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Hemolysate-induced release of prostaglandinlike substances from the canine cerebral arteries
Surg Neurol 1985;23:421-4
421
Hemolysate-induced Release of Prostaglandinlike Substances from the Canine Cerebral Arteries Shinichiro Okamoto,
M.D., Hajime
Handa, M.D., and Yutaka Handa, M.D.
Department of Neurosurgery, Kyoto University Medical School, Kyoto, Japan
Okamoto S, Handa H, Handa Y. Hemolysate-induced release of prostaglandinlike substances from the canine cerebral arteries. Surg Neurol I985;23:421-4.
The release of prostaglandinlike substances from canine pial arteries that was induced by exposure of the pial arteries to red blood cell hemolysate was estimated by using a superfusion technique for prostaglandin bioassay. The assay organs used were strips of rat stomach for prostaglandin E2- or prostaglandin Fz~-like substances, strips of dog coronary artery for prostaglandin I2-1ike substance, and strips of dog ileum for prostaglandin Fz,,-like substance. The substances released from the canine pial arteries induced contractions in rat stomach strips and relaxations in canine coronary arterial strips, whereas they did not induce any response in canine ileal strips. The equivalent prostaglandin E2 doses for the contractions of the rat stomach strips and the equivalent prostaglandin I2 doses for the relaxations of the canine coronary arterial strips were 125.2 -+ 19.4 (n = 8) and 59.5 - 16.4 (n = 6) pmol/g wet wt -+ SEM, respectively. Cerebral artery; Cerebral vasospasm; Prostaglandin; Subarachnoid hemorrhage KEY W O R D S :
Among the candidates to be included as a causative substance of cerebral vasospasm after a subarachnoid hemorrhage are the breakdown products of red blood cells [8]. This possibility may explain the delayed onset of vasospasm after a hemorrhagic insult when the incubation period that is necessary for the subarachnoid blood clot to cause hemolysis is taken into consideration. The good correlation between the sites o f vasospasm and those of subarachnoid hematoma on computed tomographic scanning [2] is another basis of this concept. It was suggested that the intrinsic arachidonate metabolites of the cerebral artery have a role in the cerebral vasoconstriction induced by the red blood cell hemolysate [7]. We have examined further the release of the Address reprint requests to: Dr. Hajime Handa, Department of Neurosurgery, Kyoto University Hospital, 54 Shogoin Kawaharacho, Sakyoku, Kyoto 606, Japan.
© 1985 by Elsevier Science Publishing Co., Inc.
prostaglandinlike substances from the cerebral artery in an in vitro experimental condition by using a superfusion technique for prostaglandin bioassay. Materials and Methods Mongrel dogs of either sex, weighing 8 - 1 2 kg, were anesthetized with intraperitoneal injections of thiopental sodium (50 mg/kg) and sacrificed by rapid exsanguination from the common carotid arteries. The brain, heart, and ileum were rapidly removed. The pial arteries from two canine brains including basilar artery and anterior, middle, and posterior cerebral arteries, as well as their main branches, were dissected free and placed in a small chamber. The chamber was continuously irrigated with modified Ringer-Locke solution at a flow rate of 3.3 mL/min using an infusion pump. The solution overflowing from the chamber was then collected and introduced to the bioassay cascade and superfused on assay organs (Figure 1). The composition of irrigating solution was in millimolar: Na + 139.7, K ÷ 5.4, Ca ++ 2.2, Mg *+ 1.0, Cl " 131.5, HCO~; 20.0, and glucose 5.6. The stock solution was maintained at 38°C and continuously bubbled by a gas mixture of 9 5 % 02 and 5% COe. The p H of the solution was approximately 7.3. The assay organs used were rat stomach strips, dog ileal strips, and dog coronary arterial strips. The rat stomach strip was prepared from the fundus of the stomach of a Kyoto-Wistar rat as described by Vane [12]. A segment of dog ileum about 3 cm long was opened longitudinally, the mucosa and connective tissue were removed, and then the circular muscle layer was cut into a strip about 10 cm in length. The proximal portion of the dog coronary artery was dissected free as long as 15 mm and then cut helically into a strip. Each strip of the assay organs was grasped by small clips at both ends and vertically mounted. The lower end was fixed to a rigid, immobilized arm, and the upper end was connected to the lever of a transducer. An isotonic transducer (model TD-112S, Nihon Kohden Kogyo Co., Ltd., Tokyo, Japan) was used for both the rat stomach strip and the dog ileal strip, and a strain gauge transducer (model T70090-3019/85/$~.30
422
Surg Neurol 1985;23:421-4
Okamoto et al
antagonists
i ndomethacin
Ir D
',
95% 02 5% CO2
m
7 °C
Figure 1. Diagram of the apparatus for superfusion experiment. Dog pial arteries were placed in a small chamber which was continuously irrigated with modified Ringer-Locke solution. The solution that overflowed from the chamber was collected and superfused on the assay organs. The hemolysate was at first injected into the stream at the point indicated by the right arrow to confirm that it induces no direct response in the assay organs; then it was injected at the point indicated by the left arrow to challenge the pial arteries. The pial arteries were challenged before and after the treatment of them with indomethacin.
8-240, Toyo-Baldwin Inc., Tokyo, Japan) for the dog coronary arterial strip. The resting tensions applied were 2.5 g for both the rat stomach strip and the dog ileal strip and 1.5 g for the dog coronary arterial strip. The assay organs were treated continuously with a mixture of antagonists, including atropine, an anticholinergic agent (10 -7 M, final concentration in the superfusate), chlorpheniramine, a histamine H~ blocker (10 -6 M), cinanserin, a serotonin blocker (10 -6 M), indomethacin, a prostaglandin synthesis inhibitor (3 x 10 -7 M), and propranolol, a/3-adrenergic blocker (10 6 M). In some experiments, methysergide (10 -6 M) was used instead of cinanserin. Red blood cells from anticoagulated canine arterial blood were washed with saline and hemolyzed by adding an equal volume of distilled water. The hemolyzed so-
lution was centrifuged for 20 minutes at 10,000 rpm, and the ghost-free supernatant was used as hemolysate. The concentration of the hemolysate, assayed for hemoglobin content by a hemoglobincyanide method, was adjusted to that containing 10 g/dL hemoglobin. The direct bolus application of hemolysate (40 ~L, 10 g/dL hemoglobin-containing) to assay organs induced a transient change of their length or tension. When the organs were continuously superfused with hemolysate (0.1 g/dL hemoglobin in final concentration in the superfusate), further bolus application of hemolysate to the assay organs did not induce significant change that was presumably due to the tachyphylaxis. The responses of rat stomach strip and dog ileal strip to prostaglandins were not influenced by this procedure. Dog coronary artery, however, gained some active tension after the hemolysate was superfused on it, indicating that the sensitivity of the artery to the relaxation induced by prostaglandin I2 was markedly increased. The hemolysate was applied to cerebral arteries, and isotonic length changes in either the rat stomach strip or the dog ileal strip, and isometric tension changes in dog coronary arterial strip, were recorded on ink-writing oscillographs. The concentration of the prostaglandinlike substance released from cerebral arteries was expressed as a concentration of the authentic prostaglandin that is observed to induce the same degree of response in the assay organ, being calculated from responses of the assay organ to the authentic prostaglandin in different concentrations. The drugs used were prostaglandins A2, B2, D2, E2, F2~, and I2 (Ono Pharmaceutical Co., Osaka, Japan), atropine sulfate, d-chlorpheniramine maleate, cinanserin, indomethacin, d/-propranolol hydrochloride, and methysergide (Sandoz Ltd., Basel, Switzerland). Results were expressed as mean values _ standard error of the mean, Student's t-test being used for statistical analysis. Results The responses of assay organs to the authentic prostaglandins (5-20 pmol) were as follows (Figure 2). The rat stomach strip was contracted markedly by both prostaglandins E2 and F2~, and to a much lesser extent by prostaglandin I2. The dog ileal strip was also contracted markedly by prostaglandin F2~, but it responded by slight relaxation to both prostaglandins E2 and I2. The dog coronary arterial strip was contracted by prostaglandins E1 and F2~ slightly, whereas it relaxed markedly with prostaglandin I2. Other prostaglandins, such as prostaglandins A2, B2, and D2, induced indefinite responses in some assay organs, but these responses were induced only by a very large amount of prostaglandins, which
Prostaglandins in Cerebral Arteries
PGE 2
PGF2~' PGI 2
Surg Neurol 1985;23:421-4
PERFUSATE
5 mm
DOG ILEUM
f /t/~TER~
1
5 mm
•
°
3 rain F i g u r e 2. The responses of assay organs t Oauthentic prostaglandins f20 pmol) and prostaglandinlike substances releasedfrom the dog pial arteries challenged by the hemolysate ~PERFUSATE). Note that the contraction of dog ileum and the relaxation of dog coronary artery are identical to prostaglandins F2, and I~, respectively. The perfusate induced a marked contraction in the rat stomach and a slight but definite relaxation in the dog coronary artery, whereas no response was induced in dog ileum.
means that these prostaglandins are practically not detectable with the assay organs used. A bolus injection of 4 0 / z L of hemolysate containing 10 g/dL hemoglobin to the cerebral arterial specimen induced a marked contraction of a rat stomach strip and a slight, but definite, relaxation of a dog coronary arterial strip, whereas no response was induced in dog ileal strip (Figure 2). It was apparent that these responses were induced by a certain substance(s) released from the cerebral arteries by the action of the hemolysate, because a bolus application of hemolysate to assay organs without conditioning with the arterial specimen induced no significant response in the assay organs. After the cerebral arterial specimen was treated with 3 × 10-7 M indomethacin (final concentrationin the superfusate) for 30 minutes, the responses of assay organs to the hemolysate conditioned with the cerebral arteries were abolished or markedly attenuated (Table 1), although the responses
423
of the assay organs to the authentic prostaglandins were not affected. It was thus apparent that the substance released from the arterial specimen was prostaglandin itself or a prostaglandinlike substance. The releasing action was reversed after the exposure of the arterial specimen to the control irrigating solution for 30-60 minutes. The amounts of prostaglandinlike substances released by the application of the hemolysate are summarized in Table 1. The amount of prostaglandinlike substance to be released from 1 g of the cerebral arteries was equivalent to 125.2 +_ 19.4 pmol of prostaglandin E2 (n = 8) with respect to contraction of the rat stomach strip. From the relaxation of the dog coronary arterial strip, the amount of 59.5 +- 16.4 pmol of prostaglandin 12 (n = 6) was equivalent to the prostaglandin I2-1ike substance to be released from 1 g of the cerebral arteries. The prostaglandin F2~-like substance was not detected (n = 3).
Discussion The present study clearly shows that canine cerebral arteries release prostaglandinlike substances into the bathing medium in response to hemolysate application. The superfusion technique used in this study was originally described by Gaddum [3], and many fruitful experiments have been performed so far with this technique in the field of prostaglandin investigation [6]. This technique is especially useful to estimate, as in this study, a substance that is released from a specimen challenged by some kind of stimulus. If the specimen continuously releases the substance in its basal status, only the increase from the basal value induced by the stimulus is detected by this method, so that the variability of the basal value can be left out of consideration. The combination of responses in the assay organs that were induced by the hemolysate conditioned with the cerebral arteries does not correspond to any single sort of prostaglandin. The contractile response in the rat stomach strip can be induced not only by prostaglandins E2 and Fe~ but also, though to much lesser extent, by prostaglandin I2 or thromboxane A2. However, it may not be prostaglandin F2, that induced the response in the rat stomach strip, because the dog ileal strip that responded significantly only to prostaglandin F2,, but
Table 1. Amount of Prostaglandinlike Substances Releasedfrom Control and Indomethacin-treated Dog Pial Arteries Substance
Control
lndomethacin-treated
P r o s t a g l a n d i n E2-1ike P r o s t a g l a n d i n 12-like P r o s t a g l a n d i n F2~-like
125.2 -+ 19.4 ~ (n = 8) 59.5 -+ 16.4 (n = 6) N o t d e t e c t e d (n = 3)
26.5 +- 6.2 (n = 7) h 19.0 +- 12.0 (n = 6) b
~pmol/g w e t w e i g h t . M e a n -+ SEM. ~Significantly different from control, p < 0.05.
424
Surg Neurol 1985;23:421-4
not to prostaglandins E2 or I2, did not respond to the conditioned hemolysate. We confirmed that the contractile responses in canine cerebral arterial strip induced by hemolysate (10 -4 to 1.0 g/dL hemoglobincontaining) are not attenuated by the treatment of the artery with OKY-046 (Kissei Pharmaceutical Co., Ltd., Matsumoto, Japan), a thromboxane A2 synthetase inhibitor (Handa Y, Okamoto S, Handa H [1983], unpublished data). This suggests that thromboxane A2 has no role in the cerebral vasoconstriction induced by the hemolysate. From these facts, it is reasonably implied that the contraction of the rat stomach strip induced by the conditioned hemolysate is mediated mainly by a prostaglandin E2-1ike substance released from the cerebral arteries. The relaxation of the dog coronary arterial strip can be interpreted to be due to the prostaglandin I2-1ike substance released from the cerebral arteries, because the combination of responses in other assay organs is not qualitatively contradictory to the interpretation. To summarize, the dog cerebral arteries release, responding to the hemolysate, both a prostaglandin E2-1ike substance and a prostaglandin I2-1ike substance, whereas a prostaglandin F2,-like substance is not released in a detectable amount. It has been described in detail that arterial walls including the cerebral artery are able to metabolize the extrinsic arachidonate into both prostaglandins E2 and I2 [4,5,10]. The prostaglandin profile in a vascular tissue examined with the extrinsic [t4C]arachidonate appears to be disproportionately deviated to prostaglandin I2 [1], and it is quite different from the present results. Although the quantitative analysis in this study is a preliminary one, it may be possible that the prostaglandin profile resulting from the metabolism of the intrinsic arachidonate is different from that of the extrinsic arachidonate. Indeed, the details of the prostaglandin profile in the cerebral artery metabolizing the intrinsic arachidonate are still to be elucidated. The profile may also be different between the basal status and in a response to some kind of stimulus, as well as between different kinds of stimuli [9]. The present results are compatible with our previous
Okamoto et al
report that the cerebral vasoconstriction induced by the hemolysate is inhibited by aspirin, a prostaglandin synthetase inhibitor, which suggested that the intrinsic arachidonate metabolite has a role in vasoconstriction induced by the hemolysate [7]. It may be inferred that the prostaglandin involved in the hemolysate-induced cerebral vasoconstriction is prostaglandin E2. Prostaglandin E2 contracted a dog cerebral artery in an in vitro experiment [11]. A similar mechanism may be involved in the cerebral vasospasm after a subarachnoid hemorrhage, at least in the experimental vasospasm of dogs, in which the cerebral arteries may be immersed in hemolyzing blood clots. References 1. Abdel-Halim MS, yon Hoist H, Meyerson B, Sachs C, Anggard E. Prostaglandin profiles in tissue and blood vessels from human brain. J Neurochem 1980;34:1331-3. 2. Fisher CM, Kistler JP, Davis JM. Relation of cerebral vasospasm to subarachnoid hemorrhage visualized by computerized tomographic scanning. Neurosurgery 1980;6:1-9. 3. Gaddum JH. The technique of superfusion. Br J Pharmacol 1953;8:321-6. 4. Hagen AA, White RP, Robertson JT. Synthesis of prostaglandins and thromboxane B2 by cerebral arteries. Stroke 1979;10:306-9. 5. Maeda Y, Tani E, Miyamoto T. Prostaglandin metabolism in experimental cerebral vasospasm. J Neurosurg 1981 ;779-85. 6. Moncada S, Ferreira SH, Vane JR. Bioassay ofprostaglandins and biologically active substances derived from arachidonic acid. In: Frilich JC, ed. Advances in Prostaglandin and Thromboxane Research, Vol 5. New York: Raven Press, 1978:211-36. 7. Okamoto S, Handa H, Toda N. Role of intrinsic arachidonate metabolites in the vascular action oferythrocyte breakdown products. Stroke 1984; 15:60-4. 8. Osaka K. Prolonged vasospasm produced by the breakdown products of erythrocytes. J Neurosurg 1977;47:403-11. 9. Sametz W, Juan H. Release of different prostaglandins from vascular tissue by different stimulators. Prostaglandins Leukotrienes Med 1982;9:593-602. 10. Sasaki T, Murota S, Wakai S, Asano T, Sano K. Evaluation of prostaglandin biosynthetic activity in canine basilar artery following subarachnoid injection of blood. J Neurosurg 1981 ;55:771-8. 11. Toda N, Miyazaki M. Response of isolated dog cerebral and peripheral arteries to prostaglandins after application of aspirin and polyphloretin phosphate. Stroke 1978;9:490-8. 12. Vane JR. A sensitive method for assay of 5-hydroxytryptamine. Br J Pharmacol 1957;12:344-9.
421
Hemolysate-induced Release of Prostaglandinlike Substances from the Canine Cerebral Arteries Shinichiro Okamoto,
M.D., Hajime
Handa, M.D., and Yutaka Handa, M.D.
Department of Neurosurgery, Kyoto University Medical School, Kyoto, Japan
Okamoto S, Handa H, Handa Y. Hemolysate-induced release of prostaglandinlike substances from the canine cerebral arteries. Surg Neurol I985;23:421-4.
The release of prostaglandinlike substances from canine pial arteries that was induced by exposure of the pial arteries to red blood cell hemolysate was estimated by using a superfusion technique for prostaglandin bioassay. The assay organs used were strips of rat stomach for prostaglandin E2- or prostaglandin Fz~-like substances, strips of dog coronary artery for prostaglandin I2-1ike substance, and strips of dog ileum for prostaglandin Fz,,-like substance. The substances released from the canine pial arteries induced contractions in rat stomach strips and relaxations in canine coronary arterial strips, whereas they did not induce any response in canine ileal strips. The equivalent prostaglandin E2 doses for the contractions of the rat stomach strips and the equivalent prostaglandin I2 doses for the relaxations of the canine coronary arterial strips were 125.2 -+ 19.4 (n = 8) and 59.5 - 16.4 (n = 6) pmol/g wet wt -+ SEM, respectively. Cerebral artery; Cerebral vasospasm; Prostaglandin; Subarachnoid hemorrhage KEY W O R D S :
Among the candidates to be included as a causative substance of cerebral vasospasm after a subarachnoid hemorrhage are the breakdown products of red blood cells [8]. This possibility may explain the delayed onset of vasospasm after a hemorrhagic insult when the incubation period that is necessary for the subarachnoid blood clot to cause hemolysis is taken into consideration. The good correlation between the sites o f vasospasm and those of subarachnoid hematoma on computed tomographic scanning [2] is another basis of this concept. It was suggested that the intrinsic arachidonate metabolites of the cerebral artery have a role in the cerebral vasoconstriction induced by the red blood cell hemolysate [7]. We have examined further the release of the Address reprint requests to: Dr. Hajime Handa, Department of Neurosurgery, Kyoto University Hospital, 54 Shogoin Kawaharacho, Sakyoku, Kyoto 606, Japan.
© 1985 by Elsevier Science Publishing Co., Inc.
prostaglandinlike substances from the cerebral artery in an in vitro experimental condition by using a superfusion technique for prostaglandin bioassay. Materials and Methods Mongrel dogs of either sex, weighing 8 - 1 2 kg, were anesthetized with intraperitoneal injections of thiopental sodium (50 mg/kg) and sacrificed by rapid exsanguination from the common carotid arteries. The brain, heart, and ileum were rapidly removed. The pial arteries from two canine brains including basilar artery and anterior, middle, and posterior cerebral arteries, as well as their main branches, were dissected free and placed in a small chamber. The chamber was continuously irrigated with modified Ringer-Locke solution at a flow rate of 3.3 mL/min using an infusion pump. The solution overflowing from the chamber was then collected and introduced to the bioassay cascade and superfused on assay organs (Figure 1). The composition of irrigating solution was in millimolar: Na + 139.7, K ÷ 5.4, Ca ++ 2.2, Mg *+ 1.0, Cl " 131.5, HCO~; 20.0, and glucose 5.6. The stock solution was maintained at 38°C and continuously bubbled by a gas mixture of 9 5 % 02 and 5% COe. The p H of the solution was approximately 7.3. The assay organs used were rat stomach strips, dog ileal strips, and dog coronary arterial strips. The rat stomach strip was prepared from the fundus of the stomach of a Kyoto-Wistar rat as described by Vane [12]. A segment of dog ileum about 3 cm long was opened longitudinally, the mucosa and connective tissue were removed, and then the circular muscle layer was cut into a strip about 10 cm in length. The proximal portion of the dog coronary artery was dissected free as long as 15 mm and then cut helically into a strip. Each strip of the assay organs was grasped by small clips at both ends and vertically mounted. The lower end was fixed to a rigid, immobilized arm, and the upper end was connected to the lever of a transducer. An isotonic transducer (model TD-112S, Nihon Kohden Kogyo Co., Ltd., Tokyo, Japan) was used for both the rat stomach strip and the dog ileal strip, and a strain gauge transducer (model T70090-3019/85/$~.30
422
Surg Neurol 1985;23:421-4
Okamoto et al
antagonists
i ndomethacin
Ir D
',
95% 02 5% CO2
m
7 °C
Figure 1. Diagram of the apparatus for superfusion experiment. Dog pial arteries were placed in a small chamber which was continuously irrigated with modified Ringer-Locke solution. The solution that overflowed from the chamber was collected and superfused on the assay organs. The hemolysate was at first injected into the stream at the point indicated by the right arrow to confirm that it induces no direct response in the assay organs; then it was injected at the point indicated by the left arrow to challenge the pial arteries. The pial arteries were challenged before and after the treatment of them with indomethacin.
8-240, Toyo-Baldwin Inc., Tokyo, Japan) for the dog coronary arterial strip. The resting tensions applied were 2.5 g for both the rat stomach strip and the dog ileal strip and 1.5 g for the dog coronary arterial strip. The assay organs were treated continuously with a mixture of antagonists, including atropine, an anticholinergic agent (10 -7 M, final concentration in the superfusate), chlorpheniramine, a histamine H~ blocker (10 -6 M), cinanserin, a serotonin blocker (10 -6 M), indomethacin, a prostaglandin synthesis inhibitor (3 x 10 -7 M), and propranolol, a/3-adrenergic blocker (10 6 M). In some experiments, methysergide (10 -6 M) was used instead of cinanserin. Red blood cells from anticoagulated canine arterial blood were washed with saline and hemolyzed by adding an equal volume of distilled water. The hemolyzed so-
lution was centrifuged for 20 minutes at 10,000 rpm, and the ghost-free supernatant was used as hemolysate. The concentration of the hemolysate, assayed for hemoglobin content by a hemoglobincyanide method, was adjusted to that containing 10 g/dL hemoglobin. The direct bolus application of hemolysate (40 ~L, 10 g/dL hemoglobin-containing) to assay organs induced a transient change of their length or tension. When the organs were continuously superfused with hemolysate (0.1 g/dL hemoglobin in final concentration in the superfusate), further bolus application of hemolysate to the assay organs did not induce significant change that was presumably due to the tachyphylaxis. The responses of rat stomach strip and dog ileal strip to prostaglandins were not influenced by this procedure. Dog coronary artery, however, gained some active tension after the hemolysate was superfused on it, indicating that the sensitivity of the artery to the relaxation induced by prostaglandin I2 was markedly increased. The hemolysate was applied to cerebral arteries, and isotonic length changes in either the rat stomach strip or the dog ileal strip, and isometric tension changes in dog coronary arterial strip, were recorded on ink-writing oscillographs. The concentration of the prostaglandinlike substance released from cerebral arteries was expressed as a concentration of the authentic prostaglandin that is observed to induce the same degree of response in the assay organ, being calculated from responses of the assay organ to the authentic prostaglandin in different concentrations. The drugs used were prostaglandins A2, B2, D2, E2, F2~, and I2 (Ono Pharmaceutical Co., Osaka, Japan), atropine sulfate, d-chlorpheniramine maleate, cinanserin, indomethacin, d/-propranolol hydrochloride, and methysergide (Sandoz Ltd., Basel, Switzerland). Results were expressed as mean values _ standard error of the mean, Student's t-test being used for statistical analysis. Results The responses of assay organs to the authentic prostaglandins (5-20 pmol) were as follows (Figure 2). The rat stomach strip was contracted markedly by both prostaglandins E2 and F2~, and to a much lesser extent by prostaglandin I2. The dog ileal strip was also contracted markedly by prostaglandin F2~, but it responded by slight relaxation to both prostaglandins E2 and I2. The dog coronary arterial strip was contracted by prostaglandins E1 and F2~ slightly, whereas it relaxed markedly with prostaglandin I2. Other prostaglandins, such as prostaglandins A2, B2, and D2, induced indefinite responses in some assay organs, but these responses were induced only by a very large amount of prostaglandins, which
Prostaglandins in Cerebral Arteries
PGE 2
PGF2~' PGI 2
Surg Neurol 1985;23:421-4
PERFUSATE
5 mm
DOG ILEUM
f /t/~TER~
1
5 mm
•
°
3 rain F i g u r e 2. The responses of assay organs t Oauthentic prostaglandins f20 pmol) and prostaglandinlike substances releasedfrom the dog pial arteries challenged by the hemolysate ~PERFUSATE). Note that the contraction of dog ileum and the relaxation of dog coronary artery are identical to prostaglandins F2, and I~, respectively. The perfusate induced a marked contraction in the rat stomach and a slight but definite relaxation in the dog coronary artery, whereas no response was induced in dog ileum.
means that these prostaglandins are practically not detectable with the assay organs used. A bolus injection of 4 0 / z L of hemolysate containing 10 g/dL hemoglobin to the cerebral arterial specimen induced a marked contraction of a rat stomach strip and a slight, but definite, relaxation of a dog coronary arterial strip, whereas no response was induced in dog ileal strip (Figure 2). It was apparent that these responses were induced by a certain substance(s) released from the cerebral arteries by the action of the hemolysate, because a bolus application of hemolysate to assay organs without conditioning with the arterial specimen induced no significant response in the assay organs. After the cerebral arterial specimen was treated with 3 × 10-7 M indomethacin (final concentrationin the superfusate) for 30 minutes, the responses of assay organs to the hemolysate conditioned with the cerebral arteries were abolished or markedly attenuated (Table 1), although the responses
423
of the assay organs to the authentic prostaglandins were not affected. It was thus apparent that the substance released from the arterial specimen was prostaglandin itself or a prostaglandinlike substance. The releasing action was reversed after the exposure of the arterial specimen to the control irrigating solution for 30-60 minutes. The amounts of prostaglandinlike substances released by the application of the hemolysate are summarized in Table 1. The amount of prostaglandinlike substance to be released from 1 g of the cerebral arteries was equivalent to 125.2 +_ 19.4 pmol of prostaglandin E2 (n = 8) with respect to contraction of the rat stomach strip. From the relaxation of the dog coronary arterial strip, the amount of 59.5 +- 16.4 pmol of prostaglandin 12 (n = 6) was equivalent to the prostaglandin I2-1ike substance to be released from 1 g of the cerebral arteries. The prostaglandin F2~-like substance was not detected (n = 3).
Discussion The present study clearly shows that canine cerebral arteries release prostaglandinlike substances into the bathing medium in response to hemolysate application. The superfusion technique used in this study was originally described by Gaddum [3], and many fruitful experiments have been performed so far with this technique in the field of prostaglandin investigation [6]. This technique is especially useful to estimate, as in this study, a substance that is released from a specimen challenged by some kind of stimulus. If the specimen continuously releases the substance in its basal status, only the increase from the basal value induced by the stimulus is detected by this method, so that the variability of the basal value can be left out of consideration. The combination of responses in the assay organs that were induced by the hemolysate conditioned with the cerebral arteries does not correspond to any single sort of prostaglandin. The contractile response in the rat stomach strip can be induced not only by prostaglandins E2 and Fe~ but also, though to much lesser extent, by prostaglandin I2 or thromboxane A2. However, it may not be prostaglandin F2, that induced the response in the rat stomach strip, because the dog ileal strip that responded significantly only to prostaglandin F2,, but
Table 1. Amount of Prostaglandinlike Substances Releasedfrom Control and Indomethacin-treated Dog Pial Arteries Substance
Control
lndomethacin-treated
P r o s t a g l a n d i n E2-1ike P r o s t a g l a n d i n 12-like P r o s t a g l a n d i n F2~-like
125.2 -+ 19.4 ~ (n = 8) 59.5 -+ 16.4 (n = 6) N o t d e t e c t e d (n = 3)
26.5 +- 6.2 (n = 7) h 19.0 +- 12.0 (n = 6) b
~pmol/g w e t w e i g h t . M e a n -+ SEM. ~Significantly different from control, p < 0.05.
424
Surg Neurol 1985;23:421-4
not to prostaglandins E2 or I2, did not respond to the conditioned hemolysate. We confirmed that the contractile responses in canine cerebral arterial strip induced by hemolysate (10 -4 to 1.0 g/dL hemoglobincontaining) are not attenuated by the treatment of the artery with OKY-046 (Kissei Pharmaceutical Co., Ltd., Matsumoto, Japan), a thromboxane A2 synthetase inhibitor (Handa Y, Okamoto S, Handa H [1983], unpublished data). This suggests that thromboxane A2 has no role in the cerebral vasoconstriction induced by the hemolysate. From these facts, it is reasonably implied that the contraction of the rat stomach strip induced by the conditioned hemolysate is mediated mainly by a prostaglandin E2-1ike substance released from the cerebral arteries. The relaxation of the dog coronary arterial strip can be interpreted to be due to the prostaglandin I2-1ike substance released from the cerebral arteries, because the combination of responses in other assay organs is not qualitatively contradictory to the interpretation. To summarize, the dog cerebral arteries release, responding to the hemolysate, both a prostaglandin E2-1ike substance and a prostaglandin I2-1ike substance, whereas a prostaglandin F2,-like substance is not released in a detectable amount. It has been described in detail that arterial walls including the cerebral artery are able to metabolize the extrinsic arachidonate into both prostaglandins E2 and I2 [4,5,10]. The prostaglandin profile in a vascular tissue examined with the extrinsic [t4C]arachidonate appears to be disproportionately deviated to prostaglandin I2 [1], and it is quite different from the present results. Although the quantitative analysis in this study is a preliminary one, it may be possible that the prostaglandin profile resulting from the metabolism of the intrinsic arachidonate is different from that of the extrinsic arachidonate. Indeed, the details of the prostaglandin profile in the cerebral artery metabolizing the intrinsic arachidonate are still to be elucidated. The profile may also be different between the basal status and in a response to some kind of stimulus, as well as between different kinds of stimuli [9]. The present results are compatible with our previous
Okamoto et al
report that the cerebral vasoconstriction induced by the hemolysate is inhibited by aspirin, a prostaglandin synthetase inhibitor, which suggested that the intrinsic arachidonate metabolite has a role in vasoconstriction induced by the hemolysate [7]. It may be inferred that the prostaglandin involved in the hemolysate-induced cerebral vasoconstriction is prostaglandin E2. Prostaglandin E2 contracted a dog cerebral artery in an in vitro experiment [11]. A similar mechanism may be involved in the cerebral vasospasm after a subarachnoid hemorrhage, at least in the experimental vasospasm of dogs, in which the cerebral arteries may be immersed in hemolyzing blood clots. References 1. Abdel-Halim MS, yon Hoist H, Meyerson B, Sachs C, Anggard E. Prostaglandin profiles in tissue and blood vessels from human brain. J Neurochem 1980;34:1331-3. 2. Fisher CM, Kistler JP, Davis JM. Relation of cerebral vasospasm to subarachnoid hemorrhage visualized by computerized tomographic scanning. Neurosurgery 1980;6:1-9. 3. Gaddum JH. The technique of superfusion. Br J Pharmacol 1953;8:321-6. 4. Hagen AA, White RP, Robertson JT. Synthesis of prostaglandins and thromboxane B2 by cerebral arteries. Stroke 1979;10:306-9. 5. Maeda Y, Tani E, Miyamoto T. Prostaglandin metabolism in experimental cerebral vasospasm. J Neurosurg 1981 ;779-85. 6. Moncada S, Ferreira SH, Vane JR. Bioassay ofprostaglandins and biologically active substances derived from arachidonic acid. In: Frilich JC, ed. Advances in Prostaglandin and Thromboxane Research, Vol 5. New York: Raven Press, 1978:211-36. 7. Okamoto S, Handa H, Toda N. Role of intrinsic arachidonate metabolites in the vascular action oferythrocyte breakdown products. Stroke 1984; 15:60-4. 8. Osaka K. Prolonged vasospasm produced by the breakdown products of erythrocytes. J Neurosurg 1977;47:403-11. 9. Sametz W, Juan H. Release of different prostaglandins from vascular tissue by different stimulators. Prostaglandins Leukotrienes Med 1982;9:593-602. 10. Sasaki T, Murota S, Wakai S, Asano T, Sano K. Evaluation of prostaglandin biosynthetic activity in canine basilar artery following subarachnoid injection of blood. J Neurosurg 1981 ;55:771-8. 11. Toda N, Miyazaki M. Response of isolated dog cerebral and peripheral arteries to prostaglandins after application of aspirin and polyphloretin phosphate. Stroke 1978;9:490-8. 12. Vane JR. A sensitive method for assay of 5-hydroxytryptamine. Br J Pharmacol 1957;12:344-9.