Oxyhemoglobin enhancement of vasopressin-induced constriction in rat cerebral arterioles

Life Sciences, Vol. 56, No. 5 pp. PL 123-127, 1995 Copyright o 1994 Elsevier Science Ltd Printed in the USA. All rights reserved

Pergamon

0024-32o5/95$95o+ .00

0024-3205(94)00460-9 PHARMACOLOGY LETTERS Accelerated Communication

O X Y H E M O G L O B I N E N H A N C E M E N T OF VASOPRESSIN-INDUCED CONSTRICTION IN R A T C E R E B R A L A R T E R I O L E S Masakazu Takayasu a, Yasukazu Kajita a, Yoshio Suzuki a, Yoshimasa Mori a, Masato Shibuya a, Kenichiro Sugita a and Hiroyoshi Hidaka b Department of Neurosurgery a and Pharmacology b, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, 466, Nagoya, Japan (Submitted September 16, 1994; accepted October 6, 1994; received in final form Novmeber 14, 1994) Abstract: This study was undertaken to determine the effects of oxyhemogloblin on vasopressininduced responses in cerebral arterioles. Rat intracerebral arterioles about 60 pm in diameter were isolated and cannulated using pipettes. Changes in diameter secondary to the extraluminal application of drugs were monitored through a video micro-scaler. Vasopressin produced a triphasic, dose-dependent response consisting of vasodilation (10-11 M), vasoconstriction (10-910-8 M) and a decrease in vasoconstriction (10-7-10-6 M). Pretreatment with oxyhemoglobin (10-5 M) abolished the vasodilation induced by the lower dose vasopressin and doubled the vasoconstriction induced by the higher dose. The combination of L-arginine (10-4 M) and superoxide dismutase (600 U) restored low-dose vasopressin vasodilation and suppressed highdose vasoconstriction in oxyhemoglobin-pretreated arterioles, while they showed little effect when used singly. This study indicates that oxyhemoglobin enhances vasopressin-induced constriction of intracerebral arterioles and these effects can be inhibited by the combination of L-arginine and superoxide dismutase. Key Words: cerebral microcirculation,L-ar#nlne, oxyhemoglobin,supero~idedi~mutase, vasopressin

Introduction Oxyhemoglobin (OxyHb) is thought to be one of the principle pathogenetic agents causing cerebral vasospasm following subarachnoid hemorrhage (1). This substance has a variety of mechanisms by which it may cause this occurrence (2). These include the release of free radicals, vasoactive eicosanoids and endothelin, the inhibition of endothelium-dependent relaxation, the induction of structural damage in the arterial wall, etc (3-6). The direct vasoconstrictive action of OxyHb is not potent, but it may enhance vasoconstriction by modifying the action of other vasoactive substances. Vasopressin, a circulating hormone and neuropeptide, may be one such endogenous vasoactive substance. We have shown in rat intracerebral arterioles that increasing concentrations of vasopressin induced a triphasic response: vasodilation, vasoconstriction and a decrease in vasoconstriction (7). It has also been shown that the vasodilation elicited by the lower doses of vasopressin is mediated through endothelium-dependent relaxation (nitric oxide; NO), while the vasoconstriction elicited by the higher doses of vasopressin is produced by direct action on smooth muscle cells. The purpose of this study was to investigate the effects of oxyhemoglobin on the vasopressin-induced vasomotor responses in intracerebral arterioles, and subsequently to determine whether Dretreatment with a orecursor of NO. L-areinine. and a orotector of NO inactivation. Correspondence; Masakazu Takayasu, MD, Deaprtment of Neurosurgery, Nagoya University School of Medicine, 65 Tsurumai, showa, 466, Nagoyam Japan. Tel 81-52-741-2111, FAX 8152-731-0638

PL-124

OxyHb & Vasopressin-indueed Vasoconstriction

Vol. 56, No. 5, 1995

superoxide dismutase, could modify these effects. An in vitro study was undertaken using isolated, cannulated rat intracerebral arterioles (8-10). Methods Pret~aration of arterioles: Animal experimentation conformed with the American Physiological Society's "Guiding Principles in the Care and Use of Animals". Intracerebral arterioles were isolated and cannulated in an organ bath (described below). The changes in vessel diameter in response to the extraluminal application of agents were measured as previously reported in detail (8-10). Briefly, arterioles were surgically isolated at 4°C from the brains of pentobarbital-anesthetized male SpragueDawley rats weighing 300 to 400 gms. Cerebral parenchymal arterioles, 50 to 80 Ixm in diameter, were obtained from the first (M-l) portion of the middle cerebral arteries. The vessels were transferred to a temperature-controlled chamber (25 °C) on the stage of an Olympus inverted microscope and were cannulated with glass pipettes. The inner diameter of the vessel was determined using a video micro-scaler (FOR.A Model IV-550, Tokyo, Japan). The transmural pressure was set and maintained throughout the experimental protocol at 60 mm Hg via the cannulating pipette. The bath temperature was raised to 37.5 °C and the vessels were allowed to equilibrate for 45 minutes in an extraluminal bath with a pH of 7.3. During the 30 minute equilibration period, the vessels developed tone and contracted to approximately 70% of their maximum passive diameter. Vessel responsiveness was then assessed by changing the extraluminal pH from 7.3 to 6.8 or to 7.6. The physiological salt solution (PSS) used in the intraluminal preparation was a modified Ringer's, composed of: 144 mM NaCI, 3.0 mM KCI, 2.5 mM CaCI2, 1.4 mM MgSO4, 5.0 mM glucose, 2.0 mM pyruvate, 0.02 mM ethylenediaminetetraacetic acid (EDTA), 2.0 mM 3-[N-morpholino] propanesulfonic acid (MOPS), 1.21 mM NaH2PO4, and 0.91.0 g/100 ml bovine serum albumin. The extraluminal solution was PSS with no albumin. Superoxide dismutase (SOD) was obtained from Asahi Chemical Industry (Tokyo, Japan); Synthetic vasopressin (Arg-vasopressin: AVP) was obtained from Peptide Institute Inc. (Osaka, Japan). Oxyhemoglobin was prepared from purified crystalline human hemoglobin (Sigma Chemical Co., St. Louis, MO) according to the method of Martin et al (4). All the other chemicals were of reagent grade. The agents were dissolved in PSS and applied to the extraluminal side_of the arterioles after a pH adjustment to 7.3. Protocol: The experiments were divided into two parts. In the first part, increasing concentrations (10-13 - 10-6 M) of AVP were sequentially applied to six arterioles pretreated with OxyHb (10-5 M) to determine the dose-response curve. This was compared with the responses obtained from six control arterioles which had no pretreatment. In the second part, the effects of L-arginine (10-4 M, n = 5) and SOD (600 U, n = 5) on the vasopressin dose-response curves were examined in OxyHbpretreated arterioles. L-arginine and SOD were used singly or in combination and were applied to the arterioles simulatneously with OxyHb (n ---5 in each group). Statistical analvsis: Vessel diameters at each agonist dose in the dose-response sequence were expressed as a percent of the control vessel diameter. The magnitude of vasoconstriction and vasodilation was expressed as the percent change in diameter from the control vessel diameter. These data were reported as mean + S.E.M.. The significance of differences among a set of three or more samples was evaluated by one-way analysis of variance (ANOVA) with the Fisher Protected Least Significant Difference (PLSD) multiple range test as post-ANOVA test. The significance of differences in vessel diameter at a given dose of AVP between control and oxyhemoglobinpretreated arterioles was evaluated by an unpaired t-test. We considered differences significant at p < 0.05. Baseline characteristics of the arterioles: The arterioles used in each series of experiments had similar baseline characteristics (the control vessel diameter and their response to pH 6.8 and 7.6 before the application of drugs) without statistical differences (ANOVA, p=0.09 to 0.47). Therefore, any differences in response to vasopressin in each group of arterioles can attribute to differences in effects of agents.

Vol. 56, No. 5, 1995

OxyHb & Vasopressin-inducedVasoconstriction

PL-125

Results As shown in a previous study (7), vasopressin produced the triphasic response of vasodilation (10-12 -10-11 M), vasoconstriction (10-9 - 10-8 M) and a decrease in vasoconstriction (10-7 - 10- 6 M) in six control intracerebral arterioles (Fig. 1A). Change in mean arteriolar diameter was statistically significant at AVP doses of 10-11M (increase in diameter) and 10-9-10-7M (decrease in diameter). Following pretreatment with oxyhemoglobin (10-5 M), the vessel showed a small constriction of 5.9 :t: 2.5 % (p < 0.05). Then, increasing concentrations of AVP was applied to the vessels, which showed a biphasic response of increasing vasoconstriction (10-13 -10-8 M) and a decrease in vasoconstriction (10-7 - 10-OM) (Fig. 1A). The vessel diameters of the control and the oxyhemoglobin-pretreated arterioles were significantly different at each AVP dose except for those at a dose of 10-6 M (Fig. 1A). In the presence of oxyhemoglobin, even physiological concentrations of AVP (10-11 M) produced a vasoconstriction of 11.4 + 3.8 %, in contrast to a vasodilation observed in the control arterioles (11.6 + 1.7 % dilation). The maximal contraction induced by AVP application after pretreatment with oxyhemoglobin was about double that produced without pretreatment ( 61.6 + 3.9 % vs. 32.4 + 7.4 %).

A 120

. I

t

,OOl

B I

o--

120

. 0 o° . 0 . 100 p

g

E

80

80

60

~

60

40

E

40

ZO

OxyHb . . . . -12 -10 -8 Vasopressin Dose (log M)

¢w

I

-6

20 -14

OxyHb "~,. - / +L-arg "v t +SOD +L-arg+SOD

• I

I

I

!

-1Z -10 -8 -6 Vasopressin Dose (log M)

Fig. 1A & 1B Dose-response curve to vasopressin (AVP) in control and oxyhemoglobinpretreated intracerebral arterioles from rats (n=6 in each group) (A). Vasopressin dose-response curves in pretreated rat intracerebral arterioles (n=5 in each group) (B). Each point represents the percentage of change from control vessel diameter immediately before application of AVP (mean + S.E). * Significantly difference in the mean diameter between the control and oxyhemoglobin-pretreated arterioles at each AVP dose (p < 0.05). Control: control arterioles without any pretreatment; OxyHb: arterioles pretreated with oxyhemoglobin; +L-arg: arterioles treated with Larginine after pretreatment with oxyhemoglobin; +SOD: arterioles treated with superoxide dismutase after pretreatment with oxyhemoglobin; +L-arg+SOD: arterioles treated with combination of L-arginine and superoxide dismutase after pretreatment with oxyhemoglobin. :The effects of L-arginine (10-4 M) and SOD (600 U) on the vasopressin dose-response curves were then examined in OxyHb-pretreated arterioles (Fig. 1B). Simultaneous pretreatment of L-arginine or SOD with oxybemoglobin showed a small, but significant vasoconstriction of 5.6 + 1.9 % and 5.6 + 2.3 %, respectively. Both L-arginine and SOD showed only a small effect on oxyhemoglobin enhancement of AVP-induced contraction when they were used singly. The maximum vasoconstriction produced at 10-8 M AVP was slightly less with each drug (L-arginine: 40.4 + 6.7 %, SOD: 40.3 + 7.4 %) than that in the oxyhemoglobin-pretreated control vessels (Table 1). The vasodilation produced by low-dose AVP in control arterioles did not appear after a single

PL-126

OxyHb & Vasopressin-induced Vasoconstriction

Vol. 56, No. 5, 1995

treatment with these agents; instead, 10-11 M AVP still produced vasoconstriction of 11.6 + 7,4 % (L-arginine) and 8.9 + 4.2 % (SOD). These vessel diameters were significantly different from those seen in control arterioles (Table 1). On the other hand, the combination of L-arginine and SOD produced much greater effects. The simultaneous pretreatment of the combination of L-arginine and SOD with oxyhemoglobin did not produce significant constriction of the arterioles (1.5 + 0.9 % constriction), while single pretreatment of these agents with oxyhemoglobin showed significant vasoconstriction. The effect on oxyhemoglobin enhancement of AVP-induced constriction was also greater in the combination therapy. The vasodilation produced by 10-11 M AVP reappeared (7.5 + 1.4 %) and the vasoconstriction at 10-8 M was reduced (25.2 + 3.4 %). The dose-response curve was almost identical to that seen in control vessels (Fig. 1B). There was no significant difference in vessel diameters at any dose of AVP between control arterioles and arterioles receiving combination treatment (Table 1).

TABLE I-Differences o f Arteriolar Diameters in Pretreated Arterioles AVP dose (M) 10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 I

Difference from control Arg + + + SOD + + + Arg+SOD II Difference from OxyHb-pretreated control Arg SOD Arg+SOD + + +

+ -

-

+

+ + +

+

+

4-

Statistical significance of response to each dose of AVP in variously pretreated arterioles I: from control arterioles and II: from Oxy-Hb pretreated arterioles. AVP, arginine vasopressin; Arg, arginine; SOD, superoxide dismutase; OxyHb, oxyhemoglobin; +, significantly different (ANOVA, P<0.05); -, not significantly different (ANOVA, P>0.05).

Discussion This study demonstrated that oxyhemoglobin could significantly enhance vasopressininduced vasoconstriction of intracerebral arterioles in the cerebral microcirculation, and that these effects could be inhibited by the combined application of L-arginine and superoxide dismutase. The mechanism by which OxyHb abolishes the vasodilation produced by low doses of vasopressin and enhances the vasoconstriction at higher doses is probably due to inhibition of the vasodilatory effects of nitric oxide (NO), as a number of in vitro and in vivo studies have demonstrated that OxyHb is a potent inhibitor of nitric oxide, i.e., endothelium-derived relaxing factor (EDRF) (11-13). This notion is also supported by our previous study which demonstrated similar effects on the vasopressin-induced response following pretreatment with NG-monomethyl-Larginine (L-NMMA), a specific inhibitor of NO (7). The inhibitory effects of oxyhemoglobin on nitric oxide may act through two different mechanisms: a decrease in nitric oxide synthesis following damage to the arterial endothelium, and inactivation of nitric oxide either following high-affinity binding or through the generation of oxygen free radicals (6,14-16). The direct vasoconstrictor effect of OxyHb may be small (maximum vasocontraction of 5.9 + 2.5 % in the present study), but it may potentiate vasoconstriction by modifying the action of other endogenous vasoactive substances, such as vasopressin, abolishing the endothelium-derived relaxation so enhancing vasoconstriction.

Vol. 56, No. 5, 1995

OxyHb & Vasopressin-indueedV~nstrietion

PL-127

The combination of L-arginine and SOD produced significant effects, restoring vasodilation by low-dose AVP and suppressing vasoconstriction by high-dose AVP. When used singly, Larginine (10-4 M) and SOD (600 U) produced little effect on OxyHb enhancement of (,asopressininduced vasoconstriction. L-arginine and SOD are thought to restore NO function by countering the two different actions of oxyhemoglobin. Although L-arginine lacks vasomotor activity, it can activate the synthesis of nitric oxide by supplying enough substrate for its formation (17). SOD, a scavenger of free radicals, protects NO from inactivation and increases its half-life (18). When individually applied, neither L-arginine nor SOD is effective in restoring nitric oxide function fully. Macdonald et al were not able to demonstrate the effectiveness of intrathecal SOD and catalase, a free radical scavenging enzyme, on oxyhemoglobin-induced vasospasm in monkeys (13). The combination of L-arginine and SOD may result in sufficient recovery of nitric oxide function. In the present study, 10-5 M oxyhemoglobin pretreatment produced a small, constriction (5.9 + 2.5 %) in arterioles. The combination pretreatment of L-arginine and SOD with oxyhemoglobin inhibited this constriction, while single pretreatment of L-arginine or SOD did not (data are not shown). Such enhanced effects of combination therapy on spastic basilar arteries were also shown in our previous study in the dog subarachnoid hemorrhage model (19). Thus, this combination therapy may have therapeutic implications in the treatment of cerebral vasospasm after subarachnoid hemorrhage, by restoring nitric oxide function in both the cerebral microcirculation and the cerebral macrocirculation. In conclusion, we have shown that oxyhemoglobin modifies the effects of endogenous vasoactive substances such as vasopressin by enhancing vasopressin-induced vasoconstriction of intracerebral arterioles in the cerebral microcirculation. This enhanced arteriolar contraction may reduce cerebral microcirculation and worsen cerebral ischemia. These unfavorable effects can be inhibited by a combination of L-arginine and superoxide dismutase, suggesting a therapeutic strategy for managing cerebral vasospasm after subarachnoid hemorrhage. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

B.K.A.WEIR, Aneurvsms Affectin~ the Nervous System, 518-539,Williams & Wilkins Co, Baltimore, USA (198~/). R.L.MACDONALD and B.K.A.WEIR, Stroke 22 971-982 (1991). T.ASANO, T.TANISHIMA, T.SASAKI and K.SANO, Cerebral Arterial Spasm, R.H.Wilkins (Ed), 190-201, Williams &Wilkins Co., Baltimore, USA (1980). W.MARTIN, G.M.VILLANI, D.JOTHIANANDAN and R.F.FURCHGOTT, J. Pharmacol. Exp.Ther. 232 708-716 (1985). E.H.OHLSTEIN and B.L.STORER, J. Neurosurg. 77 274-278 (1992). H.OKADA, S.ENDO, K.KAMIYAMA and J.SUZUKI, Neurol. Med. Chir. (Tokyo) 20 573582 (1980). M.TAKAYASU, Y.KAJ1TA, Y.SUZUKI, M.SHIBUYA, K.SUGITA, T.ISHIKAWA and H.HIDAKA, J. Cereb. Blood Flow Metab. 13 303-3091993. R.G.Jr.DACEY and B.R.DULING, Am. J. Physiol. 243 H592-H606 (1982). B.R.DULING, R.W.GORE, R.G.Jr.DACEY and D.N.DAMON, Am. J. Physiol. 241 H108Hl16 (1981). M.TAKAYASU and R.G.Jr.DACEY, Stroke 20 778-782 (1989). F.COSENTINO, J.C.SILL and Z.S.KATUSIC, Am. J. Physiol. 264 H413-H418 (1993). Z.S.KATUSIC, Am. J. Physiol. 262 H1557-H1562 (1992). R.L.MACDONALD, B,K.A. WEIR, T.D.RUNZER, M.G.A.GRACE and M.J.POZNANSKY, Neurosurgery 30 529-539 (1992). J.M.FINDLAY, B.K.A.WEIR, K.KANAMARU, F.ESPINOSA, Neurosurgery 25 736-746 (1989). Q.H.GIBSON and F.J.W.ROUGHTON, J. Physiol. 1~6 507-526 (1957). J.A.STEELE, N.STOCKBRIDGE, G.MALJKOVIC, B.WEIR, Cir Res. 68 416-423 (1991). F.M.FARACI, Am. J. Physiol. 259 H1216-H1221 (1990). R.M.J.PALMER, A.G.FERRIGE and S.MONCADA, Nature ~27 524-526 (1987). Y.KAJ1TA, Y.SUZUKI, H.OYAMA, T.TANAZAWA, M.TAKAYASU, M.SHIBUYA and K.SUGITA, J. Neurosurg. 80 476-483 (1984).