Br. J. Pharmacol. (1992), 105, 925-928
'."
Macmillan Press Ltd, 1992
Differences in neurokinin receptor pharmacology between rat and guinea-pig superior cervical ganglia 'G.R. Seabrook, M. Main, B. Bowery, N. Wood & R.G. Hill Department of Pharmacology, Merck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR 1 The depolarizations elicited by seven neurokinin receptor agonists were examined in both rat and guinea-pig superior cervical ganglia by use of grease-gap methodology in the presence of tetrodotoxin (0.1 pM). Responses were normalised with respect to 1 AM eledoisin. 2 The rank order of agonist potency in the rat ganglia was senktide > substance P > substance P methyl ester = eledoisin = Sar-Met-substance P > neurokinin B > neurokinin A, whereas in guinea-pig superior cervical ganglion (SCG) the rank order was senktide > Sar-Met-substance P > neurokinin B = eledoisin = substance P methyl ester. The concentration-effect curves for substance P and neurokinin A in guinea-pig ganglia were biphasic which precluded the determination of meaningful potency values. 3 The maximal depolarization achieved by subtype selective ligands was different between these two species. On rat and guinea-pig SCG, the NK3-selective ligand, senktide, produced a maximal depolarization of 27% and 274% respectively, whereas the NK,-selective ligand, substance P methyl ester, produced depolarizations of 77% and 64% respectively. 4 The depolarizations induced by substance P methyl ester and senktide in either species were unaffected by atropine (1 pM), suggesting a lack of involvement of presynaptic neurokinin receptors in the generation of the response. 5 The potency of substance P methyl ester, senktide, and neurokinin A were unaffected by pretreating ganglia with the peptidase inhibitors bacitracin (40pgmlm'), leupeptin (4fpgmhl'), and chymostatin (2 g ml-1). Similarly, these peptidase inhibitors had no effect on the maximal depolarizations achieved by any of these agonists. 6 It is evident that rat and guinea-pig superior cervical ganglia possess both NK, and NK3 receptors, but that their net contribution to depolarizations are different between the two species. The depolarizations in guinea-pig SCG are mediated predominantly by an NK3 subtype and in rat SCG by an NK, receptor subtype. Keywords: Substance P; senktide; sympathetic ganglia; tachykinin; neurokinin receptors
Introduction Substance P is an eleven amino acid peptide discovered by von Euler & Gaddum in 1931, and subsequently sequenced from bovine hypothalamus by Chang & Leeman in 1970. In mammals receptors to substance P have since been shown to be widely distributed, in the central nervous system, auto-
nomic ganglia and peripheral musculature. Consequently activation of these receptors elicits a diversity of physiological effects. Such responses are principally excitatory, and include the increase in firing rate of monoamine neurones in the CNS (Guyenet & Aghajanian, 1977), the depolarization of autonomic ganglia (Dun & Karczmar, 1979; Konishi et al., 1985), and contraction of smooth muscle (Lee et al., 1982). The coupling of these receptors to physiological responses involves G proteins, usually linked to the inhibition of one or more potassium conductances (Nakajima et al., 1988; Stanfield et al., 1985; Nowak & Macdonald, 1982). In sympathetic ganglia substance P is localized in ganglionic cell bodies and fibres of intrinsic interneurones (Hokfelt et al., 1977; Robinson et al., 1980; Dalsgaard et al., 1982). When released by either electrical or chemical stimulation this peptide elicits a postsynaptic depolarization caused partly by the inhibition of postsynaptic potassium conductances which includes, in some ganglia, the voltage-dependent M-current (Adams et al., 1983; Konishi et al., 1985).
'Author for correspondence.
At least three neurokinin receptor subtypes have been identified in the mammalian nervous system (reviewed in Iversen et al., 1990). To date their characterization has been based principally on the potency of the following agonists, substance P, neurokinin A, and neurokinin B, which are the prototypic NK1, NK2 and NK3 receptor selective ligands respectively. Recently, some important progress has been made in the development of specific NK1 and NK2 receptor antagonists (Williams et al., 1988; McKnight et al., 1988b; Ward et al., 1990; Snider et al., 1991). However, currently no selective NK3 antagonist is available, and thus agonist pharmacology still remains an important criterion in receptor classification. More than one receptor subtype can presumably coexist within any one tissue and the majority of tachykinin agonists can act upon more than one subtype; thus definitive studies into neurokinin receptor subtypes required full evaluation of both the potency and efficacy of a range of neurokinin agonists. It has been suggested that in a number of tissues the neurokinin agonist pharmacology does not conform to described receptor subtypes. These tissues include guinea-pig trachea where the existence of an NK4 receptor subtype has been suggested (McKnight et al., 1988a), although, with the use of NK1- and NK2-selective antagonists it is clear that more than one neurokinin receptor subtype may be present in this tissue (Ireland et al., 1991). In rat superior cervical ganglia an anomalous agonist profile has also been reported (Brown et al., 1983). In this study we present evidence for a heterogeneity of neurokinin receptors in rat and guinea-pig
926
G.R. SEABROOK et al.
superior cervical ganglia, demonstrating that their relative contribution to the maximal depolarization which can be elicited with tachykinins, exhibits differences between these two mammalian species.
Methods
a
t
t
Superior cervical ganglia were excised from male SpragueDawley rats (150-250g), and male Dunkin Hartley guineapigs (200-300g), which had previously been concussed and then exsanguinated. Ganglia were desheathed and then placed in a grease-gap recording chamber (Brown et al., 1980) through which warm physiological salt solution was continually perfused (25C, 1-2 ml min-'). The saline contained (in mM): NaCl 125, KCl 5, KH2PO4 1, CaCl2 2.5, MgSO4 1, NaHCO3 25, glucose 10 and tetrodotoxin 0.1 gM and was gassed with 95% 02: 5% CO2. The potential difference between the ganglion cell body (earthed) and the postganglionic trunk was monitored via Ag/AgCl electrodes connected via a d.c. amplifier to a chart recorder. Drugs were applied to the ganglion cell body for 1 min, at 40 min intervals to minimize receptor desensitization. Ganglia were allowed to stabilize until consecutive applications of either eledoisin (1 1AM) or senktide (1 g1M) produced depolarizations of equilavent amplitude and then the test ligand was applied in increasing concentration. The absolute potential changes were dependent upon the electrical seal and cell density of each preparation, and therefore data were normalized to a control application of eledoisin (1 MM) in each ganglion. This concentration produced submaximal depolarizations in ganglia of both species, and as such minimized errors associated with receptor desensitization. Eledoisin was used as the standard, in preference to substance P, because it produced large depolarizations in both rat and guinea-pig ganglia. Concentration-effect curves were fitted by least-squares analysis of variance to the equation Y = Ymax/1( + (EC50/ agonist concentration)nH), where EC50 is the half maximally effective concentration and nH is the Hill coefficient, using an iterative procedure on a VAX computer with Research System 1 software (BBN Software Products Corporation). Data are expressed as the mean ± standard error. Drugs were obtained from the following companies: neurokinin A (NKA), neurokinin B (NKB), senktide (SE), and Sar-Met-substance P (Sar-Met-SP) from Cambridge Research Biochemicals; eledoisin (E), substance P (SP), substance Pmethyl ester (SPOMe), tetrodotoxin (TTX), atropine sulphate, and bacitracin from Sigma; chymostatin and leupeptin from Bachem. Drugs were dissolved in distilled water, except for senktide which was dissolved in dimethylsulphoxide. Peptide stock solutions were stored in aliquots at -70'C.
Results Both rat and guinea-pig superior cervical ganglia were depolarized by the application of a range of neurokinin agonists. However, the relative potency and maximal ability of these ligands to depolarize ganglia was observed to vary between these two species.
Agonist pharmacology of rat ganglia In rat superior cervical ganglia, eledoisin was observed to be highly efficacious neurokinin agonist (Figure la), with an EC50 of 37 nM. The NK3-selective ligand, senktide (Wormser et al., 1986; Guard et al., 1990) proved to be the most potent ligand examined with an EC50 of 4 nM, although the maxi-
a
mum
depolarization produced in rat ganglia
was
only 27%
that of eledoisin and smaller than that of alternative agonists (Figure la and b). Similarly the NKI-selective ligands, SP, Sar-Met-SP and SPOMe, all exhibited maximal depolariza-
t
SE
E
NKA
t
t
E
SPO
0.2 mV
10 min
.E
b
.O 150
NKA
0
'a
a)
SPOMe
100
.-
o
0
Zs(a
50
Senktide
w7-
N
o0
-;i
10
100
1000 10 000
log [Agonist] (nM)
Figure 1 The depolarization of rat superior cervical ganglia by subtype selective neurokinin agonists. (a) At the time points indicated by the arrows the following neurokinin agonists were applied (3 1M) for a I min period in the presence of atropine (1 gM) and tetrodotoxin (0.1 AM): eledoisin (E), senktide (SE), neurokinin A (NKA), and substance P methyl ester (SPO). (b) The resultant depolarizations in the same ganglia were normalized to the control response with eledoisin. These normalized depolarizations, from a number of ganglia, were used to calculate concentration-effect curves for each agonist (see Table 1). Eledoisin proved to be the most efficacious compound at a concentration of 1 AM, with SPOMe and senktide producing maximal depolarizations of 72% and 27% respectively.
tions of only 72-80% that of eledoisin (Table 1). The potency of substance P was unaffected during continuous perfusion of senktide (1 MM; n = 4) which desensitized NK3 receptors. Pretreatment of rat ganglia with the peptidase inhibitors bacitracin (40 Lg ml'), chymostatin (2pg ml-') and leupeptin (4 Mg ml-') had no effect upon the potency or efficacy of SPOMe (36 ± 13 nM; 117 + 7%, n = 7) or neurokinin A (288 ± 95 nM, 119 ± 7%, n = 6). Both neurokinin A and neurokinin B, peptides which are preferentially, although not exclusively, selective for NK2 and NK3 receptors respectively, both exhibited weak potencies and had Hill slopes significantly less than one. The maximal responses obtained with NKA and NKB at high concentrations were greater than those obtained with eledoisin (Table 1).
Agonist pharmacology of guinea-pig ganglia In guinea-pig superior cervical ganglia, in contrast to the rat, senktide proved to be the most active compound in terms of both efficacy and potency (Figure 2a and b). The maximal depolarization achieved by the NK, selective ligand SPOMe was only 23% that of senktide, and 64% that of 1 fM eledoisin (Table 2). With NKA and SP, the concentrationeffect curves were clearly biphasic, suggestive of an interaction at more than one receptor type. However, the lack of a distinct maximal depolarization with these weaker agonists prevented a reasonable fit to either a single- or two-site logistic model of ligand-receptor interaction. The depolariza-
927
NEUROKININ RECEPTORS IN SYMPATHETIC GANGLIA Table 1 Neurokinin receptor agonist potency and ability to depolarize rat superior cervical ganglia
Ligand
Senktide Substance P SPOMe Eledoisin Sar-Met-SP NKB NKA
Table 2 Neurokinin receptor agonist potency and ability to depolarize guinea-pig superior cervical ganglia
EC50 (nM)
Max (% I fiM eledoisin)
Hill slope
n
Ligand
4±2 12±3 30±6 33±9 39±4 117 ± 65 268 ± 35
27 ± 3
0.99 ± 0.45 0.94 ± 0.16
7 8 8 9 7 7 7
37 ± 14 274 ± 24 102 ± 8 90 ± 26 NKB 259 ± 20 1473 ± 545 273 ± 5 1918 ± 97 Eledoisin 64 ± 41 2052 ± 4552 SPOMe Indeterminate 26% at I pIM NKA Substance P Indeterminate 25% at 1 jM
Max (% I FM
72±4 77 ± 5 103 ± 8 80± 3 132 ± 18 140 ± 5
1.04±0.17 1.06 ± 0.21 1.52 ± 0.22 0.67 ± 0.15 0.76 ± 0.04
SPOMe = substance P methyl ester; NKA, NKB = neurokinins A and B; Sar-Met-SP = Sar-Met-subtance P.
Senktide Sar-Met-SP
EC50 (nM)
eledoisin)
Hill slope
n
0.74 ± 0.12 0.89 ± 0.23 0.53 ± 0.06 0.85 ± 0.02 0.67 ± 0.47 Biphasic Biphasic
8 4 6 9 9 8
3
Sar-Met-SP = Sar-Met-substance P; SPOMe = substance P methyl ester; NKA = neurokinin A.
statin (2 jig ml-') and leupeptin (4 Ag ml ') had no significant effect upon the potency or efficacy of SPOMe (2.9 + 2.1 AM, 44 ± 18%, n = 4), senktide (23 ± 4 nM, 200 ± 9%, n = 4), or neurokinin A (biphasic, 15% at 1 g1M).
a
II
Discussion
1< t
E
t
t
t
rNNKA SPO
SE
t E
0.2 mV 10 min C ._
b
.
(1
a1
SPOMe 10
100
1000
10 000 100 000
log [Agonist] (nM) Figure 2 The depolarization of guinea-pig superior cervical ganglia by subtype selective neurokinin agonists. (a) At the time points indicated by the arrows the following neurokinin agonists were applied (3 pM) for a 1 min period in the presence of atropine (1 AM) and tetrodotoxin (0.1 Mm): eledoisin (E), senktide (SE), neurokinin A (NKA), and substance P methyl ester (SPO). The slower rate of decay of these traces relative to Figure la reflect differences in flow rate of the drug solutions through the bath. (b) The normalized depolarizations in a number of ganglia were used to calculate concentration-effect curves for each agonist (see Table 2). Senktide proved to be the most efficacious agonist, the maximal depolarization achieved by SPOMe was 23% that of senktide. The concentration-effect relationship with neurokinin A was biphasic, and the depolarization was not maximal at 30 MM.
tions elicited by senktide or Sar-Met-SP were not blocked by application of atropine (Figure 2a), and similarly the potency of senktide was unaffected by continuous perfusion of substance P (1 gM; n = 4) which desensitizes NK, receptors. As was seen in rat tissue, pretreatment of guinea-pig ganglia with the peptidase inhibitors bacitracin (40 Mg ml'), chymo-
This study demonstrates that neurokinin receptor pharmacology differs in rat and guinea-pig superior cervical ganglia. These differences exist not only in terms of agonist potency, but also in terms of relative agonist efficacy. It has previously been reported that substance P depolarizes rat superior cervical ganglia (Hawcock et al., 1982), but that it is a relatively weak agonist with an EC50 of 297 nm (Brown et al., 1983). In the present study, substance P was found to be 20 times more potent. The rank order of agonist potency in this tissue is therefore consistent with an NK, receptor as the predominant receptor population. The lack of involvement of NK2 receptors was indicated by the absence of a response to low concentrations of NKA, and the insensitivity of the SPOMe-mediated depolarizations to the NK2 selective antagonist L-659,877 (data not shown). Senktide binds selectively to both central and peripheral NK3 receptors with high affinity (Guard et al., 1990). The high potency and relatively weak efficacy of senktide in rat ganglia thus provides evidence for the presence of an NK3 receptor subtype. Similarly the greater efficacy of eledoisin relative to NK, selective ligands suggests that the response to this ligand is a consequence of activation of both NK, and NK3 receptors. The depolarization caused by the NK, receptor agonist SPOMe (72%) and the NK3 receptor agonist senktide (27%) were equieffective to the maximal depolarization achieveable with the non-selective agonist, eledoisin (see Figures lb, 2b). In guinea-pig ileum, both NK1 and NK3 receptors contribute to the contraction of smooth muscle (Lee et al., 1982). These NK3 receptors are located on the myenteric plexus and mediate their effects by the release of acetylcholine. The NK3-mediated contraction can be blocked by incubation of the tissue in atropine to block the postjunctional muscarinic receptors. However, unlike guinea-pig ileum, application of atropine did not antagonize the neurokinin-mediated depolarizations in rat ganglia (Figure la), which suggests the absence of presynaptic neurokinin receptors in this response. The variation in efficacy of neurokinin agonists in this tissue can be explained by receptor heterogeneity, and/or by partial agonism. The possibility of heterogeneity of receptor subtypes is difficult to test due to the lack of a selective NK3 receptor antagonist. However, as previously discussed there is some evidence for such heterogeneity in rat ganglia based upon the relative potency of senktide. Also consistent with this interpretation is the observation that desensitization of presumptive NK3 receptors with prolonged application of senktide did not reduce the maximal depolarization obtain-
928
G.R. SEABROOK et al.
able with substance P. During this experiment the potency of substance P remained unaffected, therefore it is unlikely that senktide acted as partial agonist at an identical receptor population. It is not clear as to whether both NKI and NK3 receptors are located on common neuronal cell bodies, or whether their distribution is more discrete, in that the receptor distribution reflects separate neurokinin pathways through the ganglia. It is interesting to note that the pharmacology of superior cervical ganglia may be different between species for 5-hydroxytryptamine (Newberry et al., 1991). Indeed in the present study the response to neurokinin receptor agonists was also found to differ between species. In guinea-pig superior cervical ganglia, in contrast to the rat, the majority of ligands tested were only weak agonists with EC50s typically greater than 1 pM (Table 2). The most potent ligand senktide, 37 nM, also exhibited the most efficacious response, whereas substance P was only weakly active in this species. It is unlikely that NK3 receptors are the exclusive neurokinin receptor in guinea-pig superior cervical ganglia as biphasic concentra-
tion-effect curves were observed with NKA and substance P. The binding of senktide to neurokinin receptors in cerebral cortex is similar for both rats and guinea-pigs (Renzetti et al., 1991), whereas NKB binding was shown to be 27 fold weaker in guinea-pig tissue; this may indicate subtle differences in the agonist recognition site between the two species. In conclusion we have shown that neurokinin receptor agonists depolarize both rat and guinea-pig superior cervical ganglia, but that the potency and efficacy of these ligands differ between these two species. Rat ganglia appear to express multiple neurokinin receptor subtypes, the predominant receptor population being of an NKI subtype, whereas in guinea-pig ganglia NK3 receptors appear to be the principal subtype involved. The development of a selective NK3 receptor antagonist will help classify the receptor subtypes present in this tissue. We would like to thank Dr J.A. Kemp for helpful comments on this manuscript.
References ADAMS, P.R., BROWN, D.A. & JONES, S.W. (1983). Substance P inhibits the M-current in bullfrog sympathetic neurones. Br. J. Pharmacol., 79, 330-333.
LEE, C., IVERSEN, L.L., HANLEY, M.R. & SANDBERG, B.E.B. (1982).
BROWN, D.A., FATHERAZI, S., GARTHWAITE, J. & WHITE, R.D.
MCKNIGHT, A.T., MAGUIRE, J.J., WILLIAMS, B.J. & IVERSEN, L.L.
(1980). Muscarinic receptors in rat sympathetic ganglia. Br. J. Pharmacol., 70, 577-592.
(1988a). Characterisation of tachykinin receptors using selectivity of agonists and antagonists: evidence for an NK-4 type. Reg. Pept., 22, 126.
BROWN, J.R., HAWCOCK, A.B., HAYES, A.G., HILL, R.G. & TYERS,
M.B. (1983). The depolarising actions of substance P and other tachykinins on the isolated superior cervical ganglion of the rat. J. Physiol., 334, 91P. CHANG, M.M. & LEEMAN, S.E. (1970). Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterisation as substance P. J. Biol. Chem., 245, 4784-4790.
The possible existence of multiple receptors for substance P. Naunyn-Schmiedebergs Arch. Pharmacol., 318, 281-287.
MCKNIGHT, A.T., MAGUIRE, J.J., WILLIAMS, B.J., FOSTER, A.C.,
EMSON, P. (1982). Substance P-containing primary sensory neurons projecting to the inferior mesenteric ganglion: Evidence from combined retrograde tracing and immunohistochemistry. Neuroscience, 7, 647-654. DUN, N.J. & KARCZMAR, A.G. (1979). Actions of substance P on sympathetic neurons. Neuropharmacology, 18, 215-218. EULER, U.S. VON & GADDUM, J.H. (1931). An unidentified depressor substance in certain tissue extracts. J. Physiol., 72, 74-87.
TRIDGETT, R. & IVERSEN, L.L. (1988b). Pharmacological specificity of synthetic peptides as antagonists at tachykinin receptors. Reg. Pept., 22, 127. NAKAJIMA, Y., KAKAJIMA, S. & INOUE, M. (1988). Pertussis toxininsensitive G protein mediates substance P-induced inhibition of potassium channels in brain neurons. Proc. Nati. Acad. Sci. U.S.A., 85, 3643-3647. NEWBERRY, N.R., CHESHIRE, S.H. & GILBERT, M.J. (1991). Evidence that the 5-HT3 receptors of the rat, mouse and guinea-pig superior cervical ganglion may be different. Br. J. Pharmacol., 102, 615-620. NOWAK, L.M. & MACDONALD, R.L. (1982). Substance P: Ionic basis for depolarising responses of mouse spinal cord neurons in cell culture. J. Neurosci., 2, 1119-1128.
GUARD, S., WATSON, S.P., MAGGIO, J.E., PHOON TOO, H. & WATL-
RENZETTI, A.R., BARSACCHI, P., CRISCUOLI, M. & LUCACCHINI, A.
ING, K.J. (1990). Pharmacological analysis of [3H]senktide binding to NK3 tachykinin receptors in guinea-pig ileum longitudinal muscle-myenteric plexus and cerebral cortex membranes. Br. J. Pharmacol., 99, 767-773. GUYENET, P.G. & AGHAJANIAN, G.K. (1977). Excitation of neurons in the nucleus locus coeruleus by substance P and related peptides. Brain Res., 136, 178-184. HAWCOCK, A.B., HAYES, A.G. & TYERS, M.B. (1982). Agonists effects of [D-Pro2, D-Phe7, D-Trp9]substance P - evidence for different receptors. Eur. J. Pharmacol., 80, 135-138.
(1991). Characterisation of NK3 binding sites in rat and guineapig cortical membranes by the selective ligand [3H]senktide. Neuropeptides, 18, 107-114. ROBINSON, S.E., SCHWARTZ, J.P. & COSTA, E. (1980). Substance P in the superior cervical ganglion and the submaxillary gland of the rat. Brain Res., 182, 11-17.
DALSGAARD, C.-J., HOKFELT, T., ELFVIN, L.-G., SKIRBOLL, L. &
HOKFELT, T., ELFVIN, L.-G., SCHULTZBERG, M., GOLDSTEIN, M. &
NILSSON, G. (1977). On the occurrence of substance P-containing fibers in sympathetic ganglia: immunocytological evidence. Brain Res., 132, 29-41. IRELAND, S.J., BAILEY, F., COOK, A., HAGAN, R.M., JORDAN, C.C. & STEPHENS-SMITH, M.L. (1991). Receptors mediating tachykinin-induced contractile responses in guinea-pig trachea. Br. J. Pharmacol., 103, 1463-1469. IVERSEN, L.L., MCKNIGHT, A.T., FOSTER, A.C., YOUNG, S.C. &
WILLIAMS, B.J. (1990). Pharmacology of the tachykinin system. In Neuropeptides and Their Receptors. ed. Schwartz, T.W., Hilsted, L.M. & Rehfeld, J.F. pp. 363-372. Copenhagen: Munks-
gaard. KONISHI, S., OKAMOTO, T. & OTSUKA, M. (1985). Substance P as a neurotransmitter released from peripheral branches of primary afferent neurones producing slow synaptic excitation in autonomic ganglion cells. In Substance P: Metabolism and Biological Actions. ed. Jordan, C.C. & Oeheme, P. pp. 121-136. London: Taylor Francis.
SNIDER, R.M., CONSTANTINE, J.W., LOWE, J.A., LONGO, K.P., LEBEL, W.S., WOODY, H.A., DROZDA, S.E., DESAI, M.C., VINICK,
F.J., SPENCER, R.W. & HESS, H.-J. (1991). A potent nonpeptide antagonist of the substance P (NK1) receptor. Science, 251, 435-437. STANFIELD, P.R., NAKAJIMA, Y. & YAMAGUCHI, K. (1985). Sub-
stance P raises neuronal excitability by reducing inward rectification. Nature, 315, 498-501. WARD, P., EWAN, G.B., JORDAN, C.C., IRELAND, S.J., HAGAN, R.M.
& BROWN, J.R. (1990). Potent and highly selective neurokinin antagonists. J. Med. Chem., 33, 1848-1851. WILLIAMS, B.J., CURTIS, N.R., McKNIGHT, A.T., FOSTER, A.C. & TRIDGETT, R. (1988). Development of NK-2 selective antagonists. Reg. Pept., 22, 189. WORMSER, U., LAUFER, R., HART, Y., CHOREV, M., GILON, C. & SELINGER, Z. (1986). Highly selective agonists for substance P receptor subtypes. EMBO J., 5, 2805-2808.
(Received October 3, 1991 Revised December 6, 1991 Accepted December 23, 1991)
'."
Macmillan Press Ltd, 1992
Differences in neurokinin receptor pharmacology between rat and guinea-pig superior cervical ganglia 'G.R. Seabrook, M. Main, B. Bowery, N. Wood & R.G. Hill Department of Pharmacology, Merck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR 1 The depolarizations elicited by seven neurokinin receptor agonists were examined in both rat and guinea-pig superior cervical ganglia by use of grease-gap methodology in the presence of tetrodotoxin (0.1 pM). Responses were normalised with respect to 1 AM eledoisin. 2 The rank order of agonist potency in the rat ganglia was senktide > substance P > substance P methyl ester = eledoisin = Sar-Met-substance P > neurokinin B > neurokinin A, whereas in guinea-pig superior cervical ganglion (SCG) the rank order was senktide > Sar-Met-substance P > neurokinin B = eledoisin = substance P methyl ester. The concentration-effect curves for substance P and neurokinin A in guinea-pig ganglia were biphasic which precluded the determination of meaningful potency values. 3 The maximal depolarization achieved by subtype selective ligands was different between these two species. On rat and guinea-pig SCG, the NK3-selective ligand, senktide, produced a maximal depolarization of 27% and 274% respectively, whereas the NK,-selective ligand, substance P methyl ester, produced depolarizations of 77% and 64% respectively. 4 The depolarizations induced by substance P methyl ester and senktide in either species were unaffected by atropine (1 pM), suggesting a lack of involvement of presynaptic neurokinin receptors in the generation of the response. 5 The potency of substance P methyl ester, senktide, and neurokinin A were unaffected by pretreating ganglia with the peptidase inhibitors bacitracin (40pgmlm'), leupeptin (4fpgmhl'), and chymostatin (2 g ml-1). Similarly, these peptidase inhibitors had no effect on the maximal depolarizations achieved by any of these agonists. 6 It is evident that rat and guinea-pig superior cervical ganglia possess both NK, and NK3 receptors, but that their net contribution to depolarizations are different between the two species. The depolarizations in guinea-pig SCG are mediated predominantly by an NK3 subtype and in rat SCG by an NK, receptor subtype. Keywords: Substance P; senktide; sympathetic ganglia; tachykinin; neurokinin receptors
Introduction Substance P is an eleven amino acid peptide discovered by von Euler & Gaddum in 1931, and subsequently sequenced from bovine hypothalamus by Chang & Leeman in 1970. In mammals receptors to substance P have since been shown to be widely distributed, in the central nervous system, auto-
nomic ganglia and peripheral musculature. Consequently activation of these receptors elicits a diversity of physiological effects. Such responses are principally excitatory, and include the increase in firing rate of monoamine neurones in the CNS (Guyenet & Aghajanian, 1977), the depolarization of autonomic ganglia (Dun & Karczmar, 1979; Konishi et al., 1985), and contraction of smooth muscle (Lee et al., 1982). The coupling of these receptors to physiological responses involves G proteins, usually linked to the inhibition of one or more potassium conductances (Nakajima et al., 1988; Stanfield et al., 1985; Nowak & Macdonald, 1982). In sympathetic ganglia substance P is localized in ganglionic cell bodies and fibres of intrinsic interneurones (Hokfelt et al., 1977; Robinson et al., 1980; Dalsgaard et al., 1982). When released by either electrical or chemical stimulation this peptide elicits a postsynaptic depolarization caused partly by the inhibition of postsynaptic potassium conductances which includes, in some ganglia, the voltage-dependent M-current (Adams et al., 1983; Konishi et al., 1985).
'Author for correspondence.
At least three neurokinin receptor subtypes have been identified in the mammalian nervous system (reviewed in Iversen et al., 1990). To date their characterization has been based principally on the potency of the following agonists, substance P, neurokinin A, and neurokinin B, which are the prototypic NK1, NK2 and NK3 receptor selective ligands respectively. Recently, some important progress has been made in the development of specific NK1 and NK2 receptor antagonists (Williams et al., 1988; McKnight et al., 1988b; Ward et al., 1990; Snider et al., 1991). However, currently no selective NK3 antagonist is available, and thus agonist pharmacology still remains an important criterion in receptor classification. More than one receptor subtype can presumably coexist within any one tissue and the majority of tachykinin agonists can act upon more than one subtype; thus definitive studies into neurokinin receptor subtypes required full evaluation of both the potency and efficacy of a range of neurokinin agonists. It has been suggested that in a number of tissues the neurokinin agonist pharmacology does not conform to described receptor subtypes. These tissues include guinea-pig trachea where the existence of an NK4 receptor subtype has been suggested (McKnight et al., 1988a), although, with the use of NK1- and NK2-selective antagonists it is clear that more than one neurokinin receptor subtype may be present in this tissue (Ireland et al., 1991). In rat superior cervical ganglia an anomalous agonist profile has also been reported (Brown et al., 1983). In this study we present evidence for a heterogeneity of neurokinin receptors in rat and guinea-pig
926
G.R. SEABROOK et al.
superior cervical ganglia, demonstrating that their relative contribution to the maximal depolarization which can be elicited with tachykinins, exhibits differences between these two mammalian species.
Methods
a
t
t
Superior cervical ganglia were excised from male SpragueDawley rats (150-250g), and male Dunkin Hartley guineapigs (200-300g), which had previously been concussed and then exsanguinated. Ganglia were desheathed and then placed in a grease-gap recording chamber (Brown et al., 1980) through which warm physiological salt solution was continually perfused (25C, 1-2 ml min-'). The saline contained (in mM): NaCl 125, KCl 5, KH2PO4 1, CaCl2 2.5, MgSO4 1, NaHCO3 25, glucose 10 and tetrodotoxin 0.1 gM and was gassed with 95% 02: 5% CO2. The potential difference between the ganglion cell body (earthed) and the postganglionic trunk was monitored via Ag/AgCl electrodes connected via a d.c. amplifier to a chart recorder. Drugs were applied to the ganglion cell body for 1 min, at 40 min intervals to minimize receptor desensitization. Ganglia were allowed to stabilize until consecutive applications of either eledoisin (1 1AM) or senktide (1 g1M) produced depolarizations of equilavent amplitude and then the test ligand was applied in increasing concentration. The absolute potential changes were dependent upon the electrical seal and cell density of each preparation, and therefore data were normalized to a control application of eledoisin (1 MM) in each ganglion. This concentration produced submaximal depolarizations in ganglia of both species, and as such minimized errors associated with receptor desensitization. Eledoisin was used as the standard, in preference to substance P, because it produced large depolarizations in both rat and guinea-pig ganglia. Concentration-effect curves were fitted by least-squares analysis of variance to the equation Y = Ymax/1( + (EC50/ agonist concentration)nH), where EC50 is the half maximally effective concentration and nH is the Hill coefficient, using an iterative procedure on a VAX computer with Research System 1 software (BBN Software Products Corporation). Data are expressed as the mean ± standard error. Drugs were obtained from the following companies: neurokinin A (NKA), neurokinin B (NKB), senktide (SE), and Sar-Met-substance P (Sar-Met-SP) from Cambridge Research Biochemicals; eledoisin (E), substance P (SP), substance Pmethyl ester (SPOMe), tetrodotoxin (TTX), atropine sulphate, and bacitracin from Sigma; chymostatin and leupeptin from Bachem. Drugs were dissolved in distilled water, except for senktide which was dissolved in dimethylsulphoxide. Peptide stock solutions were stored in aliquots at -70'C.
Results Both rat and guinea-pig superior cervical ganglia were depolarized by the application of a range of neurokinin agonists. However, the relative potency and maximal ability of these ligands to depolarize ganglia was observed to vary between these two species.
Agonist pharmacology of rat ganglia In rat superior cervical ganglia, eledoisin was observed to be highly efficacious neurokinin agonist (Figure la), with an EC50 of 37 nM. The NK3-selective ligand, senktide (Wormser et al., 1986; Guard et al., 1990) proved to be the most potent ligand examined with an EC50 of 4 nM, although the maxi-
a
mum
depolarization produced in rat ganglia
was
only 27%
that of eledoisin and smaller than that of alternative agonists (Figure la and b). Similarly the NKI-selective ligands, SP, Sar-Met-SP and SPOMe, all exhibited maximal depolariza-
t
SE
E
NKA
t
t
E
SPO
0.2 mV
10 min
.E
b
.O 150
NKA
0
'a
a)
SPOMe
100
.-
o
0
Zs(a
50
Senktide
w7-
N
o0
-;i
10
100
1000 10 000
log [Agonist] (nM)
Figure 1 The depolarization of rat superior cervical ganglia by subtype selective neurokinin agonists. (a) At the time points indicated by the arrows the following neurokinin agonists were applied (3 1M) for a I min period in the presence of atropine (1 gM) and tetrodotoxin (0.1 AM): eledoisin (E), senktide (SE), neurokinin A (NKA), and substance P methyl ester (SPO). (b) The resultant depolarizations in the same ganglia were normalized to the control response with eledoisin. These normalized depolarizations, from a number of ganglia, were used to calculate concentration-effect curves for each agonist (see Table 1). Eledoisin proved to be the most efficacious compound at a concentration of 1 AM, with SPOMe and senktide producing maximal depolarizations of 72% and 27% respectively.
tions of only 72-80% that of eledoisin (Table 1). The potency of substance P was unaffected during continuous perfusion of senktide (1 MM; n = 4) which desensitized NK3 receptors. Pretreatment of rat ganglia with the peptidase inhibitors bacitracin (40 Lg ml'), chymostatin (2pg ml-') and leupeptin (4 Mg ml-') had no effect upon the potency or efficacy of SPOMe (36 ± 13 nM; 117 + 7%, n = 7) or neurokinin A (288 ± 95 nM, 119 ± 7%, n = 6). Both neurokinin A and neurokinin B, peptides which are preferentially, although not exclusively, selective for NK2 and NK3 receptors respectively, both exhibited weak potencies and had Hill slopes significantly less than one. The maximal responses obtained with NKA and NKB at high concentrations were greater than those obtained with eledoisin (Table 1).
Agonist pharmacology of guinea-pig ganglia In guinea-pig superior cervical ganglia, in contrast to the rat, senktide proved to be the most active compound in terms of both efficacy and potency (Figure 2a and b). The maximal depolarization achieved by the NK, selective ligand SPOMe was only 23% that of senktide, and 64% that of 1 fM eledoisin (Table 2). With NKA and SP, the concentrationeffect curves were clearly biphasic, suggestive of an interaction at more than one receptor type. However, the lack of a distinct maximal depolarization with these weaker agonists prevented a reasonable fit to either a single- or two-site logistic model of ligand-receptor interaction. The depolariza-
927
NEUROKININ RECEPTORS IN SYMPATHETIC GANGLIA Table 1 Neurokinin receptor agonist potency and ability to depolarize rat superior cervical ganglia
Ligand
Senktide Substance P SPOMe Eledoisin Sar-Met-SP NKB NKA
Table 2 Neurokinin receptor agonist potency and ability to depolarize guinea-pig superior cervical ganglia
EC50 (nM)
Max (% I fiM eledoisin)
Hill slope
n
Ligand
4±2 12±3 30±6 33±9 39±4 117 ± 65 268 ± 35
27 ± 3
0.99 ± 0.45 0.94 ± 0.16
7 8 8 9 7 7 7
37 ± 14 274 ± 24 102 ± 8 90 ± 26 NKB 259 ± 20 1473 ± 545 273 ± 5 1918 ± 97 Eledoisin 64 ± 41 2052 ± 4552 SPOMe Indeterminate 26% at I pIM NKA Substance P Indeterminate 25% at 1 jM
Max (% I FM
72±4 77 ± 5 103 ± 8 80± 3 132 ± 18 140 ± 5
1.04±0.17 1.06 ± 0.21 1.52 ± 0.22 0.67 ± 0.15 0.76 ± 0.04
SPOMe = substance P methyl ester; NKA, NKB = neurokinins A and B; Sar-Met-SP = Sar-Met-subtance P.
Senktide Sar-Met-SP
EC50 (nM)
eledoisin)
Hill slope
n
0.74 ± 0.12 0.89 ± 0.23 0.53 ± 0.06 0.85 ± 0.02 0.67 ± 0.47 Biphasic Biphasic
8 4 6 9 9 8
3
Sar-Met-SP = Sar-Met-substance P; SPOMe = substance P methyl ester; NKA = neurokinin A.
statin (2 jig ml-') and leupeptin (4 Ag ml ') had no significant effect upon the potency or efficacy of SPOMe (2.9 + 2.1 AM, 44 ± 18%, n = 4), senktide (23 ± 4 nM, 200 ± 9%, n = 4), or neurokinin A (biphasic, 15% at 1 g1M).
a
II
Discussion
1< t
E
t
t
t
rNNKA SPO
SE
t E
0.2 mV 10 min C ._
b
.
(1
a1
SPOMe 10
100
1000
10 000 100 000
log [Agonist] (nM) Figure 2 The depolarization of guinea-pig superior cervical ganglia by subtype selective neurokinin agonists. (a) At the time points indicated by the arrows the following neurokinin agonists were applied (3 pM) for a 1 min period in the presence of atropine (1 AM) and tetrodotoxin (0.1 Mm): eledoisin (E), senktide (SE), neurokinin A (NKA), and substance P methyl ester (SPO). The slower rate of decay of these traces relative to Figure la reflect differences in flow rate of the drug solutions through the bath. (b) The normalized depolarizations in a number of ganglia were used to calculate concentration-effect curves for each agonist (see Table 2). Senktide proved to be the most efficacious agonist, the maximal depolarization achieved by SPOMe was 23% that of senktide. The concentration-effect relationship with neurokinin A was biphasic, and the depolarization was not maximal at 30 MM.
tions elicited by senktide or Sar-Met-SP were not blocked by application of atropine (Figure 2a), and similarly the potency of senktide was unaffected by continuous perfusion of substance P (1 gM; n = 4) which desensitizes NK, receptors. As was seen in rat tissue, pretreatment of guinea-pig ganglia with the peptidase inhibitors bacitracin (40 Mg ml'), chymo-
This study demonstrates that neurokinin receptor pharmacology differs in rat and guinea-pig superior cervical ganglia. These differences exist not only in terms of agonist potency, but also in terms of relative agonist efficacy. It has previously been reported that substance P depolarizes rat superior cervical ganglia (Hawcock et al., 1982), but that it is a relatively weak agonist with an EC50 of 297 nm (Brown et al., 1983). In the present study, substance P was found to be 20 times more potent. The rank order of agonist potency in this tissue is therefore consistent with an NK, receptor as the predominant receptor population. The lack of involvement of NK2 receptors was indicated by the absence of a response to low concentrations of NKA, and the insensitivity of the SPOMe-mediated depolarizations to the NK2 selective antagonist L-659,877 (data not shown). Senktide binds selectively to both central and peripheral NK3 receptors with high affinity (Guard et al., 1990). The high potency and relatively weak efficacy of senktide in rat ganglia thus provides evidence for the presence of an NK3 receptor subtype. Similarly the greater efficacy of eledoisin relative to NK, selective ligands suggests that the response to this ligand is a consequence of activation of both NK, and NK3 receptors. The depolarization caused by the NK, receptor agonist SPOMe (72%) and the NK3 receptor agonist senktide (27%) were equieffective to the maximal depolarization achieveable with the non-selective agonist, eledoisin (see Figures lb, 2b). In guinea-pig ileum, both NK1 and NK3 receptors contribute to the contraction of smooth muscle (Lee et al., 1982). These NK3 receptors are located on the myenteric plexus and mediate their effects by the release of acetylcholine. The NK3-mediated contraction can be blocked by incubation of the tissue in atropine to block the postjunctional muscarinic receptors. However, unlike guinea-pig ileum, application of atropine did not antagonize the neurokinin-mediated depolarizations in rat ganglia (Figure la), which suggests the absence of presynaptic neurokinin receptors in this response. The variation in efficacy of neurokinin agonists in this tissue can be explained by receptor heterogeneity, and/or by partial agonism. The possibility of heterogeneity of receptor subtypes is difficult to test due to the lack of a selective NK3 receptor antagonist. However, as previously discussed there is some evidence for such heterogeneity in rat ganglia based upon the relative potency of senktide. Also consistent with this interpretation is the observation that desensitization of presumptive NK3 receptors with prolonged application of senktide did not reduce the maximal depolarization obtain-
928
G.R. SEABROOK et al.
able with substance P. During this experiment the potency of substance P remained unaffected, therefore it is unlikely that senktide acted as partial agonist at an identical receptor population. It is not clear as to whether both NKI and NK3 receptors are located on common neuronal cell bodies, or whether their distribution is more discrete, in that the receptor distribution reflects separate neurokinin pathways through the ganglia. It is interesting to note that the pharmacology of superior cervical ganglia may be different between species for 5-hydroxytryptamine (Newberry et al., 1991). Indeed in the present study the response to neurokinin receptor agonists was also found to differ between species. In guinea-pig superior cervical ganglia, in contrast to the rat, the majority of ligands tested were only weak agonists with EC50s typically greater than 1 pM (Table 2). The most potent ligand senktide, 37 nM, also exhibited the most efficacious response, whereas substance P was only weakly active in this species. It is unlikely that NK3 receptors are the exclusive neurokinin receptor in guinea-pig superior cervical ganglia as biphasic concentra-
tion-effect curves were observed with NKA and substance P. The binding of senktide to neurokinin receptors in cerebral cortex is similar for both rats and guinea-pigs (Renzetti et al., 1991), whereas NKB binding was shown to be 27 fold weaker in guinea-pig tissue; this may indicate subtle differences in the agonist recognition site between the two species. In conclusion we have shown that neurokinin receptor agonists depolarize both rat and guinea-pig superior cervical ganglia, but that the potency and efficacy of these ligands differ between these two species. Rat ganglia appear to express multiple neurokinin receptor subtypes, the predominant receptor population being of an NKI subtype, whereas in guinea-pig ganglia NK3 receptors appear to be the principal subtype involved. The development of a selective NK3 receptor antagonist will help classify the receptor subtypes present in this tissue. We would like to thank Dr J.A. Kemp for helpful comments on this manuscript.
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(Received October 3, 1991 Revised December 6, 1991 Accepted December 23, 1991)
