Brain Research, 517 (1990) 341-343 Elsevier
341
BRES 24093
Occurrence of neutral endopeptidase activity in the cat carotid body and its significance in chemoreception Ganesh K. Kumar 1, M. Runold 1, R. D. Ghai 2, Neil S. Cherniack I and N a n d u r i R. P r a b h a k a r 1 1Department of Medicine, Case Western Reserve University, Cleveland, OH 44106 (U.S.A.) and 2Research Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, Summit, NJ 07901 (U.S.A.)
(Accepted 30 January 1990) Key words: Neutral endopeptidase; Carotid body; Chemoreception; Hypoxia; Hypercapnia
The carotid body contains both tachykinins and enkephalins. Neutral endopeptidase (NEP, E.C. 3.4.24.11), has been suggested to involve in the metabolism of these neuropeptides in several organs. In the present study we determined neutral endopeptidase activity of the cat carotid body and assessed its significance in chemoreception. The cytosolic and membrane fractions of the carotid body contained NEP-fike activity whereas it occurred only in the membrane fractions of the superior cervical and the nodose ganglia. Phosphoramidon, thiorphan and metal ion chelators inhibited NEP-like activity of all the 3 tissues studied; other protease inhibitors, however, were ineffective. Close carotid body administration of phosphoramidon significantly potentiated the carotid body response to low PO2 but not to hypercapnia. The enhanced response to hypoxia following phosphoramidon was further augmented by naloxone, an enkephalin antagonist. These results demonstrate that the glomus tissue contains detectable amounts of NEP-like activity and its inhibition selectively affects the hypoxic response of the carotid body.
The neuropeptides, tachykinins and enkephalins, are present in the carotid body 1. Exogenous enkephalins inhibit the chemosensory discharge and naloxone, an enkephalin antagonist, augments the hypoxic response of the carotid body 1°. Tachykinins, i.e. substance p7,8,14 and neurokinin A 14, on the other hand, augment the chemosensory discharge and antagonists specific to tachykinins abolish or attenuate the carotid body response to low PO2, but not that produced by CO212"13. These observations suggest that tachykinins and enkephalins are of physiological importance in the expression of carotid body response to hypoxia. In the brain and kidney, the neutral endopeptidase (also termed as enkephalinase; E.C. 3.4.24.11) is involved in the metabolism of tachykinins and enkephalins 3'15. The purposes of the present study are to determine whether neutral endopeptidase is present in the carotid body and to evaluate its possible significance in chemoreception. Experiments were performed on 24 cats, anaesthetized by pentobarbital sodium (35 mg/kg). An arterial catheter was used for monitoring blood pressure and sampling blood, to determine arterial blood gases. The trachea was intubated and the carotid bifurcation exposed. All animals were ventilated with a respirator after spontaneous breathing movements were stopped by the injection of
additional doses of the anaesthetic (10-15 mg/kg i.v.). End tidal PCO 2 was continuously monitored (Beckman infrared CO 2 analyzer). Arterial blood gases and p H were measured (Instrumentation Laboratory system 1303). The body temperature was kept constant at 38 ° + 1 °C. The carotid body, the superior cervical ganglion and the nodose ganglion were removed for biochemical analysis from 14 cats, artificially ventilated with room air (PaO 2 105 + 4 mmHg; PaCO 2 32 + 1 mmHg; mean + S.E.M.). The tissues were washed with saline and stored at -70 °C till further analysis. Cell-free extract was prepared by sonicating the tissues in 50 mM Tris-HC1 buffer, pH 7.4 (600/A) at 4 °C for 30 s. The membrane fractions of the carotid body, the superior cervical ganglion and the nodose ganglion were prepared at 4 °C according to the procedure of Maeda et al s. Neutral endopeptidase activity was determined by following the procedure of Orlowski and Wilk 9. One unit of neutral endopeptidase is defined as 1 nmol of 2-naphthylamine released per h at 37 °C. Specific activity of the neutral endopeptidase is expressed in units/mg tissue protein. Protein concentration was determined by the Rose Bengal assay 2 using bovine serum albumin as the standard. In additional experiments, aliquots of the tissue ex-
Correspondence: G.K. Kumar, Department of Medicine, Pulmonary Division, University Hospitals, 2074 Abington Road, Cleveland, OH 44106, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
342 tracts were incubated with protease inhibitors (phosphoramidon, thiorphan, ethylenediaminetetraacetate (EDTA), 1,10-phenanthroline, captopril, soybean trypsin inhibitor, pepstatin and leupeptin) for 60 min at 25 °C. At the end of the incubation period, NEP activity was measured and compared with the untreated controls. Afferent nerve activity of the carotid body was measured in 10 cats as described previously 13'14. Close carotid body injections of chemicals were accomplished via a catheter placed in the lingual artery. The ganglioglomerular nerves were cut to avoid the influence of sympathetic nerve activity on carotid body discharge. Chemoreceptor activity was recorded while ventilating the animals with 100% O2, and then with 12% O2H-N 2 and/or 7% CO 2 in 93% 0 2. The durations of hypoxic and CO 2 challenges were 4 and 5 min, respectively. Arterial blood gas and pH measurements were made during the last 30 s of gas challenges. Phosphoramidon was administered close to the carotid body over 3 min at a concentration of 46 ~M/min and the responses to hypoxia and CO 2 were determined and compared with control infusion of saline containing the vehicle. In two additional cats, the effect of phosphoramidon on hypoxic and CO 2 responses were assessed after intracarotid administration of naloxone (0.4 mg), an enkephalin antagonist. Chemoreceptor activity was averaged over the last 30 s of a gas challenge and expressed as Aimp/s (testcontrol). The data were averaged and expressed as mean + S.E.M. The statistical significance was evaluated with paired 't'-test. The average values of the neutral endopeptidase activity are summarized in Table I. Neutral endopeptidase activity of the carotid body and the nodose ganglion were 5-fold higher than the superior cervical ganglion. Neutral endopeptidase activity in the carotid body was distributed equally in cytosolic and membrane fractions (Table I), but was present only in the membrane fractions
60
o O.
_E
50
p < 0.001 i
30 2o
I I
:
-r-
10 !
!
Pre-Phos
Post-Phos
Fig. 1. Individual data of carotid chemoreceptor responses to hypoxia pre-phosphoramidon (Pre-Phos) and after administration of phosphoramidon (Post-Phos) from 8 cats (10 observations). The data presented are chemoreceptor activity expressed as/limp/s, i.e. hypoxic response (hypoxia-hyperoxia). * represents the cats in which both the right and the left carotid bodies were studied.
of the superior cervical and the nodose ganglia. In all the 3 tissues, 10 /~M of either thiorphan or phosphoramidon, completely inhibited neutral endopeptidase activity. Similar inhibition was also seen with metal ion chelators such as EDTA, and 1,10-phenanthroline (1 mM). Other protease inhibitors like soybean trypsin inhibitor, captopril, leupeptin and pepstatin had no significant effect. The significance of neutral endopeptidase activity in the carotid body chemoreception was assessed using phosphoramidon, an inhibitor of neutral endopeptidase. Data from 10 experiments (8 cats) assessing the carotid body response to hypoxia are presented in Fig. 1. Prior to the administration of phosphoramidon, lowering PO 2 from 424 + 41 mmHg to 36.0 + 8.0 mmHg increased the
20
E
A comparison of neutral endopeptidase activity of the cat carotid body (CB), the superior cervical ganglion (SCG) and the nodose ganglion
(NG)
CB SCG NG
"~
Q,
TABLE I
Tissue
70
& .0 .o r-
Activity* (units per mg protein)
n
Cell-free extract
Cytosolic fraction
Membrane fraction
3.80 0.82 3.86
1.64 n.d. n.d.
1.53 0.71 3.72
* Neutral endopeptidase activity is expressed in units per mg protein. One unit is equivalent to 1 nmol 2-naphthylamine formed per h at 37 °C. Data are from pooled tissues from 6 cats. Average values from triplicate determinations are shown, n.d., not detected.
o
10 n.s
I---I
--
--
*
H
J
o
i
Pre-Phos
!
Post-Phos
Fig. 2. Individual data of carotid chemoreceptor responses to 7% CO2+93% O z before (Pre-Phos) and after administration of phosphoramidon (Post-Phos) from 8 cats (10 observations). The data represent chemoreceptor activity expressed as Aimp/s. (Hypercapnic response, i.e. hypercapnia-normocapnia), n.s., not significant; p > 0.05; paired 't'-test. For other details see legend to Fig. 1.
343 chemoreceptor activity in every cat on an average by +22 + 4 imp/s. The increase after phosphoramidon was +35 + 6 imp/s and is 59 + 8 % higher than the controls (P < 0.001). Resting chemoreceptor activity was unaffected (control, 2.0 + 0.9 imp/s vs post-phosphoramidon, 2.3 _+ 0.8 imp/s). The effect of phosphoramidon on chemoreceptor response to elevated arterial CO2 was also tested in the same animals. As can be seen from Fig. 2, in 7 of the 10 experiments the CO 2 response was either the same or less than the pre-phosphoramidon controls. Elevating the arterial CO 2 from 37 + 3.5 m m H g to 60 + 2.5 m m H g augmented chemoreceptor activity by +6 + 1.5 imp/s. The increase after phosphoramidon was +6.1 + 1.6 imp/s which was not statistically different from the controls (P
> 0.05). Naloxone, an enkephalin antagonist (0.4 mg), was administered close to the carotid body in two additional cats. At this concentration, chemosensory inhibition of Met-enkephalin (10 gg) was abolished by naloxone. In both the experiments, following naloxone, phosphoramidon further increased hypoxic response by 48% and 39%, respectively. The present study shows that neutral endopeptidase activity is present in the cat carotid body both in the cytosolic and membrane fractions. It has been reported that neutral endopeptidase (E.C. 3..4.24.11) occurs as a transmembrane enzyme 3"15. The present results with the superior cervical and the nodose ganglia are in accord with the observations from other organs. The fact that the carotid body has, in addition, a cytosolic component suggests that the distribution of the neutral endopeptidase in this tissue is rather unique. Although the 1 Chen, I.V., Yates, R.D. and Hansen, J.T., Substance P-like immunoreactivity in rat and cat carotid bodies: light and electron microscopic studies, Histol. Histopathol., 1 (1986) 203-212. 2 Elliot, J.I. and Brewer, J.M., The inactivation of yeast enolase by 2,3-butanedione, Arch. Biochem. Biophys., 190 (1978) 351-357. 3 Erdos, E.G. and Skidgel, R.A., Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones, FASEB J., 3 (1989) 145-151. 4 Kumar, G.K., Prabhakar, N.R. and Cherniack, N.S., In vitro studies on the hydrolysis of substance P by proteases of carotid body, Regul. Pept., 22 (1988) 107. 5 Maeda, T., Balakrishnan, K. and Mehdi, S.Q., A simple and rapid method for the preparation of plasma membranes, Biochim. Biophys. Acta, 731 (1983) 115-121. 6 Matsas, R., Kenny, A.J. and Turner, A.J., The metabolism of neuropeptides: the hydrolysis of peptides, including enkephalins, tachykinins and their analogues by endopeptidase-24.11, Biochem. J., 223 (1984) 433-440. 7 McQueen, D.S., Effects of substance P on carotid chemoreceptor activity in the cat, J. Physiol. (Lond.), 302 (1980) 31-47. 8 Monti-Bloch, L. and Eyzaguirre, C., Effects of methionineenkephalin and substance P on the chemosensory discharge of the cat carotid body, Brain Research, 338 (1985) 297-307. 90rlowski, M. and Wilk, S., Purification and specificity of a
distribution pattern of NEP is different in the carotid body, its inhibition characteristics resemble that of the superior cervical and the nodose ganglia. The inhibitory effects of phosphoramidon, thiorphan and metal ion chelators observed in these tissues are in accord with those reported in other organs 3'15. In this study, close carotid body administration of phosphoramidon potentiated the chemoreceptor response to hypoxia but not to CO 2. Preliminary in vitro studies have shown that substance P and enkephalins are degraded by the carotid body cell free extract and phosphoramidon prevents this degradation 4 (Kumar, unpublished results). Therefore, the augmented response to low P O E after phosphoramidon may be due to altered levels of tachykinins and/or enkephalins. Enkephalins inhibit 1° whereas exogenous substance P augments the chemosensory discharge of the carotid body 7"8'14. Substance P has been shown to potentiate the response of the carotid body to hypoxia 11. Since phosphoramidon induced potentiation of hypoxic response was further enhanced following an enkephalin antagonist, this augmentation probably does not involve enkephalin degradation. Moreover, substance P is a preferred substrate for neutral endopeptidase (Kin, 31.9 gM) than enkephalins 6 (Kin, 62 and 88/zM for Met- and Leu-enkephalins, respectively). These observations taken together suggest that substance P and/or a related tachykinin contributes to the phosphoramidon induced potentiation of the hypoxic response of the carotid body. Supported in part by grants from American Lung Association (G.K.K), National Institutes of Health, Heart and Lung Institute, HL-38986 (N.R.P) and HL-25830, HL-39921 (N.S.C). membrane bound metalloendopeptidase from bovine pituitary, Biochemistry, 20 (1981) 4942-4950. 10 Pokorski, M. and Lahiri, S., Effects of naloxone on carotid body chemoreception and ventilation in the cat, J. Appl. Physiol., 51 (1981) 1533-1538. 11 Prabhakar, N.R. and Cherniack, N.S., Importance of tachykinin peptides in hypoxic ventilatory drive. In S. Lahiri (Ed.), Chemoreceptors and Reflexes in Breathing, Oxford, New York, 1988, pp. 57-63. 12 Prabhakar, N.R., Runold, M., Yamamoto, Y., Lagergrantz, H. and Von Euler, C., Effect of substance P antagonist on the hypoxia induced carotid chemoreceptor activity, Acta. Physiol. Scand., 121 (1984) 301-303. 13 Prabhakar, N.R., Mitra, J. and Cherniack, N.S., Role of Substance P in hypercapnic excitation of carotid chemoreceptors, J. Appl. Physiol., 63 (1987) 2418-2425. 14 Prabhakar, N.R., Landis, S.C., Kumar, G.K., Mullikin-Kilpatrick, D., Chemiack, N.S. and Leeman, S.E., Substance P and neurokinin A in the cat carotid body: localization, exogenous effects and changes in content in response to arterial PO2, Brain Research, 481 (1989) 205-214. 15 Turner, A.J., Endopeptidase-24.11 and neuropeptide metabolism. In A. J. Turner (Ed.), Neuropeptides and Their Peptidases, Ellis-Horwood, Chichester, 1987, pp. 183-201.
341
BRES 24093
Occurrence of neutral endopeptidase activity in the cat carotid body and its significance in chemoreception Ganesh K. Kumar 1, M. Runold 1, R. D. Ghai 2, Neil S. Cherniack I and N a n d u r i R. P r a b h a k a r 1 1Department of Medicine, Case Western Reserve University, Cleveland, OH 44106 (U.S.A.) and 2Research Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, Summit, NJ 07901 (U.S.A.)
(Accepted 30 January 1990) Key words: Neutral endopeptidase; Carotid body; Chemoreception; Hypoxia; Hypercapnia
The carotid body contains both tachykinins and enkephalins. Neutral endopeptidase (NEP, E.C. 3.4.24.11), has been suggested to involve in the metabolism of these neuropeptides in several organs. In the present study we determined neutral endopeptidase activity of the cat carotid body and assessed its significance in chemoreception. The cytosolic and membrane fractions of the carotid body contained NEP-fike activity whereas it occurred only in the membrane fractions of the superior cervical and the nodose ganglia. Phosphoramidon, thiorphan and metal ion chelators inhibited NEP-like activity of all the 3 tissues studied; other protease inhibitors, however, were ineffective. Close carotid body administration of phosphoramidon significantly potentiated the carotid body response to low PO2 but not to hypercapnia. The enhanced response to hypoxia following phosphoramidon was further augmented by naloxone, an enkephalin antagonist. These results demonstrate that the glomus tissue contains detectable amounts of NEP-like activity and its inhibition selectively affects the hypoxic response of the carotid body.
The neuropeptides, tachykinins and enkephalins, are present in the carotid body 1. Exogenous enkephalins inhibit the chemosensory discharge and naloxone, an enkephalin antagonist, augments the hypoxic response of the carotid body 1°. Tachykinins, i.e. substance p7,8,14 and neurokinin A 14, on the other hand, augment the chemosensory discharge and antagonists specific to tachykinins abolish or attenuate the carotid body response to low PO2, but not that produced by CO212"13. These observations suggest that tachykinins and enkephalins are of physiological importance in the expression of carotid body response to hypoxia. In the brain and kidney, the neutral endopeptidase (also termed as enkephalinase; E.C. 3.4.24.11) is involved in the metabolism of tachykinins and enkephalins 3'15. The purposes of the present study are to determine whether neutral endopeptidase is present in the carotid body and to evaluate its possible significance in chemoreception. Experiments were performed on 24 cats, anaesthetized by pentobarbital sodium (35 mg/kg). An arterial catheter was used for monitoring blood pressure and sampling blood, to determine arterial blood gases. The trachea was intubated and the carotid bifurcation exposed. All animals were ventilated with a respirator after spontaneous breathing movements were stopped by the injection of
additional doses of the anaesthetic (10-15 mg/kg i.v.). End tidal PCO 2 was continuously monitored (Beckman infrared CO 2 analyzer). Arterial blood gases and p H were measured (Instrumentation Laboratory system 1303). The body temperature was kept constant at 38 ° + 1 °C. The carotid body, the superior cervical ganglion and the nodose ganglion were removed for biochemical analysis from 14 cats, artificially ventilated with room air (PaO 2 105 + 4 mmHg; PaCO 2 32 + 1 mmHg; mean + S.E.M.). The tissues were washed with saline and stored at -70 °C till further analysis. Cell-free extract was prepared by sonicating the tissues in 50 mM Tris-HC1 buffer, pH 7.4 (600/A) at 4 °C for 30 s. The membrane fractions of the carotid body, the superior cervical ganglion and the nodose ganglion were prepared at 4 °C according to the procedure of Maeda et al s. Neutral endopeptidase activity was determined by following the procedure of Orlowski and Wilk 9. One unit of neutral endopeptidase is defined as 1 nmol of 2-naphthylamine released per h at 37 °C. Specific activity of the neutral endopeptidase is expressed in units/mg tissue protein. Protein concentration was determined by the Rose Bengal assay 2 using bovine serum albumin as the standard. In additional experiments, aliquots of the tissue ex-
Correspondence: G.K. Kumar, Department of Medicine, Pulmonary Division, University Hospitals, 2074 Abington Road, Cleveland, OH 44106, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
342 tracts were incubated with protease inhibitors (phosphoramidon, thiorphan, ethylenediaminetetraacetate (EDTA), 1,10-phenanthroline, captopril, soybean trypsin inhibitor, pepstatin and leupeptin) for 60 min at 25 °C. At the end of the incubation period, NEP activity was measured and compared with the untreated controls. Afferent nerve activity of the carotid body was measured in 10 cats as described previously 13'14. Close carotid body injections of chemicals were accomplished via a catheter placed in the lingual artery. The ganglioglomerular nerves were cut to avoid the influence of sympathetic nerve activity on carotid body discharge. Chemoreceptor activity was recorded while ventilating the animals with 100% O2, and then with 12% O2H-N 2 and/or 7% CO 2 in 93% 0 2. The durations of hypoxic and CO 2 challenges were 4 and 5 min, respectively. Arterial blood gas and pH measurements were made during the last 30 s of gas challenges. Phosphoramidon was administered close to the carotid body over 3 min at a concentration of 46 ~M/min and the responses to hypoxia and CO 2 were determined and compared with control infusion of saline containing the vehicle. In two additional cats, the effect of phosphoramidon on hypoxic and CO 2 responses were assessed after intracarotid administration of naloxone (0.4 mg), an enkephalin antagonist. Chemoreceptor activity was averaged over the last 30 s of a gas challenge and expressed as Aimp/s (testcontrol). The data were averaged and expressed as mean + S.E.M. The statistical significance was evaluated with paired 't'-test. The average values of the neutral endopeptidase activity are summarized in Table I. Neutral endopeptidase activity of the carotid body and the nodose ganglion were 5-fold higher than the superior cervical ganglion. Neutral endopeptidase activity in the carotid body was distributed equally in cytosolic and membrane fractions (Table I), but was present only in the membrane fractions
60
o O.
_E
50
p < 0.001 i
30 2o
I I
:
-r-
10 !
!
Pre-Phos
Post-Phos
Fig. 1. Individual data of carotid chemoreceptor responses to hypoxia pre-phosphoramidon (Pre-Phos) and after administration of phosphoramidon (Post-Phos) from 8 cats (10 observations). The data presented are chemoreceptor activity expressed as/limp/s, i.e. hypoxic response (hypoxia-hyperoxia). * represents the cats in which both the right and the left carotid bodies were studied.
of the superior cervical and the nodose ganglia. In all the 3 tissues, 10 /~M of either thiorphan or phosphoramidon, completely inhibited neutral endopeptidase activity. Similar inhibition was also seen with metal ion chelators such as EDTA, and 1,10-phenanthroline (1 mM). Other protease inhibitors like soybean trypsin inhibitor, captopril, leupeptin and pepstatin had no significant effect. The significance of neutral endopeptidase activity in the carotid body chemoreception was assessed using phosphoramidon, an inhibitor of neutral endopeptidase. Data from 10 experiments (8 cats) assessing the carotid body response to hypoxia are presented in Fig. 1. Prior to the administration of phosphoramidon, lowering PO 2 from 424 + 41 mmHg to 36.0 + 8.0 mmHg increased the
20
E
A comparison of neutral endopeptidase activity of the cat carotid body (CB), the superior cervical ganglion (SCG) and the nodose ganglion
(NG)
CB SCG NG
"~
Q,
TABLE I
Tissue
70
& .0 .o r-
Activity* (units per mg protein)
n
Cell-free extract
Cytosolic fraction
Membrane fraction
3.80 0.82 3.86
1.64 n.d. n.d.
1.53 0.71 3.72
* Neutral endopeptidase activity is expressed in units per mg protein. One unit is equivalent to 1 nmol 2-naphthylamine formed per h at 37 °C. Data are from pooled tissues from 6 cats. Average values from triplicate determinations are shown, n.d., not detected.
o
10 n.s
I---I
--
--
*
H
J
o
i
Pre-Phos
!
Post-Phos
Fig. 2. Individual data of carotid chemoreceptor responses to 7% CO2+93% O z before (Pre-Phos) and after administration of phosphoramidon (Post-Phos) from 8 cats (10 observations). The data represent chemoreceptor activity expressed as Aimp/s. (Hypercapnic response, i.e. hypercapnia-normocapnia), n.s., not significant; p > 0.05; paired 't'-test. For other details see legend to Fig. 1.
343 chemoreceptor activity in every cat on an average by +22 + 4 imp/s. The increase after phosphoramidon was +35 + 6 imp/s and is 59 + 8 % higher than the controls (P < 0.001). Resting chemoreceptor activity was unaffected (control, 2.0 + 0.9 imp/s vs post-phosphoramidon, 2.3 _+ 0.8 imp/s). The effect of phosphoramidon on chemoreceptor response to elevated arterial CO2 was also tested in the same animals. As can be seen from Fig. 2, in 7 of the 10 experiments the CO 2 response was either the same or less than the pre-phosphoramidon controls. Elevating the arterial CO 2 from 37 + 3.5 m m H g to 60 + 2.5 m m H g augmented chemoreceptor activity by +6 + 1.5 imp/s. The increase after phosphoramidon was +6.1 + 1.6 imp/s which was not statistically different from the controls (P
> 0.05). Naloxone, an enkephalin antagonist (0.4 mg), was administered close to the carotid body in two additional cats. At this concentration, chemosensory inhibition of Met-enkephalin (10 gg) was abolished by naloxone. In both the experiments, following naloxone, phosphoramidon further increased hypoxic response by 48% and 39%, respectively. The present study shows that neutral endopeptidase activity is present in the cat carotid body both in the cytosolic and membrane fractions. It has been reported that neutral endopeptidase (E.C. 3..4.24.11) occurs as a transmembrane enzyme 3"15. The present results with the superior cervical and the nodose ganglia are in accord with the observations from other organs. The fact that the carotid body has, in addition, a cytosolic component suggests that the distribution of the neutral endopeptidase in this tissue is rather unique. Although the 1 Chen, I.V., Yates, R.D. and Hansen, J.T., Substance P-like immunoreactivity in rat and cat carotid bodies: light and electron microscopic studies, Histol. Histopathol., 1 (1986) 203-212. 2 Elliot, J.I. and Brewer, J.M., The inactivation of yeast enolase by 2,3-butanedione, Arch. Biochem. Biophys., 190 (1978) 351-357. 3 Erdos, E.G. and Skidgel, R.A., Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones, FASEB J., 3 (1989) 145-151. 4 Kumar, G.K., Prabhakar, N.R. and Cherniack, N.S., In vitro studies on the hydrolysis of substance P by proteases of carotid body, Regul. Pept., 22 (1988) 107. 5 Maeda, T., Balakrishnan, K. and Mehdi, S.Q., A simple and rapid method for the preparation of plasma membranes, Biochim. Biophys. Acta, 731 (1983) 115-121. 6 Matsas, R., Kenny, A.J. and Turner, A.J., The metabolism of neuropeptides: the hydrolysis of peptides, including enkephalins, tachykinins and their analogues by endopeptidase-24.11, Biochem. J., 223 (1984) 433-440. 7 McQueen, D.S., Effects of substance P on carotid chemoreceptor activity in the cat, J. Physiol. (Lond.), 302 (1980) 31-47. 8 Monti-Bloch, L. and Eyzaguirre, C., Effects of methionineenkephalin and substance P on the chemosensory discharge of the cat carotid body, Brain Research, 338 (1985) 297-307. 90rlowski, M. and Wilk, S., Purification and specificity of a
distribution pattern of NEP is different in the carotid body, its inhibition characteristics resemble that of the superior cervical and the nodose ganglia. The inhibitory effects of phosphoramidon, thiorphan and metal ion chelators observed in these tissues are in accord with those reported in other organs 3'15. In this study, close carotid body administration of phosphoramidon potentiated the chemoreceptor response to hypoxia but not to CO 2. Preliminary in vitro studies have shown that substance P and enkephalins are degraded by the carotid body cell free extract and phosphoramidon prevents this degradation 4 (Kumar, unpublished results). Therefore, the augmented response to low P O E after phosphoramidon may be due to altered levels of tachykinins and/or enkephalins. Enkephalins inhibit 1° whereas exogenous substance P augments the chemosensory discharge of the carotid body 7"8'14. Substance P has been shown to potentiate the response of the carotid body to hypoxia 11. Since phosphoramidon induced potentiation of hypoxic response was further enhanced following an enkephalin antagonist, this augmentation probably does not involve enkephalin degradation. Moreover, substance P is a preferred substrate for neutral endopeptidase (Kin, 31.9 gM) than enkephalins 6 (Kin, 62 and 88/zM for Met- and Leu-enkephalins, respectively). These observations taken together suggest that substance P and/or a related tachykinin contributes to the phosphoramidon induced potentiation of the hypoxic response of the carotid body. Supported in part by grants from American Lung Association (G.K.K), National Institutes of Health, Heart and Lung Institute, HL-38986 (N.R.P) and HL-25830, HL-39921 (N.S.C). membrane bound metalloendopeptidase from bovine pituitary, Biochemistry, 20 (1981) 4942-4950. 10 Pokorski, M. and Lahiri, S., Effects of naloxone on carotid body chemoreception and ventilation in the cat, J. Appl. Physiol., 51 (1981) 1533-1538. 11 Prabhakar, N.R. and Cherniack, N.S., Importance of tachykinin peptides in hypoxic ventilatory drive. In S. Lahiri (Ed.), Chemoreceptors and Reflexes in Breathing, Oxford, New York, 1988, pp. 57-63. 12 Prabhakar, N.R., Runold, M., Yamamoto, Y., Lagergrantz, H. and Von Euler, C., Effect of substance P antagonist on the hypoxia induced carotid chemoreceptor activity, Acta. Physiol. Scand., 121 (1984) 301-303. 13 Prabhakar, N.R., Mitra, J. and Cherniack, N.S., Role of Substance P in hypercapnic excitation of carotid chemoreceptors, J. Appl. Physiol., 63 (1987) 2418-2425. 14 Prabhakar, N.R., Landis, S.C., Kumar, G.K., Mullikin-Kilpatrick, D., Chemiack, N.S. and Leeman, S.E., Substance P and neurokinin A in the cat carotid body: localization, exogenous effects and changes in content in response to arterial PO2, Brain Research, 481 (1989) 205-214. 15 Turner, A.J., Endopeptidase-24.11 and neuropeptide metabolism. In A. J. Turner (Ed.), Neuropeptides and Their Peptidases, Ellis-Horwood, Chichester, 1987, pp. 183-201.
