Effect of dopamine-beta-hydroxylase inhibition on cerebral vasospasm in the cat

Transcription

1 Effect of dopamine-beta-hydroxylase inhibition on cerebral vasospasm in the cat ARTHUR F. BATTISTA,.D., EUGENE S. FLA,.D., ENEK GOLDSTEIN, PH.D., AND LEWIS S. FREEDAN, PH.D. Departments of Neurosurgery and Psychiatry, New York University edical Center, New York, New York Inhibitors of dopamine beta hydroxylase (DBH) administered intraperitoneally were shown to prevent spasm of the basilar artery caused by blood application in cats. A low or nondetectable blood DBH activity level may be related to the absence of spasm. In several instances, however, moderate spasm did occur with a low blood DBH activity level. Possible mechanisms for these findings are discussed. KEYWORDS 9 dopamine-beta-hydroxylase inhibitors 9 disulfiram 9 fusaric acid 9 cerebral vasospasm 9 subarachnoid hemorrhage T HE presence of adrenergic nerve plexuses within the walls of cerebral vessels in animals has been demonstrated by the fluorescence staining method?,le arkedly diminished or absent fluorescence after cerebral vasospasm produced in animals by blood application or direct mechanical pressure has also been reported. 6'~7 It has been proposed that the noradrenergic fiber plexuses in cerebral vessels may be involved In the mechanism of cerebral vasospasm produced by various substances. Catecholamines have been implicated in vasospasm of cerebral vessels and have been examined in animals and in man by the use of various alpha and beta adrenergic receptor blocking agents. However, the results when adrenergic receptor blocking agents are used to relieve vasospasm in the experimental animal and in man, have been inconsistent, x'5.e'13'17 By means of immunofluorescent techniques, Hartman and Udenfriend 9 found that the enzyme dopamine beta hydroxylase (DBH) was localized in the nerve endings of large vessels at the base of the rat's brain. We used a quantitative chemical method to show the presence of DBH in the cerebral vessels of the cat, monkey, and man? ~ The enzyme DBH is required for the synthesis of norepinephrine. It converts dopamine to norepinephrine and is the only significant physiological pathway for the hydroxylation of dopamine to norepinephrine. 18 Therefore, we investigated the relationship of the noradrenergic cerebral vessel innervation to cerebral vasospasm. The effect of inhibiting "168 J. Neurosurg. / Volume 44 / February, 1976

2 Inhibiting DBH in cerebral vasospasm in cats the DBH enzyme on cerebral vasospasm produced by blood application to the basilar artery of the cat was studied? aterials and ethods Adult cats weighing 1.4 to 3.2 kg were anesthetized with intraperitoneal barbiturate (25 mg/kg). A tracheal cannula was inserted and the animal allowed to breathe spontaneously. Femoral arterial and venous catheters were inserted to record blood pressure and to obtain blood samples. Blood PaCO2 and PaOe were monitored. Rectal temperature was measured by an indwelling thermistor, and the body temperature was maintained within 1 ~ of 37 ~ C by a heating pad. We used an operating microscope and approached the basilar artery transclivally. 2,4," Hemostasis was meticulously obtained before the dura was opened. The arachnoid adjacent to the basilar artery was opened, and care was taken not to strip the arachnoid from the vessel. Isotonic saline at body temperature was used to cover the artery during the dissection. Sufficient time was allowed for all mechanically produced spasm to disappear. Then, through the operating microscope, photographs of the vessels were obtained at 25 magnification. Spasm was produced by the application of fresh arterial blood obtained from the experimental animal or from a normal, unanesthetized, untreated animal. After 3 minutes the blood was removed by gentle suction and photographs were taken. Changes in vessel size were measured from these photographs by projecting the slides and tracing the images. Disulfiram,.7'14 or the derivative of fusaric acid, 5(n-butyl-picolinamide), 15 both powerful DBH inhibitors, was injected intraperitoneally 1 to 18 hours prior to blood application. Because disulfiram and the fusaric acid derivative are poorly soluble in most physiological solvents, an emulsion of the drugs in saline was used. ore recently, the fusaric acid derivative has been found to be very soluble in dimethyl sulfoxide (DSO), *Disulfiram (tetraethylthiuram disulfide) in vivo becomes reduced to diethyl-dithiocarbamate which acts as the inhibitor of dopamine beta hydroxylase. TABLE 1 Effect of disulfiram on vasospasm of the basilar artery produced by application of blood Cat Dose Time Blood Applied No. Sex (mg/kg) (Hrs after DBH Spasm Injection) F F F %* o 7 F ~o* ~o* * Segmental spasm measured at point of maximal focal narrowing. and this has been used as the solvent. Ten cats received disulfiram in doses ranging from 100 to 500 mg/kg, 1 to 18 hrs before the application of blood to the basilar artery. Seven cats were given the fusaric acid derivative (90 to 120 mg/kg), 1 to 4 hrs prior to blood application. Activity of DBH in the serum was determined by the method used by Goldstein, et al. 8 Results Blood applied to the basilar artery of the cat produced spasm which narrows the lumen of the vessel approximately 40% to 50% for 20 to 30 minutes.' In 10 untreated control animals there was a mean decrease in the caliber of the basilar artery of 44% with a range of 36% to 55% after the application of blood. Of the 10 cats pretreated with disulfiram, six showed no spasm following the application of blood to the basilar artery. Three developed a small amount of segmental spasm and one showed marked spasm (Table 1). Blood pressure, pulse, and respiration, as well as blood gases, were stable prior to and after blood application. Because four of the 10 cats studied showed some spasm, another DBH inhibitor, the fusaric derivative 5 (n-butylpicolinamide) was used. In these animals, the serum prior to and after the administration of the drug was analyzed for DBH activity. Seven cats were studied in this manner, and the results are summarized in Table 2. In Cats 1 and 2, the absence of vasospasm was J. Neurosurg. / Volume 44 / February,

3 A. F. Battista, E. S. Flamm,. Goldstein and L. S. Freedman FIG. 1. Basilar artery of the cat, 12. Left: Before application of blood. Right: Four hours after 120 mg/kg of fusaric acid derivative given intraperitoneally, and 10 minutes after blood application. No spasm is seen. associated with a marked decrease in the serum DBH activity. Cats 3 and 4 showed a mild amount of spasm, and a marked decrease in serum DBH activity. In Cats 5, 6, and 7, the fusaric acid derivative was given in a solution with DSO as the solvent. All three cats showed a marked decrease in the serum DBH activity and showed any spasm following the application of blood to the basilar artery (Fig. 1). To exclude the possibility that the blood of the cats treated with either disulfiram or the fusaric acid derivative was incapable of producing spasm, blood from untreated cats was also used. No difference was found (Fig. 2). Control studies using DSO intraperitoneally without any DBH blocking agent failed to prevent spasm after blood application (Fig. 3). Discussion FIG. 2. Basilar artery of the cat, 12. Upper." Before application of blood. Center." Four hours after 100 mg/kg of fusaric acid derivative given intraperitoneally, and 10 minutes after blood application. No spasm is seen. Lower."five hours after administration of the fusaric acid derivative, and 10 minutes after blood application (blood from a nontreated normal cat). No spasm is seen. 170 It has been reported that the distribution of the DBH enzyme as determined by immunofluorescence technique corresponds well with the localization of norepinephrine as determined by the formaldehyde fluorescence method?,1~ The possible involvement of norepinephrine in cerebral vasospasm has been proposed. 6'18'~7 It is known that DBH is present in the nerve ending varicosities which contain norepinephrine. Upon stimulation, norepinephrine as well as DBH are liberated from the vesicles. T Since a high level of DBH activity indicates the possibility of a greater production of norepinephrine (NE), the response of the NE receptor in the vessel wall is probably related to the concentration gradient of the transmitter substance. The presence, therefore, of a high or low level of DBH activity may reflect the concentration of NE and thus be related to the receptor response shown by the amount of vessel con- J. Neurosurg. / Volume 44 / February, 1976

4 Inhibiting DBH in cerebral vasospasm in cats Fm 3. Basilar artery of the cat, 10. Left: Before blood application. Right: Two and a half hours after administration intraperitoneally of DSO (control for fusaric acid derivative solvent), and 10 minutes after blood application. DSO does not prevent the marked spasm produced by blood. TABLE 2 Effect of fi4saric acid opt vasospasm of the basilar artery produced by application of blood Serum DBH Activity* Cat Dose No. Sex (mg/kg) Prefusaric Postfusaric Spasm 1 hr 2 hr 4 hr t 0.43t ND ND t 3 F ND 1.15~ 38~ 4 5 F ND -- ND t t 15% t 0.36t * In units (1 unit= m~ol of ~4C product/ml serum/hr. ND-nondetectable. Time blood applied to basilar artery. striction on blood application. The present data suggest a relationship between the development of vasospasm in this experimental model and the serum level of DBH activity. However, more direct evidence will be required to clarify this hypothesis. Activity of DBH in the serum does not necessarily reflect the activity of the DBH enzyme in the vessel wall. This relationship could explain the results in the cats that developed spasm at a time when serum DBH activity was not detectable or low. Activity of DBH may be present in the vessel wall with no activity in the serum. When we determine the actual DBH activity level in the wall of the basilar artery, we will be able to detect any direct relationship between DBH activity and basilar artery vasospasm on blood application. Another important variable which we are also determining is the presence of any stored NE in the vessel wall and its relationship to basilar artery spasm on blood application. Stored NE would not be affected by a DBH inhibitor which blocks only the synthesis of new norepinephrine. By its release, norepinephrine stored in the vessel wall could play a part in vasospasm of the basilar artery after application of blood even if the serum and the vessel wall DBH activity levels were low or nondetectable. Such information which is currently under study will help to determine the importance of norepinephrine in the etiology of cerebral vasospasm, and whether clinical evaluation of DBH blocking agents for cerebral vasospasm in man is warranted. References 1. Cummins BH, Griffith HB: Intracarotid phenoxybenzamine for cerebral arterial spasm. Br ed J 1: , Echlin FA: Spasm of basilar and vertebral arteries caused by experimental subarachnoid hemorrhage. J Neurosurg 23:1-11, Falck B: Observations on the possibilities of the cellular localization of monoamines by a fluorescent method. Acta Physioi Scand [Suppl] 197:1-25, 1962 J. Neurosurg. / Volume 44 / February,

5 A. F. Battista, E. S. Flamm,. Goldstein and L. S. Freedman 4. Flamm ES, Ya~argil G, Ransohoff J: Alteration of experimental cerebral vasospasm by adrenergic blockage. J Neurosurg 37: , Flamm ES, Ya~argil G, Ransohoff J: Control of cerebral vasospasm by parenteral phenoxybenzamine. Stroke 3: , Fraser RAR, Stein B, Barrett RE, et al: Noradrenergic mediation of experimental cerebral vasospasm. Stroke 1: , Goldstein, Anagnoste B, Lauber E, et al: Inhibition of dopamine-/3-hydroxylase by disulfiram. Life Sei 3: , Goldstein, Freedman LS, Boonay : An assay for dopamine-fl-hydroxylase activity in tissues and serum. Experientia 27: , Hartman BK, Udenfriend S: Immunofluorescent localization of dopamine-/3- hydroxylase in tissues. oi Pharmaeol 6:35-94, Hartman BK, Zide D, Udenfriend S: The use of dopamine-/3-hydroxylase as a marker for the central noradrenergic nervous system in rat brain. Proe Natl Aead Sei USA 69: , Kapp J, ahaley S Jr, Odom GL: Cerebral arterial spasm, Parts 1-3. J Neurosurg 29: , Levin EY, Levenberg B, Kaufman S: The enzymatic conversion of 3,4 dihydroxyphenylethylamine to norepinephrine. J Bioi Chem 235: , Lieberman AN, Gardner AL, Goodgold AL, et al: Chronic vasospasm following subarachnoid hemorrhage: treatment with phenoxybenzamine. Presented at the combined meeting of the New England and New York Neurological Societies, ount Sinai Hospital, New York, Nov. 13, ussachio J, Kopin I J, Snyder S: Effects of disulfiram on tissue norepinephrine content and subcellular distribution of dopamine, tyramine, and their /3-hydroxylated metabolites. Life Sei 3: , Nagatsu T, Hidaka H, Kuzuya H, et al: Inhibition of dopamine-/3-hydroxylase by fusaric acid (5-butylpicolinic acid) in vitro and in vivo. Bioehem Pharmaeol 19:35-44, Nielsen KC, Owman C: Adrenergic innervation of pial arteries related to the circle of Willis in the cat. Brain Res 6: , Peerless S J, Ya~argil G: Cerebral micro- circulation: adrenergic innervation of the cerebral blood vessels in the rabbit. J Neurosurg 35: , Potter LT, Axelrod J: Properties of norepinephrine storage particles of the rat heart. J Pharmaeol Exp Ther 142: , Roffman, Freedman LS, Battista AF, et al: Dopamine-~3-hydroxylase activity in the vasculature. Fed Proe 32:707, 1973 (Abstract 2788) This paper was presented at the annual meeting of the American Association of Neurological Surgeons, St. Louis, issouri, April 21-25, This work was supported in part by USPHS Grant NA Address reprint requests to: Arthur F. Battista,.D., Department of Neurosurgery, New York University edical Center, 550 First Avenue, New York, New York J. Neurosurg. / Volume 44 / February, 1976