Spasmogenic qualities of prostaglandin in the cat JOHN P. KAPP, M.D., PH.D., JAMES T. ROBERTSON, M.D., AND RICHARD P. WHITE, PH.D. Department of Neurosurgery and Pharmacology, University of Tennessee Medical Units and Semmes-Murphey Clinic, Memphis, Tennessee ~," Available data indicate that the concentration of prostaglandin F2~ required to produce arterial spasm in an experimental model is approximately a thousand-fold higher than the concentration that occurs under physiological conditions. The spasmogenic platelet factor which has been previously described is shown to be a substance other than prostaglandin F~, because of differences in susceptibility of the two substances to enzymatic digestion. Key WORDS
cerebralvasospasm
A
COMPONENT of platelets has been described which is highly spasmogenic when applied topically to the feline basilar artery? This material is stable to boiling and dialyzable through cellophane. It may be separated from serotonin by chromatography. It may be differentiated from angiotensin, vasopressin, and substance P (a vasoactive polypeptide 1~ since these materials are digested by trypsin, while the spasmogenic platelet factor is not. It is not bradykinin, adenosine diphosphate, epinephrine, norepinephrine, or histamine, since these substances are not spasmogenic. It is completely inactivated by the nonspecific bacterial proteolytic enzyme pronase. Because of its digestibility by a proteolytic enzyme and because of its apparent small molecular size, as estimated by gel filtration studies, it appeared that the spasmogenic platelet factor was a peptide. Recent studies in this laboratory have demonstrated that significant spasm follows the subarachnoid injection of prostaglandin J. Neurosurg. / Volume 44 / February, 1976
9 prostaglandin F2.
(PG) F~,, especially when mixed with blood? Prostaglandin F~ is known to be present in both brain tissue and platelets; it is stable to boiling and is dialyzable. Because of the possible similarity, the present study was undertaken to ascertain the relationship of PGF2~ to the spasmogenic platelet factor. This study consisted of two parts. First, a dose-response curve was obtained to determine in what concentration PGF2~ is an effective spasmogenic agent in the cat. Second, studies were done to determine if PGF~ would be inactivated by incubation with pronase. If it were not inactivated, the evidence would indicate that the platelet spasmogenic factor and PGF~ were distinctly different substances. Materials and Methods
Studies were conducted that used the transorally exposed basilar artery of cats anesthetized with pentobarbital sodium (25 mg/kg). The basilar artery was bathed by a "173
j. P. K a p p , J. T. R o b e r t s o n a n d R. P. W h i t e assay the pH was adjusted with phosphate buffer to the pH of Ringer's solution. Control solutions of PGF2, were treated in the same way except that no enzyme was added. Reactivity of the artery to platelets was determined after assay of the various other fractions. As a control on the quality of the enzyme and the technique of digestion, a parallel study was made which used angiotensin, a known spasmogenic agent which is pronase digestible. Results
The results of the dilution studies are shown in Fig. 1. No significant constriction was produced by concentrations less than 0.1 ~zg/ml. Constriction was dose-related to a F~G. 1. Dose-response curve for prostaglandin concentration of 100 ug/ml. Similar results F~, as spasmogenicagent when applied topically to were obtained in the other study. In each of the enzymatic digestion experiments, the the basilar artery of the cat. spasmogenic activity of angiotensin was destroyed by incubation with pronase. Table TABLE 1 1 summarizes the data. Constriction averagPronase digestion of spasmogenic platelet factor ing 36% was produced by PGF2~ incubated (SPF) and prostaglandin t72 alpha with pronase. This was essentially the same as Change in Vessel Diameter (7o) the 32% produced by untreated PGF~. Enzyme Substrate Study 1 Study 2 Study 3 Study 4 Previous investigations8 have shown that pronase completely inactivates the spasmopronase F2a --44 -- 38 --40 --22 genic platelet factor. none F2a --37 -26 -44 --21 pronase SPF --5 +7 0 --3 Discussion none SPF --29 --17 --53 --15
continuous irrigation of Ringer's solution, to which solutions to be assayed for spasmogenic activity were added. Photographs of the exposed artery were made with a camera attached to the operative microscope. The diameter of the artery was measured from the photographs. The data were expressed as percent change relative to the control diameter? Dilution studies were carried out in two animals with PGF2~ in Ringer's solution used in concentrations ranging from IO s to 100 ~tg/ml. The stability of PGF2~ to digestion with pronase was studied. Mixtures of pronase, 0.2 mg/cc, and PGF~, 10 ~g/cc, were incubated at 37 ~ C for 15 hours at pH 6.1. Subsequently, an additional 0.2 mg/cc of pronase was added to the mixture, and the mixture was incubated an additional 3 hours. The solutions were boiled for 3 minutes to inactivate the enzyme and then frozen. Prior to ]74
Prostaglandin F2~ is known to be synthesized in brain tissue and in platelets. Its exact concentration in platelets is not known. Assays of platelet suspensions for total prostaglandin-like activity in smooth muscle strips 9 have given values about a thousand times less than the concentrations of PGF2~ required in our dilution studies to produce significant spasm. The possibility that prostaglandins may be released from the cerebral tissue into the subarachnoid space in response to subarachnoid hemorrhage is intriguing. The recent study by La Torre, et al., 7 suggests that this may occur. The F type prostaglandins in brain may be regionally as high as 389 ng/gm of tissue4 and are reportedly released into the cerebral ventricles at about 75 ng/hr in dogs~ and from cat cortex at about 1.3 ng/cm ~ surface/min. 1 Furthermore, the spasmogenic agent serotonin increases fourfold the release of prostaglandins from dog brain? Although these amounts are far less than those required to produce spasm in our studies, it is possible that prostaglandins J. Neurosurg. / Volume 44 / February, 1976
Spasmogenic platelet factor and PGF2~ may be released and progressively accumulate in the subarachnoid space following subarachnoid hemorrhage. It is of interest, however, that the highest cerebrospinal fluid concentration of PGF2~ found in La Torre's series of patients with subarachnoid hemorrhage was 1.4 ng/ml, or a concentration which is a thousandfold less than we found necessary to induce spasm in our experimental model. It is possible that PGF2, and other spasmogenic substances in blood or cerebral tissue may act synergistically in the production of spasm as suggested by previous reports. T M Further studies will be required to clarify this point. These data indicate that the spasmogenic platelet factor is not PGF2,, but do not exclude the role of prostaglandins in the genesis of arterial spasm. References l. Bradley PB, Samuels GMR, Shaw JE: Correlation of prostaglandin release from cerebral cortex of cats with the electrocorticogram, following stimulation of the reticular formation. Br J Pharmacol 37: 151-157, 1969 2. Holmes SW: The spontaneous release of prostaglandins into the cerebral ventricles of the dog and the effect of external factors on this release. Br J Pharmaeol 38:653-658, 1970 3. Holmes SW, Horton EW: The identification of four prostaglandins in dog brain and their regional distribution in the central nervous system. J Physiol (Lond) 195:731-741, 1968 4. Holmes SW, Horton EW: Prostaglandins and the central nervous system, in Ramwell PW, Shaw JE (eds): Prostaglandin Symposium of the Worehester Foundation for Experimental Biology. New York: Interscience, 1968, pp 21-36
J. Neurosurg. / Volume 44 / February, 1976
5. Kapp J, Mahaley MS Jr, Odom GL: Cerebral arterial spasm. Part 1: Evaluation of experimental variables affecting the diameter of the exposed basilar artery. J Neurosurg 29:331-338, 1968 6. Kapp J, Mahaley MS Jr, Odom GL: Cerebral arterial spasm. Part 3: Partial purification and characterization of a spasmogenic substance in feline platelets. J Neurosurg 29:350-356, 1968 7. La Torre E, Patrono C, Fortuna A, et al: Role of prostaglandin F~ alpha in human cerebral vasospasm. J Neurosurg 41:293-299, 1974 8. Pennink M, White RP, Crockarell JR, et al: Role of prostaglandin F2 alpha in the genesis of experimental cerebral vasospasm. Angiographic study in dogs. J Neurosurg 37: 398-406, 1972 9. Smith JB, Willis AL: Aspirin selectively inhibits prostaglandin production in human platelets. Nature [New Bioi] 231:235-237, 1971 10. yon Euler US: Substance P in subcellular particles in peripheral nerves. Ann NY Acad Sei 104:449-463, 1963 11. White RP, Pennink M, Robertson JT: Prostaglandin F2 alpha and experimental cerebral vasospasm in dogs. Pharmacologist 13:292, 1971 (Abstract #560)
This investigation was supported in part by the Cerebrovascular Research Center Grants USPHS NS06826 and NS05651 from the National Institutes of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland. Address reprint requests to: John P. Kapp, M.D., Ph.D., 717 East 7th Street, Panama City, Florida 32401.
175
cerebralvasospasm
A
COMPONENT of platelets has been described which is highly spasmogenic when applied topically to the feline basilar artery? This material is stable to boiling and dialyzable through cellophane. It may be separated from serotonin by chromatography. It may be differentiated from angiotensin, vasopressin, and substance P (a vasoactive polypeptide 1~ since these materials are digested by trypsin, while the spasmogenic platelet factor is not. It is not bradykinin, adenosine diphosphate, epinephrine, norepinephrine, or histamine, since these substances are not spasmogenic. It is completely inactivated by the nonspecific bacterial proteolytic enzyme pronase. Because of its digestibility by a proteolytic enzyme and because of its apparent small molecular size, as estimated by gel filtration studies, it appeared that the spasmogenic platelet factor was a peptide. Recent studies in this laboratory have demonstrated that significant spasm follows the subarachnoid injection of prostaglandin J. Neurosurg. / Volume 44 / February, 1976
9 prostaglandin F2.
(PG) F~,, especially when mixed with blood? Prostaglandin F~ is known to be present in both brain tissue and platelets; it is stable to boiling and is dialyzable. Because of the possible similarity, the present study was undertaken to ascertain the relationship of PGF2~ to the spasmogenic platelet factor. This study consisted of two parts. First, a dose-response curve was obtained to determine in what concentration PGF2~ is an effective spasmogenic agent in the cat. Second, studies were done to determine if PGF~ would be inactivated by incubation with pronase. If it were not inactivated, the evidence would indicate that the platelet spasmogenic factor and PGF~ were distinctly different substances. Materials and Methods
Studies were conducted that used the transorally exposed basilar artery of cats anesthetized with pentobarbital sodium (25 mg/kg). The basilar artery was bathed by a "173
j. P. K a p p , J. T. R o b e r t s o n a n d R. P. W h i t e assay the pH was adjusted with phosphate buffer to the pH of Ringer's solution. Control solutions of PGF2, were treated in the same way except that no enzyme was added. Reactivity of the artery to platelets was determined after assay of the various other fractions. As a control on the quality of the enzyme and the technique of digestion, a parallel study was made which used angiotensin, a known spasmogenic agent which is pronase digestible. Results
The results of the dilution studies are shown in Fig. 1. No significant constriction was produced by concentrations less than 0.1 ~zg/ml. Constriction was dose-related to a F~G. 1. Dose-response curve for prostaglandin concentration of 100 ug/ml. Similar results F~, as spasmogenicagent when applied topically to were obtained in the other study. In each of the enzymatic digestion experiments, the the basilar artery of the cat. spasmogenic activity of angiotensin was destroyed by incubation with pronase. Table TABLE 1 1 summarizes the data. Constriction averagPronase digestion of spasmogenic platelet factor ing 36% was produced by PGF2~ incubated (SPF) and prostaglandin t72 alpha with pronase. This was essentially the same as Change in Vessel Diameter (7o) the 32% produced by untreated PGF~. Enzyme Substrate Study 1 Study 2 Study 3 Study 4 Previous investigations8 have shown that pronase completely inactivates the spasmopronase F2a --44 -- 38 --40 --22 genic platelet factor. none F2a --37 -26 -44 --21 pronase SPF --5 +7 0 --3 Discussion none SPF --29 --17 --53 --15
continuous irrigation of Ringer's solution, to which solutions to be assayed for spasmogenic activity were added. Photographs of the exposed artery were made with a camera attached to the operative microscope. The diameter of the artery was measured from the photographs. The data were expressed as percent change relative to the control diameter? Dilution studies were carried out in two animals with PGF2~ in Ringer's solution used in concentrations ranging from IO s to 100 ~tg/ml. The stability of PGF2~ to digestion with pronase was studied. Mixtures of pronase, 0.2 mg/cc, and PGF~, 10 ~g/cc, were incubated at 37 ~ C for 15 hours at pH 6.1. Subsequently, an additional 0.2 mg/cc of pronase was added to the mixture, and the mixture was incubated an additional 3 hours. The solutions were boiled for 3 minutes to inactivate the enzyme and then frozen. Prior to ]74
Prostaglandin F2~ is known to be synthesized in brain tissue and in platelets. Its exact concentration in platelets is not known. Assays of platelet suspensions for total prostaglandin-like activity in smooth muscle strips 9 have given values about a thousand times less than the concentrations of PGF2~ required in our dilution studies to produce significant spasm. The possibility that prostaglandins may be released from the cerebral tissue into the subarachnoid space in response to subarachnoid hemorrhage is intriguing. The recent study by La Torre, et al., 7 suggests that this may occur. The F type prostaglandins in brain may be regionally as high as 389 ng/gm of tissue4 and are reportedly released into the cerebral ventricles at about 75 ng/hr in dogs~ and from cat cortex at about 1.3 ng/cm ~ surface/min. 1 Furthermore, the spasmogenic agent serotonin increases fourfold the release of prostaglandins from dog brain? Although these amounts are far less than those required to produce spasm in our studies, it is possible that prostaglandins J. Neurosurg. / Volume 44 / February, 1976
Spasmogenic platelet factor and PGF2~ may be released and progressively accumulate in the subarachnoid space following subarachnoid hemorrhage. It is of interest, however, that the highest cerebrospinal fluid concentration of PGF2~ found in La Torre's series of patients with subarachnoid hemorrhage was 1.4 ng/ml, or a concentration which is a thousandfold less than we found necessary to induce spasm in our experimental model. It is possible that PGF2, and other spasmogenic substances in blood or cerebral tissue may act synergistically in the production of spasm as suggested by previous reports. T M Further studies will be required to clarify this point. These data indicate that the spasmogenic platelet factor is not PGF2,, but do not exclude the role of prostaglandins in the genesis of arterial spasm. References l. Bradley PB, Samuels GMR, Shaw JE: Correlation of prostaglandin release from cerebral cortex of cats with the electrocorticogram, following stimulation of the reticular formation. Br J Pharmacol 37: 151-157, 1969 2. Holmes SW: The spontaneous release of prostaglandins into the cerebral ventricles of the dog and the effect of external factors on this release. Br J Pharmaeol 38:653-658, 1970 3. Holmes SW, Horton EW: The identification of four prostaglandins in dog brain and their regional distribution in the central nervous system. J Physiol (Lond) 195:731-741, 1968 4. Holmes SW, Horton EW: Prostaglandins and the central nervous system, in Ramwell PW, Shaw JE (eds): Prostaglandin Symposium of the Worehester Foundation for Experimental Biology. New York: Interscience, 1968, pp 21-36
J. Neurosurg. / Volume 44 / February, 1976
5. Kapp J, Mahaley MS Jr, Odom GL: Cerebral arterial spasm. Part 1: Evaluation of experimental variables affecting the diameter of the exposed basilar artery. J Neurosurg 29:331-338, 1968 6. Kapp J, Mahaley MS Jr, Odom GL: Cerebral arterial spasm. Part 3: Partial purification and characterization of a spasmogenic substance in feline platelets. J Neurosurg 29:350-356, 1968 7. La Torre E, Patrono C, Fortuna A, et al: Role of prostaglandin F~ alpha in human cerebral vasospasm. J Neurosurg 41:293-299, 1974 8. Pennink M, White RP, Crockarell JR, et al: Role of prostaglandin F2 alpha in the genesis of experimental cerebral vasospasm. Angiographic study in dogs. J Neurosurg 37: 398-406, 1972 9. Smith JB, Willis AL: Aspirin selectively inhibits prostaglandin production in human platelets. Nature [New Bioi] 231:235-237, 1971 10. yon Euler US: Substance P in subcellular particles in peripheral nerves. Ann NY Acad Sei 104:449-463, 1963 11. White RP, Pennink M, Robertson JT: Prostaglandin F2 alpha and experimental cerebral vasospasm in dogs. Pharmacologist 13:292, 1971 (Abstract #560)
This investigation was supported in part by the Cerebrovascular Research Center Grants USPHS NS06826 and NS05651 from the National Institutes of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland. Address reprint requests to: John P. Kapp, M.D., Ph.D., 717 East 7th Street, Panama City, Florida 32401.
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