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Physiological chemoreceptor stimulation decreases enkephalin and substance P in the carotid body
Peptides, Vol. 7, pp. 767-769, 1986. ©Ankho InternationalInc. Printedin the U.S.A.
0196-9781/86$3.00 + .00
Physiological Chemoreceptor Stimulation Decreases Enkephalin and Substance P in the Carotid Body G. HANSON, L. J O N E S A N D S. F I D O N E 1 D e p a r t m e n t s o f Biochemical P h a r m a c o l o g y and Toxicology, and Physiology University o f Utah, Salt L a k e City, UT 84108 R e c e i v e d 31 M a r c h 1986 HANSON, G., L. JONES AND S. FIDONE. Physiological chemoreceptor stimulation decreases enkephalin and substance P in the carotid body. PEPTIDES 7(5) 767-769, 1986.--Neuroactive peptides, including the enkephalins (Met- and Leu-enkephalin; ME, LE) and substance P (SP) are known to be present in the mammalian carotid body, an arterial chemoreceptor organ sensitive to the 02, CO2 and pH levels in blood. The principal parenchymal (type I) cells of the organ, which receive sensory innervation from the carotid sinus nerve (CSN), have been shown to contain both ME and SP; SP is also present in CSN afferent fibers. In the present study, rabbits were exposed in a chamber to a physiological chemoreceptor stimulus (5% 02 in N2) for one hour, then anesthetized during surgical removal of both carotid bodies for later RIA measurement of ME and SP levels in the tissue; control animals were exposed to air in the chamber, but otherwise treated as the hypoxic animals. Both ME and SP levels were significantly reduced (approximately 40%) in the carotid bodies from hypoxic rabbits, compared to their normoxic controls. The results suggest that these neuroactive peptides are released from carotid body elements during physiological stimulation, and consequently may play a role in the transduction of chemosensory information between the type I cells and their apposed afferent terminals. Substance P
Carotid sinus nerve
Met-enkephalin
Leu-enkephalin
Rabbits
namely, inhalation of an hypoxic gas mixture consisting of 5% O2 in N2.
THE mammalian carotid body, an arterial chemoreceptor organ sensitive to blood PO2, PCO~ and pH, contains two types of cells disposed together in groups or glomoids. The type I, or glomus, cells receive specialized synaptic-like contacts from afferent fibers of the carotid sinus nerve [3], but the precise mechanism by which the nerve endings are excited is unknown. The neuropeptides Met- and Leuenkephalin (ME, LE) have been localized to the type I cells with immunohistochemical techniques [9,16], while substance P (SP)-like immunoreactivity (SPLI) has been reportedly found in both nerve fibers [8] and 20% of the type I cells [2]. Little is known regarding the role of these substances in carotid body function (see review, [12]), but recent pharmacological studies suggest that administration of exogenous neuropeptides is able to modify chemoreceptor activity of both in vivo and in vitro carotid body preparations [ll-15], and consequently these substances are thought to play a role in the genesis of the normal chemoresponse. In studying the role of SP and enkephalin in the carotid body, an important step is to determine the response of the endogenous neuropeptides to natural stimuli which are known to alter the physiological activities of this organ. In the present study, we investigated whether the levels of SP and ME in the carotid body are responsive to a physiological stimulus,
METHOD Adult New Zealand rabbits (2-2.5 kg) were placed singly in a clear lucite chamber which was continuously flushed (4 liters/min) with either air or 5% 02 in N2 (certified gas mixture, _+0.5% of rated purity, IAP, Portland) for two 30 min periods, with 20 min interim. The rabbits were then anesthetized with pentobarbital sodium and respired with the given gas mixture during surgical removal of the carotid bodies, along with an accompanying sensory neural tissue, the nodose ganglia, which served as 'control.' Tissues were quickly cleaned of surrounding connective tissue in a bath of ice-cold modified Tyrode's medium (in mM: NaCl, 112; KC1, 4.7; CaCl2, 2.2; MgCl~, I. 1; sodium glutamate, 42; HEPES, 5; glucose, 5.6), weighed on a Cahn electrobalance, and immediately frozen to -20°C and stored at -80°C for later analysis. Tissue samples were homogenized in 0.01 N HCI in preparation for radioimmunoassay (RIA) determinations. An aliquot was removed from each sample and protein content was measured according to the method of Bradford [l]. The
tRequests for reprints should be addressed to Salvatore J. Fidone, Ph.D., Department of Physiology, University of Utah School of Medicine, 410 Chipeta Way, Research Park, Salt Lake City, UT 84108.
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FIG. 1. Effects of hypoxia on neuropeptide concentrations in the carotid body. Rabbits were exposed to two 30 rain periods of either 5% O~ (hypoxia) or air as described in the Method section. Immediately following this treatment, carotid bodies were surgically removed and assayed for MELI or SPLI. These results are the combination of two experiments; each sample consisted of 2 carotid bodies and each mean represents 7-9 samples. The control values for MELI and SPLI concentrations were 63.2 and 2.97 ng/mg protein, respectively. The means_+_S.E.M, are expressed as percent of control. *p<0.025 according to the 2-tailed Student's t-test. ()pen column: room air; dark column: hypoxia.
FIG. 2. Effects of hypoxia on neuropeptide concentrations in the nodose ganglia. Rabbits were treated identically to that described for Fig. I prior to removal and assay for MELI and SPLI concentrations in nodose ganglia. Each mean represents a total of 4-5 samples. The means+S.E.M, are expressed as percent of control. The control values for MELI and SPLI concentrations were 50 and 580 pg/mg protein, respectively. Open column: room air: dark column: hypoxia.
remaining homogenate was heated in boiling water for 10 min to denature proteases, after which the samples were centrifuged (5000 × g, 20 min) and the resulting supernatant removed and lyophilized for storage at -80°C until assayed. The lyophilized samples were reconstituted with phosphate-buffered saline, pH 7.4, containing 0.1% gelatin. The reconstituted samples were halved and assayed for either SP-like immunoreactivity (SPLI) or Met-enkephalinlike immunoreactivity (MELI). The SPLI analysis has been described in detail elsewhere [6]. The SP antiserum used in this study could reliably detect 10 pg of synthetic bovine hypothalamic SP at a 1:200,000 dilution and displayed less than 2% cross-reactivity with eledoisin, substance K and physalaemin, three peptides structurally similar to SP. Analysis tbr MEL1 was done in a manner similar to that for SPLI, with the exception that following the additions of '~I-ME (NEN) and the ME antiserum, the samples were allowed to incubate at 4°C for 24 hours, after which the charcoal separation was performed. The antiserum for ME (ImmunoNuclear Corporation, Stillwater, MN) could reliably detect 10 pg of ME and displayed insignificant cross-reactivity with Leu-enkephalin (less than 3%), substance P (less than 0.002%), beta endorphin (less than 0.002%), and porcine dynorphin' ]:~(less than 0.002%).
changes in peptide concentrations which we observed in this study are unliketv to be due to alterations in protein synthesis: Harmer and Keen [7] have shown that 8 hr after complete blockade of protein synthesis, the SP levels in dorsal root ganglia remain unchanged. Preliminary experiments have also been initiated to investigate the effects of chronic section of the carotid sinus nerve (CSN) and chronic sympathectomy on neuropeptide levels in the carotid body. Our previous studies with carotid body catecholamines had shown that these nerves exert long-term trophic effects on biosynthetic enzyme levels and transmitter release by the carotid body [4,5]. Our initial results suggest that 12-15 days following section of the CSN, the basal (unstimulated) level of SPL1 in the carotid body is significantly increased, in spite of the loss of CSN-SPLI. On the other hand, it appears that the level of MELI in the carotid body is reduced following CSN section. Following chronic sympathectomy (12-15 days), the basal peptide levels in the carotid body were likewise increased in the case of SPLI, and decreased for MELt. These preliminary results on changes in carotid body peptides following chronic denervations of the organ suggest long-term trophic effects of these nerves on transmitter regulation, and are reminiscent of similar actions we earlier reported for the trophic effects of these nerves on carotid body biogenic amines. The effects of hypoxia on neuropeptides in nodose ganglia were also examined to determine whether these substances were similarly affected in this sensory ganglion. The SPLI and MELI levels in nodose ganglia were unchanged after exposure of the animal to the hypoxic episode (Fig. 2). Although our experiments do not rule out possible non-specific effects of hypoxia on the carotid body, such as those unrelated to direct changes in chemoreception (e.g., local tissue hypoxia, blood pressure changes, hormonal and temperature effects, etc.), the lack of effect of hypoxia on neuropeptides in nodose ganglia suggests that tissue peptide stores may be resistant to such sequelae, and that changes observed in
RESULTS AND DISCUSSION As shown in Fig. 1, animals exposed to hypoxia had significantly reduced levels of both SPLI and MELI in their carotid bodies, compared to animals exposed only to room air in the chamber. These results suggest that SP and ME may be involved in the regulation and/or mediation of carotid body chemoreception. The implicit assumption is that decreases in neuropeptide levels during the relatively short period of exposure to hypoxia are indicative of increased release and subsequent rapid proteolysis. Such an assumption may not be unreasonable, since the relatively rapid
ENKEPHALIN
A N D S P IN T H E C A R O T I D B O D Y
769
c a r o t i d b o d y peptide levels reflect a true physiological res p o n s e d e p e n d e n t on a l t e r e d c h e m o r e c e p t o r drive. In s u m m a r y , o u r d a t a s u g g e s t t h a t the SP a n d M E syst e m s in the c a r o t i d b o d y are r e s p o n s i v e to n a t u r a l stimuli
w h i c h affect the p h y s i o l o g i c a l activity o f this c h e m o r e c e p t o r organ. W e r e p o r t h e r e t h a t t h e n a t u r a l stimulus, h y p o x i a , is able to a l t e r S P a n d M E levels in this s t r u c t u r e .
ACKNOWLEDGEMENTS Supported by USPHS Grants NS-12636, NS-07938, MH37762 and MH40175.
REFERENCES
1. Bradford, M. A rapid and sensitive method for the quantitation of micro organism quantities of protein utilizing the principle of protein-dye binding. Anal Bio~'hem 72: 248-254, 1976. 2. Cuello, A. C. and D. S. McQueen. Substance P: a carotid body peptide. Nettros~'i Lett 17: 215-219, 1980. 3. De Castro, F. Sur la structure et l'innervation du sinus carotidien de l'homme et des mammiferes. Nouveaux faits sur l'innervation et la fonction du glomus caroticum. Etudes anatomiques et physiologiques. 7)ab Lab Invest Biol Univ Madrid 25:331-380, 1928. 4. Fidone, S. J., C. Gonzalez and K. Yoshizaki. Effects of low oxygen on the release of dopamine from the rabbit carotid body in vitro. J Phvsiol (Lond) 333:93-110, 1982. 5. Gonzalez, C., Y. Kwok, J. W. Gibb and S. J. Fidone. Reciprocal modulation of tyrosine hydroxylase activity in rat carotid body. Brain Res 172: 572-576, 1979. 6. Hanson, G. and W. Lovenberg. Elevation of substance P-like immunoreactivity in rat central nervous system by protease inhibition. J Neurochem 35: 1370-1374, 1980. 7. Harmer, A. and P. Keen. Chemical characterization of substance P-like immunoreactivity in primary afferent neurons. Brain Res 220: 203-207, 1981. 8. Jacobowitz, D. M. and C. J. Helke. Localization of substance P immunoreactive nerves in the carotid body. Brain Res Bull 5: 195-197, 1980.
9. Lundberg, J. M., T. Hokfelt, J. Fahrenkrug, G. Nilsson and L. Terenius. Peptides in the cat carotid body (glomus caroticum): VIP-, enkephalin- and substance P-like immunoreactivity. Acta Physiol Stand 107: 279-281, 1979. 10. McQueen, D. S. Effects of substance P on carotid chemoreceptor activity in the cat. J Physiol (Lond) 302: 31-47, 1980. I I. McQueen D. S. and J. A. Ribeiro. Inhibitory actions of methionine-enkephalin and morphine on the cat carotid chemoreceptors. Br J Pharmacol 71: 297-305, 1980. 12. McQueen, D. S. Pharmacological aspects of putative transmitters in the carotid body. In: Physiology ~fthe Peripheral Arterial Chemoreceptors, edited by H. Acker and R. G. O'Regan. Amsterdam: Elsevier, 1984, pp. 149-195. 13. Monti-Bloch, L. and C. Eyzaguirre. Effects of methionineenkephalin and substance P on the chemosensory discharge of the cat carotid body. Brain Res 338: 297-307, 1985. 14. Pokorski, M. and S. Lahiri. Effects ofnaloxone on carotid body chemoreception and ventilation in the cat. J Appl Phvsiol 51: 1533-1538, 1981. 15. Prabhakar, N. R., M. Runold, Y. Yamamoto, M. Langercrantz and C. von Euler. Effects of substance P antagonist on the hypoxia-induced carotid chemoreceptor activity. A~ta Physiol S~'aml 121: 301-303, 1984. 16. Wharton, J., J. M. Polak, A. G. E. Pearse, G. P. McGregor, M. G. Bryant, S. R. Bloom, P. C. Emson, G. E. Bisgard and J. A. Will. Enkephalin-, VIP-, and substance P-like immunoreactivity in the carotid body. Natttre 284: 269-271. 1980.
0196-9781/86$3.00 + .00
Physiological Chemoreceptor Stimulation Decreases Enkephalin and Substance P in the Carotid Body G. HANSON, L. J O N E S A N D S. F I D O N E 1 D e p a r t m e n t s o f Biochemical P h a r m a c o l o g y and Toxicology, and Physiology University o f Utah, Salt L a k e City, UT 84108 R e c e i v e d 31 M a r c h 1986 HANSON, G., L. JONES AND S. FIDONE. Physiological chemoreceptor stimulation decreases enkephalin and substance P in the carotid body. PEPTIDES 7(5) 767-769, 1986.--Neuroactive peptides, including the enkephalins (Met- and Leu-enkephalin; ME, LE) and substance P (SP) are known to be present in the mammalian carotid body, an arterial chemoreceptor organ sensitive to the 02, CO2 and pH levels in blood. The principal parenchymal (type I) cells of the organ, which receive sensory innervation from the carotid sinus nerve (CSN), have been shown to contain both ME and SP; SP is also present in CSN afferent fibers. In the present study, rabbits were exposed in a chamber to a physiological chemoreceptor stimulus (5% 02 in N2) for one hour, then anesthetized during surgical removal of both carotid bodies for later RIA measurement of ME and SP levels in the tissue; control animals were exposed to air in the chamber, but otherwise treated as the hypoxic animals. Both ME and SP levels were significantly reduced (approximately 40%) in the carotid bodies from hypoxic rabbits, compared to their normoxic controls. The results suggest that these neuroactive peptides are released from carotid body elements during physiological stimulation, and consequently may play a role in the transduction of chemosensory information between the type I cells and their apposed afferent terminals. Substance P
Carotid sinus nerve
Met-enkephalin
Leu-enkephalin
Rabbits
namely, inhalation of an hypoxic gas mixture consisting of 5% O2 in N2.
THE mammalian carotid body, an arterial chemoreceptor organ sensitive to blood PO2, PCO~ and pH, contains two types of cells disposed together in groups or glomoids. The type I, or glomus, cells receive specialized synaptic-like contacts from afferent fibers of the carotid sinus nerve [3], but the precise mechanism by which the nerve endings are excited is unknown. The neuropeptides Met- and Leuenkephalin (ME, LE) have been localized to the type I cells with immunohistochemical techniques [9,16], while substance P (SP)-like immunoreactivity (SPLI) has been reportedly found in both nerve fibers [8] and 20% of the type I cells [2]. Little is known regarding the role of these substances in carotid body function (see review, [12]), but recent pharmacological studies suggest that administration of exogenous neuropeptides is able to modify chemoreceptor activity of both in vivo and in vitro carotid body preparations [ll-15], and consequently these substances are thought to play a role in the genesis of the normal chemoresponse. In studying the role of SP and enkephalin in the carotid body, an important step is to determine the response of the endogenous neuropeptides to natural stimuli which are known to alter the physiological activities of this organ. In the present study, we investigated whether the levels of SP and ME in the carotid body are responsive to a physiological stimulus,
METHOD Adult New Zealand rabbits (2-2.5 kg) were placed singly in a clear lucite chamber which was continuously flushed (4 liters/min) with either air or 5% 02 in N2 (certified gas mixture, _+0.5% of rated purity, IAP, Portland) for two 30 min periods, with 20 min interim. The rabbits were then anesthetized with pentobarbital sodium and respired with the given gas mixture during surgical removal of the carotid bodies, along with an accompanying sensory neural tissue, the nodose ganglia, which served as 'control.' Tissues were quickly cleaned of surrounding connective tissue in a bath of ice-cold modified Tyrode's medium (in mM: NaCl, 112; KC1, 4.7; CaCl2, 2.2; MgCl~, I. 1; sodium glutamate, 42; HEPES, 5; glucose, 5.6), weighed on a Cahn electrobalance, and immediately frozen to -20°C and stored at -80°C for later analysis. Tissue samples were homogenized in 0.01 N HCI in preparation for radioimmunoassay (RIA) determinations. An aliquot was removed from each sample and protein content was measured according to the method of Bradford [l]. The
tRequests for reprints should be addressed to Salvatore J. Fidone, Ph.D., Department of Physiology, University of Utah School of Medicine, 410 Chipeta Way, Research Park, Salt Lake City, UT 84108.
767
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FIG. 1. Effects of hypoxia on neuropeptide concentrations in the carotid body. Rabbits were exposed to two 30 rain periods of either 5% O~ (hypoxia) or air as described in the Method section. Immediately following this treatment, carotid bodies were surgically removed and assayed for MELI or SPLI. These results are the combination of two experiments; each sample consisted of 2 carotid bodies and each mean represents 7-9 samples. The control values for MELI and SPLI concentrations were 63.2 and 2.97 ng/mg protein, respectively. The means_+_S.E.M, are expressed as percent of control. *p<0.025 according to the 2-tailed Student's t-test. ()pen column: room air; dark column: hypoxia.
FIG. 2. Effects of hypoxia on neuropeptide concentrations in the nodose ganglia. Rabbits were treated identically to that described for Fig. I prior to removal and assay for MELI and SPLI concentrations in nodose ganglia. Each mean represents a total of 4-5 samples. The means+S.E.M, are expressed as percent of control. The control values for MELI and SPLI concentrations were 50 and 580 pg/mg protein, respectively. Open column: room air: dark column: hypoxia.
remaining homogenate was heated in boiling water for 10 min to denature proteases, after which the samples were centrifuged (5000 × g, 20 min) and the resulting supernatant removed and lyophilized for storage at -80°C until assayed. The lyophilized samples were reconstituted with phosphate-buffered saline, pH 7.4, containing 0.1% gelatin. The reconstituted samples were halved and assayed for either SP-like immunoreactivity (SPLI) or Met-enkephalinlike immunoreactivity (MELI). The SPLI analysis has been described in detail elsewhere [6]. The SP antiserum used in this study could reliably detect 10 pg of synthetic bovine hypothalamic SP at a 1:200,000 dilution and displayed less than 2% cross-reactivity with eledoisin, substance K and physalaemin, three peptides structurally similar to SP. Analysis tbr MEL1 was done in a manner similar to that for SPLI, with the exception that following the additions of '~I-ME (NEN) and the ME antiserum, the samples were allowed to incubate at 4°C for 24 hours, after which the charcoal separation was performed. The antiserum for ME (ImmunoNuclear Corporation, Stillwater, MN) could reliably detect 10 pg of ME and displayed insignificant cross-reactivity with Leu-enkephalin (less than 3%), substance P (less than 0.002%), beta endorphin (less than 0.002%), and porcine dynorphin' ]:~(less than 0.002%).
changes in peptide concentrations which we observed in this study are unliketv to be due to alterations in protein synthesis: Harmer and Keen [7] have shown that 8 hr after complete blockade of protein synthesis, the SP levels in dorsal root ganglia remain unchanged. Preliminary experiments have also been initiated to investigate the effects of chronic section of the carotid sinus nerve (CSN) and chronic sympathectomy on neuropeptide levels in the carotid body. Our previous studies with carotid body catecholamines had shown that these nerves exert long-term trophic effects on biosynthetic enzyme levels and transmitter release by the carotid body [4,5]. Our initial results suggest that 12-15 days following section of the CSN, the basal (unstimulated) level of SPL1 in the carotid body is significantly increased, in spite of the loss of CSN-SPLI. On the other hand, it appears that the level of MELI in the carotid body is reduced following CSN section. Following chronic sympathectomy (12-15 days), the basal peptide levels in the carotid body were likewise increased in the case of SPLI, and decreased for MELt. These preliminary results on changes in carotid body peptides following chronic denervations of the organ suggest long-term trophic effects of these nerves on transmitter regulation, and are reminiscent of similar actions we earlier reported for the trophic effects of these nerves on carotid body biogenic amines. The effects of hypoxia on neuropeptides in nodose ganglia were also examined to determine whether these substances were similarly affected in this sensory ganglion. The SPLI and MELI levels in nodose ganglia were unchanged after exposure of the animal to the hypoxic episode (Fig. 2). Although our experiments do not rule out possible non-specific effects of hypoxia on the carotid body, such as those unrelated to direct changes in chemoreception (e.g., local tissue hypoxia, blood pressure changes, hormonal and temperature effects, etc.), the lack of effect of hypoxia on neuropeptides in nodose ganglia suggests that tissue peptide stores may be resistant to such sequelae, and that changes observed in
RESULTS AND DISCUSSION As shown in Fig. 1, animals exposed to hypoxia had significantly reduced levels of both SPLI and MELI in their carotid bodies, compared to animals exposed only to room air in the chamber. These results suggest that SP and ME may be involved in the regulation and/or mediation of carotid body chemoreception. The implicit assumption is that decreases in neuropeptide levels during the relatively short period of exposure to hypoxia are indicative of increased release and subsequent rapid proteolysis. Such an assumption may not be unreasonable, since the relatively rapid
ENKEPHALIN
A N D S P IN T H E C A R O T I D B O D Y
769
c a r o t i d b o d y peptide levels reflect a true physiological res p o n s e d e p e n d e n t on a l t e r e d c h e m o r e c e p t o r drive. In s u m m a r y , o u r d a t a s u g g e s t t h a t the SP a n d M E syst e m s in the c a r o t i d b o d y are r e s p o n s i v e to n a t u r a l stimuli
w h i c h affect the p h y s i o l o g i c a l activity o f this c h e m o r e c e p t o r organ. W e r e p o r t h e r e t h a t t h e n a t u r a l stimulus, h y p o x i a , is able to a l t e r S P a n d M E levels in this s t r u c t u r e .
ACKNOWLEDGEMENTS Supported by USPHS Grants NS-12636, NS-07938, MH37762 and MH40175.
REFERENCES
1. Bradford, M. A rapid and sensitive method for the quantitation of micro organism quantities of protein utilizing the principle of protein-dye binding. Anal Bio~'hem 72: 248-254, 1976. 2. Cuello, A. C. and D. S. McQueen. Substance P: a carotid body peptide. Nettros~'i Lett 17: 215-219, 1980. 3. De Castro, F. Sur la structure et l'innervation du sinus carotidien de l'homme et des mammiferes. Nouveaux faits sur l'innervation et la fonction du glomus caroticum. Etudes anatomiques et physiologiques. 7)ab Lab Invest Biol Univ Madrid 25:331-380, 1928. 4. Fidone, S. J., C. Gonzalez and K. Yoshizaki. Effects of low oxygen on the release of dopamine from the rabbit carotid body in vitro. J Phvsiol (Lond) 333:93-110, 1982. 5. Gonzalez, C., Y. Kwok, J. W. Gibb and S. J. Fidone. Reciprocal modulation of tyrosine hydroxylase activity in rat carotid body. Brain Res 172: 572-576, 1979. 6. Hanson, G. and W. Lovenberg. Elevation of substance P-like immunoreactivity in rat central nervous system by protease inhibition. J Neurochem 35: 1370-1374, 1980. 7. Harmer, A. and P. Keen. Chemical characterization of substance P-like immunoreactivity in primary afferent neurons. Brain Res 220: 203-207, 1981. 8. Jacobowitz, D. M. and C. J. Helke. Localization of substance P immunoreactive nerves in the carotid body. Brain Res Bull 5: 195-197, 1980.
9. Lundberg, J. M., T. Hokfelt, J. Fahrenkrug, G. Nilsson and L. Terenius. Peptides in the cat carotid body (glomus caroticum): VIP-, enkephalin- and substance P-like immunoreactivity. Acta Physiol Stand 107: 279-281, 1979. 10. McQueen, D. S. Effects of substance P on carotid chemoreceptor activity in the cat. J Physiol (Lond) 302: 31-47, 1980. I I. McQueen D. S. and J. A. Ribeiro. Inhibitory actions of methionine-enkephalin and morphine on the cat carotid chemoreceptors. Br J Pharmacol 71: 297-305, 1980. 12. McQueen, D. S. Pharmacological aspects of putative transmitters in the carotid body. In: Physiology ~fthe Peripheral Arterial Chemoreceptors, edited by H. Acker and R. G. O'Regan. Amsterdam: Elsevier, 1984, pp. 149-195. 13. Monti-Bloch, L. and C. Eyzaguirre. Effects of methionineenkephalin and substance P on the chemosensory discharge of the cat carotid body. Brain Res 338: 297-307, 1985. 14. Pokorski, M. and S. Lahiri. Effects ofnaloxone on carotid body chemoreception and ventilation in the cat. J Appl Phvsiol 51: 1533-1538, 1981. 15. Prabhakar, N. R., M. Runold, Y. Yamamoto, M. Langercrantz and C. von Euler. Effects of substance P antagonist on the hypoxia-induced carotid chemoreceptor activity. A~ta Physiol S~'aml 121: 301-303, 1984. 16. Wharton, J., J. M. Polak, A. G. E. Pearse, G. P. McGregor, M. G. Bryant, S. R. Bloom, P. C. Emson, G. E. Bisgard and J. A. Will. Enkephalin-, VIP-, and substance P-like immunoreactivity in the carotid body. Natttre 284: 269-271. 1980.