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Neurokinin A and substance P vary independently in different regions of rat sensory neurons
Neuropeptides (1995) 28, 237-241 ,© Pearson Professional Ltd 1995
Neurokinin A and Substance P Vary Independently in Different Regions of Rat Sensory Neurons Y. S. BAKHLE* and C. BELL1-
*Department of Applied Pharmacology, National Heart and Lung Institute, London SW3 6L Y, UK and f Department of Physiology, University of Melbourne, Victoria 3052, Australia
Abstract--Neurokinin A (NKA) and substance P (SP) are often co-localized in small sensory neurons and have been suggested to subserve similar roles. We have now compared tissue levels of NKA and SP in rat cervical dorsal root ganglia with those in the central and peripheral terminations of the same neurons. We found that N KA content was less that that of SP in dorsal root neuron perikarya and in two peripheral tissues (superior cervical ganglion and ear skin) containing SP axon terminals from cervical spinal ganglia; in a third peripheral tissue, trachea, equal amounts of NKA and SP were present. By contrast, in the spinal cord containing the central terminals of these sensory neurons there was almost twice as much NKA as SP. Our results indicate that, although NKA and SP are co-localized in sensory neurons, their levels vary independently, suggesting distinct functional roles.
Introduction The tachykinin SP is thought to be one of the main neurotransmitters transducing nociceptive sensory information. 1'2 The peripheral endings of SP-containing sensory nerves are widely distributed in peripheral tissue elements such as glands, blood vessels and skin and, at these sites, SP has been implicated in generation of antidromic vasodilation and fluid extravasation 3'4 SP-containing sensory axons are also seen within sympathetic ganglia, where they
Date received 16 August 1994 Date accepted 19 September 1994 Correspondence to Dr Y. S. Bakhle, Department of Applied Pharmacology, National Heart and Lung Institute, London SW3 6LY, UK.
form contacts on the sympathetic motoneurons and have been suggested to modulate synaptic transmission. 4 Numerous reports have documented the apparent co-localization of N K A with SP in sensory neurons (e.g. refs 5-8) and in neurons of the enteric nervous system. 7,9 These tachykinins are closely similar in structure, and exhibit similar functional properties in a variety of situations. 7,1° Furthermore, they have a c o m m o n gene (PPT1) and comm o n derived m R N A s coding for the tachykinin precursors fl and 7 preprotachykinin (fi and 7 PPT), each containing one copy of SP and one of NKA.I~,~2 In view of these similarities, the general presumption has been that N K A and SP are coreleased and fulfil similar neurotransmitter roles. However, no data appear to be available on the
237
238 intracellular distribution and handling of the two tachykinins within the sensory neuron. In this study, we have used radioimmunoassay (RIA) to measure tissue levels of N K A and SP in peripheral and central regions of sensory neurons in normal rats and in a related strain of rats (GH Wistar) in which there is a genetic neurotrophic defect resulting in an increase of SP-containing spinal sensory neurones. 13,14
NEUROPEPTIDES USA). The methanolic extracts were evaporated under reduced pressure and re-dissolved in 100 #l RIA buffer and assays were performed as prescribed in the kits. Cross-reactivities between the antisera used are anti-SP vs N K A or NKB < 0.5%; anti-NKA vs SP 0.01%. The N K A antiserum is also reported by the suppliers to cross-react strongly with N K B (80%).
Statistics Materials and methods
Animals The rats used in this study were bred in the University of Melbourne from stock obtained in 1984 from the Wellcome Medical Research Institute, University of Otago. All experimental procedures were in accord with the guidelines laid down by the National Health & Medical Research Council of Australia and were approved in advance by the University of Melbourne Animal Experimentation Ethics Committee.
Tissue preparation Young adult (6--8 weeks old) male animals of either strain were killed with an overdose of sodium pentobarbitone intraperitoneally (i.p.) and the C6 spinal cord segment and C7 dorsal root ganglia were removed, together with the superior cervical ganglia and samples of ear and trachea. All samples were placed on pre-weighed squares of aluminium foil on ice, weighed and stored at - 30°C until assayed.
Assays Tissues were heated in hot 1 M acetic acid (boiling water bath) for 10 rain and then homogenized, cooled and centrifuged. Supernatants were evaporated to dryness under reduced pressure and reconstituted in 1 ml of 4% (v/v) aqueous acetic acid. ~5 This extract was passed through a SepPak cartridge (C18, Waters, Mass, USA) and washed with 2 x 5 ml of 4% acetic acid. The absorbed peptide was eluted with 2 ml of 90% methanol in (v/v) 4% acetic acid? 5 Assays for immunoreactive SP (ir-SP) and N K A (ir-NKA) were carried out by RIA using kits supplied by Peninsula Laboratories Inc. (Belmont, CA,
Results are given as mean + SEM values from 56 tissues assayed. Comparisons of means were made using an unpaired two-tailed Student's t-test, and a value of P <0.05 was taken as indicating a significant difference.
Results
The content of immunoreactive N K A (ir-NKA) and ir-SP in three peripheral tissues receiving SP sensory neurons, the superior cervical ganglion, ear skin and trachea, are summarized in Figure 1. In normal Wistar rats (control), for two tissues (superior cervical ganglion and ear skin), the content of ir-NKA was about half that ofir-SP whereas in the trachea approximately equal amounts of the two tachykinins were found. In the G H rats, where there is exaggerated developmental survival of a subpopulation of SP sensory neurons sensitive to an unidentified neurotrophin, 13"16tissue content of N K A was not changed either in the superior cervical ganglion and trachea, which have higher than normal contents of SP, or in the ear skin. The tachykinin levels in perikarya and the central terminals of the same sensory neurons are shown in Figure 2. The cell bodies in the dorsal root ganglia contained about half as much ir-NKA as ir-SP, in both strains. By contrast, in spinal cord where both tachykinins represent the terminations of spinal sensory neurons, the concentrations of ir-NKA in normal animals was about 50% higher than that of ir-SP. Furthermore, unlike the results from the peripheral axon terminals where increased SP innervation density was accompanied by an increase of tissue ir-SP but not of ir-NKA, the spinal cord of G H animals contained elevated levels of both ir-NKA and ir-SP.
239
RAT PERIPHERAL NEUROKININS
15
DRG
.
5
;Z F(>
500-
.
EAR
~ 400c
300-
2004a
, g lo0VO
TRAOHEA -I-
II
FControl
Control
GH
Fig. 2 ir-NKA and ir-SP in dorsal root ganglia and spinal cord from normal Wistar (control) and the related GH strain (GH) rats. The content of ir-NKA ([7) and ir-SP (N) are shown as the mean (_+ SEM) values from 5-6 animals for either strain of rat. *Significantly different from value for ir-NKA; P < 0.05. + Significantly different from value in control strain; P < 0.05.
GH
Fig. 1 Content of ir-NKA and ir-SP in peripheral tissues from normal Wistar (control) and the related GH strain (GH) rats. The mean (+_ SEM) values of ir-NKA (D) and ir-SP (gt) from tissues from 5-6 animals for either strain are shown. *Significantly different from value for ir-NKA; P < 0.05. +Significantly different from value in control strain; P < 0.05.
Discussion
Although ir-SP and i r - N K A are both synthesised and stored in the same sensory neurons and can both be released in response to electrical activation
of these neurons, 7,I7,18 our results show that there are striking differences between the amounts of these two tachykinins in different parts of sensory neurons. Measurement of these tachykinins in tissues containing peripheral axons originating from SP-containing sensory neurons revealed that in two tissues, the superior cervical ganglion and ear, levels of i r - N K A were only about half those of ir-SP. In the superior cervical ganglion, the sympathetic neurons receive synaptic inputs from collaterals of cervical sensory axons projecting to the ear and other targets in head and neck. 14 In the third tissue examined, trachea, there was no difference between the amounts of the two tachykinins, a finding comparable to that of H u a & Yaksh. 19 In this tissue a considerable proportion
240 of the sensory innervation is provided by axons running along the vagus. 2° As a consequence, any particular tachykinin ratio associated with the spinal sensory fibres could be obscured by the tachykinin ratio in those sensory neurons derived from vagal ganglia. Some support for the possibility of a uniform ratio is given by the values for the dorsal root ganglia containing the cell bodies of these sensory neurons; thus the perikarya and two loci of peripheral terminals exhibit the same ratio of tachykinin content, i.e. less i r - N K A than ir-SP. However this postulate is not supported by the excess of ir-NKA over ir-SP found in the central terminals of the sensory neurons in the spinal cord. Whereas an excess of SP might have reflected the presence of e PPT, the precursor containing only SP and not N K A , "'12 there is no comparable form of PPT containing only the sequence for N K A without SP, that might give rise to such a preponderance of N K A . This distinction, i.e., an excess of N K A over SP, was also observed in the G H rats where the amounts of N K A were unchanged in all tissues examined, except the spinal cord. Here the increase in ir-SP characteristic of this strain, ~3'~6 was accompanied by an increase in ir-NKA, maintaining the excess observed in normal Wistar rats. It is clear that there was no fixed ratio of tachykinin content either within a strain or between strains, implying the influence of factors other than those predictable from the postulate of co-localization and the structure of the predominant tachykinin precursors, fi and ~ PPT. 11'12 Such a variety of tachykinin content might suggest an equivalent variety in the tachykinin released. However, the relation between the amounts of SP and of N K A released from nerves and the functional responses to nerve stimulation has been difficult to assess. One confounding factor is that both peptides are full agonists at NK1 or N K 2 receptors, with only a potency difference to distinguish them. ~° Also, in many tissues the effects of SP and N K A are identical and the response to a small amount of one can be mimicked by that to a larger amount of the other. Nevertheless, some studies do support independent actions of the two substances. For example, selective antibodies against either SP or N K A have been shown to only partially inhibit responses to nerve stimulation, while tachykinin receptor antagonists provided
NEUROPEPTIDES complete inhibition. 17'18 Furthermore, in lung, SP selectively acts as a secretagogue of mucus while N K A selectively acts as a bronchoconstrictor. ~° Such differences could be reflected in a particular content of tachykinins related to the target tissue. Since the N K A antiserum we used had extensive cross-reactivity with N K B (80%), some of the irN K A we measured may have represented NKB. Against this possibility is the generally low level of N K B in rat tissues; a wide range of peripheral tissues and dorsal root ganglia have all been found to contain no detectable N K B and it is less than 10% the level of N K A in spinal cord. 21'22Overall, therefore, our results reinforce the view that N K A and SP may have distinct functional roles within the sensory nervous system and that this is reflected in distinctive patterns of distribution.
Acknowledgements This work was supported by grants from the National Health & Medical Research Councilof Australia and the Helen M. Schutt Trust, Y.S.B. was a University of Melbourne Visiting Fellow and also thanks the WellcomeTrust and Draco AB for support. We are grateful to Boon-TiongTan for assistance with some experiments and to Richard Davies for co-ordinationof Qantas cargo shipments.
References 1. Duggan,A. W., Hendry, I. A., Morton, C. R., Hutchinson, W. D. and Zhao, Z. Q. Cutaneous stimuli releasing immunoreactive substance P in the dorsal horn of the cat. Brain Res. 1988;451: 261-273. 2. Otsuka, M. and Konishi, S. Electrophysiologicaland neurochemical evidence for substance P as a transmitter of primary sensory neurons. In: U.S. Von Euler, B. Pernow, eds. Substance P. Nobel Symposium37. New York: Raven Press, 1977:207-214. 3. Chahl, L. A. Antidromic vasodilatation and neurogenic inflammation. In: C. Bell, ed. Novel Peripheral Neurotransmitters. New York: PergamonPress, 1991: 161-192. 4. Holzer, P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 1988;24: 739-768. 5. Hua, X-Y. Theodorsson-Norheim, E., Brodin, E., Lundberg, J. and HOkfelt, T. Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in capsaicin-sensitive sensory neurons in the guinea-pig. Regul. Pept. 1985; 13: 119. 6. Martling, C-R., Theodorsson-Norheim, E. and Lundberg, J. Occurrenceand effectsof multiple tachykinins: substance P, neurokinin A and neuropeptide K in human lower airways. Life Sci. 1987;40: 1633-1643.
RAT PERIPHERAL NEUROKININS 7. Otsuka, M. and Yoshioka, K. Neurotransmitter functions of mammalian tachykinins. Physiol. Rev. 1993; 73: 229-308. 8. Dalsgaard, C. J., Haegerstrand, A., Theodorsson-Norheim, E., Brodin, E. and H6kfelt, T. Neurokinin A-like immunoreactivity in rat primary sensory neurons; co-existence with substance P. Histochemistry 1985; 83:37 39. 9. Deacon, C. L. and Conlon, J. M. Biosynthesis of substance P and neurokinin A in the enteric nervous system. In: J. L. Henry, R. Contnre, A. C. Cnello, G. Pelletier, R. Quirion and D. Regoli, eds. Substance P and Neurokinins. New York: Springer-Verlag, 1989; 26-29. 10. Maggi, C. A., Patacchini, R., Rovero, P. and Giachetti, A. Tachykinin receptors and tachykinin receptor antagonists, J. Auton. Pharmacol. 1993; 13: 23-93, 11. Helke, C. J., Krause, J. E., Mantyh, P, W., Couture, R. and Bannon, M. J. Diversity in mammalian tachykinin peptidergic neurons: multiple peptides. FASEB J. 1990; 4: 16061615. 12. Nawa, H., Kotani, H. and Nakanishi, S. Tissue-specific generation of two preprotachykinin mRNAs from one gene by alternative RNA splicing. Nature 1984; 312: 729-734. 13. Bakhle, Y. S. and Bell, C. Increased numbers of substance P-containing sensory neurons in a rat strain with a genetic neurotrophic defect. Neuropeptides (1994) in press. 14. Gurusinghe, C. J. and Bell, C. Different patterns of immunolocalization of calcitonin gene-related peptide and substance P in sympathetic ganglia of normotensive and genetically hypertensive rats. Neurosci. Lett. t989; 106:89 94.
241 15. Gepetti, P., Maggi, C. A., Zecchi-Orlandini, S. et al. Substance P-like immunoreactivity in capsaicin-sensitive structures of the thymus. Regul. Pept. 1987; 18: 321-329. 16. Messina, A. and Bell, C. Are genetically hypertensive rats deficient in nerve growth factor? Neuroreport 1991; 2: 4548. 17. Maggi, C. A., Patacchini, R., Baroldi, P., Theodorsson, E. and Meli, A. Immunoblockade by a specific tachykinin antiserum of the non-cholinergic contractile responses in the guinea pig isolated bronchus. J. Auton. Pharmacol. 1990; 10:173 179. I8. Beding-Barnekow, B., Brodin, E. and Hakanson, R. Substance P, neurokinin A and neurokinin B in the ocular response to injury in the rabbit. Br. J. Pharmacol. 1988; 95: 259-267. 19. Hua, X-Y. and Yaksh, T. L. Release of calcitonin generelated peptide and tachykinins from rat trachea, Peptides 1992; 13: 113-120. 20. Lundberg, J. M., Hokfelt, T., Martling, C-R., Saria, A. and Cuello, C. Substance P-immunoreactive sensory nerves in the lower respiratory tract of various mammals including man. Cell Tissue Res. 1984; 235: 251-261. 21. Moussaoni, S. M., LePrado, N., Bonici, B. et al. Distribution of neurokinin B in rat spinal cord and peripheral tissues: comparison with neurokinin A and substance P and effects of neonatal capsaicin treatment. Neuroscience 1992; 48: 969-978. 22. Too, H. P. and Maggio, J. E. Immunocytochemical localization of neuromedin K (neurokinin B) in rat spinal ganglia and cord. Peptides 1991; 12: 431-443.
Neurokinin A and Substance P Vary Independently in Different Regions of Rat Sensory Neurons Y. S. BAKHLE* and C. BELL1-
*Department of Applied Pharmacology, National Heart and Lung Institute, London SW3 6L Y, UK and f Department of Physiology, University of Melbourne, Victoria 3052, Australia
Abstract--Neurokinin A (NKA) and substance P (SP) are often co-localized in small sensory neurons and have been suggested to subserve similar roles. We have now compared tissue levels of NKA and SP in rat cervical dorsal root ganglia with those in the central and peripheral terminations of the same neurons. We found that N KA content was less that that of SP in dorsal root neuron perikarya and in two peripheral tissues (superior cervical ganglion and ear skin) containing SP axon terminals from cervical spinal ganglia; in a third peripheral tissue, trachea, equal amounts of NKA and SP were present. By contrast, in the spinal cord containing the central terminals of these sensory neurons there was almost twice as much NKA as SP. Our results indicate that, although NKA and SP are co-localized in sensory neurons, their levels vary independently, suggesting distinct functional roles.
Introduction The tachykinin SP is thought to be one of the main neurotransmitters transducing nociceptive sensory information. 1'2 The peripheral endings of SP-containing sensory nerves are widely distributed in peripheral tissue elements such as glands, blood vessels and skin and, at these sites, SP has been implicated in generation of antidromic vasodilation and fluid extravasation 3'4 SP-containing sensory axons are also seen within sympathetic ganglia, where they
Date received 16 August 1994 Date accepted 19 September 1994 Correspondence to Dr Y. S. Bakhle, Department of Applied Pharmacology, National Heart and Lung Institute, London SW3 6LY, UK.
form contacts on the sympathetic motoneurons and have been suggested to modulate synaptic transmission. 4 Numerous reports have documented the apparent co-localization of N K A with SP in sensory neurons (e.g. refs 5-8) and in neurons of the enteric nervous system. 7,9 These tachykinins are closely similar in structure, and exhibit similar functional properties in a variety of situations. 7,1° Furthermore, they have a c o m m o n gene (PPT1) and comm o n derived m R N A s coding for the tachykinin precursors fl and 7 preprotachykinin (fi and 7 PPT), each containing one copy of SP and one of NKA.I~,~2 In view of these similarities, the general presumption has been that N K A and SP are coreleased and fulfil similar neurotransmitter roles. However, no data appear to be available on the
237
238 intracellular distribution and handling of the two tachykinins within the sensory neuron. In this study, we have used radioimmunoassay (RIA) to measure tissue levels of N K A and SP in peripheral and central regions of sensory neurons in normal rats and in a related strain of rats (GH Wistar) in which there is a genetic neurotrophic defect resulting in an increase of SP-containing spinal sensory neurones. 13,14
NEUROPEPTIDES USA). The methanolic extracts were evaporated under reduced pressure and re-dissolved in 100 #l RIA buffer and assays were performed as prescribed in the kits. Cross-reactivities between the antisera used are anti-SP vs N K A or NKB < 0.5%; anti-NKA vs SP 0.01%. The N K A antiserum is also reported by the suppliers to cross-react strongly with N K B (80%).
Statistics Materials and methods
Animals The rats used in this study were bred in the University of Melbourne from stock obtained in 1984 from the Wellcome Medical Research Institute, University of Otago. All experimental procedures were in accord with the guidelines laid down by the National Health & Medical Research Council of Australia and were approved in advance by the University of Melbourne Animal Experimentation Ethics Committee.
Tissue preparation Young adult (6--8 weeks old) male animals of either strain were killed with an overdose of sodium pentobarbitone intraperitoneally (i.p.) and the C6 spinal cord segment and C7 dorsal root ganglia were removed, together with the superior cervical ganglia and samples of ear and trachea. All samples were placed on pre-weighed squares of aluminium foil on ice, weighed and stored at - 30°C until assayed.
Assays Tissues were heated in hot 1 M acetic acid (boiling water bath) for 10 rain and then homogenized, cooled and centrifuged. Supernatants were evaporated to dryness under reduced pressure and reconstituted in 1 ml of 4% (v/v) aqueous acetic acid. ~5 This extract was passed through a SepPak cartridge (C18, Waters, Mass, USA) and washed with 2 x 5 ml of 4% acetic acid. The absorbed peptide was eluted with 2 ml of 90% methanol in (v/v) 4% acetic acid? 5 Assays for immunoreactive SP (ir-SP) and N K A (ir-NKA) were carried out by RIA using kits supplied by Peninsula Laboratories Inc. (Belmont, CA,
Results are given as mean + SEM values from 56 tissues assayed. Comparisons of means were made using an unpaired two-tailed Student's t-test, and a value of P <0.05 was taken as indicating a significant difference.
Results
The content of immunoreactive N K A (ir-NKA) and ir-SP in three peripheral tissues receiving SP sensory neurons, the superior cervical ganglion, ear skin and trachea, are summarized in Figure 1. In normal Wistar rats (control), for two tissues (superior cervical ganglion and ear skin), the content of ir-NKA was about half that ofir-SP whereas in the trachea approximately equal amounts of the two tachykinins were found. In the G H rats, where there is exaggerated developmental survival of a subpopulation of SP sensory neurons sensitive to an unidentified neurotrophin, 13"16tissue content of N K A was not changed either in the superior cervical ganglion and trachea, which have higher than normal contents of SP, or in the ear skin. The tachykinin levels in perikarya and the central terminals of the same sensory neurons are shown in Figure 2. The cell bodies in the dorsal root ganglia contained about half as much ir-NKA as ir-SP, in both strains. By contrast, in spinal cord where both tachykinins represent the terminations of spinal sensory neurons, the concentrations of ir-NKA in normal animals was about 50% higher than that of ir-SP. Furthermore, unlike the results from the peripheral axon terminals where increased SP innervation density was accompanied by an increase of tissue ir-SP but not of ir-NKA, the spinal cord of G H animals contained elevated levels of both ir-NKA and ir-SP.
239
RAT PERIPHERAL NEUROKININS
15
DRG
.
5
;Z F(>
500-
.
EAR
~ 400c
300-
2004a
, g lo0VO
TRAOHEA -I-
II
FControl
Control
GH
Fig. 2 ir-NKA and ir-SP in dorsal root ganglia and spinal cord from normal Wistar (control) and the related GH strain (GH) rats. The content of ir-NKA ([7) and ir-SP (N) are shown as the mean (_+ SEM) values from 5-6 animals for either strain of rat. *Significantly different from value for ir-NKA; P < 0.05. + Significantly different from value in control strain; P < 0.05.
GH
Fig. 1 Content of ir-NKA and ir-SP in peripheral tissues from normal Wistar (control) and the related GH strain (GH) rats. The mean (+_ SEM) values of ir-NKA (D) and ir-SP (gt) from tissues from 5-6 animals for either strain are shown. *Significantly different from value for ir-NKA; P < 0.05. +Significantly different from value in control strain; P < 0.05.
Discussion
Although ir-SP and i r - N K A are both synthesised and stored in the same sensory neurons and can both be released in response to electrical activation
of these neurons, 7,I7,18 our results show that there are striking differences between the amounts of these two tachykinins in different parts of sensory neurons. Measurement of these tachykinins in tissues containing peripheral axons originating from SP-containing sensory neurons revealed that in two tissues, the superior cervical ganglion and ear, levels of i r - N K A were only about half those of ir-SP. In the superior cervical ganglion, the sympathetic neurons receive synaptic inputs from collaterals of cervical sensory axons projecting to the ear and other targets in head and neck. 14 In the third tissue examined, trachea, there was no difference between the amounts of the two tachykinins, a finding comparable to that of H u a & Yaksh. 19 In this tissue a considerable proportion
240 of the sensory innervation is provided by axons running along the vagus. 2° As a consequence, any particular tachykinin ratio associated with the spinal sensory fibres could be obscured by the tachykinin ratio in those sensory neurons derived from vagal ganglia. Some support for the possibility of a uniform ratio is given by the values for the dorsal root ganglia containing the cell bodies of these sensory neurons; thus the perikarya and two loci of peripheral terminals exhibit the same ratio of tachykinin content, i.e. less i r - N K A than ir-SP. However this postulate is not supported by the excess of ir-NKA over ir-SP found in the central terminals of the sensory neurons in the spinal cord. Whereas an excess of SP might have reflected the presence of e PPT, the precursor containing only SP and not N K A , "'12 there is no comparable form of PPT containing only the sequence for N K A without SP, that might give rise to such a preponderance of N K A . This distinction, i.e., an excess of N K A over SP, was also observed in the G H rats where the amounts of N K A were unchanged in all tissues examined, except the spinal cord. Here the increase in ir-SP characteristic of this strain, ~3'~6 was accompanied by an increase in ir-NKA, maintaining the excess observed in normal Wistar rats. It is clear that there was no fixed ratio of tachykinin content either within a strain or between strains, implying the influence of factors other than those predictable from the postulate of co-localization and the structure of the predominant tachykinin precursors, fi and ~ PPT. 11'12 Such a variety of tachykinin content might suggest an equivalent variety in the tachykinin released. However, the relation between the amounts of SP and of N K A released from nerves and the functional responses to nerve stimulation has been difficult to assess. One confounding factor is that both peptides are full agonists at NK1 or N K 2 receptors, with only a potency difference to distinguish them. ~° Also, in many tissues the effects of SP and N K A are identical and the response to a small amount of one can be mimicked by that to a larger amount of the other. Nevertheless, some studies do support independent actions of the two substances. For example, selective antibodies against either SP or N K A have been shown to only partially inhibit responses to nerve stimulation, while tachykinin receptor antagonists provided
NEUROPEPTIDES complete inhibition. 17'18 Furthermore, in lung, SP selectively acts as a secretagogue of mucus while N K A selectively acts as a bronchoconstrictor. ~° Such differences could be reflected in a particular content of tachykinins related to the target tissue. Since the N K A antiserum we used had extensive cross-reactivity with N K B (80%), some of the irN K A we measured may have represented NKB. Against this possibility is the generally low level of N K B in rat tissues; a wide range of peripheral tissues and dorsal root ganglia have all been found to contain no detectable N K B and it is less than 10% the level of N K A in spinal cord. 21'22Overall, therefore, our results reinforce the view that N K A and SP may have distinct functional roles within the sensory nervous system and that this is reflected in distinctive patterns of distribution.
Acknowledgements This work was supported by grants from the National Health & Medical Research Councilof Australia and the Helen M. Schutt Trust, Y.S.B. was a University of Melbourne Visiting Fellow and also thanks the WellcomeTrust and Draco AB for support. We are grateful to Boon-TiongTan for assistance with some experiments and to Richard Davies for co-ordinationof Qantas cargo shipments.
References 1. Duggan,A. W., Hendry, I. A., Morton, C. R., Hutchinson, W. D. and Zhao, Z. Q. Cutaneous stimuli releasing immunoreactive substance P in the dorsal horn of the cat. Brain Res. 1988;451: 261-273. 2. Otsuka, M. and Konishi, S. Electrophysiologicaland neurochemical evidence for substance P as a transmitter of primary sensory neurons. In: U.S. Von Euler, B. Pernow, eds. Substance P. Nobel Symposium37. New York: Raven Press, 1977:207-214. 3. Chahl, L. A. Antidromic vasodilatation and neurogenic inflammation. In: C. Bell, ed. Novel Peripheral Neurotransmitters. New York: PergamonPress, 1991: 161-192. 4. Holzer, P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 1988;24: 739-768. 5. Hua, X-Y. Theodorsson-Norheim, E., Brodin, E., Lundberg, J. and HOkfelt, T. Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in capsaicin-sensitive sensory neurons in the guinea-pig. Regul. Pept. 1985; 13: 119. 6. Martling, C-R., Theodorsson-Norheim, E. and Lundberg, J. Occurrenceand effectsof multiple tachykinins: substance P, neurokinin A and neuropeptide K in human lower airways. Life Sci. 1987;40: 1633-1643.
RAT PERIPHERAL NEUROKININS 7. Otsuka, M. and Yoshioka, K. Neurotransmitter functions of mammalian tachykinins. Physiol. Rev. 1993; 73: 229-308. 8. Dalsgaard, C. J., Haegerstrand, A., Theodorsson-Norheim, E., Brodin, E. and H6kfelt, T. Neurokinin A-like immunoreactivity in rat primary sensory neurons; co-existence with substance P. Histochemistry 1985; 83:37 39. 9. Deacon, C. L. and Conlon, J. M. Biosynthesis of substance P and neurokinin A in the enteric nervous system. In: J. L. Henry, R. Contnre, A. C. Cnello, G. Pelletier, R. Quirion and D. Regoli, eds. Substance P and Neurokinins. New York: Springer-Verlag, 1989; 26-29. 10. Maggi, C. A., Patacchini, R., Rovero, P. and Giachetti, A. Tachykinin receptors and tachykinin receptor antagonists, J. Auton. Pharmacol. 1993; 13: 23-93, 11. Helke, C. J., Krause, J. E., Mantyh, P, W., Couture, R. and Bannon, M. J. Diversity in mammalian tachykinin peptidergic neurons: multiple peptides. FASEB J. 1990; 4: 16061615. 12. Nawa, H., Kotani, H. and Nakanishi, S. Tissue-specific generation of two preprotachykinin mRNAs from one gene by alternative RNA splicing. Nature 1984; 312: 729-734. 13. Bakhle, Y. S. and Bell, C. Increased numbers of substance P-containing sensory neurons in a rat strain with a genetic neurotrophic defect. Neuropeptides (1994) in press. 14. Gurusinghe, C. J. and Bell, C. Different patterns of immunolocalization of calcitonin gene-related peptide and substance P in sympathetic ganglia of normotensive and genetically hypertensive rats. Neurosci. Lett. t989; 106:89 94.
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