Endothelin-1 modulates cardiorespiratory control by the central nervous system

Neuro~hem lnt Vol 18, No 4, pp 51%524, 1991 Printed m Great Britain All rights reserved

0197-0186/91 $3 00+0 00 Copyright (~ 1991 Pergamon Press plc

ENDOTHELIN-1 MODULATES CARDIORESPIRATORY C O N T R O L BY THE C E N T R A L N E R V O U S SYSTEM TOMOYUKI KUWAKI, I NAOHIRO KOSHIYA,2 NAOHITO TERUI 2 a n d MAMORU KUMADAI* tDepartment of Physiology, Faculty of Medicine, The University of Tokyo, Tokyo 113, Japan 2Institute of Basic Medical Sciences, The University of Tsukuba, Tsukuba 305, Japan (Recewed 31 May 1990, accepted 23 August 1990)

urethane-anesthetlzed, vagotomlzed and immoblhzed rats under artificial ventilation, an mtraclsternal rejection of 0 1 pmol of endothehn-I resulted m Immediate increases, lasting for 3-15 mm, m arterial pressure, heart rate and renal sympathetic nerve activity Phrenlc nerve actlvlty and the rate of its burst actwlty (burst rate) also increased initially but subsequently decreased for 5 ~ 0 mm At doses of 1 or I0 pmol, the initial increases (phase I) were followed by a period of decreases in all variables, that lasted for 2(~80 mm, below the pre-lnjection level (phase II) Phrenlc nerve actwlty often disappeared completely All the variables usually returned to, or often exceeded, pre-inlectlon levels (phase III). However, arterial pressure sometimes remained below control for at least 2 h Topical application of endothehn-1 to the ventral surface of the medulla produced the same pattern of changes as with intracisternal injection This pamcular response pattern was not generated by local administration to any other brain sites examined In conclusion, mtraclsternally administered endothehn-1 modulates cardloresplratory control by the central nervous system The effect on the central respiratory control was especially powerful. The ventral surface of the medulla appears to play a crucial role m this modulation Abstract--In

Several hnes o f evidence s u p p o r t the view t h a t endothelin (ET) in the central nervous system (CNS) has functional roles. Ftrst, ET-1 a n d ET-3, two o f the m e m b e r s o f the ET family, are b o t h present in the C N S ( M a t s u m o t o et al., 1989). Second, binding sites for ET-1 a n d ET-3 were f o u n d in the b r a i n (Fuxe et a l , 1989b; Kosekl et al., 1989) Third, messenger R N A o f ET has been d e m o n s t r a t e d by m sttu hybridization in n e u r o n s o f h u m a n spinal cord (Glald et a l , 1989) a n d porcine p a r a v e n t r l c u l a r nucleus (Yoshlzawa et al., 1990). Fmally, ET-11ke l m m u n o reactive p r o d u c t s in the posterior pituitary o f the rat were depleted in response to water deprivation (Yoshlzawa et al , 1990). We previously reported that, in urethane-anesthetlzed rats, lntraclsternal a d m i n i s t r a t i o n o f ET-3 ehclted a p a t t e r n of changes, c o m p o s e d typically o f three phases, in arterial pressure (AP), h e a r t rate (HR), renal sympathetic nerve activity ( R S N A ) a n d phrenlc nerve activity ( P N A ) a n d m o d u l a t e d the arterial b a r o r e c e p t o r - s y m p a t h e t l c v a s o m o t o r reflex

(Kuwakl et a l , 1990). F u r t h e r m o r e , topical apphcation o f ET-3 to the ventral surface o f the medulla (VSM) p r o d u c e d the same p a t t e r n o f cardiovascular a n d sympathetic changes as with lntraclsternal administration suggesting t h a t the V S M played a crucial role m the a b o v e - m e n t i o n e d effect o f ET-3 As a sequel to t h a t report, the present study is intended to demo n s t r a t e t h a t ET-1 affects central neural control of the circulation a n d respiration.

EXPERIMENTAL PROCEDURES

Experiments were performed on 47 male Spragu~Dawley rats (300-450 g) anesthetized, unless otherwise noted, with urethane (mmally 1 g/kg, 1.p ), lmmoblhzed by gallamme tnethlodlde (Initially l0 mg per rat i v , thereafter 6-8 mg/h i v ), and artificially ventdated by oxygen-enriched room air End-tidal Pc% was maintained at 3~4 5%. The vagus nerve was cut bdaterally in the neck so that the central respiratory rhythm generator was not influenced by the afferent signal of this nerve The aortic and carotid sinus nerves remained intact Methods for (a) preparation and maintenance of the ammals, (b) mtracisternal injection of ET, (c) recording of AP, HR, RSNA and PNA, (d) precolhcular decerebratlon, (e) appllcaUon of ET to the VSM, (f) data analysis, and

*Author to whom all correspondence should be addressed 519

TOMOYUKI KUWAKI ('l a/

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(g) hlstolog,cal reconstruction of inJection sites have been described m detail in our previous paper (Kuwakl et a l , 1990) In short, ET-1 (Peptlde Institute, Osaka) was dissolved m l0 /J1 of artificial cerebrosplnal fluid (pH = 7 4) and was reJected Into the clsterna magna at doses of 0 1, 1 and 10 pmol The left renal nerve was approached retroperltoneally through a left flank lnclsmn and prepared for recording from near the renal artery The right or left phrenlc nerve was prepared by separating neck muscles Multlfiber discharges of renal sympathetic and phrenlc nerves were rectified, rotegrated over a 10-s interval and stored m a tape recorder At the time of reproduction for further data processing, the instrumentation noise was subtracted from the nerve signal and RSNA and PNA were determined The number of bursts per mln of phremc nerve &scharges (burst rate) was calculated directly from the unprocessed multlfiber discharges It has been known that multnfiber phrenlc nerve d]scharges increase abruptly at the onset of the lnsplratory phase from a background ofhttle or no activity, keep increasing and then rapidly diminish at the termination of this phase, although a much smaller portion of them sometnmes continues Into the postmsplratory phase (Richter, 1982, Berger and Hornbeln, 1989) In the present experiments in vagotomlzed rats, PNA was thus used to assess activity of the central respiratory rhythm generator Likewise, RSNA was used as a measure of sympathetm vasomotor actlwty, since it accurately reflects activity of sympathetic vasoconstrictor fibers (Dorward et al, 1986)

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Precolhcular transection ~as perlormed in 6 rats under halothane anesthesia A hole was perforated at a point approx 1 mm rostral to the lambdo)d suture and 1 mm lelt to the mldhne of the parietal bone The hole was then enlarged and part of the occipital lobe was aspirated so that a spatula could be easily inserted into the mldbraln After the precolhcular transection, the anesthesia was dlscontmued and a 2-h mtermlssmn was interposed to eliminate the effect of hatothane In 10 rats, the VSM was approached ventrally The ammal's head was fixed to a stereotaxlc frame in a supine pos> lion The basdar part of the occipital bone was exposed b) removing the trachea, esophagus, muscles and soft hssues covering the bone and was carefully removed under the dissection microscope The exposed VSM was chemically stimulated by topical appllcatmn of ET-t dissolved m artlfic,al cerebrospmal fluid and soaked into a small piece of filteI paper (I , I ram) and apphed unilaterally to the area oxel the root of the Xllth cranial nerve Statistical analysis was carried out using Student's t-tesl for paired data Differences m data were considered to be significant when P < 0 05

RESULTS A n lntraclsternal mjectnon o f 0 1 p m o l o f ET-1 resulted m a n ~mmedlate a n d t r a n s i e n t m c r e a s e m AP,

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Endothehn-1 and central cardloresplratory control HR, RSNA, PNA and burst rate that lasted for 3-15 mln [Fig. I(A) and Table 1]. This period of inlnal increases was termed phase I. After the increases subs~ded, P N A and burst rate usually fell below the premjecnon level for the next 5-20 mln At a dose of I or 10 pmol, the phase I was followed by falls in all the variables below the control level [phase II, F~g I(B)] Especially striking was the inhibition of respiration. Namely, in most experiments, PNA and ~ts burst rate were completely eliminated for a while, demonstrating disappearance of the centrally generated respiratory rhythm, Phase II was terminated usually m 20-80 mln and turned Into phase III during which these vanables returned to or exceeded the control levels However, AP, PNA and burst rate sometimes remained below control throughout the 2-h observation period. For example, at a dose of 1 pmol, AP remained below control in 3 out of 6 rats. Thus, the most prominent result of lntraclsternal ET-1 is its Inhibitory action on the central respiratory actiwty, although it also affected cardiovascular and sympathetic activity to a lesser extent (Table 1). Actually, on administration of I pmol of ET- 1, mean values of the peak decrease from control during phase II in RSNA, PNA and burst rate were 43, 70 and 80%, respectively On the other hand, when arnficml cerebrosplnal fluid was injected as vehicle in 10 rats, none of the variables exhibited statistically sigmficant peak changes during the 2-h observation period (data shown in the row corresponding to the dose of 0 pmol m T a b l e 1). In order to examine whether cardloresplratory responses to mtracisternally administered ET-1 were seriously affected by urethane, they were ehclted in 6 unanesthetlzed precolhcular decerebrated rats One pmol of lntraclsternal ET-1 resulted in a pattern of card~oresplratory responses similar in ~ts time course (Fig. 2) and comparable, by and large, in the magnitude of peak changes (Table 1) to those in urethaneanesthetized animals. This result demonstrated that urethane did not seriously distort the pattern of cardloresplratory changes in response to lntraclsternal ET-I For th~s reason, urethane was used for general anesthesia in the following experiments We next sought to locate the CNS s~te responsible for the modulatory action of ET-1 on the cardlorespiratory control Since precolhcular decerebranon did not appreciably affect responses to ET-1, the active site was expected to be located along the neuraxls at or caudal to the mldbraln Our previous study disclosed that topical apphcatlon of ET-3 to the VSM resulted in a pattern of cardiovascular and

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Fig 2 EffectoflntraclsternaladmmlstratIonofl p m o l o f E T - I munanesthetlzedprecolhcular decerebrated rats on mean arterml pressure (MAP), RSNA, PNA, burst rate of PNA and H R Responses observed in Individual animals are indicated by dotted hnes and the average values are represented by heavy lines Data are expressed as percent of prelnjectlon control values

sympathetic changes very similar to that following lntraclsternal administration (Kuwakl et al, 1990) On the basis of this finding, the VSM was exposed in 10 rats and a piece of filter paper (1 x 1 ram) soaked with 1 pmol of ET-1 dissolved in artificial cerebrospinal fluid was apphed to the VSM rostral to the root of the hypoglossal nerve. This area corresponds approximately to the intermediate (or S) and caudal half of the rostral (or M) areas of the VSM (Trouth et al, 1973) As expected, the pattern of changes in AP, HR, R S N A , P N A and burst rate were essentially identical to that following lntraclsternal administration (Fig. 3 and Table 1) Simultaneously injected Evans Blue on the occasion of lntraclsternal administration of ET-1 always reached the VSM This result suggests that the VSM is involved in the modulatory action of intraclsternally administered ET-1 on the central cardlorespiratory control DISCUSSION

The present study demonstrated that lntraclsternal administration of ET-1 modulated cardloresplratory control by altering AP, HR, RSNA, P N A and burst

rate Especially powerful was the inhibitory effect on respiration. Namely, at a dose as low as 0 1 pmol, ET-l caused a transient Increase followed by longerlasting decreases in P N A and burst rate Since the Inhibition occurred in vagotomized rats, the observed changes were not secondary to the vagally mediated respiratory reflex but a result of a direct action on the CNS. In line with our observations, Fuxe et al (1989a) reported apnoea in rats anesthetized with ~-chloralose on lntraclsternal administration of 200 pmol of ET- 1 In our study, Inhibition of respiration occurred at doses much smaller than that As mentioned in our previous paper (Kuwakl et al., 1990), mtraclsternal ET-3 also caused an inhibition of P N A However, thc dose required for this effect was 100 pmol In other words, as far as the modulation of central respiratory control ns concerned, ET-1 was more potent than ET3 by a factor of about 1000 lntraclsternally administered ET may cause vasoconstriction of cerebral arteries (Ide et al, 1989) and ensuing cerebral lschemla might have resulted in the observed cardloresplratory changes However, thc acute effect of cerebral lschemla is a powerful pressor response with sympathoexcltatlon and acceleration of

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Fag. 3. (A and B) Effects of topical appllcatmn of ET-1 to the ventral surface of the medulla (VSM) on MAP, RSNA and HR (A) or MAP, PNA and burst rate (B). Recordings an (A) and (B) were obtained from two dafferent rats A paece of filter paper soaked with 1 pmol of ET-1 was placed on the exposed VSM as shown by the dotted area m (C) which illustrates the ventral surface of the lower bramstem Abbreviations are as follows. Basdar a., basflar artery, Pyr, pyramadal tract, V, trlgemmal nerve, X, vagus nerve, XII, hypoglossal nerve

respiration (Dampney et aL, 1979) rather than the complex cardiorespiratory changes observed in the present study On topical application to the VSM, ET-1 elicited a pattern of cardiorespiratory changes similar to that following an intracisternal injection. The VSM has been regarded as the site of central chemoreceptors which is responsive to a variety of chemical agents in addition to CO2 and H + (Bruce and Chernlack, 1987) Furthermore, the VSM was found, both functionally and neuroanatomlcally, to be associated very closely with the rostral ventrolateral medulla (Benarroch et al., 1986), a key CNS site with respect to maintenance of tonic sympathetic vasomotor discharges (Ross et al., 1984). When the VSM was stimulated by inhalation of 5% CO2 in room air for 3 mln before and after lntracisternal administration of ET-l in two rats, reflex increases in PNA and burst rate were suppressed during phase II (data not shown). This observation also suggests involvement of the VSM in the cardloresplratory responses to lntraclsternal administration of ET- 1

On the other hand, cardioresplratory responses to a local injection into the nucleus tractus sohtarius (NTS) or intrathecal administration of ET-1 were not identical to those following intracisternal administration (see Kuwaki et a l , 1990, for the method of local administration). Namely, a unilateral microlnjectlon of ET-1 (4 pmol) in 6 rats into the NTS, usually coverlng its medial and ventrolateral subnuclel (Kaha et al., 1984), caused augmentation of PNA and burst rate that lasted for more than 45 min with small changes in RSNA and AP (data not shown). Intrathecal administration of 100 pmol of ET-1 in 3 rats produced only minute changes in the five variables measured (data not shown). In agreement with these results, local injection of ET-3 into the NTS or lntrathecal administration of it did not bring about changes in AP, H R and RSNA which were similar to those followlng lntracisternal administration (Kuwakl et al., 1990) Collectively, these findings support the view that the VSM is involved critically in the modulatory action of lntracisternally administered ET-1 on the central cardIorespIratory control

TOMOYUKI KUWAKI el a/

524

4~l~nowledqement,s This research was supported by GrantsIn-Aid (62570038) for Sclenhfic Research from the Ministry of Educahon, Soence and Culture. Japan• and grants from the Ministry of Health and Welfare• Japan and the lch~ro Kanehara Foundation

REFERENCES

Benarroch E E . G r a n a t a A R• Rugglero D A . Park D H and Rels D J 11986) Neurons of C~ area mediate cardmvascular responses initiated from ventral medullary surface 4m J Ph.vs'tol 250 (Regul htteq; ~omp Ph~s'tol 19), R932 R945 Berger A J and Hornbem T F 11989) Control of Resplranon In Tevthoolt o[ Phy,swloqy (Patton H D , Fuchs A G , Hllle B • Scher A M and Sterner R . eds). 21st edn. pp 1026 1045 Saunders, Philadelphia Bruce E N and Chernmck N S 11987) Central chemoreceptors J appl Phvswl 62, 389 4(12 Dampney R A L, Kumada M and Rels D J (1979) Central neural mechamsms of the cerebral ~schemtc response C.~ Res 44,48 62 Dorward P K . B u r k e S L . J a m g W a n d C a s e l l J 11986) Reflex responses to baroreceptor, chemoreceptor and nocJceptor inputs in single renal sympathenc neurones In the rabNt and the effects of anaesthesia on them J 4utonom Nett' S~st 18, 39 54 Fuxe K , Andbjel B, Kaha M and Agnan L F 11989a) Centrally admmtstered endothehn-I produces apnoea m the u-chloralose-anaesthenzed male rat 4~ta phvstol ~,aml 137. 157 158 Fuxe K , Anggfird E, Lundgren K • Cintra A , Agnat~ L F • Galton S and Vane J (1989b) Locahzatmn of [~2'llendothehn-I and [~Z~l]endothelln-3 binding s~tes m the rat btam 4~ta ph~,stol ~ a m l 137, 563 S64 G~ad A . G ~ b s o n S J , lbrahm3 N B N • Legon S. Bloom S R . Yanagtsawa M . Masak~ T . Varndell 1 M and Polak J M 11989) Endothehn I. an endothehum-derlved pephde, ~s e~pressed m neurons of the human spinal cord

and dorsal root gangha Pm~ naln A~ad 5~; l 5 4 86, 7634 7638 lde K , Yamakawa K . Nakagoml T• Sasakl T , Salto I Kunhara H , Yoshlzuml M , Yazakl Y and Takakura K 11989) The role of endothehn in the pathogenesls of ~asospasm following subarachnoJd hemorrhage Nemol Re,s I I , 101 104 Kaha M• Fuxe K , Hokfelt l', Johansson O , Lang R Ganten D , Cuello C and Teremus L 11984) Distribution ol ncuropeptlde lmmunoreactlve nerve terminals Vvlthln the subnucleJ of the nucleus of the tractus sohtarms of the rat J ~omp A~'uml 222, 409 444 Ko',ekl C . Imam M , Hlrata Y . "Yanaglsawa M and Masakl T 11989) Autoradlographlc locahzatlon of [~2'l]-endothchn-I binding sites m rat brain Netoos~; Re,~ 6, 581 585 Kuwakl "l, Koshlya N , 1-akahashl 11 Terul N and Kumada M 11991)) Modulatory effect,, of rat endothehn in central cardlo,,ascular control m lats Jpn J P h ~ m l 40,97 116 Matsumoto H ,Suzuki N ,Onda It and Fujunoto M 11989) ~bundance of endothehn-3 m rat intestine, pltmtary gland dud brain Bto~hem htophvs Res Commun 164, 74 80 Richter D ~ 11982) Generahon and maintenance ol" the resplrator~ rh~,thm J exp Bud 100, 93 107 Ross C A . Ruggzero D A . F'ark D H . Joh T H • S~ed A F . Fernandez-Padal I. Saa~cdra I M and Rels D J (1984) Tome vasomotol control by the rostral '~entxolateral medulla effect ol electrical or chemical .,tmml.mon of the area containing C1 adrenahne neulons on arterial pres',ure, heart rate and plasma catecholamme,, and va~,opressm J Netoos~t 4, 479 494 Trouth C O . Loeschcke H H and Berndt J (1973) superficml substrate on the ~ennal surface of the medulla oblongata influencing respmmon Plluqets 4t~h 339, 135 152 Yo~lnzawa 1", Shmml O , (slald A , ~ auaglsawa M . Gibson S I Klmura S, Uchlyama Y . Polak J M , Masakl T and Kana/awa I 11990) Fndothehn a novel pephde m the posterior pltmtar~ system S~wme 247, 462 464