. RESULTS Gastric Submucosal Arteriolar Response to Prostaglandin Ez Significant dose-related dilatation of gastric submucosal arterioles occurred with 2.8 x 10 -9 to 2.8 x 10 -6 M PGE2 (P < 0.05, sign test) (Fig. 1). The vehicle in which the PGE2 was dissolved had no effect by itself on arteriolar diameter. Arterioles selected for measurement in this part of the investigation had a mean
PROSTANOIDS AND GASTRIC MICROCIRCULATION
339
160.
_j 150. o n,, I.zo 140. ID I.L 0
130.
I w
I10.
IO0 2.8 X -" I~I°M
IOL9M
10:8 M
PROSTAGLANDIN
10:7M
10'-6M
10'-5M
E2
FIG. 1. Log dose-response curve of maximum response of gastric submucosal arterioles to increasing doses of prostaglandin Ez. Maximum dilatation occurred within 15-30 sec of prostaglandin applications. The control diameter averaged 25.3 _+ 2.4 Ixm (n = 6). Results in this and subsequent figures are expressed as mean _+ standard error.
luminal diameter of 25.3 + 2.4 ~xm. Dilatation of the arterioles was prompt, maximal dilatation occurring within 15 to 30 sec of prostaglandin application.
Effect of lndomethacin on Arteriolar Constrictor Response to Norepinephrine The maximum constriction of arterioles to topically applied norepinephrine before and after injection of indomethacin is presented in Fig. 2. Arterioles selected for study had a mean luminal diameter of 38.8 -+ 0.9 p~m. Maximum norepinephrine vasoconstriction occurred within the same time period both before and after indomethacin, i.e., 15 to 30 sec. Before inhibition of prostanoid synthesis there was a dose-related vasoconstriction (P < 0.05) of the arterioles in response to norepinephrine. Following indomethacin injection alone, the mean arteriolar diameter was significantly reduced (P < 0.05, Wilcoxon sum of ranks test) to 20.1 ___ 0.8 p~m. Dose-related vasoconstriction (P < 0.05) to norepinephrine was still observed after indomethacin. The vasoconstrictor effect, when expressed as percentage of control diameter, appeared much greater for postindomethacin arterioles and the log dose-response curve was shifted to the left. The data suggested that endogenous prostanoids attenuated the constrictor response to norepinephrine. However, when the same data were analyzed with vessel diameter expressed in micrometers instead of as percentage of control another explanation became apparent (Fig. 3). Although there was still a log dose response for norepinephrine constriction, the pre- and postindomethacin regression lines were parallel and the difference in response of the vessels after indomethacin could be entirely accounted for by the decrease in control diameter when prostanoid synthesis was inhibited. If the log dose-response curves were
340
GUTH AND MOLER
~oo ._1 O n~
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X PRE-INDOMETHACIN
20.
O POST- INDOMETHACIN
i
i
I x 106M
i
2 5 x 10"6M
NOREPINEPHRINE
5x IC) 6M
(log DOSE)
FIG. 2. Log dose response of gastric submucosal arterioles to topically applied norepinephrine before and after inhibition of prostanoid synthesis by 10 mg kg -I indomethacin iv. Vessel diameter is expressed as percentage of control. The control diameter averaged 38.8 ___ 0.9 ixm before, and 20.1 - 0.8 Ixm after, indomethacin (n = 6).
plotted as micrometer change from the respective control diameters, the two lines would be identical.
Effect of lndomethacin on Arteriolar Dilator Response to Histamine Prior to indomethacin administration there was a dose-related vasodilatation (P < 0.06) in response to histamine (Fig. 4). As was the case with the other vasoactive substances, arterioles reached peak diltation within 15 to 30 sec after stimulation with histamine. Following indomethacin alone mean basal vessel 40. :3 I
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~
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,
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J
IxliD6M
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FIG. 3. Log dose response of gastric submucosal arterioles to topically applied norepinephrine before and after injection of indomethacin. Same data as in Fig. 2 except vessel diameter is expressed in micrometers. ** = significantly different from preindomethacin value at P < 0.01, t test for correlated means.
341
PROSTANOIDS AND GASTRIC MICROCIRCULATION
40_
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nw
i - 20_ LLI ~E
o
I0_
0
X O
,
CONTROL
PRE - INDOMETHACIN POST- INDOMETHACIN
tl
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10"4M
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HISTAMINE
FIG. 4. Log dose response of gastric submucosalarterioles to topicallyapplied histaminebefore and after injection of indomethacinin six rats. *,** = significantlydifferentfrom preindomethacin value at P < 0.05 and 0.01, t test for correlated means.
diameter decreased significantly (P < 0.05) from 20.8 ___ 0.9 to 14 ___ 0.6 ~m. The arterioles still demonstrated prompt dose-related dilatation (P < 0.05) in response to histamine. The pre- and postindomethacin regression lines for 105-10-4 M histamine were parallel. The significantly smaller final diameters for the postindomethacin regression could be entirely accounted for by the decrease in control diameter when prostanoid synthesis was inhibited. If the responses were plotted as micrometer change from the respective control diameters, the two lines would be identical. The loss of the dilating effect of naturally occurring prostanoid on basal diameter was overcome with 10 -3 M histamine which caused near-maximum vasodilatation and there was no significant difference between pre- and postindomethacin vessel size.
Effect of Indomethacin and Histamine Receptor Blockade on Escape from Norepinephrine-Induced Vasoconstriction The effect of indomethacin administration on escape from adrenergic vasoconstriction is shown in Fig. 5. The arterioles selected had an average luminal diameter of 41.5 - 2.9 ~m. Because of the importance of expressing results as micrometer change from control demonstrated in the first part of this investigation, all results are expressed in this manner. Before inhibition of prostanoid synthesis, 2.5 × 10-6 M norepinephrine produced a constriction with the arteriolar diameters decreasing an average of 12.9 ~m. Despite the continued presence of norepinephrine, the arterioles began to dilate and after 3 min the average vessel diameter was 5.8 txm less than control values. A diameter measurement taken 1 min following removal of the norepinephrine indicated that the vessels had dilated slightly above control values--a postconstriction dilatation. Following injection of indomethacin, the basal vessel size decreased to 25.5 --- 1.3 txm; however, inhibition of prostanoid synthesis by indomethacin had no effect on the escape phenomenon. After indomethacin the arterioles constricted
342
GUTH AND MOLER
*10
[]
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[]
Post-Indomethacin
*& 0 -5-
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~ -25
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Constriction
2.5 x ~ - ~
Maximum
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No,e~.~i.e
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Escape
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5 xlO-6M Norepinephrine
FIG. 5. Effect of inhibition of prostanoid synthesis by indomethacin on escape from norepinephrine-induced vasoconstriction in six rats. In this figure and in Figs. 6 and 7 results are expressed as change in micrometers from control diameter. Maximum constriction occurred 15-30 sec after the application of norepinephrine. The escape diameter was measured at the end of the 3-min application of norepinephrine. The postnorepinephfine diameter was measured 1 min after removal of the norepinephrine solution.
in response to norepinephrine, but still escaped and dilated significantly (P < 0.5, t test for correlated means) toward control values in spite of the continued presence of norepinephrine. After removal of norepinephrine there again was a slight dilatation above control values. The same response was seen with both doses of norepinephrine. There were no significant differences (t test for correlated means) in the magnitude of the constriction, escape, or postnorepinephrine dilatation responses before and after indomethacin. The effect of histamine receptor blockade on escape from norepinephrine vasoconstriction is shown in Fig. 6. The arterioles selected had an average control luminal diameter of 42.4 ___ 4.4 Ixm in the preblockade phase and 40.2 ___ 4.9 p,m in the blockade phase of this study. There was no significant difference in norepinephrine-induced vasoconstriction, escape from vasoconstriction, or postnorepinephrine dilatation between norepinephrine alone and with H1 + Hz histamine receptor antagonists. The effect of indomethacin administration plus histamine receptor blockade is shown in Fig. 7. The arterioles selected had an average luminal diameter of 39.7 ___ 1.7 t~m before and 25.6 ___ 1.8 txm after indomethacin. The pattern of response was similar to that observed in the previous two studies. In spite of inhibition of prostanoid synthesis plus histamine receptor blockade, the vessels still escaped from norepinephrine-induced vasoconstriction and showed a slight postnorepinephrine dilatation. Norepinephrine-induced vasoconstriction, however, was significantly less after indomethacin plus histamine blockade (P < 0.05), although escape and postnorepinephrine dilatation were not.
343
PROSTANOIDS AND GASTRIC MICROCIRCULATION
+10-
[]
NE alone
[]
NE +HIA*H2A
T
+50 -,5-10i I'~-a~
< N-25<1
-35-40-
T
Maximum
Escape
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Constriction 2.5x10-6M Norepinephrine CNE)
Maximum
Escape
Post NE
Constriction 5 x 10-6 M Norepinephrine
F]6. 6. Effect of H1- and H2-histamine receptor blockade on escape from norepinephrine-induced vasoconstriction in six rats. H~A = H r h i s t a m i n e receptor antagonist, m e p y r a m i n e 10 5 M, and HzA = Hz-histamine receptor antagonist, metiamide 10 -5 M. The times for taking diameter m e a s u r e m e n t s were the same as in Fig. 5.
DISCUSSION Prostaglandins have been shown to play an important role in the regulation of microcirculatory blood flow (3,16,17). Our previous in vivo microscopic studies of the gastric microcirculation have demonstrated that local control of blood flow to the gastric mucosa is exerted via constriction or dilatation of submucosal [ ] Pre-lndomethacin ,lO
[ ] Post- Indornethacin.HIA+H2A
+5
o -5
E
-10
'
-15
wl
< -25 Z~ N
-30 -35-
-40
Maximum Constriction
Escape
Post NE
2~5x10-6M Norepinephrine ~NE)
Maximum Escape Constriction
Post NE
5x110-6MNorepinephrine
FIG. 7. Effect of indomethacin plus histamine receptor blockade on escape from norepinephrineinduced vasoconstriction. The times for taking diameter m e a s u r e m e n t s were the same as in Fig. 5. * ** = significantly different from postindomethacin + H~A + H2A value at P < 0.05 and 0.01, t test.
346
GUTH AND MOLER
5. DEBIAK, D. M., MILLER, E. R., HUSLIG, R. L., AND SMITH, W. L. (1975). Distribution of prostaglandin-forming cyclooxygenase in the porcine stomach. Fed. Proc. 38, 884. 6. FERREIRA,S. H., AND MONCADA,S. (1971). Inhibition of prostaglandin synthesis augments the effects of sympathetic nerve stimulation of the cat spleen. Br. J. Pharmacol. 43, 419-420. 7. GERKINS,J. F., STRAND,D. G., FLEXNER, C., NIES, A., OATES, J., AND DATA, J. L. (1977). Effect of indomethacin and aspirin on gastric blood flow and acid secretion. J. Pharmacol. Exp. Ther. 203, 646-652. 8. GUTH, P. H., AND SMITH, E. (1975). Neural control of gastric mucosal blood flow in the rat. Gastroenterology 69, 935-940. 9. GUTH, P. H., AND SMITH, E. (1975). Escape from vasoconstriction in the gastric microcirculation. Amer. J. Physiol. 228, 1893-1895. 10. GUTH, P. H., MOLER, T. L., AND SMITH, E. (1980). H~ and H2 histamine receptors in rat gastric submucosal arterioles. Microvasc. Res. 19, 320-328. ll. HEDQVlST,P. (1977). Basic mechanism of prostaglandin action on autonomic neurotransmission. Annu, Rev. Pharmacol. Toxicol. 17, 259-279. 12. KAUFFMAN,G. L., JR., AURES, D., AND GROSSMAN,M. I. (1980). Intravenous indomethacin and aspirin reduce basal gastric mucosal blood flow in dogs. Amer. J. Physiol. 238, G131-G134. 13. KONTUREK,S. J., LANCASTER,C., HANCHAK,A. J., NEZAMIS,J. E., AND ROBERT, A. (1979). The influence of prostacyclin on gastric mucosal blood flow in resting and stimulated canine stomach. Gastroenterology 76, 1173. 14. MAIN, I. H. M., AND WHITTLE, B. J. R. (1973). The effects of E and A prostaglandins on gastric mucosal blood flow and acid secretion in the rat. Br. J. Pharmacol. 49, 428-436. 15. MAIN, I. H. M., AND WHITTLE, B. J. R. (1975). Investigation of the vasodilator and antisecretory role of prostaglandins in the rat gastric mucosa by use of non-steroidal anti-inflammatory drugs. Br. J. Pharmacol. 53, 217-224. 16. MESSINA, E. J., WEINER, R., AND KALEV, G. (1974). Microcirculatory effects of prostaglandins E , E2, and A~ in the rat mesentery and cremaster muscle. Microvasc. Res. 8, 77-89. 17. MESSlNA, E. J., WEINER, R., AND KALEY, G. (1975). Inhibition of bradykinin vasodilation and potentiation of norepinephrine and angiotensin vasoconstriction by inhibitors of prostaglandin synthesis in skeletal muscle of the rat. Circ. Res. 37, 430-437. 18. MONCADA,S., SALMON,J. A., VANE, J. R., AND WHITTLE, B. J. R. (1977). Formation of prostacyclin (PGI2) and its product, 6-oxo-PGFla, by the gastric mucosa of several species. J. Physiol. 275, 4-5P. 19. PESKAR,B. M. (1978). Regional distribution of prostaglandin-metabolizing enzymes in the mucosa of the human upper gastrointestinal tract. Acta Hepato-Gastroenterol. 25, 49-51. 20. ROSENBER6,A., AND GUTH, P. H. (1970). A method for the in vivo study of the gastric microcirculation. Microvasc. Res. 2, 111-112. 21. Ross, G. (1971). Escape of mesenteric Vessels from adrenergic and nonadrenergic vasoconstriction. Amer. J. Physiol. 221, 1217-1222. 22. SELVE, H. (1965). "The Mast Cells," p. 385. Butterworths, London. 23. ZIMMERMAN,B. G., RYAN, M. I., GOMER, S., AND KRAFT, E. (1973). Effect of the prostaglandin synthesis inhibitors indomethacin and eieosa-5-8-11-14-tetraynoic acid on adrenergic responses in dog cutaneous vasculature. J. Pharmaeol. Exp. Ther. 187, 315-323.