Effect of hypoxia on the contractile response to KC1,

Transcription

1 J Neurosurg 67: , 1987 Effect of hypoxia on the contractile response to KC1, prostaglandin F2., and hemoglobin TADAYOSHI NAKAGOMI, M.D., D.M.Sc., NEAL F. KASSELL, M.D., TOMIO SASAKI, M.D., D.M.Sc., SHIGERU FUJIWARA, M.D., D.M.Sc., R. MICHAEL LEHMAN, B.A., HIROO JOHSHITA, M.D., D.M.Sc., AND JAMES C. TORNER, PH.D. Department oj Neurological Surgery, University of Virginia School of Medicine, Charlottesville, Virginia ~," The purpose of this experiment was to evaluate the effect of hypoxia on the in vitro contractile responses of canine basilar artery to KCI, prostaglandin (PG) F2,~, and hemoglobin. Hypoxia was induced by changing the bubbling gas mixture in the chamber from 95% 02/5% CO2 to 95% N2/5% CO2. Hypoxia augmented the contractile response developed at 95% 02 to 25 mm and 50 mm KC1, 3 x 10-7 M and 10-5 M PGF2~, and 10-6 M hemoglobin. No significant alteration of the hypoxic augmentation in any preparation exposed to 25 mm KC1, M PGF2,, or 10-6 M hemoglobin was observed with guanethidine (10-5 M), prazosin (10-5 M), methysergide (10-5 M), or diphenhydramine (10-~ M). Endothelial denudation did not affect hypoxic augmentation. Hypoxia did not cause any alteration of the contractile response to 10-6 M PGF2, in Ca media. Pretreatment with a calcium channel blocker, nicardipine, significantly inhibited the hypoxic potentiation of the contractile response to 25 mm KC1, 3 x 10-7 M PGF2,, and 10-6 M hemoglobin. These results suggest that hypoxia augments the contractile response to these agonists by a direct action on the smooth-muscle cells, facilitating the transmembrane influx of extracellular calcium. Hypoxia of smoothmuscle cells in the major cerebral arteries might be involved in the pathogenesis of vasospasm. KEY WORDS 9 hypoxia 9 vasospasm 9 prostaglandin 9 calcium channel blocker 9 subarachnoid hemorrhage T HERE is a close association between the development of vasospasm and the amount of blood in the subarachnoid space.12,22,34,35 Due to the lack of vasa vasorum in cerebral arteries, 19,45 the smoothmuscle cells of arteries surrounded by thick subarachnoid clots may be rendered hypoxic as a result of the clogging of adventitial pores with fibrin and red blood cellsy Therefore, it is quite important to know the effect of hypoxia on the contractile response of cerebral arteries to vasoconstrictive agents, especially to possible spasmogenic substances. However, only a few studies have been done on hypoxia and the responsiveness of cerebral arteries, and the results are not consistent. Van Nueten, et al., 39 have demonstrated that hypoxia induced contractions in the presence of serotonin in canine basilar artery. On the other hand, Simeone and VinalP 3 have reported that hypoxia has a direct inhibitory effect on vasoconstriction in bovine middle cerebral arteries. The purposes of the present study were: 1 ) to evaluate the effect of hypoxia on the contractile responses to possible spasmogens, prostaglandin (PG) F2~, and hemoglobin, and to a non-physiological test activator, KC1; and 2) to investigate the mechanism of hypoxic alteration of the vasoactivity in canine basilar artery in vitro. Preparation of Arteries Materials and Methods Adult mongrel dogs of either sex, weighing 14 to 20 kg, were anesthetized with sodium pentobarbital (30 mg/kg) and sacrificed by exsanguination from the femoral artery. These procedures were performed with the approval of the University of Virginia Animal Research Committee, and all guidelines for animal care and handling were met. The brain with the basilar artery in situ was removed and placed in a dissecting chamber filled with a modified Krebs bicarbonate solution (in ram: NaC1 120; KC1 4.5; MgSO, 1.0; NaHCO3 27.0; J. Neurosurg. / Volume 6 7/October,

2 T. Nakagomi, et al. KH2PO4 1.0; CaC12 2.5; and dextrose 10.0). The basilar artery was dissected free under magnification and 4 mm-long ring segments were prepared. Each specimen was suspended between t_-shaped stainless steel rods (diameter 0.32 mm) in an organ bath with a 10-ml working volume, which was bubbled with 95% 02/5% CO2. The ph of the solution ranged from 7.40 to The preparations were allowed to equilibrate at 37~ for 60 minutes before use. Resting tension was adjusted to 1.0 gm, since it was confirmed in preliminary experiments that, under hypoxic conditions, consistent results could be obtained at this resting tension. Contractile force was recorded isometrically using a forcedisplacement transducer and was displayed on a polygraph.* In order to evaluate the contractile activity of each specimen, the contractile response to 40 mm KC1 was first determined on each ring segment. Only the specimens that showed good response to 40 mm KC1 were used for the hypoxia study. The contractile responses to 25 mm and 50 mm KC1, 3 x 10-7 M and 10-5 M PGF2,, and 10-6 M hemoglobin were then obtained. The different concentrations of both KC1 and PGF2, were used in order to study the hypoxic alteration of the contractile response to low and high concentrations of the agonists. When the contractile responses to these agonists became stable, hypoxia was induced by changing from a gas mixture containing 95% 02/5% CO2 to one containing 95% N2/5% CO2. The po2 in the organ bath was checked using a ph/blood gas analyzer.? Hypoxia was discontinued after 10 minutes by changing back to a gas mixture of 95 % 02/5 % CO2. In some experiments, hypoxia was discontinued following a shorter time interval, but only after the alteration in vasoactivity had reached its maximal point. Alterations of the vasoactivity during and after hypoxia were expressed as a percentage of the stable contraction induced by the agonists before induction of hypoxia. Initial results demonstrated that hypoxia augments contractions induced by 25 mm and 50 mm KC1, 3 x 10-7 M and 10-5 M PGF2,, or 10-6 M hemoglobin in canine basilar arteries. Consequently, three other experiments were performed in order to investigate the mechanism by which hypoxia augments contractions induced by these three agonists. The first study was designed to evaluate the action of norepinephrine released from adrenergic nerve endings and the participation of the specific receptors for agonists, serotonin, and histamine in hypoxic potentiation. Preparations were exposed to 10-5 M guanethidine, 10-5 M prazosin, 10-5 M methysergide, or 10-5 M diphenhydramine for * Force-displacement transducer, Model FT 0.03, manufactured by Grass Instrument Co., Quincy, Massachusetts; polygraph, Model 3418, manufactured by Soltec Corp., Sun Valley, California. t ph/blood gas analyzer, Model 158, manufactured by Coming Medical, Coming Glass Works, Medfield, Massachusetts mrnh 60O (~ D. 02 N '" " 50 " 0 t., C)... 9 : I ~ [ [ Time rnln FIG. 1. Time course of po2 change in the organ bath. At zero time, the gas was switched from 95% 02/5% CO2 to 95% NJ5% CO2. Five minutes after switching the gas mixture, po2 decreased from 620 to 10 mm Hg, then returned to the control level 2 minutes after reintroduction of oxygen. Results are expressed as means _+ standard error of the means. Each data point represents 10 measurements. 30 minutes before the addition of 25 mm KC1, 3 x 10-7 M PGF2,, or 10-6 M hemoglobin. In a second experiment, the possibility that the endothelium plays a role in the augmentation by hypoxia was tested. The endothelium was removed by the following procedure. The basilar artery was halved transversely and one-half was used as a control. The endothelium from the other half of the basilar artery was removed by the procedure described elsewhere. '3 The hypoxic alteration of the vasoactivity in an arterial ring without endothelium was compared to that of a ring with endothelium from the control half of the same basilar artery. After in vitro experiments, some of the rings, including those with and without removal of endothelium, were processed for scanning electron microscopy to investigate the presence of the endothelium. The third experiment was designed to evaluate the influence of extracellular calcium on hypoxic potentiation. After having confirmed that hypoxia augments contraction induced by 10-6 M PGF2,, the preparations were exposed to a Ca++-free medium containing 1 mm ethyleneglycol-bis (r N'-tetra-acetic acid (EGTA) for 15 minutes. Then 10-6 M PGF2~ was added, and hypoxia was induced when the preparations showed stable contraction. Ten minutes after the induction of hypoxia, 2.5 mm Ca ++ was added. Finally, the effect of nicardipine, a calcium channel blocking agent, on the hypoxic potentiation was investigated. Preparations were exposed to nicardipine (10-9 to 10-7 M) for 15 minutes before the addition of 25 mm KC1, M PGF2,, or 10-6 M hemoglobin. In this experiment, the effect of nicardipine on the contractile responses to these agonists was also obtained. Contractile activities of these agonists were expressed as a percentage of the contraction elicited by a standard dose of 40 mm KC J. Neurosurg. / Volume 67~October, 1987

3 Effect of hypoxia on vasoconstriction B Oz I N2 I Oz A 02 I N2 I 02 5min - - [ C 02 I N2 I mM KCI 105M PG Fa~ 104M Hb FIG. 2. Typical original recordings of arterial contraction demonstrating the hypoxic potentiation in contractile response to 25 mm KCI (A), 10-5 M PGF2,, (B), and 10-6 M hemoglobin (C). Hypoxia was induced when the contractile response to the agonist became stable. When the gas in the organ bath was returned to 95% oxygen, the arterial rings transiently relaxed and then returned to the initial contractile level. PG = prostaglandin; Hb = hemoglobin. Statistical Analysis The data were expressed as means _+ standard error of the means. Multiple comparisons of the alterations of the contractile responses to the agonists during and after hypoxia were evaluated by Schefft's test after analysis of variance. The contractile responses or hypoxic potentiation of the contractile responses between control and treatment preparations were compared for a specific antagonist concentration or for each concentration of nicardipine using a paired t-test. For graphic presentation, the control values were averaged together. Statistical comparisons between the hypoxic potentiation of the arterial rings with and without endothelium were made using Student's t-test. When p was smaller than 0.05, values were considered to be significantly different. Drugs Drugs used in this study were PGF2~, diphenhydramine hydrochloride, papaverine, EGTA, prazosin hydrochloride, methysergide maleate substance, guanethidine sulfate, and nicardipine.{ Hemoglobin solutions were prepared according to the method of Tanishima. 36 Concentration of hemoglobin was measured by the cyanomethemoglobin method? Prazosin was dissolved in a diluted ethanol solution resulting in a final concentration of ethanol in the 10-ml organ bath of 0.001%. Preliminary experiments demonstrated that, at the concentration used in the present study, this solvent did not affect the contractile responses to KCI, PGF2,, or hemoglobin. Nicardipine was dissolved in Krebs solution before use every day. All other drugs were dissolved in distilled water to make stock solutions and were then dissolved in Krebs solution before use. In 12 preliminary experiments, it was demonstrated that the 50% effective dose (EDs0) of the dose-response curve for PGF2~ was (7.1 _ 0.25) x 10-7 M. The mean absolute value of the contraction induced by 40 mm KC1 in the present experiments was 4.4 _ gm for 60 arterial rings. Nicardipine was kindly provided by Yamanouchi Pharmaceutical Co., Tokyo, Japan. Results Changing from the gas mixture containing 95% 02/ 5% CO2 to one containing 95% Nj5% CO2 produced a rapid decrease in the partial pressure of oxygen (po2) in the bath from 620 to 10 mm Hg without a significant change in ph (Fig. 1). After 10 minutes, control oxygenation was reintroduced. The po2 returned to the control level 2 minutes after reintroduction of oxygen. Effect of Hypoxia on Contractile Responses to KC1, PGF:~, and Hemoglobin The arterial rings were contracted with 25 mm and 50 mm KC1, 3 x 10-7 M and 10-5 M PGF2,, and 10-6 M hemoglobin in an organ bath. The initial contractile responses stabilized before induction of hypoxia to 25 and 50 mm KC1, 3 x 10-7 and 10-5 M PGF2,, and 10-6 M hemoglobin were 31.2% %, 107.7% %, 22.3% _+ 3.84%, 61.1% _+ 4.54%, and 19.2% %, respectively (as a percentage of the contraction elicited by the standard dose of 40 mm KC1, 12 arterial rings each). Hypoxia augmented the contractile response to each agonist (hypoxic potentiation) (Figs. 2 and 3). When the gas in the organ bath returned to 95% 02, the arterial tings that were preconstricted with KC1 and PGF2,~ transiently relaxed (post-hypoxic relaxation): the tension usually decreased rapidly below the initial control contractile response and then returned to the initial level (restabilization). This response was not seen in vessels contracted with hemoglobin. Hypoxic potentiation of the contractile responses to low concentrations of KC1 and PGFz~ was greater than to the higher concentrations of these agonists (Fig. 3). The arterial rings contracted with 10-6 M hemoglobin showed the greatest augmentation of the contractile response during hypoxia. Effect of Specific Antagonist on Hypoxic Potentiation The initial contractile responses to 25 mm KC1, 3 x 10-7 M PGF2~, or 10-6 M hemoglobin were not significantly different between each control and the treatment group with specific antagonists. Pretreatment with guanethidine, prazosin, methysergide, or diphenhydramine J. Neurosurg. / Volume 67~October,

4 T. Nakagomi, et al. s.o- Z 0 4~- hypoxlc H I potentiation post-hypoxlc relaxation T reatabillzatlon (I) Z u,i! uj ~> T I-<[..J ill 1.0-(s 25 mm KCl SO mm KCl 3x10"7 M PG-F2~ i(fs M PG'F2~ Hb FIG. 3. Summary of results showing the effect of hypoxia on the contraction induced by 25 and 50 mm KC1, 3 x 10-7 M and 10-5 M PG (prostaglandin)f2~, and 10-6 M hemoglobin (Hb). Hypoxia resulted in a further increase in tension (hypoxic potentiation). When the gas in the organ bath returned to 95% oxygen, the arterial rings relaxed (post-hypoxic relaxation) and the tension returned to the control level (restabilization). Hypoxic potentiation to low concentrations of KC1 and PGF2, was greater than that to the higher concentrations of these agonists (p < 0.01). Hypoxia caused marked potentiation in hemoglobin-induced contraction (p < 0.01 vs. 25 and 50 mm KC1, 10-5 PGF2,; and p < 0.05 vs. 3 x 10-7 M PGF2,). The changes in tension are expressed as a relative value to the stabilized contractile response before induction of hypoxia. Twelve arterial rings were used for each experiment. Results are expressed as means standard error of the means. did not affect the hypoxic p o t e n t i a t i o n of the contractile response to these agonists (Table 1). Effect o f Endothelium Removal on Hypoxic Potentiation The initial contractile responses to 25 m M KC1, 3 x 10 -v M PGF2,, or 10-6 M h e m o g l o b i n were not significantly different between the preparations with a n d without e n d o t h e l i u m. I n arterial rings without e n d o t h e l i u m, hypoxia a u g m e n t e d the contractile responses to these agonists. R e m o v a l of the e n d o t h e l i u m did not significantly affect the hypoxic p o t e n t i a t i o n of the contractile responses to these agonists (Fig. 4). Morphological studies performed with the scanning electron microscope d e m o n s t r a t e d that, after 5 to 6 hours in the organ bath, arterial rings m a i n t a i n e d intact endothelial cells over nearly all of the i n t i m a l surface. O n the other hand, after the l u m e n was rubbed with a polyethylene tube, a n arterial ring was almost completely devoid of endothelial cells ( 95% denuded) a n d there was n o notable injury to the elastic l a m i n a or smooth-muscle cells. Effect o f Ca Media or Nicardipine on Hypoxic Potentiation Hypoxia a u g m e n t e d the contractile response to 10-6 M PGF2~ (Fig. 5A). In a Ca++-free m e d i u m c o n t a i n i n g 1 m M E G T A, a slight contractile response was elicited TABLE 1 Effect of hypoxia on contractile responses to KCI, PGF2,, and hemoglobin before and after pretreatment with inhibitors* Antagonist guanethidine prazosin methysergide diphenhydramine KC1 (25 mm) PGF2~(3 x 10-7 M) Hemoglobin(10-6 M) Control With Antagonist Control With Antagonist Control With Antagonist 2.01 _ _ _ _ _ _ _ _ _ * Pretreatment with any antagonist did not affect hypoxic potentiation. Data are expressed as means + standard error of the means. Eight arterial rings were used for each experiment. PG = prostaglandin. 568 J. Neurosurg. / Volume 67 / October, 1987

5 Effect of hypoxia on vasoconstriction S'0-- [] with endothetium L T without endothellum / / / m I,'// Z / / / LM ~/~ [ _ 2.0- [,- I,r..I ~. 1, I// 25 mm 3x10-TM 10-6 M KCI PG-Fa~ H b FIG. 4. Effect of hypoxia on the arterial rings with or without endothelium. Hypoxia augmented the contractile response to 25 mm KCI, M PG (prostaglandin)f2o, and 10-6 M hemoglobin (Hb) in the arterial rings without endothelium. There were no significant differences in the hypoxic potentiation between the rings with endothelium and those without endothelium. The changes in tension are expressed as a relative value to the stabilized contractile response before induction of hypoxia. Eight arterial rings were used for each experiment. Results are expressed as means +_ standard error of the means. by 10-6 M PGF2~. However, no potentiation of the contractile response to this agonist was noticed during hypoxia in eight specimens (Fig. 5B). The addition of 2.5 mm CaC12 10 minutes after the induction of hypoxia produced a much stronger response. Initial contractile responses before induction of hypoxia to 25 mm KCI, 3 x 10-7 M PGF2~, or 10-6 M hemoglobin were inhibited by pretreatment with nicardipine in a dose-dependent manner (Figs. 6 and 7A), especially the responses to 25 mm KC1 which were almost completely abolished by 10-7 nicardipine. Pretreatment with nicardipine also inhibited hypoxic potentiation of the contractile response to KC1, PGF2,, or hemoglobin in a dose-dependent manner (Figs. 6 and A CONTROL (ldsm PG-F2~() 02 I N2 I 02 - I 5 mln B Ca+-+FREE (with lmm EGTA) O2 [ N2 2.5mM Ca'* 2g I0-6M PG-F2r...!_. ~_ t... i 2g 5 rain F Representative tracings of the effect of hypoxia on the contractile response to 10-6 M PG (prostaglandin)f2~ in normal (A) and in a Ca++-free (B) media. Hypoxia did not augment the contractile response in Ca++-free media. Adding 2.5 mm Ca ++ to the Ca+ media caused marked contraction under hypoxic conditions. EGTA = ethyleneglycol-bis (~-aminoethylether)-n, N'-tetra-acetic acid. 7B). Hypoxic potentiation to 25 mm KC1 was completely abolished by 10 -v M nicardipine, and that to 3 x 10-7 M PGF2~ was markedly inhibited. There was only a partial inhibitory effect of nicardipine on the hypoxic potentiation to 10-6 M hemoglobin. Discussion It is well documented that mechanical tension in some vascular smooth-muscle preparations is sensitive to organ bath oxygenation. Hypoxia usually depresses the reactivity of isolated arteries to vasoactive agents. 5,8J~ 27.32,33 Several studies, however, have demonstrated I A CONTROL (25rnM KCl) 02 I N2 I 02 loem NICARDIPINE O2 I N2 I 02 B CONTROL (3xldTM PG-F~) 1OZM NICARDIPINE o, I Nz t O, O2 I N2 I 02 KCI ~ 2g ~"F2I PG-F2~ S rain 5 rain 5 mln FIG. 6. Representative original tracings of the effect of hypoxia on the contractile response to 25 mm KCI (A), 3 x 10 -v M PGF2, (B), and 10-6 M hemoglobin (C) after pretreatment with a calcium channel blocker, nicardipine. Pretreatment with nicardipine abolished or decreased hypoxic potentiation of the contractile response to the agonists. PG = prostaglandin; Hb = hemoglobin. C CONTROL (ldtm Hb) 107M NICARDIPINE O, I N, f O, O, I N2 I O~ 5 mln 5 rain J. Neurosurg. / Volume 67 / October,

6 _ T. Nakagomi, et al. A A o 40-- I % Z ~- t.- u.i k- lo-..j ILl E r Z ILl I" 0 B S,O uj > I'- <C.,J LIJ r O 25 mm KCl xIO7 M PG-F2~ I /I i ct'** ld+~ ida +o-t CONTROL N I CARDI P I NE 14 O 2S mm KCI 9 311o-7 M PG-F~ D +o "s,, 14. i ~ " "''- I... S o I ][ I I I +o -o 1de lo -7 CONTROL NICARDI P I NE FIG. 7. Results of the effect of nicardipine pretreatment on the contractile responses to 25 mm KC1 (open circles), 3 x 10-7 M PGF2~ (closed circles), and 10-6 M hemoglobin (squares), and on the hypoxic potentiation to these agonists. The changes in tension are expressed as a relative value to the stabilized contractile response. Results are expressed as means _ standard error of the means. For graphic presentation, the control values were averaged together. The figures indicate the number of arterial rings examined. * = p < 0.05; ** = p < A: Pretreatment with nicardipine decreased the contractile response to the agonists in a dose-dependent manner. The contractile response to the agonists is expressed as a percentage of the contraction elicited by a standard dose of 40 mm KCI. B: Hypoxic potentiation to 25 mm KC1 was completely abolished by 10-7 M nicardipine and that to M PGF2, was markedly inhibited. There was a partial inhibitory effect of nicardipine on the hypoxic potentiation to 10-6 M hemoglobin. PG = prostaglandin; Hb = hemoglobin. that hypoxia can induce contraction in isolated arterial preparations. 6"28'29'39'4~ Different responses of vascular smooth muscles to hypoxia seem to be based on the differences in anatomical origin of the preparations 7,38 as well as on the experimental conditions, such as the vasoactive agents used or timing or duration of hypoxia imposed. The present experiments demonstrated that ss Hb hypoxia augments the contractile response to KC1, PGF2~, and hemoglobin in canine basilar artery. Our results are inconsistent with those obtained by Simeone and Vinall, 33 who demonstrated that, after 30 minutes incubation in the hypoxic medium, the contractile response to serotonin or whole blood was significantly depressed in bovine middle cerebral arteries during subsequent hypoxia. The discrepancy between these two studies is probably due to the difference in the preparations used or to the manner in which hypoxia was introduced. The experiments with different concentrations of KC1 and PGF2~ demonstrated that the hypoxic response to those agonists diminished at higher concentrations. De Mey and Vanhoutte 6 also reported that the relative amplitude of the hypoxic potentiation decreased as the normoxic responses to increasing concentrations of norepinephrine became larger. The most plausible explanation for these diminished responses is that the cellular mechanisms involved in the hypoxic potentiation may require a certain level of cell membrane polarization. 28,37 The augmentation of contractile responses during hypoxia is not due to the responses to adrenergic nerve stimulation, since guanethidine did not affect the relative magnitude of the contraction during hypoxia. Likewise, the hypoxic augmentation is not mediated by a]- adrenergic, serotonergic, or H~-histaminergic receptors, since pretreatment with prazosin (10-5 M), methysergide (10-5 M), or diphenhydramine (10-5 M) could not affect the hypoxic responses to the agonists. It has recently been reported that endothelial cells can respond to a variety of neurohumoral mediators by either inhibiting or activating the smooth-muscle cellsy 6'38 Moreover, Rubanyi and Vanhoutte 29 demonstrated that hypoxia causes the release of a diffusible vasoconstrictor substance from endothelial cells in canine coronary arteries. Therefore, the effect of hypoxia on arterial ring preparations with and without endothelium was investigated. Integrity of the endothelium was confirmed morphologically by the scanning electron microscope. Although hypoxic responses to M PGF2~ were a little depressed in the arterial tings without endothelium, the present experiments did not reveal any significant differences in the hypoxic responses to the agonists between the arterial tings with endothelium and those without endothelium. Therefore, it is concluded that hypoxia can augment contractions by a direct effect on the vascular smooth-muscle cells of the canine basilar arteries. Increase in intracellular free calcium results in contraction of the smooth muscle. There are two sources of calcium for the smooth-muscle contraction: transmembrane influx of extracellular calcium and release of intracellularly stored calcium. In the present experiments, hypoxia did not alter the contractile response to 10-6 M PGF2~ in the Ca+*-free media. The augmentation of the contractile response during hypoxia is considered to be dependent on the transmembrane influx 570 J. Neurosurg. / Volume 6 7 / October, 198 7

7 Effect of hypoxia on vasoconstriction of extracellular calcium. These results are reinforced by the fact that calcium channel blockers inhibited the potentiation of the contractile response during hypoxia both in the previous studies 2~176 and in the present study. It is well known that calcium enters the smoothmuscle cells through potential dependent (highly K + sensitive) channels and receptor-operated channels. In most vessels, calcium channel blockers effect a potent blockade of the potential dependent channels. 21 It has been reported that nicardipine possesses potent inhibitory action for the potential dependent channels in guinea pig 14 and canine basilar artery. 44 The present experiments revealed that 10-7 M nicardipine abolished the potentiation of the contractile responses to 25 mm KC1 during hypoxia and attenuated the initial contractile response. This action of nicardipine is considered to result from the complete inhibition of calcium entry through potential dependent channels. On the other hand, the inhibitory action of nicardipine on the hypoxic potentiation of the contractile response to 3 x 10-7 M PGF2, was not complete at a concentration of 10-7 M and was only partially effective on the hypoxic response to 10-6 M hemoglobin. These results suggest that hypoxia facilitates the calcium entry through the receptor-operated channels as well as through the potential dependent channels. The underlying mechanism for calcium entry into the smooth-muscle cells during hypoxia seems to be the property change of the cell membrane. However, the exact mechanism for the calcium entry during hypoxia is still unclear and requires further investigation. Hypoxia is of special interest with regard to the pathogenesis of cerebral vasospasm. Due to the lack of vasa vasorum,19,45 cerebral arteries surrounded by thick subarachnoid clots may be hypoxic as a result of the clogging of adventitial pores with fibrin and red blood cells. 23 Intimal thickening of cerebral arteries following subarachnoid hemorrhage 6'32 can also disturb oxygen supply to the smooth-muscle cells. The vasoactive substances released into the cerebrospinal fluid cause constriction of cerebral arteries ,3~ Hypoxia stimulates further constriction of cerebral arteries as shown in the present experiments. Together with impairment of vasodilatory activity of cerebral arteries following subarachnoid bleeding,13,25,31,43 these processes may contribute to the prolonged vasoconstriction. It is noteworthy that hypoxia can markedly augment contractile responses to the vasoactive agents even when the initial contractile responses to the agonists are moderate. Prevention of cerebral vasospasm after subarachnoid hemorrhage with calcium channel blockers is of contemporary interest. 1'11'17"18'2441,42 The present experiment revealed that hypoxic augmentation of the contractile response depends on the transmembrane influx of extracellular calcium. Therefore, prophylactic use of a calcium antagonist may be effective in ameliorating vasospasm, although the inhibitory effect is only partial, especially in hemoglobin-induced contraction during hypoxia. Acknowledgments The authors thank Dennis Vollmer, M.D., for reviewing the manuscript, Sarah Hudson and Helen Tuan for technical assistance, and Lucille Staiger for manuscript preparation. References 1. Allen GS, Bahr AL: Cerebral arterial spasm: Part 10. Reversal of acute and chronic spasm in dogs with orally administered nifedipine. Neurosurgery 4:43-47, Allen GS, Gross C J, French LA, et al: Cerebral arterial spasm. Part 5: In vitro contractile activity of vasoactive agents including human CSF on human basilar and anterior cerebral arteries. J Neurosurg 44: , Boullin DJ, Mohan J, Grahame-Smith DG: Evidence for the presence of a vasoactive substance (possibly involved in the aetiology of cerebral arterial spasm) in cerebrospinal fluid from patients with subarachnoid haemorrhage. J Neurol Neurosurg Psychiatry 39: , Brandt L, Ljunggren B, Anderson KE, et al: Vasoconstrictire effects of human post-hemorrhagic cerebrospinal fluid on cat pial arterioles in situ. J Neurosurg 54: , Busse R, F6rstermann U, Matsuda H, et al: The role of prostaglandins in the endothelium-mediated vasodilatory response to hypoxia. Ptlugers Arch 401:77-83, De Mey JG, Vanhoutte PM: Anoxia and endotheliumdependent reactivity of the canine femoral artery. J Physiol (Lond) 335:65-74, De Mey JG, Vanhoutte PM: Heterogeneous behavior of the canine arterial and venous wall. Importance of the endothelium. Circ Res 51: , Detar R, Bohr DF: Oxygen and vascular smooth muscle contraction. Am J Physiol 214: , Drabkin DL, Austin JH: Spectrophotometric studies. II. Preparations from washed blood cells; nitric oxide hemoglobin and sulfhemoglobin. J Biol Chem 112:51-65, Ebeigbe AB: Influence of hypoxia on contractility and calcium uptake in rabbit aorta. Experientia 38: , Espinosa F, Weir B, Overton T, et al: A randomized placebo-controlled double-blind trial of nimodipine after SAH in monkeys. Part 1: Clinical and radiological findings. J Neurosnrg 60: , Fisher CM, Kistler JP, David JM: Relation of cerebral vasospasm to subarachnoid hemorrhage visualized by computerized tomographic scanning. Nenrnsurgery 6: 1-9, Fujiwara S, Kassell NF, Sasaki T, et al: Selective hemoglobin inhibition of endothelium-dependent vasodilation of rabbit basilar artery. J Neurosurg 64: , Fujiwara S, Kuriyama H: Nicardipine actions on smooth muscle cells and neuromuscular transmission in the guinea-pig basilar artery. J Pharmacol Exp Ther 225: , Furchgott RF: Metabolic factors that influence contractility of vascular smooth muscle. Bull NY Acad Med 42: , Furchgott RF, Zawadzki JV: The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: , Gioia AE, White RP, Bakhtian B, et al: Evaluation of the efficacy of intrathecal nimodipine in canine models of chronic cerebral vasospasm. J Neurosurg 62: , Grotenhuis JA, Bettag W, Fiebach BJO, et al: Intracarotid slow bolus injection of nimodipine during angiography J. Neurosurg. / Volume 67~October,

8 T. Nakagomi, et al. for treatment of cerebral vasospasm after SAH. A preliminary report. J Neurosurg 61: , Hughes JT, Schianchi PM: Cerebral artery spasm. A histological study at necropsy of the blood vessels in cases of subarachnoid hemorrhage. J Neurosurg 48: , Karmazyn M, Beamish RE, Dhalla NS: Involvement of calcium in coronary vasoconstriction due to prolonged hypoxia. Am Heart J 107: , Kazda S: Pharmacology of nimodipine, a calcium antagonist with preferential cerebrovascular activity. Neuroehirurgia (Stottg) 28:70-73, Kistler JP, Crowell RM, Davis KR, et al: The relation of cerebral vasospasm to the extent and location of subarachnoid blood visualized by CT scan: a prospective study. Neurology 33: , Liszczak TM, Varsos VG, Black PMcL, et al: Cerebral arterial constriction after experimental subarachnoid hemorrhage is associated with blood components within the arterial wall. J Neurosurg 58:18-26, Ljunggren B, Brandt L, S~iveland H, et al: Outcome in 60 consecutive patients treated with early aneurysm operation and intravenous nimodipine. J Neurosurg 61: , Maeda Y, Tani E, Miyamoto T: Prostaglandin metabolism in experimental cerebral vasospasm. J Neurosurg 55: , Okwuasaba FK, Weir BKA, Cook DA, et al: Effects of various intracranial fluids on smooth muscle. Neurosurgery 9: , Roberts AM, Messina E J, Kaley G: Prostacyclin (PGI2) mediates hypoxic relaxation of bovine coronary arterial strips. Prostaglandins 21: , Rubanyi GM, Paul RJ: Two distinct effects of oxygen on vascular tone in isolated porcine coronary arteries. Circ Res 56:1-10, Rubanyi GM, Vanhoutte PM: Hypoxia releases a vasoconstrictor substance from the canine vascular endothelium. J Physiol (Lond) 364:45-56, Sasaki T, Asano T, Takakura K, et al: Nature of the vasoactive substance in CSF from patients with subarachnoid hemorrhage. J Neurosurg 60: , Sasaki T, Murota S, Wakai S, et al: Evaluation of prostaglandin biosynthetic activity in canine basilar artery following subarachnoid injection of blood. J Neurosurg 55: , Shibata S, Briggs AJ: Mechanical activity of vascular smooth muscle under anoxia. Am J Physiol 212: , Simeone FA, Vinall PE: Effect of oxygen and glucose deprivation on vasoactivity and calcium movement in isolated middle cerebral arteries, in Wilkins RH (ed): Cerebral Arterial Spasm. Baltimore: Williams & Wilkins, 1980, pp Suzuki J, Komatsu S, Sato T, et al: Correlation between CT findings and subsequent development of cerebral infarction due to vasospasm in subarachnoid hemorrhage. Aeta Neurochir 55:63-70, Symon L, Bell BA, Kendall B: Relationship between effused blood and clinical course and prognosis in aneurysmal subarachnoid hemorrhage: a preliminary computerized tomograpby scan study, in Wilkins RH (ed): Cerebral Arterial Spasm. Baltimore: Williams & Wilkins, 1980, pp Tanishima T: Cerebral vasospasm: contractile activity of hemoglobin in isolated canine basilar arteries. J Neurosurg 53: , Vanhoutte PM: Effects of anoxia and glucose depletion on isolated veins of the dog. Am J Physiol 230: , Vanhoutte PM, Miller VM: Heterogeneity of endothelium-dependent responses in mammalian blood vessels. J Cardiovase Pharmacol 7 (Suppl 3):S12-$23, Van Nueten JM, De Ridder W, Van Beek J: Hypoxia and spasms in the cerebral vasculature. J Cereb Blood Flow Metab 2 (Sop# 1):$29-$31, Van Nueten JM, Vanhoutte PM: Effect of the Ca 2+ antagonist lidoflazine on normoxic and anoxic contractions of canine coronary arterial smooth muscle. Eur J Pharmacol 64: , Varsos VG, Liszczak TM, Han DH, et al: Delayed cerebral vasospasm is not reversible by aminophylline, nifedipine, or papaverine in a "two-hemorrhage" canine model. J Neurosurg 58:11-17, Voldby B, Petersen OF, Buhl M, et al: Reversal of cerebral arterial spasm by intrathecal administration of a calcium antagonist (nimodipine). Acta Neurochir 70: , Walker V, Pickard JD, Smythe P, et al: Effects of subarachnoid haemorrhage on intracranial prostaglandins. J Neuroi Neurosurg Psyehiatry 46: , Yamamoto M, Ohta T, Toda N: Mechanisms of relaxant action of nicardipine, a new Ca++-antagonist, on isolated dog cerebral and mesenteric arteries. Stroke 14: , Zervas NT, Liszczak TM, Mayberg MR, et al: Cerebrospinal fluid may nourish cerebral vessels through pathways in the adventitia that may be analogous to systemic vasa vasorum. J Neurosurg 56: , 1982 Manuscript received July 18, Accepted in final form March 10, Address reprint requests to." Tadayoshi Nakagomi, M.D., Department of Neurosurgery, University of Tokyo Hospital, Tokyo 113, Japan. 572 J. Neurosurg. / Volume 67~October, 1987