Pharmacology oE the Shydroxytryptamine-induced of the ferret vagus nerve in vitro
Nigcl R. Newberry, Glare J. Watkins, D. John M. Reynolds, Ronald A. Leslie and David G. Grahamc-Smith
Received 2X July lYY7. acccptrd 4 August tYY2
Grcasc-gap recordings rcvcalcd that S-hydroxytryptaminc 6HT) dcpolarizcd the fcrrct vagus n~rvc (pECS,, = 4.9). This rcsponsc was mimicked hy ?-methyl-S-HT and I-phcnylbiguanidc, hut not by S-carhoxamidotryptaminc. Paroxctinc (I PM) or kctnminc (IO PM) did not potcntiatc the rcsponsc. Kctanscrin (I PM) did not rzducc the depolarization. hut four S-HT, rcccptor antagonists did. It is concluded that S-HT dcpolarizcs the fcrrct vagus ncrvc via S-HT, rcccptors, hut thcsc receptors may differ pharnlaa~logically from those in other spccics. Vagus ncrvc: S-HT (S-hydroxytryptaminc, scrotonin). S-HT, rcccptors; Emcsis; (Fcrrct)
emcsis due to cylotoxic emetic response involves
In 108h, Richardson tcncc
and Engcl proposed the cxisof S-HT, rcccptors based on
diffcrcnccs in the affinities of S-HT,3 rcccptor antagonists for S-HT, rcccptor-mcdiatcd rcsponscs in diffcrcnt tissues. Subsequently. scvcral groups have reported that the S-HT, rcccptors on rhc same preparation taken from diffcrcnt spccics can bc distinguished pharmacologically. The S-HT, rcccptors of the rat and guinea-pig vagus ncrvc wcrc shown lo bc pharmacologically distinct (Butler ct al., :YYO), as wcrc the rcccptot-s on rabbit and guinea-pig nodosc ganglion neuroncs (Malone ct al., IYYI). S -HT, rcccptors in the superior cervical ganglion of rat, mouse and guinea-pig
drugs such as cisplatin. This the activation of vagal 5-HT,
rcccptors (Reynolds ct al., IYY I). To date, however, the pharmacology of the S-HT, receptors on the ferret vagus ncrvc has not been directly studied. Our aims wcrc to cxaminc the cffcct of S-HT on the fcrrct vagus ncrvc using a simple electrophysiological method. to determine whether S-HT, receptors were prcscnt on thcsc axons and, if so, whcthcr they diffcrcd pharmacologically from those in other species.
2. Materials and methods
ccptors in a given spccics appear to hc pharmacologically and clcctrophysiologically similar (Butler cl al.,
Thirteen fcmalc fcrrcts (0.7-1.0 kg) wcrc killed by an intraperitoneal injection of 200 mg/kg pcntobarbitone. The two cervical trunks of vagus nerve were cxciscd, dcshcathcd and cut into two equal lengths (ca. I.5 cm). Each preparation was then set up in a threecompartment bath at 25°C. The experimental methods and supcrfusing medium wcrc the same as previously
1YW . Malone et al., IYYl). The fcrrct is widely used to dcmonstratc the in vivo efficacy of S-HT, rcccptor antagonists as inhibitors of
used for the superior cervical ganglion (Newberry et al., IYYI). Agonists were applied for I-min periods with IO-20-min intervals bctwccn applications. With high
wcrc also shown to differ, particularly with the wide variation among thcsc spccics in the antagonist affinity of t-t I-tubocurarinc (Ncwbcrry cl al., 1991). In contrast to the differences among species, the 5HI’, rc-
agonist concentrations, the agonist application was terminated. if the rcsponsc began to return to baseline C’orrcspontlcncc to: N.R. Ncwlxmy. Oxtiml Univrrsity. SmithKlinr I3CCChi~~~l Ccntrc tbr At~plicd Ncuropsycllohiclloey. IIcp;lrt~llcnt 01 (‘linicd Pll;lrrll;lcd~~gy, Riddit’t’c I~~l’ir~ll;l~. Woorlsio~k Ro;I~I. OXlilrd. OX?. hill:. IJK. Td. (44.X65) 703417, hx (44.X05) 7Yt712.
during its supcrfusion. At the beginning of an cxpcrimcnt, S-HT was applied to each vagus ncrvc at Wmin intervals until two consccutivc responses wcrc obtained which varied by less than 10%. Subscqucnt peak rc-
sp0n.se amplitudes on thr preparation were normalized to the last of these responses. A conccntration-rc-
spon:w curie was determined before and after a l-h superfusion with (or without) an antagonist. unless otherwise stated. Alternatively. the concentration of an antagonist (superfused
for 4S min) necessary to reduce
by half the response to IOU CM S-HT was determined. its IC,,. The Ix,tcncy of an agonist was individually determined for each vagus and expressed as its pEC>,, value ( - log,,,(EC,,,). The antagonist potency of tubocurarine was estimated from the equation pA 2 = Iog,,,( DR-I I-log,,,(molar concentration of tuhocurarine). where pAz is the negative logarithm of the antagonist concentration
giving a DR (dose ratio) of 2.
Fig. I. Depol;wizing
rcsponscs IO 5-HT.
The DR was determined at the level of half of the maximum rcsponsc in the prcscncc of antagonist. The
min. ;II arrow) was applied
number (n) refers to numbers of separate
on a ferret
in vitro and their
f I h). Each agonist f 100 PM,
hy 3 PM f + btubocurarinr
in the absence and prcsencc
animals. Vagi from male Sprague-Dawlcy
rats were also stud-
ied. using methods
compare the pharmacology in the two species (cf. Ircland and Tyers. IYS7). In particular. two rats (214 and ___ “’ g) were killed with pentoharbitonc (X(10- 1000
The median response induced was 0.3 (range 0.1-0.34) compared
to 100 PM
On the rat vagus nerve,
mg/kg i-p). to determine whether the method of killing affected the potencies of S-HT or ( + )-tuhocurarinc. The drug sources were as foilows: S-hydroxytrypta-
100 ,uM phenylbiguanide (pECso = 5.6 k 0.2; evoked a maximum response that was IO-IS% less than the
mine hydrochloride (S-HT) (Sigma): S-carboxamidotryptamine maleatc (S-CT). 2-methyl-S-HT maleate and ICS 2OS-930 (Rcscarch Biochcmicals Incorporated); Iphenylbiguanide (Aldridge); ketanscrin tartrate (Janssen); ( + )-tubocurarine chloride and pargyline hydro-
Tyers, 1987). The concentration-response fcrrct vagus was reproducible.
chloride (Sigma); paroxctine hydrochloride, granisetron (BRL 43694) and ondansctron (GR380.32F) (SmithKline-Beecham Pharmaceuticals).
1.12, n = 4) was obtained. The S-HT,/S-HT,,rcccptor antagonist ketanserin (I PM, 30 min) did not reduce
3. Results S-HT ( 100 PM) reproducibly depolarized the ferret vagus nerve preparation by 0.30 f 0.02 mV (mean k S.E.M.. n = 34 vagi). Its pEC5,, was 4.89 + 0.05 (n = I3 vagi). In contrast to S-HT. the potent S-HT, rcccptorsclectivc compound S-carboxamidotryptaminc had no effect at I PM (n = 3). We assessed the actions of two relatively selective S-HT, rcccptor agonists. l-mcthylS-HT and phcnylhiguanidc (XC fig. I ). The conccntration-response curve to 2-methyl-S-HT was nearly superimposable on that of S-HT with 2-methyl-S-HT having a similar maximum response and a pEC,,, of 5.18 &- 0.04 (n = 5). This contrasted with the rat vagus nerve where 2-methyl-S-HT (pEC5,, of 5.2 f 0.2, n = 4) had a maximum rcsponsc which was 20% smaller than S-HT and where S-HT was clearly the more potent agonist (pEC,,, of 6.2 f 0.04. n = 38). Phenylbiguanide was only tested at IO0 PM on four ferret vagus nerves.
(n = 7; cf. lreland
curve to S-HT on the When it was redeter-
mined, after superfusing antagonist-free medium for 1 h, a median concentration ratio of O.YS (range 0.74-
the response to 100 PM S-HT (n = 3). The response to IO PM S-HT was not potcntiated by the S-HT uptake inhibitor paroxetine (I PM, 30-60 min, n = 3). Ketamine (IO PM, 5 min) which has been reported to potcntiatc the S-HT response of rabbit nodosc ganglion neuroncs (Malone et al., 19Yl), had no effect on the response to 10 PM S-HT (n = 3). Incidental!y, kctaminc
min) did nrlt pohentiatc
S-HT-induced depolarization on the rat vagus (n = 3). We determined the effects of four S-HT, receptor antagonists on the response to I()(! PM S-HT on the ferret vagus. Their ICso values, obtained on vagi from different ferrets. were as follows: granisetron (BRL 43694, Sangcr and Nelson, 1989) I.0 and 1.3 nM; ondansetron (GR38032F, see Butler et al., 1990) 4.2, IO and IO nM; ICS 205-930 (see Richardson and Engel, 3986) 6.3 and IO nM; ( + I-tubocurarinc (e.g. Newbcrry et al., 1991) 3 and 4.3 FM. We determined the effect of two of these compounds on the concentration-response curve to S-HT of granisetron maximum
(see fig. 2). The most noticeable effect (I nM, n = 5) was a reduction of the
response to S-HT by SO%. By contrast, ( + )-
and 3 PM,
n = 2 and 5, rcspcctively)
Fig. 2. The effect of (+)-luhocur~inc (3 PM) and granisetron (I nM) on the concentration-response curve to S-HT on the ferret v:tgus nerve in vitro. The data were fitted iteratively (MurquardtLevenberg method on RSI softwurel to the equation y = m;lximum/ (I +(EC,,,/Xl* * Nl. For the control curve (CON, open circles, I2 vagi) the solution was: maximum = 1.07, EC,,, = 14 FM. N = 1.21; for (+ )-tuhocurarine (TC. filled squares, five vagi): maximum = 0.8X. EC5,, = 114 FM. N = I.14 und for granisetron (GRAN. filled triangles, five vagi): maximum = 0.53, EC,,, = 27 PM, N = 0.86. For comparison. but not shown for clarily, the values for 2-methyl-S-IIT in the absence of sntagonist (five vagi) were: N = I .W, ECs,, = 9.2 pM, N = I.1 I. The relative responses (shown as geometric meim and S.E.M.) were relilled to that to the depolarization to I00 FM S-HT at the start of the experimrnl (see Materials and methods). The insurmountable action of the umagonists. parliculurly evidcm with grunisetron. could he due to pseudo-irreversible inhibition or allosteric receptor modulation. The presrnt evidence does not allow us IO differentiate between such possibilities.
produced a clear rightward shift of the concentrationresponse curve to 5-HT, but only reduced its maximum response by 18% (fig. 2). The estimated pAz for tubocurarine against 5-HT was 6.64 + 0.05 (n = 7). A limited Schild plot of these data had a slope of 0.997 and indicated a pA, of 6.66. Tubocurarine (1 and 3 PM) also shifted the concentration-response curve of 2-methyl-5-HT to the right with an estimated pA2 of 6.60 * 0.09 (n = 3). Since we were comparing the pharmacology of the rat vagus with that of the ferret vagus, it was necessary to determine whether the method of sacrifice could affect the results since rats were usually killed by concussion followed by exsanguination, and the ferrets by an overdose of pentobarbitone. From the vagi of the two rats killed with pentobarbitone. however, it appeared that the potency of 5-HT (EC,,,s of 0.65 and 0.62 PM) and the affinity of (+I-tubocurarine (pA,s of 7.2 and 7.2) were unaffected by the method of sacrifice (cf. Ncwberry ct al., 1992). By contrast with the ferret vagus, however, on the rat vagus we have not observed a consistent reduction by tubocurarine of the maximum response to 5-HT.
We have shown that 5-HT depolarizes the ferret vagus nerve in vitro. The pharmacology of this depolarization indicates that it is mediated by 5-HT, recep_ m-s. When compared with data from other species, these ferret %-IT, receptors appear pharmacologically similar to rat 5-f-U, receptors, but there are some differences, as discussed below. The depolarizing response to 5-HT did not appear to be mediated by 5-HT, or 5-HT2 receptors because the 5-HT, -selective agonist 5-carboxamidotryptamine had no effect and the 5-HT,/5-HT,, rec;ptor antagonist ketanserin did not reduce the response. However, it was likely mediated by 5-HT, receptors since the response was mimicked by two selective agonists and reduced by four 5-HT, receptor antagonists (Bradley et al., 1986). Tubocural ine and granisetron affected the concentration-response curve to 5-HT differently. In the presence of (+ )-tubocurarine, the ECS,, for 5-HT was clearly increased and there was a small reduction in the maximum response to 5-HT. In contrast, granisetron had littlc effect on the ECS,, for 5-HT but reduced the maximum response to 5-HT by half. We conclude that the modes of action of these two antagonists are diffcrcnt, a similar situation to that observed on the rat vagus nerve (Newberry et al., 1992). It has previously been reported that (+ )-tubocurarinc antagonizes the 5-HT, rcccptor-mcdiatcd responses in different species with distinctly different potencies. pA, values of 8.1, 7.1 and 4.8 were obtained on the mouse, rat and guinea-pig superior cervical ganglion, respectively (Newberry et al., 1991). The pA2 for tubocurarine on the rat vagus (7.2) was similar to that found on the rat superior cervical ganglion indicating similarity among 5-HT, receptors in different tissues from this animal (see also Butler et al., 1990). It would therefore seem that the affinity of tubocurarine for the ferret vagal 5-HT, receptor (PA, = 6.6) is close to, but not identical with, its affinity for the rat 5-HT, rcccptor. Thcrc are other similarities in the pharmacology of the 5-HT, receptors in the rat and ferret. The pcrccntagc reduction by I nM granisetron of the maximum response to 5-HT was similar on the vagus nerve preparation from these species Ica. 50%: cf. Newberry ct al., I!%&?). Furthcrmorc, phenylbiguanide (100 PM) is an agonist on the ferret and rat vagus but not on 5-HT, receptors from the guinea-pig (Butler et al., 1990: Newberry et al., 1991). Finally, ihe potentiation by kctaminc of the 5-HT, response seen in rabbit nodosc ganglion neurones (Malone et al., l99l) was not obscrvcd on the fcrrct or rat vagus. Thcrc wcrc some differcnccs in the agonist pharmacology of ahe ferret and rat vagus. Whereas there was a clear diffcrcncc in potency between 5-HT and 2-
A.. C‘.J. I3swood.
7iilionb abf 5-l IT3 Iiws Ircland.
the ferret vagus
nerve via S-HT, recepfors. The phurmac&~~~ of tht’sc S-HT, rcccptors most closely rcsemhles those in the rat (cf. mouse. rat. guinea-pig ilDd rilbbit). The relative potencies of the agtnists. however. suggest that the ferret SHT, rcccptor may hc p~i~rmilcologicaliy distinct. Given fhc species difference:: in SHT, receptor pharmacology and the recent sequ,:ncing of one of thc.sc rcccptors (Maricq ct al.. 1991). it is now important tl) determine which species hiis SHT, rcccptors like iho.sc in the human so an intelligent choice of the best animal model for in vivo studies c;ln hc mrldc.
Iissucs. Hr. J. Pharm;d.
I I.M.. J.A. Pctcrs ;d
M;lricq. A.V.. lYYl,
and T.S. Spmsen.
IYY I. Cispl;IIin-w&cd
of the fcrrct: 5-IlTa
IYYI, Evidence 1h;11
Br. J. Pharmacd.
of Ihc S-FIT,
of IIIC rat. mouse and gkwa-pig
Myers and D. Julius.
lYY3, BRL 4b470A
superior IIP. 615.
responses on ~hc r;tl vagus
and on NGIOS- I5 cells. Br. J. Ph;wmacol. Kcynolds.
- ;I palch-clamp
ganglion may hc diffcrcnt.
IYY I. Phy~id~~gicd itnd
A.J. Brdx. d
~hc 5-tlT, ccrvkxl
of S-I II’,
of the rat isolated
Br. 3. Pharmacol.
IYX7, Ph;lrnl;lcolopic;II ch;lr;lclerization
We conclude that 5-HT
I1.T. Bunce. G.J.
in three different
S.J. and M.B. Tyers.
antI M.B. Tysrs. IYYO. The pharmacologicd
of c-jks protein
R.A. in the
vagotomy and Ihc
Brain Rcs. SbS. 131.
and G. Engel.
tion of 5-11T,x rcccptors. Brdlry. D.N.