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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 Elsevier
205
BRE 14252
Research Repods
Substance P and neurokinin A in the cat carotid body: localization, exogenous effects and changes in content in response to arterial pO 2 Nanduri R. Prabhakar 1, Story C. Landis 2, Ganesh K. Kumar 1, Debra Mullikin-Kilpatrick 3, Neil S. Cherniack I and Susan Leeman 3 Departments of 1Medicineand 2pharmacology, Case Western Reserve University, Cleveland, OH 44106 (U.S.A.) and 3Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01605 (U.S.A.) (Accepted 26 July 1988)
Key words." Tachykinin; Substance P; Neurokinin A; Hypoxia; Carotid body; Chemoreceptor
In the present work we studied the occurrence of substance P (SP) and neurokinin A (NKA) in the carotid bodies of cats by means of immunocytochemistry and radioimmunoassay (RIA). We also compared the exogenous effects of SP and NKA on carotid body sensory discharge. SP- and NKA-like immunoreactivities (SP-LI; NKA-LI) were seen in many glomus cells and in a sparse plexus of fine fibers. The SP-LI containing glomus cells and fibers also exhibited NKA-LI, suggesting that both these tachykinins coexist in the carotid body. Chemoreceptor discharge increased both by SP and NKA in a dose-dependent manner. The peak excitation produced by SP and NKA was the same when the effects were compared on an equimolar basis. The tachykinin content of the carotid bodies varied with changes in arterialpO:. During normoxia, SP and NKA levels were 57 + 8 and 85 + 14 fmol/mg, respectively. When the animals were exposed for 1 h to 100% 02, SP content was unchanged (51 + 4 fmol/mg), whereas NKA levels were significantly lower than during normoxia (29 + 3 fmol/mg, P < 0.01). Following 1 h of hypoxia, SP content of the carotid body was 146 + 20 fmol/mg, a value higher than that obtained during normoxia and hyperoxia. NKA levels, on the other hand, were not significantly different from normoxic values. These results indicate that the cat carotid body (1) contains both SP and NKA, (2) both peptides augment neural discharge of the carotid body and (3) their levels in the carotid body are substantially altered by arterial oxygen, the natural stimulus to the chemoreceptors. INTRODUCTION The ventilatory increase that occurs during hypoxia arises from stimulation of arterial chemoreceptors, in particular the carotid bodies. Several lines of evidence suggest that the n e u r o p e p t i d e substance P (SP) is involved in the production of the hypoxic response. First, SP-like immunoreactivity (SP-LI) is present in glomus cells and some afferent nerve terminals in the carotid body 2'3'7'18. Second, exogenous SP augments the sensory discharge of the carotid body both in vivo and in vitro s'9. Third, the antagonists specific for SPlike neuropeptides attenuate or abolish the hypoxic
but not the CO2-induced excitation of carotid chemoreceptors 13'14. It has been r e p o r t e d recently that SP content of carotid bodies decreases during hypoxic exposure, possibly due to enhanced release 4. SP and neurokinin A ( N K A ) belong to a group of structurally related n e u r o p e p t i d e s called tachykinins. In m a m m a l i a n tissues, SP and N K A are derived from a single preprotachykinin gene (PPT) 6A1, raising the possibility that SP, as well as N K A are present in the carotid body and might play a physiological role in chemoreception. W e therefore examined the localization of SP and N K A in the cat carotid body, studied their effects on neural discharge of chemore-
Correspondence: N.R. Prabhakar, Department of Medicine, Pulmonary Division, University Hospitals, 2074 Abington Road, Cleveland, OH 44106, U.S.A. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
206 ceptors and determined levels of these tachykinins in carotid bodies of cats breathing different concentrations of 0~. MATERIALS AND METHODS Three groups of experiments were performed on 32 adult cats of either sex weighing 2.1 to 4.3 kg. They were anesthetized with i.p. injections of pentobarbital sodium (35 mg/kg), supplemented as necessary. In all experiments, routine surgical procedures included tracheal intubation and cannulation of a femoral artery and vein. The arterial catheter was used for monitoring arterial blood pressure and for sampling blood for determinations of blood gasses. Systemic administration of fluids or drugs was accomplished through the venous catheter. To expose the carotid bifurcation, the trachea and esophagus were ligated above the site of tracheal cannula, sectioned and retracted rostrally. The exposed tissues were covered with cotton gauze soaked in saline. End tidal p C O 2 was continuously monitored with an infrared CO2 analyzer (Beckman LB-2). The body temperature of the animals was kept at 38 °C (+ 1 °C) by means of a heating blanket governed by a rectal thermistor probe.
Immunocytochemical studies The localization of SP and NKA was examined in the carotid bodies of 5 cats using immunocytochemical techniques. Anesthetized animals were artificially ventilated with room air with a positive pressure respirator (Harvard Apparatus Model 665). Heparin (1000 units/kg) was administered i.v. 15 min before the beginning of perfusion. After opening the thorax, the ascending aorta was cannulated and connected to reservoirs containing perfusion medium. An opening was made in the right atrium for drainage of fluids. Heparinized saline was perfused for 15 min until the blood vessels were free of blood. Thereafter the perfusion medium was switched to 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3, for 30 min at room temperature. The carotid bodies were removed with the bifurcation of the carotid artery, rinsed with phosphate buffer, infiltrated with 30% sucrose in 0.1 M phosphate buffer and stored frozen in sucrose at -70 °C.
Cryostat sections, 8/~m-thick, were cut and thawmounted on gelatin-coated slides. The sections were allowed to air dry for 30 min and were then either stained immediately or stored frozen at -20 °C for staining at a later time. In most cases, sections were stained for a single neuropeptide, either SP or NKA, using rabbit antisera as described below. Sections were incubated for 1 h in a buffer containing 0.5 M NaC1, 0.01 M phosphate buffer, pH 7.3, 3% bovine serum albumin, 0.3% Triton X-100 and 0.1% sodium azide and then overnight in humid chambers in primary antiserum diluted in the same buffer. The sections were then rinsed with phosphate buffered saline, incubated for 2 h in a secondary goat anti-rabbit antiserum conjugated with tetramethyl rhodamine isothiocyanate (Tago) in incubation buffer, rinsed with phosphate buffered saline, mounted with glycerin in phosphate-buffered saline (1:1 by volume) and examined. Sections were stained for both SP and NKA according to the elution technique of Tramu et al. 17. In brief, 8-~m sections were incubated with antiserum to NKA (1:1000) and processed for indirect immunofluorescence as described above. After examinations of the sections for NKA-like immunoreactivity and photography, the slides were incubated in a solution of potassium permanganate and sulfuric acid (2 ml 2.5% KMnO4 and 2% H2SO 4 in 50 ml distilled water) for 90 s. The sections were then incubated with antiserum to SP and processed for indirect immunofluorescence. The sections were again photographed and the pattern of SP-like immunoreactivity was compared with the distribution of NKA-like immunoreactivity. Labelling was examined with a Zeiss ICM 405 equipped with epifluorescence. Several antisera were used in these studies. A rabbit antiserum generated against SP was obtained from Immunonuclear (Chicago, IL, U.S.A.). One of us (S.L.) has generated antisera which specifically recognize either NKA or SP 5'1°. Preabsorption of the SP antisera with 10~g/ml of SP (Sigma Chemicals, St. Louis, MO) completely abolished labelling, whereas preabsorption with 10 ~g/ml of NKA (Sigma) did not detectably diminish labelling. Similarly, preabsorption of the antiserum directed against NKA with 10 ~g/ml of NKA abolished labelling, while preabsorption with SP did not detectably diminish staining.
207
Recording of chemoreceptor activity Seven cats were studied in this group of experiments. The detailed method for recording the chemoreceptor activity has been described previously 13,14. In brief, after exposing the carotid bifurcation, the left carotid sinus nerve was isolated and cut close to the junction with the glossopharyngeal nerve. By raising the skin flaps of the incision, a pool of paraffin oil was formed to prevent drying of the nerve. The paraffin oil was kept warm by an infrared lamp (temp. 37 °C). The nerve was desheathed and split into filaments. The electrical afferent activity of single or multiple chemoreceptor units (1-4 active units) was recorded using bipolar platinum-iridium electrodes and an AC amplifier (Grass Instruments Model PR 121). Chemoreceptor units were identified by their (1) spontaneous sporadic discharge, (2) increase in activity with hypoxia, and (3) reduced activity during ventilation with 100% 02. The unit activity was converted to standard pulses (Winston Rad II) which were fed to a rate meter (Frederick Haer) to give signals quantitatively related to their discharge rate. Close carotid body injections of SP and NKA were accomplished via a catheter in the lingual artery, so that the tip was within the common carotid artery 1 cm below the carotid sinus region. The ganglioglomerular nerves were cut to avoid the influence of sympathetic discharge on carotid body activity. The stock solutions of SP and NKA (Sigma) were prepared in 0.01 M acetic acid (1 M solutions). Before each experiment the stock solutions were diluted in saline (pH 7.4). The following protocols were used. To maintain constant levels of arterial blood gasses, anesthetized animals were artificially ventilated with room air during the experimental period after paralyzing them with intravenous injections of gallamine triethiodide (Flaxedil 3-4 mg/kg). Arterial pO 2 and pCO2 were kept close to 100 and 35 mm Hg respectively by adjusting the stroke volume and rate of the respirator. Chemosensory discharge was recorded 1 min prior to and 2 min following the administration of different doses of NKA and SP. The injection volumes were 0.3 ml. Injections of the same volume of saline served as controls. Fifteen min were allowed between tests. In 3 cats, dose responses to NKA were assessed first and followed by doses of SP. In the other 4 cats, the order of SP and NKA injections were reversed. All
variables including the arterial pressure, tidal pCO 2, chemoreceptor activity from the ratemeter output were recorded both on an oscilloscopic recorder (Electronics for Medicine), and on magnetic tape (Hewlett-Packard). Chemoreceptor activity (impulses/s) was averaged over a period of 30 s during the control period, i.e. before peptide injection and every 10 s following the injection of the tachykinin. Statistical comparisons were made with one-way analysis of variance and paired t test.
Radioimmunoassay measurements of tachykinins The tachykinin content (SP and NKA) of the carotid bodies was assessed in 20 cats by radioimmunoassay (RIA). To maintain constant blood gas levels, all animals were ventilated with a respirator after spontaneous respiratory movements were stopped with additional doses of anesthetic (10-15 mg/kg). Ventilation was adjusted to maintain arterial pCO 2 of about 30 mm Hg. The following protocols were employed. One group of animals was ventilated with room air (n = 7 cats); the second group with 100% oxygen (n = 6); and the third group with a hypoxic gas mixture (12% 02 + 88% N 2, n = 7 cats). After 1 h, an arterial blood sample was collected to determine blood gas levels. After ligating nearby blood vessels and clearing the connective tissue, one carotid body (i.e. either the left or the right carotid body) was removed and the remaining carotid body 10 min later. The average time from the point of ligating the blood vessels to removal of the carotid body was 129 + 7 s (mean + S.E.M.). The carotid bodies were placed in vials, weighed and frozen at -70 °C until further analysis. The carotid bodies were sonicated in presence of 600/A of a medium containing 0.02 M fl-mercaptoethanol and 2.0 M acetic acid per tissue for 30 s at 4 °C using a sonicator cell disrupter. After sonication, the probe was rinsed with 250pl of the medium. The homogenates were centrifuged at 20,000 rpm for 5 min at 4 °C. The supernatant was transferred to a vial and the pellet was washed with another 600 #1 of the medium and again centrifuged. The supernatants were pooled and lyophilized. The lyophilized samples were taken up in 1 ml of the assay buffer and analyzed for SP and NKA with radioimmunoassay techniques as described previously5,1°. The following variables were analyzed: (1) SP and NKA values ex-
F
209 RESULTS
Immunocytochemical localization of substance P and neurokinin A in the carotid body Many glomus cells exhibited immunoreactivity for SP and NKA (Fig. la,b). Although most cells containing immunoreactivity were intensely immunofluorescent, a range of immunofluorescence from very bright to barely detectable was evident. Most labelled cells lacked processes but a small percentage had short intensely immunoreactive processes (Fig. la,b). In addition, a relatively sparse plexus of fibers possessing immunoreactivity for either SP or NKA was present. Often these fibers appeared to contact
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Fig. 2. Example of an experiment illustrating the carotid chemoreceptor response to (A) saline; (B) neurokinin A (NKA 50 nmol); and (C) substance P (SP 50 nmol) administered close to the carotid body (arrow). The volume of injections was 0.3 ml. Imp/sec, rate meter output; CA, chemoreceptor activity; BP, arterial blood pressure (mm Hg). Note the augmented chemoreceptor discharge with NKA and SP, but not with the control saline injections.
glomus cells. When sections were stained sequentially for SP and NKA using the elution technique of Tramu et al.17, the glomus cells that contained NKA immunofluorescence also contained SP. To determine the proportion of glomus cells that contained SP and NKA, glomus cells in sections taken from several levels of carotid bodies from 5 cats were scored as positive or negative for NKA. We found that approximately 40% of glomus cells contained NKA. Since the elution studies indicated in a smaller sample of cells that all NKA-LI cells contained SP-LI, this suggests that almost half the glomus cells contained these two tachykinins.
Effect of intracarotid administration of substance P and neurokinin A on carotid chemoreceptor activity An experiment illustrating the effects of SP and NKA on chemosensory discharge is shown in Fig. 2. Control saline injections had no detectable excitatory effect on carotid body activity, whereas following administration of tachykinins, clear augmentation of sensory discharge was regularly seen. The analysis of time course of carotid body discharge with 3 doses of NKA is presented in Fig. 3. The peak excitation of carotid body activity occurred at approximately 20 s, then activity declined gradually towards control levels. The discharge frequency returned to preinjection control levels within 60 s following injection of 20 nmol of NKA, whereas it remained elevated within this period with 50 and 100 nmol (P < 0.02). In 3 experiments, the NKA-induced excitation was preceded by a transient inhibition. In the same experiments, however, control vehicle injections also produced similar depression of discharge frequency. Average values at peak excitation with all 3 doses of NKA are summarized in Table I. All the doses of NKA significantly stimulated carotid body activity. As shown in Fig. 4, the magnitude of excitation was related to the dose of NKA. However, the maximum increase caused by 100 nmol of NKA (maximum dose tested) was nearly the same as that obtained with 50 nmol, suggesting that the response reached a plateau (P > 0.05). The time course of SP-induced chemoreceptor response closely resembled that of NKA (Fig. 3). During the initial 10 s, the magnitude of the response produced by 50 and 100 nmol of SP was significantly
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Fig. 3. Time course of chemoreceptor activity following 3 concentrations of neurokinin A (NKA) and substance P (SP). Chemoreceptor activity was analyzed every 10 s for 60 s and expressed as a percent of preinjection controls. The dotted line indicates control activity. Data are mean + S.E.M. from 7 cats.
higher compared to NKA (P < 0.01). On the other hand, the peak excitation produced by both the tachykinins was the same (P > 0.05) (Fig. 4).
The arterial blood pressure fell following intracarotid administration of NKA and SP. All 3 doses of tachykinins produced a significant fall in blood pres-
TABLE I
Effect of neurokinin A and substance P on carotid chemoreceptor activity and arterial blood pressure Data are mean + S.E.M. from 7 cats. Paired t test.
Dose (nmol)
Neurokinin A 20 50 100 Substance P 20 50
100
Chemoreceptor discharge Control activity (imp~s)
Peak activity (imp~s)
Blood pressure P
Control B P (mm Hg)
B P trough (ram Hg)
2.8 + 0.9 3.0 + 1.0 3.2 _+ 1.0
5.2 + 1.3 8.5 + 1.2 9.7 + 1.9
<0.01 <0.01 <0.01
170 + 17 167 + 15 167 + 16
143 + 10 150 + 12 150 + 14
<0.01 <0.01 <0.01
2.7 + 1.3 2.8 + 1.4 2.9 + 1.5
5.7 + 1.7 9.8 + 2.0 10.0 ___ 1.5
<0.01 <0.01 <0.01
170 + 13 175 + 14 163 + 11
137 + 6 156 + 13 147 + 11
<0.01 <0.01 <0.01
211
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levels t e n d e d to be higher than SP, the difference was
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not statistically significant ( P > 0.05). A f t e r 1 h of hyperoxic ventilation, the average SP content of the carotid bodies was 51 + 4 fmol/mg, a value same as in r o o m air ventilation (Fig. 5). By contrast, N K A content was 29 + 3 fmol/mg, which was 68% less than the value o b t a i n e d during normoxia ( P < 0.01). Thus during hyperoxia, N K A levels of the carotid body were significantly less than SP (P < 0.01). As a consequence, the S P / N K A r a t i o in-
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Fig. 4. Comparison of chemoreceptor and arterial blood pressure changes caused by neurokinin A (NKA, open columns) with substance P (SP, hatched bars). Data are mean + S.E.M. from 7 cats. Peak responses caused by NKA were the same as that of SP (not significant P > 0.05). Note the lack of correlation between blood pressure and chemoreceptor responses.
O.~
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SP-LI frnol/mg I00-
sure (Table I) and the magnitude of fall in b l o o d pressure was inversely related to the concentration of the peptides (Fig. 4).
50-
Tachykinin content of carotid bodies with varying levels of arterial p 02 A t any level of arterial oxygen, the absolute tachykinin levels and the ratio of S P / N K A between the left and right carotid bodies did not differ significantly ( P > 0.05) indicating that variations in assay m e t h o d were minimal. H o w e v e r , as can be seen from Fig. 5, the tachykinin content of the carotid bodies varied with different levels of arterial pO 2. U n d e r normoxia, the average SP and N K A contents were 57 + 8 and 85 + 14 fmol/mg, respectively. The ratio of S P / N K A was 0.8 + 0.07. Though N K A
O-
I | I 1 I I HYPOXIA NORMOXIA HYPEROXIA
Fig. 5. Carotid body content of neurokinin A and substance P with 3 different arterial oxygen levels. NKA-LI and SP-LI, neurokinin A and substance P-like immunoreactivity measured by radioimmunoassay. Note increased SP and SK levels with hypoxia compared to hyperoxia. Data are mean + S.E.M.n.s., not significant P > 0.05, Kruskal-Wallis analysis of variance. Hyperoxia = p~O2 438 + 7 mm Hg;paCO 2 31 _+7 mm Hg; pH a 7.43 + 0.01; B.P 141 + 8 mm Hg. Normoxia =PaO2 101 + 2 mm Hg; PaCO 2 31 _+0.7 mm Hg; pHa 7.42 __.0.02; B.P 142 + 7 mm Hg; Hypoxia = PaO2 26 -+ 1 mm Hg; paCO2 31 _+ 0.9 mm Hg; pH a 7.40 _+0.02 mm Hg; B.P 141 + 10 mm Hg.
212 creased to 1.9 _+ 0.3 as compared to a room air value of 0.8 + 0.07 (P < 0.01). SP content of the carotid body after 1 h of hypoxic challenge was 146 _+ 20 fmol/mg, a value higher than both normoxic and hyperoxic conditions. The NKA content of the carotid body was 98 + 15 fmol/mg and was not significantly different from the value obtained during normoxia (P > 0.05), but substantially greater than hyperoxia. The ratio of SP/NKA was 1.5 + 0.1. During hypoxia, SP content was greater than NKA, but the difference was not statistically significant (P > 0.05). DISCUSSION The results of the present study demonstrate that the cat carotid body contains not only SP but also NKA, another neuropeptide of the tachykinin family. Both these peptides augment chemosensory activity of the carotid body when administered exogenously. Variations in arterial pO2, the natural stimulus to arterial chemoreceptors, altered the levels of both tachykinins in the carotid body. Confirming previous observations by others, we found SP-like immunoreactivity (SP-LI) in glomus cells of the cat carotid body 2'3,v and in a small number of nerve fibers associated with glomus cells and adjacent to blood vessels TM. The localization of NKA-like immunoreactivity (NKA-LI) appeared very similar to SP-LI. Not all glomus cells contained detectable tachykinins, a finding consistent with other reports 2' 3.7. It is not clear whether the tachykinin-containing cells represent a functionally different class of glomus cells or reflect local differences in physiological conditions. Elution and sequential labelling studies verified the coexistence of SP-LI and NKA-LI in glomus cells and fibers. Colocalization of SP and NKA has been demonstrated in other tissues including the striatonigral system 12. We have previously shown that chronic afferent and sympathetic denervation abolishes the tachykinin immunoreactivity of nerve fibers, whereas that of glomus cells is unaffected 15. These observations indicate that both SP and NKA are synthesized within glomus cells, whereas the nerve fibers and the tachykinins they contain originate outside the carotid body. As reported by others s, we consistently observed
an increase in carotid body discharge in response t o SP. In addition, we also demonstrated that NKA augments carotid body activity. Thus, our results indicate not only that both peptides are present in the carotid body, but also that they augment its sensory discharge. Since NKA and SP produced a fall in blood pressure, the increased sensory discharge we observed could have been secondary to vascular effects. Such a possibility, however, seems unlikely because the changes in blood pressure do not appear causally related to chemosensory responses (see Fig. 4). Moreover, SP has been shown to increase chemosensory discharge of the carotid body in vitro where any vascular effects of the peptide would be eliminated 9. Therefore, it is likely that tachykinins (i.e. SP and NKA) augment chemosensory discharge by acting directly on the sensory elements. Though the peak excitation produced by both peptides is similar, SP appears to induce excitation more quickly than NKA. Pharmacological studies have indicated the existence of multiple tachykinin receptor sites in a variety of tissues (e.g. ref. 12). The different response rates to SP and NKA in the carotid body could therefore reflect differences in the receptor affinity and/or the number of receptor sites for the two tachykinins. Alternatively, the mechanism(s) by which SP and NKA augment chemosensory discharge may differ. That is, one may act directly, whereas the other acts indirectly by causing the release of other excitatory neuroactive substance(s). Previous studies have examined the SP content of the cat carotid body. Wharton et al. ~s reported 54.6 _+ 16.8 fmol of SP/mg of wet wt. We found similar values, i.e., 57 + 8 fmol/mg, under normoxic conditions. Substantial species variations in the SP content of carotid body may exist. In a recent study, Hansen et al. 4 reported the SP content of rabbit carotid body to be 2.97 ng/mg of protein. If the protein content is approximately 10% of wet wt., then the SP content of the rabbit carotid body is 220 fmol/mg of wet wt., approximately 4 times greater than that of the cat carotid body. We found that under normoxic conditions, the NKA content of the carotid body was 85 + 15 fmol/mg. Whether NKA is also present in the carotid bodies of other species remains to be determined. Of considerable interest is the finding that both the content of the two tachykinins and the ratio of SP/ NKA varied substantially with changes in arterial
213 p O 2. Both SP and NKA levels were significantly greater in hypoxic conditions than in hyperoxic ventilation (Fig. 5). It seems unlikely that the observed increases in SP and NKA are artifactual, since various factors such as arterial blood pressure, blood gas tensions, and handling of the carotid body tissue which might have contributed to the variations in tachykinin levels, were kept constant in all the groups studied. The present results differ from those obtained by Hansen et al. 4 who found that the SP content of rabbit carotid bodies diminished with exposure to intermittent hypoxia (5% O 2 + 95% N2). A number of experimental differences are likely to account for this discrepancy. The tachykinin levels of cats and rabbits may respond differently to hypoxia. Moreover, in contrast to the present study in which cats were anesthetized and then exposed to hypoxia, Hansen and colleagues 4 exposed unanesthetized rabbits to hypoxic challenge and then removed carotid bodies after anesthetizing them. In fact, an increase in SP content of substantia nigra has been demonstrated in chronically hypoxic rats 1. The tissue tachykinin content will be affected by various factors which include rate of synthesis of the precursor peptide, processing from the precursor molecule, release and subsequent degradation of the peptide. The increase in tachykinin levels during hypoxia could have occurred if lowered p O 2 enhanced either synthesis or processing of peptides from the precursor molecule. Alternatively, the enzymatic degradation could have been affected by hypoxia leading to elevated tachykinin levels. Deviations from normoxia, i.e. either hyper- or hypoxia, caused an increase in the ratio of SP/NKA. This could be due to changes in synthesis and/or release of these peptides. Three rat PPT m R N A ' s de-
REFERENCES 1 Arregui, A., Barer, G.R. and Emson, P.C., Neurochemical studies in the hypoxic brain: substance P, Met-enkephalin, GABA and angiotensin converting enzyme, Life Sci., 28 (1981) 2925-2929. 2 Chen, I.-U., Yates, R.D. and Hansen, J.T., Substance Plike immunoreactivity in rat and cat carotid bodies: light and electron microscopic studies, Histol. Histopathol., 1 (1986) 203-212. 3 Cuello, A.C. and McQueen, D.S. Substance P: a carotid body peptide, Neurosci. Lett., 17 (1980) 215-219.
rived from a single gene encode the peptides SP and NKA; a-PPT m R N A encodes only SP, whereas r and y-PPT mRNA's encode both for SP and N K A 6,n. Although the rapidity of the changes makes it seem somewhat unlikely, it is possible that differential m R N A processing and/or translation is involved in this response. The fact that exogenous NKA augments the sensory discharge of carotid body and that its content is increased by low pO2 but decreased during hyperoxia suggests that NKA may play a physiological role in chemoreception. Possibly both SP and NKA may serve as neuromodulators in chemotransduction. However, each of these tachykinins may operate over a different range o f p O 2. In summary, we have shown that cat carotid body in addition to SP also contains NKA, another structurally related peptide. Both these tachykinins are predominantly localized in glomus cells, the putative receptor element of the carotid body. In addition, both tachykinins when administered close to the carotid body augmented the chemosensory discharge. Finally, changes in arterial p O 2 substantially altered the carotid body levels of both NKA and SP, suggesting that these tachykinins may be of importance in chemotransduction.
ACKNOWLEDGEMENTS The NKA peptides were generous gifts from Drs. Angeliki Buku and Irving Schwartz from Mt. Sinai School of Medicine, New York. This work was supported in part by grants from National Heart and Lung Blood Institute (HL-38986; HL-39921) and V.A. Merit Reviews.
4 Hansen, G., Jones, L. and Fidone, S., Physiological chemoreceptor stimulation decreases enkephalin and substance P in the carotid body, Peptides, 7 (1986) 767-769. 5 Jonassen, J.A., Mullikin-Kilpatrick, D., McAdam, A. and Leeman, S.E., Thyroid hormone status regulates preprotachykinin gene expression in male rats, Endocrinology, 12 (1987) 1555-1561. 6 Krause, J.E., Chirgwin, J.M., Carter, M.S., Xu, Z.S. and Hershey, A.D., Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 881-885. 7 Lundberg, J.M., H6kfelt, T., Fahrenkrug, J., Nilsson, G.
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8
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11
12 13
and Terenius, L., Peptides in the carotid body (glomus caroticum): VIP-enkephalin and substance P-like immunoreactivity, Acta Physiol. Scand., 107 (1979) 281-297. McQueen, D.S., Effects of substance P on carotid chemoreceptor activity in the cat, J. Physiol. (Lond.), 302 (1980) 31-47. 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. Mroz, E.A. and Leeman, S.E., Substance P. In R.M. Jaffe and H.R. Berhaman (Eds.), Methods of Hormone Radioimmunoassay, Academic, New York, 1979, pp. 121-137. Nawa, H., Hirose, T., Takashima, H., Inayama, S. and Nakanishi, S., Nucleotide sequences of cloned cDNAs for two types of bovine brain substance P precursor, Nature (Lond.), 306 (1983) 32-36. Pernow, B., Substance P, Pharmacol. Rev., 35 (1983) 85-141. Prabhakar, N.R., Runold, M., Yamamoto, Y., Lagercrantz, H. and Von Euler, C., Effect of substance P antagonist on the hypoxia-induced carotid chemoreceptor ac-
tivity, Acta Physiol. Scand., 121 (1984) 301-393. 14 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. 15 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 University Press, New York, 1988, pp. 57-63. 16 Shults, C.W., Yajima, H., Gullner, G.-G., Chase, T.N. and O'Donohue, T.L., Demonstration and distribution of kassinin-like material (substance K) in the rat central nervous system, J. Neurochem., 45 (1985)552-558. 17 Tramu, G., Pilez, A. and Leonardelli, J., An efficient method of antibody elution for the successive or simultaneous localization of two antigens by immunocytochemistry, J. Histochem. Cytochem., 26 (1978) 322-324. 18 Wharton, J., Polak, J.M., Pearse, A.G.E., McGregor, G.P., Bryant, M.G., Bloom, S.R., Emson, P.C., Bisgard, G.E. and Will, J.A., Enkephalin-, VIP-, and substance Plike immunoreactivity in the carotid body, Nature (Lond.), 284 (1980) 269-271.
205
BRE 14252
Research Repods
Substance P and neurokinin A in the cat carotid body: localization, exogenous effects and changes in content in response to arterial pO 2 Nanduri R. Prabhakar 1, Story C. Landis 2, Ganesh K. Kumar 1, Debra Mullikin-Kilpatrick 3, Neil S. Cherniack I and Susan Leeman 3 Departments of 1Medicineand 2pharmacology, Case Western Reserve University, Cleveland, OH 44106 (U.S.A.) and 3Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01605 (U.S.A.) (Accepted 26 July 1988)
Key words." Tachykinin; Substance P; Neurokinin A; Hypoxia; Carotid body; Chemoreceptor
In the present work we studied the occurrence of substance P (SP) and neurokinin A (NKA) in the carotid bodies of cats by means of immunocytochemistry and radioimmunoassay (RIA). We also compared the exogenous effects of SP and NKA on carotid body sensory discharge. SP- and NKA-like immunoreactivities (SP-LI; NKA-LI) were seen in many glomus cells and in a sparse plexus of fine fibers. The SP-LI containing glomus cells and fibers also exhibited NKA-LI, suggesting that both these tachykinins coexist in the carotid body. Chemoreceptor discharge increased both by SP and NKA in a dose-dependent manner. The peak excitation produced by SP and NKA was the same when the effects were compared on an equimolar basis. The tachykinin content of the carotid bodies varied with changes in arterialpO:. During normoxia, SP and NKA levels were 57 + 8 and 85 + 14 fmol/mg, respectively. When the animals were exposed for 1 h to 100% 02, SP content was unchanged (51 + 4 fmol/mg), whereas NKA levels were significantly lower than during normoxia (29 + 3 fmol/mg, P < 0.01). Following 1 h of hypoxia, SP content of the carotid body was 146 + 20 fmol/mg, a value higher than that obtained during normoxia and hyperoxia. NKA levels, on the other hand, were not significantly different from normoxic values. These results indicate that the cat carotid body (1) contains both SP and NKA, (2) both peptides augment neural discharge of the carotid body and (3) their levels in the carotid body are substantially altered by arterial oxygen, the natural stimulus to the chemoreceptors. INTRODUCTION The ventilatory increase that occurs during hypoxia arises from stimulation of arterial chemoreceptors, in particular the carotid bodies. Several lines of evidence suggest that the n e u r o p e p t i d e substance P (SP) is involved in the production of the hypoxic response. First, SP-like immunoreactivity (SP-LI) is present in glomus cells and some afferent nerve terminals in the carotid body 2'3'7'18. Second, exogenous SP augments the sensory discharge of the carotid body both in vivo and in vitro s'9. Third, the antagonists specific for SPlike neuropeptides attenuate or abolish the hypoxic
but not the CO2-induced excitation of carotid chemoreceptors 13'14. It has been r e p o r t e d recently that SP content of carotid bodies decreases during hypoxic exposure, possibly due to enhanced release 4. SP and neurokinin A ( N K A ) belong to a group of structurally related n e u r o p e p t i d e s called tachykinins. In m a m m a l i a n tissues, SP and N K A are derived from a single preprotachykinin gene (PPT) 6A1, raising the possibility that SP, as well as N K A are present in the carotid body and might play a physiological role in chemoreception. W e therefore examined the localization of SP and N K A in the cat carotid body, studied their effects on neural discharge of chemore-
Correspondence: N.R. Prabhakar, Department of Medicine, Pulmonary Division, University Hospitals, 2074 Abington Road, Cleveland, OH 44106, U.S.A. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
206 ceptors and determined levels of these tachykinins in carotid bodies of cats breathing different concentrations of 0~. MATERIALS AND METHODS Three groups of experiments were performed on 32 adult cats of either sex weighing 2.1 to 4.3 kg. They were anesthetized with i.p. injections of pentobarbital sodium (35 mg/kg), supplemented as necessary. In all experiments, routine surgical procedures included tracheal intubation and cannulation of a femoral artery and vein. The arterial catheter was used for monitoring arterial blood pressure and for sampling blood for determinations of blood gasses. Systemic administration of fluids or drugs was accomplished through the venous catheter. To expose the carotid bifurcation, the trachea and esophagus were ligated above the site of tracheal cannula, sectioned and retracted rostrally. The exposed tissues were covered with cotton gauze soaked in saline. End tidal p C O 2 was continuously monitored with an infrared CO2 analyzer (Beckman LB-2). The body temperature of the animals was kept at 38 °C (+ 1 °C) by means of a heating blanket governed by a rectal thermistor probe.
Immunocytochemical studies The localization of SP and NKA was examined in the carotid bodies of 5 cats using immunocytochemical techniques. Anesthetized animals were artificially ventilated with room air with a positive pressure respirator (Harvard Apparatus Model 665). Heparin (1000 units/kg) was administered i.v. 15 min before the beginning of perfusion. After opening the thorax, the ascending aorta was cannulated and connected to reservoirs containing perfusion medium. An opening was made in the right atrium for drainage of fluids. Heparinized saline was perfused for 15 min until the blood vessels were free of blood. Thereafter the perfusion medium was switched to 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3, for 30 min at room temperature. The carotid bodies were removed with the bifurcation of the carotid artery, rinsed with phosphate buffer, infiltrated with 30% sucrose in 0.1 M phosphate buffer and stored frozen in sucrose at -70 °C.
Cryostat sections, 8/~m-thick, were cut and thawmounted on gelatin-coated slides. The sections were allowed to air dry for 30 min and were then either stained immediately or stored frozen at -20 °C for staining at a later time. In most cases, sections were stained for a single neuropeptide, either SP or NKA, using rabbit antisera as described below. Sections were incubated for 1 h in a buffer containing 0.5 M NaC1, 0.01 M phosphate buffer, pH 7.3, 3% bovine serum albumin, 0.3% Triton X-100 and 0.1% sodium azide and then overnight in humid chambers in primary antiserum diluted in the same buffer. The sections were then rinsed with phosphate buffered saline, incubated for 2 h in a secondary goat anti-rabbit antiserum conjugated with tetramethyl rhodamine isothiocyanate (Tago) in incubation buffer, rinsed with phosphate buffered saline, mounted with glycerin in phosphate-buffered saline (1:1 by volume) and examined. Sections were stained for both SP and NKA according to the elution technique of Tramu et al. 17. In brief, 8-~m sections were incubated with antiserum to NKA (1:1000) and processed for indirect immunofluorescence as described above. After examinations of the sections for NKA-like immunoreactivity and photography, the slides were incubated in a solution of potassium permanganate and sulfuric acid (2 ml 2.5% KMnO4 and 2% H2SO 4 in 50 ml distilled water) for 90 s. The sections were then incubated with antiserum to SP and processed for indirect immunofluorescence. The sections were again photographed and the pattern of SP-like immunoreactivity was compared with the distribution of NKA-like immunoreactivity. Labelling was examined with a Zeiss ICM 405 equipped with epifluorescence. Several antisera were used in these studies. A rabbit antiserum generated against SP was obtained from Immunonuclear (Chicago, IL, U.S.A.). One of us (S.L.) has generated antisera which specifically recognize either NKA or SP 5'1°. Preabsorption of the SP antisera with 10~g/ml of SP (Sigma Chemicals, St. Louis, MO) completely abolished labelling, whereas preabsorption with 10 ~g/ml of NKA (Sigma) did not detectably diminish labelling. Similarly, preabsorption of the antiserum directed against NKA with 10 ~g/ml of NKA abolished labelling, while preabsorption with SP did not detectably diminish staining.
207
Recording of chemoreceptor activity Seven cats were studied in this group of experiments. The detailed method for recording the chemoreceptor activity has been described previously 13,14. In brief, after exposing the carotid bifurcation, the left carotid sinus nerve was isolated and cut close to the junction with the glossopharyngeal nerve. By raising the skin flaps of the incision, a pool of paraffin oil was formed to prevent drying of the nerve. The paraffin oil was kept warm by an infrared lamp (temp. 37 °C). The nerve was desheathed and split into filaments. The electrical afferent activity of single or multiple chemoreceptor units (1-4 active units) was recorded using bipolar platinum-iridium electrodes and an AC amplifier (Grass Instruments Model PR 121). Chemoreceptor units were identified by their (1) spontaneous sporadic discharge, (2) increase in activity with hypoxia, and (3) reduced activity during ventilation with 100% 02. The unit activity was converted to standard pulses (Winston Rad II) which were fed to a rate meter (Frederick Haer) to give signals quantitatively related to their discharge rate. Close carotid body injections of SP and NKA were accomplished via a catheter in the lingual artery, so that the tip was within the common carotid artery 1 cm below the carotid sinus region. The ganglioglomerular nerves were cut to avoid the influence of sympathetic discharge on carotid body activity. The stock solutions of SP and NKA (Sigma) were prepared in 0.01 M acetic acid (1 M solutions). Before each experiment the stock solutions were diluted in saline (pH 7.4). The following protocols were used. To maintain constant levels of arterial blood gasses, anesthetized animals were artificially ventilated with room air during the experimental period after paralyzing them with intravenous injections of gallamine triethiodide (Flaxedil 3-4 mg/kg). Arterial pO 2 and pCO2 were kept close to 100 and 35 mm Hg respectively by adjusting the stroke volume and rate of the respirator. Chemosensory discharge was recorded 1 min prior to and 2 min following the administration of different doses of NKA and SP. The injection volumes were 0.3 ml. Injections of the same volume of saline served as controls. Fifteen min were allowed between tests. In 3 cats, dose responses to NKA were assessed first and followed by doses of SP. In the other 4 cats, the order of SP and NKA injections were reversed. All
variables including the arterial pressure, tidal pCO 2, chemoreceptor activity from the ratemeter output were recorded both on an oscilloscopic recorder (Electronics for Medicine), and on magnetic tape (Hewlett-Packard). Chemoreceptor activity (impulses/s) was averaged over a period of 30 s during the control period, i.e. before peptide injection and every 10 s following the injection of the tachykinin. Statistical comparisons were made with one-way analysis of variance and paired t test.
Radioimmunoassay measurements of tachykinins The tachykinin content (SP and NKA) of the carotid bodies was assessed in 20 cats by radioimmunoassay (RIA). To maintain constant blood gas levels, all animals were ventilated with a respirator after spontaneous respiratory movements were stopped with additional doses of anesthetic (10-15 mg/kg). Ventilation was adjusted to maintain arterial pCO 2 of about 30 mm Hg. The following protocols were employed. One group of animals was ventilated with room air (n = 7 cats); the second group with 100% oxygen (n = 6); and the third group with a hypoxic gas mixture (12% 02 + 88% N 2, n = 7 cats). After 1 h, an arterial blood sample was collected to determine blood gas levels. After ligating nearby blood vessels and clearing the connective tissue, one carotid body (i.e. either the left or the right carotid body) was removed and the remaining carotid body 10 min later. The average time from the point of ligating the blood vessels to removal of the carotid body was 129 + 7 s (mean + S.E.M.). The carotid bodies were placed in vials, weighed and frozen at -70 °C until further analysis. The carotid bodies were sonicated in presence of 600/A of a medium containing 0.02 M fl-mercaptoethanol and 2.0 M acetic acid per tissue for 30 s at 4 °C using a sonicator cell disrupter. After sonication, the probe was rinsed with 250pl of the medium. The homogenates were centrifuged at 20,000 rpm for 5 min at 4 °C. The supernatant was transferred to a vial and the pellet was washed with another 600 #1 of the medium and again centrifuged. The supernatants were pooled and lyophilized. The lyophilized samples were taken up in 1 ml of the assay buffer and analyzed for SP and NKA with radioimmunoassay techniques as described previously5,1°. The following variables were analyzed: (1) SP and NKA values ex-
F
209 RESULTS
Immunocytochemical localization of substance P and neurokinin A in the carotid body Many glomus cells exhibited immunoreactivity for SP and NKA (Fig. la,b). Although most cells containing immunoreactivity were intensely immunofluorescent, a range of immunofluorescence from very bright to barely detectable was evident. Most labelled cells lacked processes but a small percentage had short intensely immunoreactive processes (Fig. la,b). In addition, a relatively sparse plexus of fibers possessing immunoreactivity for either SP or NKA was present. Often these fibers appeared to contact
A
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Fig. 2. Example of an experiment illustrating the carotid chemoreceptor response to (A) saline; (B) neurokinin A (NKA 50 nmol); and (C) substance P (SP 50 nmol) administered close to the carotid body (arrow). The volume of injections was 0.3 ml. Imp/sec, rate meter output; CA, chemoreceptor activity; BP, arterial blood pressure (mm Hg). Note the augmented chemoreceptor discharge with NKA and SP, but not with the control saline injections.
glomus cells. When sections were stained sequentially for SP and NKA using the elution technique of Tramu et al.17, the glomus cells that contained NKA immunofluorescence also contained SP. To determine the proportion of glomus cells that contained SP and NKA, glomus cells in sections taken from several levels of carotid bodies from 5 cats were scored as positive or negative for NKA. We found that approximately 40% of glomus cells contained NKA. Since the elution studies indicated in a smaller sample of cells that all NKA-LI cells contained SP-LI, this suggests that almost half the glomus cells contained these two tachykinins.
Effect of intracarotid administration of substance P and neurokinin A on carotid chemoreceptor activity An experiment illustrating the effects of SP and NKA on chemosensory discharge is shown in Fig. 2. Control saline injections had no detectable excitatory effect on carotid body activity, whereas following administration of tachykinins, clear augmentation of sensory discharge was regularly seen. The analysis of time course of carotid body discharge with 3 doses of NKA is presented in Fig. 3. The peak excitation of carotid body activity occurred at approximately 20 s, then activity declined gradually towards control levels. The discharge frequency returned to preinjection control levels within 60 s following injection of 20 nmol of NKA, whereas it remained elevated within this period with 50 and 100 nmol (P < 0.02). In 3 experiments, the NKA-induced excitation was preceded by a transient inhibition. In the same experiments, however, control vehicle injections also produced similar depression of discharge frequency. Average values at peak excitation with all 3 doses of NKA are summarized in Table I. All the doses of NKA significantly stimulated carotid body activity. As shown in Fig. 4, the magnitude of excitation was related to the dose of NKA. However, the maximum increase caused by 100 nmol of NKA (maximum dose tested) was nearly the same as that obtained with 50 nmol, suggesting that the response reached a plateau (P > 0.05). The time course of SP-induced chemoreceptor response closely resembled that of NKA (Fig. 3). During the initial 10 s, the magnitude of the response produced by 50 and 100 nmol of SP was significantly
210
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o 200
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Fig. 3. Time course of chemoreceptor activity following 3 concentrations of neurokinin A (NKA) and substance P (SP). Chemoreceptor activity was analyzed every 10 s for 60 s and expressed as a percent of preinjection controls. The dotted line indicates control activity. Data are mean + S.E.M. from 7 cats.
higher compared to NKA (P < 0.01). On the other hand, the peak excitation produced by both the tachykinins was the same (P > 0.05) (Fig. 4).
The arterial blood pressure fell following intracarotid administration of NKA and SP. All 3 doses of tachykinins produced a significant fall in blood pres-
TABLE I
Effect of neurokinin A and substance P on carotid chemoreceptor activity and arterial blood pressure Data are mean + S.E.M. from 7 cats. Paired t test.
Dose (nmol)
Neurokinin A 20 50 100 Substance P 20 50
100
Chemoreceptor discharge Control activity (imp~s)
Peak activity (imp~s)
Blood pressure P
Control B P (mm Hg)
B P trough (ram Hg)
2.8 + 0.9 3.0 + 1.0 3.2 _+ 1.0
5.2 + 1.3 8.5 + 1.2 9.7 + 1.9
<0.01 <0.01 <0.01
170 + 17 167 + 15 167 + 16
143 + 10 150 + 12 150 + 14
<0.01 <0.01 <0.01
2.7 + 1.3 2.8 + 1.4 2.9 + 1.5
5.7 + 1.7 9.8 + 2.0 10.0 ___ 1.5
<0.01 <0.01 <0.01
170 + 13 175 + 14 163 + 11
137 + 6 156 + 13 147 + 11
<0.01 <0.01 <0.01
211
LI Rec.
Chemo
10-
levels t e n d e d to be higher than SP, the difference was
Activity
Imp/Sec.
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not statistically significant ( P > 0.05). A f t e r 1 h of hyperoxic ventilation, the average SP content of the carotid bodies was 51 + 4 fmol/mg, a value same as in r o o m air ventilation (Fig. 5). By contrast, N K A content was 29 + 3 fmol/mg, which was 68% less than the value o b t a i n e d during normoxia ( P < 0.01). Thus during hyperoxia, N K A levels of the carotid body were significantly less than SP (P < 0.01). As a consequence, the S P / N K A r a t i o in-
iiiii!iii!ii!!i ii~i!!i!i~i!i!ii ..,.........,., ::::::::::::::
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~
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Fig. 4. Comparison of chemoreceptor and arterial blood pressure changes caused by neurokinin A (NKA, open columns) with substance P (SP, hatched bars). Data are mean + S.E.M. from 7 cats. Peak responses caused by NKA were the same as that of SP (not significant P > 0.05). Note the lack of correlation between blood pressure and chemoreceptor responses.
O.~
150-
SP-LI frnol/mg I00-
sure (Table I) and the magnitude of fall in b l o o d pressure was inversely related to the concentration of the peptides (Fig. 4).
50-
Tachykinin content of carotid bodies with varying levels of arterial p 02 A t any level of arterial oxygen, the absolute tachykinin levels and the ratio of S P / N K A between the left and right carotid bodies did not differ significantly ( P > 0.05) indicating that variations in assay m e t h o d were minimal. H o w e v e r , as can be seen from Fig. 5, the tachykinin content of the carotid bodies varied with different levels of arterial pO 2. U n d e r normoxia, the average SP and N K A contents were 57 + 8 and 85 + 14 fmol/mg, respectively. The ratio of S P / N K A was 0.8 + 0.07. Though N K A
O-
I | I 1 I I HYPOXIA NORMOXIA HYPEROXIA
Fig. 5. Carotid body content of neurokinin A and substance P with 3 different arterial oxygen levels. NKA-LI and SP-LI, neurokinin A and substance P-like immunoreactivity measured by radioimmunoassay. Note increased SP and SK levels with hypoxia compared to hyperoxia. Data are mean + S.E.M.n.s., not significant P > 0.05, Kruskal-Wallis analysis of variance. Hyperoxia = p~O2 438 + 7 mm Hg;paCO 2 31 _+7 mm Hg; pH a 7.43 + 0.01; B.P 141 + 8 mm Hg. Normoxia =PaO2 101 + 2 mm Hg; PaCO 2 31 _+0.7 mm Hg; pHa 7.42 __.0.02; B.P 142 + 7 mm Hg; Hypoxia = PaO2 26 -+ 1 mm Hg; paCO2 31 _+ 0.9 mm Hg; pH a 7.40 _+0.02 mm Hg; B.P 141 + 10 mm Hg.
212 creased to 1.9 _+ 0.3 as compared to a room air value of 0.8 + 0.07 (P < 0.01). SP content of the carotid body after 1 h of hypoxic challenge was 146 _+ 20 fmol/mg, a value higher than both normoxic and hyperoxic conditions. The NKA content of the carotid body was 98 + 15 fmol/mg and was not significantly different from the value obtained during normoxia (P > 0.05), but substantially greater than hyperoxia. The ratio of SP/NKA was 1.5 + 0.1. During hypoxia, SP content was greater than NKA, but the difference was not statistically significant (P > 0.05). DISCUSSION The results of the present study demonstrate that the cat carotid body contains not only SP but also NKA, another neuropeptide of the tachykinin family. Both these peptides augment chemosensory activity of the carotid body when administered exogenously. Variations in arterial pO2, the natural stimulus to arterial chemoreceptors, altered the levels of both tachykinins in the carotid body. Confirming previous observations by others, we found SP-like immunoreactivity (SP-LI) in glomus cells of the cat carotid body 2'3,v and in a small number of nerve fibers associated with glomus cells and adjacent to blood vessels TM. The localization of NKA-like immunoreactivity (NKA-LI) appeared very similar to SP-LI. Not all glomus cells contained detectable tachykinins, a finding consistent with other reports 2' 3.7. It is not clear whether the tachykinin-containing cells represent a functionally different class of glomus cells or reflect local differences in physiological conditions. Elution and sequential labelling studies verified the coexistence of SP-LI and NKA-LI in glomus cells and fibers. Colocalization of SP and NKA has been demonstrated in other tissues including the striatonigral system 12. We have previously shown that chronic afferent and sympathetic denervation abolishes the tachykinin immunoreactivity of nerve fibers, whereas that of glomus cells is unaffected 15. These observations indicate that both SP and NKA are synthesized within glomus cells, whereas the nerve fibers and the tachykinins they contain originate outside the carotid body. As reported by others s, we consistently observed
an increase in carotid body discharge in response t o SP. In addition, we also demonstrated that NKA augments carotid body activity. Thus, our results indicate not only that both peptides are present in the carotid body, but also that they augment its sensory discharge. Since NKA and SP produced a fall in blood pressure, the increased sensory discharge we observed could have been secondary to vascular effects. Such a possibility, however, seems unlikely because the changes in blood pressure do not appear causally related to chemosensory responses (see Fig. 4). Moreover, SP has been shown to increase chemosensory discharge of the carotid body in vitro where any vascular effects of the peptide would be eliminated 9. Therefore, it is likely that tachykinins (i.e. SP and NKA) augment chemosensory discharge by acting directly on the sensory elements. Though the peak excitation produced by both peptides is similar, SP appears to induce excitation more quickly than NKA. Pharmacological studies have indicated the existence of multiple tachykinin receptor sites in a variety of tissues (e.g. ref. 12). The different response rates to SP and NKA in the carotid body could therefore reflect differences in the receptor affinity and/or the number of receptor sites for the two tachykinins. Alternatively, the mechanism(s) by which SP and NKA augment chemosensory discharge may differ. That is, one may act directly, whereas the other acts indirectly by causing the release of other excitatory neuroactive substance(s). Previous studies have examined the SP content of the cat carotid body. Wharton et al. ~s reported 54.6 _+ 16.8 fmol of SP/mg of wet wt. We found similar values, i.e., 57 + 8 fmol/mg, under normoxic conditions. Substantial species variations in the SP content of carotid body may exist. In a recent study, Hansen et al. 4 reported the SP content of rabbit carotid body to be 2.97 ng/mg of protein. If the protein content is approximately 10% of wet wt., then the SP content of the rabbit carotid body is 220 fmol/mg of wet wt., approximately 4 times greater than that of the cat carotid body. We found that under normoxic conditions, the NKA content of the carotid body was 85 + 15 fmol/mg. Whether NKA is also present in the carotid bodies of other species remains to be determined. Of considerable interest is the finding that both the content of the two tachykinins and the ratio of SP/ NKA varied substantially with changes in arterial
213 p O 2. Both SP and NKA levels were significantly greater in hypoxic conditions than in hyperoxic ventilation (Fig. 5). It seems unlikely that the observed increases in SP and NKA are artifactual, since various factors such as arterial blood pressure, blood gas tensions, and handling of the carotid body tissue which might have contributed to the variations in tachykinin levels, were kept constant in all the groups studied. The present results differ from those obtained by Hansen et al. 4 who found that the SP content of rabbit carotid bodies diminished with exposure to intermittent hypoxia (5% O 2 + 95% N2). A number of experimental differences are likely to account for this discrepancy. The tachykinin levels of cats and rabbits may respond differently to hypoxia. Moreover, in contrast to the present study in which cats were anesthetized and then exposed to hypoxia, Hansen and colleagues 4 exposed unanesthetized rabbits to hypoxic challenge and then removed carotid bodies after anesthetizing them. In fact, an increase in SP content of substantia nigra has been demonstrated in chronically hypoxic rats 1. The tissue tachykinin content will be affected by various factors which include rate of synthesis of the precursor peptide, processing from the precursor molecule, release and subsequent degradation of the peptide. The increase in tachykinin levels during hypoxia could have occurred if lowered p O 2 enhanced either synthesis or processing of peptides from the precursor molecule. Alternatively, the enzymatic degradation could have been affected by hypoxia leading to elevated tachykinin levels. Deviations from normoxia, i.e. either hyper- or hypoxia, caused an increase in the ratio of SP/NKA. This could be due to changes in synthesis and/or release of these peptides. Three rat PPT m R N A ' s de-
REFERENCES 1 Arregui, A., Barer, G.R. and Emson, P.C., Neurochemical studies in the hypoxic brain: substance P, Met-enkephalin, GABA and angiotensin converting enzyme, Life Sci., 28 (1981) 2925-2929. 2 Chen, I.-U., Yates, R.D. and Hansen, J.T., Substance Plike immunoreactivity in rat and cat carotid bodies: light and electron microscopic studies, Histol. Histopathol., 1 (1986) 203-212. 3 Cuello, A.C. and McQueen, D.S. Substance P: a carotid body peptide, Neurosci. Lett., 17 (1980) 215-219.
rived from a single gene encode the peptides SP and NKA; a-PPT m R N A encodes only SP, whereas r and y-PPT mRNA's encode both for SP and N K A 6,n. Although the rapidity of the changes makes it seem somewhat unlikely, it is possible that differential m R N A processing and/or translation is involved in this response. The fact that exogenous NKA augments the sensory discharge of carotid body and that its content is increased by low pO2 but decreased during hyperoxia suggests that NKA may play a physiological role in chemoreception. Possibly both SP and NKA may serve as neuromodulators in chemotransduction. However, each of these tachykinins may operate over a different range o f p O 2. In summary, we have shown that cat carotid body in addition to SP also contains NKA, another structurally related peptide. Both these tachykinins are predominantly localized in glomus cells, the putative receptor element of the carotid body. In addition, both tachykinins when administered close to the carotid body augmented the chemosensory discharge. Finally, changes in arterial p O 2 substantially altered the carotid body levels of both NKA and SP, suggesting that these tachykinins may be of importance in chemotransduction.
ACKNOWLEDGEMENTS The NKA peptides were generous gifts from Drs. Angeliki Buku and Irving Schwartz from Mt. Sinai School of Medicine, New York. This work was supported in part by grants from National Heart and Lung Blood Institute (HL-38986; HL-39921) and V.A. Merit Reviews.
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