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Nitric oxide mediates chemoreceptor inhibition in the cat carotid body
Vol. 65, No. l, pp. 217-229, 1995 Elsevier ScienceLtd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00
Neuroscience
~
Pergamon
0306-4522(94)00437-4
NITRIC O X I D E M E D I A T E S C H E M O R E C E P T O R I N H I B I T I O N IN THE CAT C A R O T I D B O D Y Z.-Z. W A N G , L. J. S T E N S A A S , B. G. D I N G E R and S. J. F I D O N E Department of Physiology, University of Utah School of Medicine, Salt Lake City, UT 84108, U.S.A. A~traet--Numerous studies have demonstrated that carotid sinus nerve fibers mediate a so-called "efferent" inhibition of carotid body chemoreceptors. However, the mechanism(s) underlying this phenomenon are not understood. Recently, it has been shown that an extensive plexus of nitric oxide synthase-containing carotid sinus nerve fibers innervate the carotid body, and that many fine, beaded fibers can be seen in close proximity to small blood vessels as well as lobules of parenchymal cells. The present study examined the effects of centrifugal neural activity in the carotid sinus nerve on the accumulation of [3H]citrulline synthesized from [3H]arginine in the cat carotid body, and the possible involvement of nitric oxide in mediating "efferent" chemoreceptor inhibition. Electrical stimulation of carotid sinus nerve C-fibers evoked an increase in [3H]citruUine accumulation in the carotid body, which was Ca2+-dependent and blocked by L-N G-nitroarginine methylester (0.1 mM), an inhibitor of nitric oxide synthase. Using a vascularly perfused in vitro carotid body preparation, chemoreceptor activity was recorded from thin nerve filaments split-off from the main trunk of the carotid sinus nerve. Electrical stimulation of the main nerve trunk at C-fiber intensities inhibited steady-state chemoreceptor discharge, and this effect was blocked by L-N °-nitroarginine methylester. However, when the organ preparation was switched to the superfuse-only mode, carotid sinus nerve stimulation failed to alter the steady-state discharge, but under these conditions, prolonged nerve stimulation (> 5 min) did attenuate the chemoreceptor response to hypoxia, an effect which was likewise blocked by L-NG-nitroarginine methylester. The present data, together with previous anatomical findings that nitric oxide synthase immunoreactivity is present in both sensory and autonomic ganglion cells innervating the carotid body, suggest that two neural mechanisms may be involved in the inhibitory neural regulation of carotid chemoreceptors. One mechanism appears to involve nitric oxide release from intralobular sensory C-fibers, which lie in close proximity to the chemoreceptor type I cells. The other mechanism involves release of nitric oxide from perivascular terminals of autonomic microganglia neurons, which control carotid body blood flow.
The m a m m a l i a n carotid body is an arterial chemoreceptor organ which monitors blood levels of 02, CO 2 and pH. Pre-neural type I (glomus) cells, the purported chemosensory elements, transduce the physico-chemical status of the blood and transmit this information to synaptically-apposed afferent terminals of carotid sinus nerve (CSN) fibers (see Refs 13 and 15). Biscoe and Sampson were the first to show that spontaneous centrifugal (efferent) neural activity could be recorded from the central cut end of the C S N . 6 Later, Neil and O ' R e g a n reported that electrical stimulation of the peripheral cut end of the C S N depressed the chemosensory discharge recorded from nerve filaments split-off from the stimulated, main nerve trunk. 24~25 Fidone and Sato confirmed these observations and showed that such "efferent" inhibition of chemoreceptor discharge was mediated by C S N C-fibers, 16 although the origin of these fibers Arg, arginine; cGMP, cyclic 3',5'-guanosine monophosphate; CNS, carotid sinus nerve; EDTA, ethylenediaminetetra-acetate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; NAME, N Gnitroarginine methylester; NMMA, N~-monomethyl arginine; NO, nitric oxide; NOS, nitric oxide synthase; PBS, phosphate-buffered saline.
Abbreviations:
217
was not identified. N u m e r o u s subsequent studies also demonstrated that this chemoreceptor inhibition could be reflexly evoked, and could alter not only basal chemoreceptor discharge but also the sensitivity of the carotid body to hypoxia, hypercapnia and acidosis (see Ref. 27 for review). However, the anatomical and physiological basis for this inhibitory phenomenon remained uncertain and controversial. Sampson suggested that chemoreceptor inhibition is mediated by centrifugal (efferent) fibers in the CSN, which somehow modulate type I cell activity to inhibit the discharge of c h e m o r e c e p t o r nerve fibers. 29'3° M c D o n a l d and Mitchell, 23 on the other hand, observed that the inhibitory effects of C S N stimulation persisted following chronic "decentralization" of the glossopharyngeal (IXth cranial) nerve, i.e. section of the nerve root central to the petrosal sensory ganglion. This procedure should have eliminated all efferent fibers to the carotid body and, consequently, these investigators hypothesized that petrosal ganglion sensory neurons, not m o t o r fibers, mediate the chemoreceptor inhibition. They pointed to the presence of reciprocal synapses between chemoreceptor nerve terminals and type I cells, and proposed that antidromic activation of these sensory
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fibers (by p r i m a r y afferent depolarization o f their central nerve terminals in the brain) would activate the type I cells to stimulate the release of inhibitory transmitter(s) a n d suppress the activity of chemoreceptor nerve endings. It has been k n o w n for m a n y years that the carotid body is innervated not only by C S N sensory fibers, but also by sympathetic fibers from the ganglioglomerular nerve o f the nearby superior cervical ganglion, as well as by p a r a s y m p a t h e t i c fibers traveling in the glossopharyngeal nerve a n d C S N . 22'27 It is also well established that these sympathetic (postganglionic) fibers innervate carotid body blood vessels, a n d can increase c h e m o r e c e p t o r discharge by decreasing blood flow t h r o u g h the microvascular bed of the organ. 5'14 P a r a s y m p a t h e t i c vascular fibers m a y also modify o r g a n b l o o d flow, and might be expected to increase flow a n d thereby reduce c h e m o r e c e p t o r discharge, s'17 However, little is k n o w n regarding how these a u t o n o m i c elements m i g h t be involved in c h e m o r e c e p t o r inhibition. Recent i m m u n o c y t o c h e m i c a l studies in o u r laboratory have delineated an extensive plexus of nitric oxide synthase (NOS)-positive nerve fibers, some of which penetrate the c h e m o s e n s o r y p a r e n c h y m a l cell lobules, while others terminate in close proximity to carotid body b l o o d vessels. 34'35 F u r t h e r m o r e , correlative physiological studies have d e m o n s t r a t e d that exogenous nitric oxide (NO) has a p o t e n t inhibitory effect on c h e m o r e c e p t o r d i s c h a r g e ) 6'37 In view of the m a n y uncertainties regarding the m e c h a n i s m s of c h e m o r e c e p t o r "efferent" inhibition, the present study was u n d e r t a k e n to examine whether centrifugal neural activity in the C S N regulates N O p r o d u c t i o n in the carotid body, a n d if N O might be a physiological m e d i a t o r o f c h e m o r e c e p t o r inhibition. It was f o u n d t h a t electrical stimulation of C S N C-fibers, but not A-fibers, elicited b o t h a Ca2+-dependent synthesis of NO, a n d a p r o f o u n d , N O - m e d i a t e d inhibition of carotid c h e m o r e c e p t o r discharge. Some preliminary findings were reported elsewhere) 036a
EXPERIMENTAL PROCEDURES
Carotid body superfusion, per~sion and electrical stimulation of the carotid sinus nerve
Domestic cats were obtained locally through the University of Utah Animal Resource Center. The carotid body and CSN were dissected from adult animals anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and transferred to ice-cold modified Tyrode's solution equilibrated with 100% 02. The composition of Tyrode's solution was (in mM): NaC1 112, KCI 4.7, CaCI 2 2.2, MgCI 2 1.1, sodium glutamate 42, HEPES 5, glucose 5.5 (pH 7.42). After careful removal of surrounding connective tissue, the carotid body was placed in a superfusion chamber containing 100% O2-equilibrated Tyrode's solution, and the superfusate passing through the chamber (0.6ml/min) was maintained at 3T'C by a heating block and a thermistor-servo mechanism. 2 The CSN was drawn up into an adjoining oil-filled compartment, placed across bipolar platinum stimulating electrodes and stimulated with rectangular pulses (20Hz, 1 ms in duration) delivered through a stimulus isolation unit. Electrical activity of the CSN (spontaneous chemosensory discharge, as well as A- and C-fiber compound action potentials) was recorded with another pair of bipolar platinum electrodes positioned near the carotid body. Maximal A-fiber and C-fiber activation was achieved by adjusting the stimulus voltage. We utilized an in vitro vascularly perfused/superfused cat carotid body modified from that developed by Belmonte and Eyzaguirre? The glossopharyngeal nerve was dissected free and transected as proximal as possible to its entry into the skull. The internal carotid, ascending pharyngeal and occipital arteries were ligated and cut, as were other small vascular branches and veins draining the carotid body. After transferring the preparation to the in vitro superfusion chamber, the common and external carotid arteries were cannulated and perfused with air-equilibrated modified Tyrode's solution containing 5% dextran (mol. wt 40,000) at 37'C and at a flow rate of I ml/min (Fig. 1). The CSN was drawn up into an adjoining oil-filled compartment, and thin nerve filaments were dissected free from the main nerve trunk for recording single chemoreceptor unit activity. One channel of an Ortec stimulator provided an isolated squarepulse stimulus (duration Ires) to the main trunk of the CSN. Stimulus artifacts were minimized utilizing a second channel of the stimulator; its output preceded that of the first channel by 0.1 ms and controlled an analog switch which interrupted the recorded signal to the amplifier for the duration of the stimulus. This circuit offset the stimulus artifact and facilitated counting of afferent impulses. 5 Stimu-
Fig, 1. Schematic diagram of perfusion superfusion system. C1, perfusion-superfusion chamber, whose temperature is maintained at 37 ± 0.2°C by a heating system (cross-hatched); C2, oil chamber; TI, temperature probe for the heated metal block; T2, temperature probe for C1; h, drain for superfusion fluid; P, pressure transducer; R, peripheral resistance: D, drop counter.
Nitric oxide in the carotid body lus current spread to the recording filament was minimized and monitored by experimental approaches described by Fidone and Sato, TM Goodman, ~7 and Belmonte and Eyzaguirre? (1) A small branch of the CSN close to the carotid body was dissected centrally along the main nerve trunk. (2) A nerve filament was separated from this branch and used for recording in order to minimize the area of contact with the stimulated main nerve trunk. (3) A small drop of Tyrode's solution was applied to the filament at its point of emergence from the main nerve trunk, and a ground lead connected to this drop, with the remaining nerve fibers immersed in warm mineral oil. (4) The refractory pause in the nerve discharge following the stimulus artifact was monitored by superposition of successive sweeps on the oscilloscope screen.~6 Perfusion pressure was monitored with a pressure transducer in the outflow tube linked to the external carotid artery, and the pressure was adjusted using a variable resistance on the outflow tube while flow rate was recorded with an electronic drop counter (Fig. l). Impulses recorded from nerve filaments were led to an a.c. preamplifier, displayed on an oscilloscope and input to an IBM-PC computer via an AD/DA converter.
[3H]Citrulline assay Carotid bodies removed from anesthetized cats were transferred to a dissection chamber containing ice-cold 100% O2-equilibrated Tyrode's solution. Connective tissue was removed, and the organs were weighed on a Cahn electrobalance equipped with a humidified chamber. Tissue samples were pre-incubated in glass minivials containing 1 ml of 100% O2-Tyrode's medium for 30min in a water bath shaker at 37°C, 32 and then incubated with 1 ml of Tyrode's solution containing L-[2,3-3H]arginine (3/iCi/ml, 56 Ci/mmol; DuPont-NEN) and equilibrated either with: (i) control (100% 02) or (ii) hypoxic gas mixtures (10% 02 in 90%N2). After 1 h incubation at 37°C, the carotid bodies were assayed for [3H]citrulline, as described below. In other experiments, the carotid body was continuously superfused during nerve stimulation with 100% O2-equilibrated media containing L-[2,3-3H]arginine (3 pCi/ml, 56 Ci/mmol; Dupont-NEN) at a flow rate of 0.6 ml/min. Following a wash in ice-cold Tyrode's media containing 4 mM EDTA and 5 mM L-arginine, the tissues were homogenized in 1 ml of 1 M trichloroacetic acid and centrifuged. Trichloroacetic acid in the supernatant was extracted three times with 2 ml diethyl ether, Aliquots of 0.5 ml of the supernatant were neutralized with 2 ml of 20 mM HEPES (pH 6.0), applied to 2-ml columns of Dowex AG50WX-8 (Na + form, BioRad) and eluted with 2 ml of water. [3H]Citrulline was quantified by liquid scintillation spectroscopy of the 4-ml eluate, 7 and the results were expressed as mean _+ S.E.M., with the data analysed using Student's t-test.
Cyclic guanosine 3',5"-monophosphate immunocytochemistry For immunocytochemistry of cyclic guanosine Y,5'monophosphate (cGMP), the tissue was continuously superfused during nerve stimulation with 100% 02 equilibrated Tyrode's medium containing theophylline (10mM) and superoxide dismutase (100 unit/ml) at a flow rate of 0.6 ml/min. Carotid bodies were fixed in ice-cold fixative (4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4) for 2 h, rinsed in cold phosphatebuffered saline (PBS; 15 min), cryoprotected in 20% sucrose (1 h at 4'C) and sectioned in a cryostat (-20°C). Frozen sections (6-8t~m thick) were thaw-mounted onto chrome alum, gelatin-coated slides and processed for cGMP immunocytochemical staining. 38 Briefly, sections were hydrated in PBS (15 min), treated with 3% hydrogen peroxide (10min) and avidin-biotin pre-blocking reagents (20 rain, Vector kit). Sections were then incubated in the following reagents, each of which was followed by a 15 min wash in PBS: (i) polyclonal cGMP antiserum (1 : I000 in PBS
219
containing 0.3% Triton X-100) overnight at 4~'C;~2"39 (ii) biotinylated goat anti-rabbit secondary antiserum (Vector, 1:400) at room temperature (30 min); (iii) avidinbiotin peroxidase complex (Vector elite kit) at room temperature (60 min); (iv) a mixture of 3,Y-diaminobenzidine tetrahydrochloride (Sigma) and hydrogen peroxide. Control sections showed no specific immunoreactivity following pre-absorption of the primary antibody with 10 3 M cGMP, or replacement by normal rabbit serum.
RESULTS
Effect o f carotid sinus nerve stimulation on nitric oxide synthesis Citrulline a c c u m u l a t i o n in the carotid body was assessed by m e a s u r i n g the conversion o f [3H]-Larginine to [3H]citrulline, a reaction t h a t stoichiometrically produces N O / T h e results were expressed b o t h as d.p.m./mg tissue/h a n d as the percentage of total radioactivity i n c o r p o r a t e d into the tissue; the latter value allowed for differences in [3H]arginine u p t a k e with different c h e m o r e c e p t o r stimuli/8 Activation of C S N A-fibers (0.7-1.6 V, 20 Hz; Fig. 2) p r o d u c e d no change in the level of [3H]citrulline f o r m a t i o n following 15 rain of nerve stimulation (Table 1). However, a significant increase in [SH]citrulline f o r m a t i o n occurred (Table 1) w h e n nerve stimulation was adjusted for m a x i m a l r e c r u i t m e n t o f C S N C-fibers (4-6 V, 20 Hz, 15 min), as m o n i t o r e d by the recorded comp o u n d action potential (Fig. 2). Superfusion media lacking Ca 2+ ( M r + = 2.2 m M ) completely blocked [3H]citrulline formation, indicating t h a t N O synthesis in the carotid b o d y is d e p e n d e n t on the availability of extracellular Ca 2+ .
Effect o f carotid sinus nerve stimulation on cyclic guanosine 3',5'-monophosphate formation It is well k n o w n t h a t N O increases c G M P production, s Consequently, we e x a m i n e d whether the increased N O synthesis from C S N stimulation would in t u r n activate guanylate cyclase a n d elevate c G M P levels in the carotid body. Figure 3 shows the time course of c G M P p r o d u c t i o n following electrical stimulation of C S N C-fibers. In control carotid bodies superfused with 100% O2-equilibrated medium, c G M P i m m u n o r e a c t i v i t y was undetectable in the tissue (Fig. 3A). Following 1 m i n of C S N stimulation (Fig. 3B), m o d e r a t e levels of c G M P i m m u n o r e a c t i v i t y a p p e a r e d in s m o o t h muscle cells of small arterial vessels, a n d after 5 min of nerve stimulation (Fig. 3C), low levels of c G M P were also a p p a r e n t in the type I cells. Prolonged C S N stimulation (15 min) markedly e n h a n c e d the level o f c G M P i m m u n o r e a c t i v i t y b o t h in blood vessels a n d in type I cells (Fig. 3D). Such changes in c G M P i m m u n o staining occurred w h e n nerve stimulation recruited C-fibers to the response, but were not observed when only A-fibers were stimulated (Fig. 4B, C). In other experiments, we examined w h e t h e r the N O S inhibitor, L-N~-nitroarginine methylester ( c - N A M E ) ,
Z.-Z. Wang et al.
220
A
A
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A
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Fig. 2. Compound action potentials evoked by stimulus parameters adjusted to recruit maximal A-fiber or C-fiber potentials while measuring [3H]citrulline formation in the in vitro cat carotid body. See also Table 1.
could block the increase in c G M P accumulation produced by C S N stimulation. As illustrated in Fig. 4D, L - N A M E (0.1 mM) effectively blocked the increase in c G M P immunoreactivity produced by 15 min of C S N stimulation. Excess L-arginine (L-Arg, l mM) reversed the block by L - N A M E (Fig. 4E), suggesting that the effect of this drug was due to competitive inhibition of endogenous N O synthesis. D-Arg, an inactive isomer of arginine, failed to overcome the inhibitory effect of L - N A M E (Fig. 4F).
Chemoreceptor inhibition in the vascularly perJused/ superfused carotid body Carotid body preparations which were both superfused and vascularly perfused (air-equilibrated media) exhibited basal levels of single-unit chemoreceptor discharge which ranged from two to 10 impulses/s. Brief hypoxic stimuli, consisting of 5% O2-equilibrated perfusion medium (30 s), elevated the chemoreceptor discharge to 30-40 impulses/s; discharge rates returned rapidly to basal levels following
Table 1. Effects of carotid sinus nerve stimulation on nitric oxide synthesis in the cat carotid body [3H]Citrulline formation (d.p.m./h per mg tissue) Control A-fiber C-fiber C-fiber/zero Ca 2.
777 + 41 808 __+67 1503 +_ 133"* 679 _ 58
Per cent of total radioactivity 5.8 + 6.3 + 12.6 ± 7.0 ±
0.85 0.69 1.03"* 0.92
6 4 6 4
n represents the number of carotid bodies. Values are mean _+ S.E.M. **P < 0.01 vs control.
Nitric oxide in the carotid body
221
12 Fig. 3. Induction of cGMP formation in cat carotid body following electrical stimulation of C-fibers of the CSN. (A) cGMP immunoreactivity is not detectable in the unstimulated control carotid body. (B) Accumulation of cGMP appears in arterial smooth muscular cells (arrows) following 1 min of nerve stimulation. (C) Low levels of cGMP immunoreactivity occur in type I cells (arrowheads) following 5 min of stimulation. (D) High levels of cGMP immunoreactivity occur in both blood vessels (arrows) and type I cells (arrowheads) following 15 rain of nerve stimulation. reintroduction of control, air-equilibrated perfusion medium. Elevation of perfusion pressure from 40 to 120mmHg reduced the discharge frequency, while abrupt reduction of pressure below 40 mmHg increased the unitary discharge. Electrical simulation of the CSN/glossopharyngeal nerve reduced the chemosensory discharge in approximately 60-70% of the units; the remaining units underwent no significant change. Although stimulus intensities of about 3 V activated some C-fibers and produced discernible chemoreceptor inhibition, chemosensory discharge was maximally attenuated by CSN stimulation at 5~5 V (10-20/s). The inhibitory effect occurred within 10s of the onset of nerve stimulation, and persisted throughout the stimulation period. Upon cessation of stimulation, inhibition occasionally lasted for more than 30 s, but ordinarily returned to basal levels within 10-20s (Fig. 5A). Superposition of successive sweeps On the oscilloscope screen failed to show a refractory pause in nerve discharge after the stimulus artifact, and nerve crush completely eliminated the electrically provoked chemoreceptor inhibition (data not shown). 16 NSC 65/I--H
The typical effect of L-NAME (0.1mM) on chemoreceptor inhibition is shown in Fig. 5B. The NOS inhibitor increased basal chemoreceptor discharge and completely blocked the inhibition induced by nerve stimulation. However, because chemoreceptor inhibition becomes less marked as basal chemosensory discharge increases, 16 we examined whether this L-NAME disinhibition was merely a consequence of increased basal chemoreceptor activity. The nerve was therefore stimulated in the presence of the drug, but after reducing chemoreceptor discharge to control levels by elevating the perfusion pressure. Under these conditions, again no marked inhibitory effect was seen (Fig. 5C). Moreover, the disinhibitory effect of L-NAME was reversed by excess L-Arg (1 mM; Fig. 5D), demonstrating competitive inhibition of endogenous NO synthesis in the carotid body. Similar results were obtained from eight preparations in five cats, as summarized in Table 2. The stereospecificity of NOS inhibitors with respect to chemoreceptor inhibition was examined using L-N °-monomethylarginine (L-NMMA). 7A° This potent NOS inhibitor clearly blocked chemo-
Z.-Z. Wang et al.
222
receptor inhibition elicited by electrical stimulation of the CSN, whereas its inactive isomer, D-N ~monomethylarginine (D-NMMA), was ineffective (Fig. 6).
Chemoreceptor inhibition in the superfused carotid body Stimulation of the CSN at 6-10 V (10-40 Hz) in the superfused carotid body preparation failed to change the basal frequency of chemoreceptor discharge. This
was true even after the rate of discharge was adjusted with 100% O2-equilibrated superfusion medium to the level recorded from the vascularly perfused preparation (Fig. 7). Nerve stimulation was also without effect on the high frequency, steady-state discharge induced by hypoxia (40% and 20% 02; Fig. 7; but see below). These findings might suggest that chemoreceptor inhibition induced by C S N stimulation is a vascular phenomenon, as originally suggested by G o o d m a n 17 and by Belmonte and Eyzaguirre. 5 However, our experiments showed that high levels of
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Fig. 4. CSN stimulation induced cGMP formation in the cat carotid body and its reversal by an NOS inhibitor, cGMP immunoreactivity is not discernible in the unstimulated control (A) and in the A-fiber stimulated (15 min) carotid body (B). High levels ofcGMP appear in arterial smooth muscle (arrowhead) and a few type I cells (arrows) following 15 min of electrical stimulation of the CSN with stimulus intensities recruiting C-fibers maximally (C). The C-fiber activation-evoked cGMP production was blocked by 0.1 mM L-NAME (D). L-Arg (1 raM) restored cGMP immunoreactivity in L-NAME-treated carotid bodies (E), and D-Arg was ineffective (F).
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223
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Fig. 5. L-NAME reverses chemoreceptor inhibition during CSN stimulation in vascularly perfused cat carotid body. (A) Single unit activity was attenuated during the nerve stimulation. (B) L-NAME (0.1 raM) elevates basal neural activity and it prevented the chemoreceptor inhibition. (C) Efferent inhibition was not elicited in the presence of L-NAME when the nerve discharge was adjusted to control levels by raising perfusion pressure. (D) L-Arg (1 mM) restored the chemoreceptor inhibition blocked by 0.1 mM L-NAME.
c G M P immunoreactivity are observed in type I cells following C S N stimulation, and it has been found that under these conditions c G M P inhibits the response to hypoxia while failing to alter basal discharge rates. 33 N o n e the less, we did find in these experiments that observable increases in tissue levels of c G M P required several minutes or more of continued CSN stimulation. Consequently, we examined whether the response to hypoxia in superfused carotid body preparations could be inhibited when C S N stimulation preceded the hypoxic stimulus. These experiments are illustrated in Fig. 8. As expected, when a short (5 min) hypoxic stimulus (20% O2-equilibrated superfusion medium) was delivered simultaneously with C S N stimulation, the chemosensory response was not markedly different
(Fig. 8B). However, the response to hypoxia was attenuated when nerve stimulation (10min) was begun 5 min prior to the start of the hypoxic episode (Fig. 8C). Maximal reduction of the chemoreceptor response occurred when C S N stimulation preceded the hypoxic episode by 10 min (Fig. 8D). This inhibition of the chemoreceptor response could be blocked by 0.1 m M L - N A M E (Fig. 8E), and the block was selectively reversed by 1 m M L-Arg (Fig. 8F). Similar results were obtained from multiple superfusion experiments, suggesting an important role for N O in chemoreceptor inhibition which is apparently independent of vascular effects (Table 3). As expected, inhibition of the hypoxic response could also be elicited in vascularly perfused preparations (Fig. 9); the magnitude and time course of the effect
Table 2. Carotid sinus nerve stimulation inhibits basal chemoreceptor activity in the perfused/superfused carotid body
CSN stimulation CSN stimulation plus 0.1 mM L-NAME CSN stimulation plus 0.1 mM L-NAME/1 mM L-Arg
Per cent of mean pre-stimulation frequency
n
61.9 + 5.9
8
97.8 4- 11.2"
6
43.2 4- 8.5
6
n represents the number of preparations; values are mean 4- S.E.M. *P < 0.05 vs CSN stimulation group.
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fused with 100% O2-equilibrated medium (Table 4). One hour of hypoxia (10% 02 in 90% N2) significantly increased [3H]citrulline levels in the carotid body. When expressed as a percentage of total radioactivity incorporated into the tissue (Table 4), the rate of [3H]citrulline formation was also elevated by hypoxia, indicating that hypoxia did not significantly change [3H]arginine uptake by the organ.
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Fig. 6. Stereospecific actions of NOS inhibitor on efferent chemoreceptor inhibition in the vascularly perfused/ superfused carotid body. Single unit discharge was reduced in control (A) and 0.1 mM D-NMMA-treated preparations during electrical stimulation of the CSN (B). L-NMMA (0.1 nM) blocked chemoreceptor inhibition (C). in perfused preparations differed little from those observed with the superfused preparation.
Effect of hypoxia on [3H]citrulline Jormation Experiments were conducted to determine the effects of hypoxia on NO synthesis. Moderate levels of [3H]citrulline were found in carotid bodies super-
The present study demonstrates that centrifugal neural activity in the CSN increases NO production in the carotid body. The elevated levels of [3H]citrulline which reflect this increase in NO synthesis occur only when CSN C-fibers are recruited by the stimulus, and only in the presence of extracellular Ca -,+ . These findings, together with our earlier immunocytochemical studies localizing NOS to unmyelinated axons in the carotid body, 34'35'37implicate CSN C-fibers as mediators of centrifugal, or "efferent," chemoreceptor inhibition. The present study further demonstrates that stimulation of CSN C-fibers induces cGMP formation in the carotid body. Our previous electrophysiological studies found that cGMP is a potent inhibitor of the hypoxia-evoked chemosensory response, 33 and that NO donors, including sodium nitroprusside and nitroglycerine, stimulate the production of cGMP in both type I cells and arterioles of the organ. 32'JT3s Taken together, these findings suggest that cGMP very likely acts as a second messenger mediator of chemoreceptor inhibition in the carotid body, triggered by the liberation of NO from NOS-positive CSN C-fibers. Stimulation of CSN C-fibers does not alter the resting chemosensory discharge of the superfused carotid body preparation, but does inhibit the chemoreceptor response to a hypoxic stimulus. Our results suggest that NOS-positive CSN sensory neurons, with cell bodies in the petrosal ganglia, mediate this non-vascular inhibitory effect through
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Nitric oxide in the carotid body
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Fig. 8. In the superfused carotid body preparation, delayed inhibition by CSN stimulation of the dynamic chemoreceptor response to hypoxia followed CSN stimulation. The chemosensory response to a 5 min hypoxic stimulus (20% O2-equilibrated superfusion medium, black bar) was not markedly changed by concomitant nerve stimulation (open bar, B). However, the hypoxic response was attenuated when the nerve stimulation (10 min) was started 5 min prior to hypoxia (C). Maximal attenuation of the response occurred with a 15 rain CSN stimulation which was started 10 min preceding hypoxia (D). The inhibition was blocked by superfusion medium containing 0.1 mM L-NAME (E). L-Arg (1 mM) reversed the effect of L-NAME (F). Note the basal chemosensory discharge was not changed by the nerve stimulation.
226
Z.-Z. Wang et al.
Table 3. Carotid sinus nerve stimulation inhibits hypoxia (20% Oj-evoked chemoreceptor response in the superfused carotid body Time of CSN stimulation 5 min 10min 15min 15 min (plus 0.1 mM L-NAME) 15min (plus 0.1 mM L-NAME/I mM L-Arg)
Per cent of control hypoxic response
tz
100.7 _+ 5.2 74.5 + 7.1" 54.3_+3.8** 106.2 _+ I0.4
5 5 5 4
44.9 _+ 4.0**
3
n represents the number of preparations; values are mean _+ S.E.M. *P < 0.05, **P < 0.01 vs control.
regulation o f c G M P levels in type I cells. Several observations support this view. First, NOS-positive nerve fibers arising from small diameter petrosal sensory neurons innervate type I cell lobules in the carotid body; 34"37 these sensory neurons also contain
Table 4. Effects of hypoxia on nitric oxide synthesis in the in vitro cat carotid body [3H]Citrulline formation (d.p.m./h per mg tissue)
Per cent of total radioactivity
n
1196 _+ 200 1784_+399"
8.9 + 1.3 14.0_+ 1.8"
6 6
100% 02 10% O~
n represents the number of carotid bodies. Values are mean + S.E.M. *P < 0.05 vs 100% O_~ group. substance P, and represent a subset o f sensory neurons distinct from the tyrosine hydroxylasepositive petrosal sensory neurons which also innervate type I cell l o b u l e s Y 4 Second, electrical stimulation o f CSN C-fibers (but not A-fibers) increases c G M P levels in type I cells (Figs 3, 4). Third, c G M P is a potent inhibitor o f the chemosensory response to hypoxia, but fails to alter basal chemo-
Perfusion
A
B
L-NAME(~.1mM 1~:...=_16,,,,i,..u ,~..,.~ ..,JHypox~ll
.__~ . . . . .
8V, 20Hz
C
,
,,°
D
.........
m
5 mJn
is0
Fig. 9. Attenuation of the chemosensory response to hypoxia by electrical stimulation of the CSN in the vascularly perfused cat carotid body. Note the basal chemosensory discharge was immediately reduced following the onset of the nerve stimulation.
Nitric oxide in the carotid body receptor discharge. 33 Fourth, the required length of time that CSN stimulation must precede an hypoxic stimulus in order to inhibit the chemosensory response corresponds to the time required for an observable accumulation of cGMP in type I cells (Figs 3, 8) finally, both chemoreceptor inhibition and cGMP formation produced by CSN stimulation in the superfused carotid body preparation are blocked by the NOS inhibitor, L-NAME. The fact that CSN stimulation inhibits steady-state chemoreceptor discharge in the vascularly perfused/ superfused carotid body preparation, but is ineffective in the superfused-only preparation, could also suggest that chemoreceptor inhibition is at least partly mediated through vascular mechanisms. This interpretation would be consistent with results from an earlier study by Belmonte and Eyzaguirre showing that activation of a vasomotor neural pathway in the CSN increased carotid body blood flow and decreased chemosensory discharge. 5 On the other hand, a decrease in total blood flow through the organ following CSN stimulation was observed by Neff and O'Regan in their original demonstration of chemoreceptor inhibition]5 and this finding was later confirmed by Acker and O'Regan. L Such discrepant findings suggest complex vascular dynamics sensitive to variable experimental conditions, and underscore the need to clarify the anatomical locations and physiological actions of vasomotor autonomic neurons controlling regional carotid body blood flow and organ chemosensitivity. This need is further emphasized by studies which show that chemoreceptor inhibition survives transection of the CSN proximal to the petrosal ganglion, a "decentralization" procedure which should eliminate all vasomotor efferents projecting from the brain to the carotid body. 23 Our studies have shown that large NOS-containing autonomic neurons occur along the CSN and within the carotid body itself,34'37 and likely correspond to the isolated groups of ganglionic neurons described by de Castro many years ago as "microganglia.''H Our immunocytochemical experiments show that these neurons are distinguishable from sensory neurons, because they exhibit neuronal processes resembling dendrites and co-stain for choline acetyltransferase and NOS. Also, these neurons can be shown to give rise to axons which innervate nearby blood vessels. Thus, these microganglia neurons appear to constitute a population of local, post ganglionic vasomotor elements in the carotid body. 34'37 Our studies using the NOS inhibitor, L-NAME, in the vascularly perfused/superfused carotid body preparation suggest that NO is a principal mediator of chemoreceptor inhibition. In this regard, it should be noted that NO is the most potent vasodilator known, and has been shown to be involved in the non-adrenergic non-cholinergic inhibition of gastrointestinal motility, as well as relaxation of vascular smooth muscle in numerous other sites, including the penile corpus cavernosum and the cerebral cortex. 8 ~0
227
NO generated by the neural innervation to vascular elements stimulates guanylate cyclase and cGMP production, leading to relaxation of smooth muscle and vascular dilatation.4'8 Because blood vessels in the carotid body are extensively innervated by NOSpositive microganglial elements, activation of these neurons should increase cGMP production in vascular smooth muscle, leading to vasodilation, increased blood flow and depression of steady-state chemosensory discharge. Our observations that CSN stimulation rapidly induces high levels of cGMP in vascular smooth muscle of the carotid body, and that L-NAME blocks this effect in the perfused/superfused organ preparation, provides compelling evidence in favor of such an NO-mediated mechanism. The physiological mechanism(s) responsible for the activation of NOS-containing sensory C-fibers in the carotid body remains an unresolved issue. One possibility is that primary afferent depolarization of the central (brainstem) terminals of these petrosal sensory neurons leads to antidromic "backfiring" of action potentials into the carotid body. 23 Another, and perhaps simpler explanation, arises as a consequence of the profuse branching of individual CSN axons within the carotid body; 19 a centripetal nerve impulse initiated within one branch of a sensory fiber could by axon reflex invade other branches, resulting in a local cascade of increased NO production within a terminal axon arbor. Evidence favoring this latter mechanism is presented in Table 4, which shows that NO production ([3H]citrulline formation) is elevated in carotid bodies superfused in vitro with low 0 2 medium. During hypoxia, the net activity of the carotid chemoreceptors very likely results from a balance between stimulatory and inhibitory processes. In this regard, previous studies have shown that centrifugal impulse traffic in the cat CSN tonically inhibits the response to both acute 26'28 and chronic 2°'21 hypoxia. The present study suggests that excitation of the carotid chemoreceptors triggers an NO-mediated inhibitory co-response, which perhaps could provide for both acute dynamic control as well as chronic adaptive adjustments within the chemoreceptor. Furthermore, such chemoreceptor inhibitory mechanisms involving neuromodulators like NO (and perhaps atrial natriuetic peptide) 33 may be important determinants in the observed blunting of ventilatory sensitivity due to chronic hypoxic exposure at high altitudes. 31 CONCLUSIONS
Our combined anatomical, neuropharmacological and electrophysiological findings support the notion that NO plays two major roles in carotid body chemoreceptor inhibition. First, acting in an anterograde fashion as a transmitter released by efferent autonomic neurons, NO has potent actions on arterial vascular elements. Second, NO liberated by
228
Z.-Z. Wang et al.
Smooth muscle Autonomic efferents (Parasympathetic)
L-Arg
NOS
~
[
C
~
[~
cGMP'~
Vascular relaxation
Flow~crease
" ~ '-'-I~" ca2. Sensory C-fibers
,I Chemosensory
Type I cell
Fig. 10. Schematic diagram depicting the two NO-mediated efferent inhibitory neural pathways that control carotid body chemoreceptors. GC, guanylate cyclase. Chemosensory discharge generated in the afferent sensory A-fibers is influenced by two separate NO-synthesizing neural pathways. Centrifugal nerve activity activates NOS in nerve terminals in the carotid body via a Ca2+-dependent mechanism. NO released from parasympathetic vasomotor fibers stimulates cGMP production in postsynaptic vascular smooth muscle and triggers vasodilation. The resultant flow increase attenuates the basal steady-state chemosensory activity. NO generated by axonal terminals of the sensory C-fibers diffuses to adjacent type I cells and stimulates cGMP accumulation, which modulates the chemoreceptor response to hypoxia.
a n t i d r o m i c activity invading the terminals o f sensory fibers (resulting either from peripheral axon reflexes or central p r i m a r y afferent depolarization) increases c G M P in c h e m o r e c e p t o r type I cells, leading to c h e m o s e n s o r y inhibition. In c o m b i n a t i o n , the vasom o t o r n e u r o n a l p a t h w a y appears to m o d u l a t e steady-state c h e m o r e c e p t o r activity, while the sensory n e u r o n a l p a t h w a y inhibits the d y n a m i c response to a hypoxic stimulus (Fig. 10). These cellular m e c h a n i s m s therefore constitute two morphologically distinct, but
functionally c o m p l e m e n t a r y , neural pathways, which together may account for the k n o w n characteristics o f the so-called "efferent" inhibitory control o f the carotid chemoreceptors (Fig. 10). Acknowledgements--This work was supported by USPHS
grants NS12636 and NS07938. Z.-Z. Wang was supported in part by a graduate research fellowship from the University of Utah. The authors wish to thank Dr J. DeVente of the Free University, Amsterdam, The Netherlands, for supplying the antiserum to cyclic GMP.
REFERENCES 1. Acker H. and O'Regan R. G. (1981) The effects of stimulation of autonomic nerves on carotid body blood flow in the cat. J. Physiol., Lond. 315, 99-I 10. 2. Almaraz L. and Fidone S. (1986) Carotid sinus nerve C-fibers release catecholamines from the cat carotid body. Neurosei. Lett. 67, 153-158. 3. Almaraz L., Wang Z.-Z., Stensaas L. J. and Fidone S. J. (1993) Release of dopamine from carotid sinus nerve fibers innervating type I cells in the cat carotid body. Biol. Signals 2, 16-26. 4. Arnold W. P, Mittal C. K., Katsuki S. and Murad F. (1977) Nitric oxide activates guanylate cyclase and increases guanosine 3":5"-cyclic monophosphate levels in various tissue preparations. Proe. natn. Acad. Sci. U.S.A. 74, 3203 3207. 5. Belmonte C. and Eyzaguirre C. (1974) Efferent influences on carotid body chemoreceptors. J. Neurophysiol. 37, 1131-1143. 6. Biscoe T. J. and Sampson S. R. (1967) Spontaneous activity recorded from the central cut end of the carotid sinus nerve of the cat. Nature, Lond. 216, 294-295. 7. Bredt D. S. and Snyder S. H. (1989) Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc. natn. Acad. Sci. U.S.A. 86, 9030-9033. 8. Bredt D. S. and Snyder S. H. (1992) Nitric oxide, a novel neuronal messenger. Neuron 8, 3-11. 9. Bult H., Boeckxstaens G. E., Pelckmans P. A., Jordaens F. H., Van Maereke Y. M. and Herman A. G. (1990) Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter. Nature 345, 346-347.
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10. Burnett A. L., Lowenstein C. J., Bredt D. S., Chang T. S. K. and Snyder S. H. (1992) Nitric oxide: a physiological mediator of penile erection. Science 257, 401-403. 11. De Castro F. (1926) Sur la structure el l'innervation de la glande intercarotidienne (glomus caroticum) de l'homme et des mammiferes, et sur un nouveau systeme d'innervation autonome du nerf glosopharyngien. Trabajos Lab. Invest. Biol. Univ. Madrid 24, 365-432. 12. De Vente J., Steinbusch H. W. M. and Schipper J. (1987) A new approach to immunocytochemistry of 3',5'-cyclic guanosine monophosphate: preparation, specificity and initial application of a new antiserum against formaldehydefixed 3',5'-cyclic guanosine monophosphate. Neuroscience 22, 361-373. 13. Eyzaguirre C., Fitzgerald R. S., Lahiri S. and Zapata P. (1983) Arterial chemoreceptors. In The Cardiovascular System. Handbook of Physiology (eds Shepperd J. T. and Abboud F.), Sect. 3, Vol. II, pp. 557q521. Am. Physiol. Soc., Bethesda, MD. 14. Eyzaguirre C. and Lewin J. (1961) The effect of sympathetic stimulation on carotid nerve activity. J. Physiol., Lond. 159, 251-267. 15. Fidone S. J. and Gonzalez C. (1986) Initiation and control of chemoreceptor activity in the carotid body. In Handbook of Physiology. The Respiratory System (eds Fishman A. P,), Sect. 3, Vol. II, Part I, pp. 247-312. Am. Physiol. Soc., Bethesda, MD. 16. Fidone S. J. and Sato A. (1970) Efferent inhibition and antidromic depression of chemoreceptor A-fibers from the cat carotid body. Brain Res. 22, 181--193. 17. Goodman N. W. (1973) Efferent control of arterial chemoreceptors mediated by glossopharyngeal fibers and artifacts introduced by stimulation techniques. J. PhysioL, Lond. 230, 295-311, 18. Kiedrowski L., Costa E. and Wroblewski J. T. (1992) Glutamate receptor agonists stimulate nitric oxide synthesis in primary cultures of cerebellar granules cells. J. Neurochem. 58, 335-341. 19. Kondo H. (1976) Innervation of the carotid body of the adult rat: a serial ultrathin section analysis. Cell Tiss. Res. 173, 1-15. 20. Lahiri S., Smatresk N., Pokorski M., Barnard P. and Mokashi A. (1983) Efferent inhibition of carotid body chemoreceptor in chronically hypoxic cats. Am. J. Physiol. 245, R678-R683. 21. Lahiri S., Smatresk N., Pokorski M., Barnard P., Mokashi A. and McGregor K. H. (1984) Dopaminergic efferent inhibition of carotid body chemoreceptors in chronically hypoxic cats. Am. J. Physiol. 247, R24-R28. 22. McDonald D. M. (1981) Peripheral chemoreceptors: structure-function relationships of the carotid body, In Lung Biology in Health and Disease. The Regulation of Breathing (ed. Hornbein T. F.), vol. 17, pp. 105-319. Dekker, New York. 23. McDonald D. M. and Mitchell R. A. (1981) The neural pathway involved in "efferent inhibition" of chemoreceptors in the cat carotid body. J. comp. Neurol. 201, 457-476. 24, Neil E. and O'Regan R. G. (1969) Effects of sinus and aortic nerve efferents on arterial chemoreceptor function. J. Physiol., Lond. 200, 69P-71P. 25. Neil E. and O'Regan R. G. (1971) The effects of electrical stimulation of the distal end of the carotid sinus and aortic nerve on peripheral arterial chemoreceptor activity in the cat. J. Physiol., Lond. 215, 15 32. 26. Neil E. and O'Regan R. G. (1971) Efferent and afferent impulse activity recorded from few-fibre preparations of otherwise intact sinus and aortic nerves. J. Physiol. 215, 33-47. 27. O'Regan R. G. and Majcherczyk S. (1983) Control of peripheral chemoreceptors by efferent nerves. In Physiology of the Peripheral Arterial Chemoreceptors (eds Acker H. and O'Regan R. G.), pp. 257-298. Elsevier, Amsterdam. 28. Sampson S. R. and Biscoe T. J. (1970) Efferent control of the carotid body chemoreceptor. Experientia 26, 261-262. 29. Sampson S. R. (1972) Mechanism of efferent inhibition of carotid body chemoreceptors in the cat. Brain Res. 45, 266-270. 30. Sampson S. R. (1975) Pharmacology of feedback inhibition of carotid body chemoreceptors in the cat. In The Peripheral Arterial Chemoreceptors (ed. Purves M. J.), pp. 207 220. Cambridge University Press, London. 31. Tatsumi K., Pickett C. K. and Weil J. V. (1991) Attenuated carotid body hypoxic sensitivity after prolonged hypoxic exposure. J. appl. Physiol. 70, 748-755. 32. Wang W.-J., Cheng G.-F,, Dinger B. G. and Fidone S. J. (1989) Effects of hypoxia on cyclic nucleotide formation in rabbit carotid body in vitro. Neurosci. Lett. 105, 164-168. 33. Wang W.-J., He L., Chen J., Dinger B. and Fidone S. (1993) Mechanisms underlying chemoreceptor inhibition induced by atrial natriuretic peptide in rabbit carotid body. J. Physiol. 460, 427-441. 34. Wang Z.-Z., Bredt D. S., Fidone S. J. and Stensaas L. J. (1993) Neurons synthesizing nitric oxide innervate the mammalian carotid body. J, comp. Neurol. 336, 419-432. 35. Wang Z.-Z., Bredt D. S., Snyder S. H., Fidone S. J. and Stensaas L. J. (1992) Nitric oxide synthase in the carotid body. Soc. Neurosci. Abstr. 18, 1197. 36. Wang Z.-Z., Dinger B., Fidone S. J. and Stensaas L. J. (1993) Physiological role of nitric oxide in the cat carotid body. Fedn Proc. Fedn Am. Socs exp. Biol. 7, A431. 36a. Wang Z.-Z., Dinger B. G., Stensaas L. J. and Fidone S. J. (1993) Nitric oxide (NO) mediates efferent neural control of carotid body chemoreceptors. Soc. Neurosci. Abstr. 19, 1429. 37. Wang Z.-Z., Stensaas L. J., Bredt D. S., Dinger B. and Fidone S. J. (1994) Localization and actions of nitric oxide in the cat carotid body. Neuroscience 60, 275-286. 38. Wang Z.-Z., Stensaas L. J., de Vente J., Dinger B. and Fidone S. J. (1991) Immunocytochemical localization of cAMP and cGMP immunoreactivity in cells of the rat carotid body following natural and pharmacological stimulation. Histochemistry 96, 523-530. 39. Wang Z.-Z., Stensaas L. J., Wang W.-J., Dinger B., de Vente J. and Fidone S. J. (1992) Atrial natriuretic peptide increases cyclic guanosine monophosphate immunoreactivity in the carotid body. Neuroscience 49, 479-486. (Accepted 27 June 1994)
Neuroscience
~
Pergamon
0306-4522(94)00437-4
NITRIC O X I D E M E D I A T E S C H E M O R E C E P T O R I N H I B I T I O N IN THE CAT C A R O T I D B O D Y Z.-Z. W A N G , L. J. S T E N S A A S , B. G. D I N G E R and S. J. F I D O N E Department of Physiology, University of Utah School of Medicine, Salt Lake City, UT 84108, U.S.A. A~traet--Numerous studies have demonstrated that carotid sinus nerve fibers mediate a so-called "efferent" inhibition of carotid body chemoreceptors. However, the mechanism(s) underlying this phenomenon are not understood. Recently, it has been shown that an extensive plexus of nitric oxide synthase-containing carotid sinus nerve fibers innervate the carotid body, and that many fine, beaded fibers can be seen in close proximity to small blood vessels as well as lobules of parenchymal cells. The present study examined the effects of centrifugal neural activity in the carotid sinus nerve on the accumulation of [3H]citrulline synthesized from [3H]arginine in the cat carotid body, and the possible involvement of nitric oxide in mediating "efferent" chemoreceptor inhibition. Electrical stimulation of carotid sinus nerve C-fibers evoked an increase in [3H]citruUine accumulation in the carotid body, which was Ca2+-dependent and blocked by L-N G-nitroarginine methylester (0.1 mM), an inhibitor of nitric oxide synthase. Using a vascularly perfused in vitro carotid body preparation, chemoreceptor activity was recorded from thin nerve filaments split-off from the main trunk of the carotid sinus nerve. Electrical stimulation of the main nerve trunk at C-fiber intensities inhibited steady-state chemoreceptor discharge, and this effect was blocked by L-N °-nitroarginine methylester. However, when the organ preparation was switched to the superfuse-only mode, carotid sinus nerve stimulation failed to alter the steady-state discharge, but under these conditions, prolonged nerve stimulation (> 5 min) did attenuate the chemoreceptor response to hypoxia, an effect which was likewise blocked by L-NG-nitroarginine methylester. The present data, together with previous anatomical findings that nitric oxide synthase immunoreactivity is present in both sensory and autonomic ganglion cells innervating the carotid body, suggest that two neural mechanisms may be involved in the inhibitory neural regulation of carotid chemoreceptors. One mechanism appears to involve nitric oxide release from intralobular sensory C-fibers, which lie in close proximity to the chemoreceptor type I cells. The other mechanism involves release of nitric oxide from perivascular terminals of autonomic microganglia neurons, which control carotid body blood flow.
The m a m m a l i a n carotid body is an arterial chemoreceptor organ which monitors blood levels of 02, CO 2 and pH. Pre-neural type I (glomus) cells, the purported chemosensory elements, transduce the physico-chemical status of the blood and transmit this information to synaptically-apposed afferent terminals of carotid sinus nerve (CSN) fibers (see Refs 13 and 15). Biscoe and Sampson were the first to show that spontaneous centrifugal (efferent) neural activity could be recorded from the central cut end of the C S N . 6 Later, Neil and O ' R e g a n reported that electrical stimulation of the peripheral cut end of the C S N depressed the chemosensory discharge recorded from nerve filaments split-off from the stimulated, main nerve trunk. 24~25 Fidone and Sato confirmed these observations and showed that such "efferent" inhibition of chemoreceptor discharge was mediated by C S N C-fibers, 16 although the origin of these fibers Arg, arginine; cGMP, cyclic 3',5'-guanosine monophosphate; CNS, carotid sinus nerve; EDTA, ethylenediaminetetra-acetate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; NAME, N Gnitroarginine methylester; NMMA, N~-monomethyl arginine; NO, nitric oxide; NOS, nitric oxide synthase; PBS, phosphate-buffered saline.
Abbreviations:
217
was not identified. N u m e r o u s subsequent studies also demonstrated that this chemoreceptor inhibition could be reflexly evoked, and could alter not only basal chemoreceptor discharge but also the sensitivity of the carotid body to hypoxia, hypercapnia and acidosis (see Ref. 27 for review). However, the anatomical and physiological basis for this inhibitory phenomenon remained uncertain and controversial. Sampson suggested that chemoreceptor inhibition is mediated by centrifugal (efferent) fibers in the CSN, which somehow modulate type I cell activity to inhibit the discharge of c h e m o r e c e p t o r nerve fibers. 29'3° M c D o n a l d and Mitchell, 23 on the other hand, observed that the inhibitory effects of C S N stimulation persisted following chronic "decentralization" of the glossopharyngeal (IXth cranial) nerve, i.e. section of the nerve root central to the petrosal sensory ganglion. This procedure should have eliminated all efferent fibers to the carotid body and, consequently, these investigators hypothesized that petrosal ganglion sensory neurons, not m o t o r fibers, mediate the chemoreceptor inhibition. They pointed to the presence of reciprocal synapses between chemoreceptor nerve terminals and type I cells, and proposed that antidromic activation of these sensory
218
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fibers (by p r i m a r y afferent depolarization o f their central nerve terminals in the brain) would activate the type I cells to stimulate the release of inhibitory transmitter(s) a n d suppress the activity of chemoreceptor nerve endings. It has been k n o w n for m a n y years that the carotid body is innervated not only by C S N sensory fibers, but also by sympathetic fibers from the ganglioglomerular nerve o f the nearby superior cervical ganglion, as well as by p a r a s y m p a t h e t i c fibers traveling in the glossopharyngeal nerve a n d C S N . 22'27 It is also well established that these sympathetic (postganglionic) fibers innervate carotid body blood vessels, a n d can increase c h e m o r e c e p t o r discharge by decreasing blood flow t h r o u g h the microvascular bed of the organ. 5'14 P a r a s y m p a t h e t i c vascular fibers m a y also modify o r g a n b l o o d flow, and might be expected to increase flow a n d thereby reduce c h e m o r e c e p t o r discharge, s'17 However, little is k n o w n regarding how these a u t o n o m i c elements m i g h t be involved in c h e m o r e c e p t o r inhibition. Recent i m m u n o c y t o c h e m i c a l studies in o u r laboratory have delineated an extensive plexus of nitric oxide synthase (NOS)-positive nerve fibers, some of which penetrate the c h e m o s e n s o r y p a r e n c h y m a l cell lobules, while others terminate in close proximity to carotid body b l o o d vessels. 34'35 F u r t h e r m o r e , correlative physiological studies have d e m o n s t r a t e d that exogenous nitric oxide (NO) has a p o t e n t inhibitory effect on c h e m o r e c e p t o r d i s c h a r g e ) 6'37 In view of the m a n y uncertainties regarding the m e c h a n i s m s of c h e m o r e c e p t o r "efferent" inhibition, the present study was u n d e r t a k e n to examine whether centrifugal neural activity in the C S N regulates N O p r o d u c t i o n in the carotid body, a n d if N O might be a physiological m e d i a t o r o f c h e m o r e c e p t o r inhibition. It was f o u n d t h a t electrical stimulation of C S N C-fibers, but not A-fibers, elicited b o t h a Ca2+-dependent synthesis of NO, a n d a p r o f o u n d , N O - m e d i a t e d inhibition of carotid c h e m o r e c e p t o r discharge. Some preliminary findings were reported elsewhere) 036a
EXPERIMENTAL PROCEDURES
Carotid body superfusion, per~sion and electrical stimulation of the carotid sinus nerve
Domestic cats were obtained locally through the University of Utah Animal Resource Center. The carotid body and CSN were dissected from adult animals anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and transferred to ice-cold modified Tyrode's solution equilibrated with 100% 02. The composition of Tyrode's solution was (in mM): NaC1 112, KCI 4.7, CaCI 2 2.2, MgCI 2 1.1, sodium glutamate 42, HEPES 5, glucose 5.5 (pH 7.42). After careful removal of surrounding connective tissue, the carotid body was placed in a superfusion chamber containing 100% O2-equilibrated Tyrode's solution, and the superfusate passing through the chamber (0.6ml/min) was maintained at 3T'C by a heating block and a thermistor-servo mechanism. 2 The CSN was drawn up into an adjoining oil-filled compartment, placed across bipolar platinum stimulating electrodes and stimulated with rectangular pulses (20Hz, 1 ms in duration) delivered through a stimulus isolation unit. Electrical activity of the CSN (spontaneous chemosensory discharge, as well as A- and C-fiber compound action potentials) was recorded with another pair of bipolar platinum electrodes positioned near the carotid body. Maximal A-fiber and C-fiber activation was achieved by adjusting the stimulus voltage. We utilized an in vitro vascularly perfused/superfused cat carotid body modified from that developed by Belmonte and Eyzaguirre? The glossopharyngeal nerve was dissected free and transected as proximal as possible to its entry into the skull. The internal carotid, ascending pharyngeal and occipital arteries were ligated and cut, as were other small vascular branches and veins draining the carotid body. After transferring the preparation to the in vitro superfusion chamber, the common and external carotid arteries were cannulated and perfused with air-equilibrated modified Tyrode's solution containing 5% dextran (mol. wt 40,000) at 37'C and at a flow rate of I ml/min (Fig. 1). The CSN was drawn up into an adjoining oil-filled compartment, and thin nerve filaments were dissected free from the main nerve trunk for recording single chemoreceptor unit activity. One channel of an Ortec stimulator provided an isolated squarepulse stimulus (duration Ires) to the main trunk of the CSN. Stimulus artifacts were minimized utilizing a second channel of the stimulator; its output preceded that of the first channel by 0.1 ms and controlled an analog switch which interrupted the recorded signal to the amplifier for the duration of the stimulus. This circuit offset the stimulus artifact and facilitated counting of afferent impulses. 5 Stimu-
Fig, 1. Schematic diagram of perfusion superfusion system. C1, perfusion-superfusion chamber, whose temperature is maintained at 37 ± 0.2°C by a heating system (cross-hatched); C2, oil chamber; TI, temperature probe for the heated metal block; T2, temperature probe for C1; h, drain for superfusion fluid; P, pressure transducer; R, peripheral resistance: D, drop counter.
Nitric oxide in the carotid body lus current spread to the recording filament was minimized and monitored by experimental approaches described by Fidone and Sato, TM Goodman, ~7 and Belmonte and Eyzaguirre? (1) A small branch of the CSN close to the carotid body was dissected centrally along the main nerve trunk. (2) A nerve filament was separated from this branch and used for recording in order to minimize the area of contact with the stimulated main nerve trunk. (3) A small drop of Tyrode's solution was applied to the filament at its point of emergence from the main nerve trunk, and a ground lead connected to this drop, with the remaining nerve fibers immersed in warm mineral oil. (4) The refractory pause in the nerve discharge following the stimulus artifact was monitored by superposition of successive sweeps on the oscilloscope screen.~6 Perfusion pressure was monitored with a pressure transducer in the outflow tube linked to the external carotid artery, and the pressure was adjusted using a variable resistance on the outflow tube while flow rate was recorded with an electronic drop counter (Fig. l). Impulses recorded from nerve filaments were led to an a.c. preamplifier, displayed on an oscilloscope and input to an IBM-PC computer via an AD/DA converter.
[3H]Citrulline assay Carotid bodies removed from anesthetized cats were transferred to a dissection chamber containing ice-cold 100% O2-equilibrated Tyrode's solution. Connective tissue was removed, and the organs were weighed on a Cahn electrobalance equipped with a humidified chamber. Tissue samples were pre-incubated in glass minivials containing 1 ml of 100% O2-Tyrode's medium for 30min in a water bath shaker at 37°C, 32 and then incubated with 1 ml of Tyrode's solution containing L-[2,3-3H]arginine (3/iCi/ml, 56 Ci/mmol; DuPont-NEN) and equilibrated either with: (i) control (100% 02) or (ii) hypoxic gas mixtures (10% 02 in 90%N2). After 1 h incubation at 37°C, the carotid bodies were assayed for [3H]citrulline, as described below. In other experiments, the carotid body was continuously superfused during nerve stimulation with 100% O2-equilibrated media containing L-[2,3-3H]arginine (3 pCi/ml, 56 Ci/mmol; Dupont-NEN) at a flow rate of 0.6 ml/min. Following a wash in ice-cold Tyrode's media containing 4 mM EDTA and 5 mM L-arginine, the tissues were homogenized in 1 ml of 1 M trichloroacetic acid and centrifuged. Trichloroacetic acid in the supernatant was extracted three times with 2 ml diethyl ether, Aliquots of 0.5 ml of the supernatant were neutralized with 2 ml of 20 mM HEPES (pH 6.0), applied to 2-ml columns of Dowex AG50WX-8 (Na + form, BioRad) and eluted with 2 ml of water. [3H]Citrulline was quantified by liquid scintillation spectroscopy of the 4-ml eluate, 7 and the results were expressed as mean _+ S.E.M., with the data analysed using Student's t-test.
Cyclic guanosine 3',5"-monophosphate immunocytochemistry For immunocytochemistry of cyclic guanosine Y,5'monophosphate (cGMP), the tissue was continuously superfused during nerve stimulation with 100% 02 equilibrated Tyrode's medium containing theophylline (10mM) and superoxide dismutase (100 unit/ml) at a flow rate of 0.6 ml/min. Carotid bodies were fixed in ice-cold fixative (4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4) for 2 h, rinsed in cold phosphatebuffered saline (PBS; 15 min), cryoprotected in 20% sucrose (1 h at 4'C) and sectioned in a cryostat (-20°C). Frozen sections (6-8t~m thick) were thaw-mounted onto chrome alum, gelatin-coated slides and processed for cGMP immunocytochemical staining. 38 Briefly, sections were hydrated in PBS (15 min), treated with 3% hydrogen peroxide (10min) and avidin-biotin pre-blocking reagents (20 rain, Vector kit). Sections were then incubated in the following reagents, each of which was followed by a 15 min wash in PBS: (i) polyclonal cGMP antiserum (1 : I000 in PBS
219
containing 0.3% Triton X-100) overnight at 4~'C;~2"39 (ii) biotinylated goat anti-rabbit secondary antiserum (Vector, 1:400) at room temperature (30 min); (iii) avidinbiotin peroxidase complex (Vector elite kit) at room temperature (60 min); (iv) a mixture of 3,Y-diaminobenzidine tetrahydrochloride (Sigma) and hydrogen peroxide. Control sections showed no specific immunoreactivity following pre-absorption of the primary antibody with 10 3 M cGMP, or replacement by normal rabbit serum.
RESULTS
Effect o f carotid sinus nerve stimulation on nitric oxide synthesis Citrulline a c c u m u l a t i o n in the carotid body was assessed by m e a s u r i n g the conversion o f [3H]-Larginine to [3H]citrulline, a reaction t h a t stoichiometrically produces N O / T h e results were expressed b o t h as d.p.m./mg tissue/h a n d as the percentage of total radioactivity i n c o r p o r a t e d into the tissue; the latter value allowed for differences in [3H]arginine u p t a k e with different c h e m o r e c e p t o r stimuli/8 Activation of C S N A-fibers (0.7-1.6 V, 20 Hz; Fig. 2) p r o d u c e d no change in the level of [3H]citrulline f o r m a t i o n following 15 rain of nerve stimulation (Table 1). However, a significant increase in [SH]citrulline f o r m a t i o n occurred (Table 1) w h e n nerve stimulation was adjusted for m a x i m a l r e c r u i t m e n t o f C S N C-fibers (4-6 V, 20 Hz, 15 min), as m o n i t o r e d by the recorded comp o u n d action potential (Fig. 2). Superfusion media lacking Ca 2+ ( M r + = 2.2 m M ) completely blocked [3H]citrulline formation, indicating t h a t N O synthesis in the carotid b o d y is d e p e n d e n t on the availability of extracellular Ca 2+ .
Effect o f carotid sinus nerve stimulation on cyclic guanosine 3',5'-monophosphate formation It is well k n o w n t h a t N O increases c G M P production, s Consequently, we e x a m i n e d whether the increased N O synthesis from C S N stimulation would in t u r n activate guanylate cyclase a n d elevate c G M P levels in the carotid body. Figure 3 shows the time course of c G M P p r o d u c t i o n following electrical stimulation of C S N C-fibers. In control carotid bodies superfused with 100% O2-equilibrated medium, c G M P i m m u n o r e a c t i v i t y was undetectable in the tissue (Fig. 3A). Following 1 m i n of C S N stimulation (Fig. 3B), m o d e r a t e levels of c G M P i m m u n o r e a c t i v i t y a p p e a r e d in s m o o t h muscle cells of small arterial vessels, a n d after 5 min of nerve stimulation (Fig. 3C), low levels of c G M P were also a p p a r e n t in the type I cells. Prolonged C S N stimulation (15 min) markedly e n h a n c e d the level o f c G M P i m m u n o r e a c t i v i t y b o t h in blood vessels a n d in type I cells (Fig. 3D). Such changes in c G M P i m m u n o staining occurred w h e n nerve stimulation recruited C-fibers to the response, but were not observed when only A-fibers were stimulated (Fig. 4B, C). In other experiments, we examined w h e t h e r the N O S inhibitor, L-N~-nitroarginine methylester ( c - N A M E ) ,
Z.-Z. Wang et al.
220
A
A
-~-
A
--
v
i~
-
2 ms
fl
PIt
o
IH
5 ms I
Fig. 2. Compound action potentials evoked by stimulus parameters adjusted to recruit maximal A-fiber or C-fiber potentials while measuring [3H]citrulline formation in the in vitro cat carotid body. See also Table 1.
could block the increase in c G M P accumulation produced by C S N stimulation. As illustrated in Fig. 4D, L - N A M E (0.1 mM) effectively blocked the increase in c G M P immunoreactivity produced by 15 min of C S N stimulation. Excess L-arginine (L-Arg, l mM) reversed the block by L - N A M E (Fig. 4E), suggesting that the effect of this drug was due to competitive inhibition of endogenous N O synthesis. D-Arg, an inactive isomer of arginine, failed to overcome the inhibitory effect of L - N A M E (Fig. 4F).
Chemoreceptor inhibition in the vascularly perJused/ superfused carotid body Carotid body preparations which were both superfused and vascularly perfused (air-equilibrated media) exhibited basal levels of single-unit chemoreceptor discharge which ranged from two to 10 impulses/s. Brief hypoxic stimuli, consisting of 5% O2-equilibrated perfusion medium (30 s), elevated the chemoreceptor discharge to 30-40 impulses/s; discharge rates returned rapidly to basal levels following
Table 1. Effects of carotid sinus nerve stimulation on nitric oxide synthesis in the cat carotid body [3H]Citrulline formation (d.p.m./h per mg tissue) Control A-fiber C-fiber C-fiber/zero Ca 2.
777 + 41 808 __+67 1503 +_ 133"* 679 _ 58
Per cent of total radioactivity 5.8 + 6.3 + 12.6 ± 7.0 ±
0.85 0.69 1.03"* 0.92
6 4 6 4
n represents the number of carotid bodies. Values are mean _+ S.E.M. **P < 0.01 vs control.
Nitric oxide in the carotid body
221
12 Fig. 3. Induction of cGMP formation in cat carotid body following electrical stimulation of C-fibers of the CSN. (A) cGMP immunoreactivity is not detectable in the unstimulated control carotid body. (B) Accumulation of cGMP appears in arterial smooth muscular cells (arrows) following 1 min of nerve stimulation. (C) Low levels of cGMP immunoreactivity occur in type I cells (arrowheads) following 5 min of stimulation. (D) High levels of cGMP immunoreactivity occur in both blood vessels (arrows) and type I cells (arrowheads) following 15 rain of nerve stimulation. reintroduction of control, air-equilibrated perfusion medium. Elevation of perfusion pressure from 40 to 120mmHg reduced the discharge frequency, while abrupt reduction of pressure below 40 mmHg increased the unitary discharge. Electrical simulation of the CSN/glossopharyngeal nerve reduced the chemosensory discharge in approximately 60-70% of the units; the remaining units underwent no significant change. Although stimulus intensities of about 3 V activated some C-fibers and produced discernible chemoreceptor inhibition, chemosensory discharge was maximally attenuated by CSN stimulation at 5~5 V (10-20/s). The inhibitory effect occurred within 10s of the onset of nerve stimulation, and persisted throughout the stimulation period. Upon cessation of stimulation, inhibition occasionally lasted for more than 30 s, but ordinarily returned to basal levels within 10-20s (Fig. 5A). Superposition of successive sweeps On the oscilloscope screen failed to show a refractory pause in nerve discharge after the stimulus artifact, and nerve crush completely eliminated the electrically provoked chemoreceptor inhibition (data not shown). 16 NSC 65/I--H
The typical effect of L-NAME (0.1mM) on chemoreceptor inhibition is shown in Fig. 5B. The NOS inhibitor increased basal chemoreceptor discharge and completely blocked the inhibition induced by nerve stimulation. However, because chemoreceptor inhibition becomes less marked as basal chemosensory discharge increases, 16 we examined whether this L-NAME disinhibition was merely a consequence of increased basal chemoreceptor activity. The nerve was therefore stimulated in the presence of the drug, but after reducing chemoreceptor discharge to control levels by elevating the perfusion pressure. Under these conditions, again no marked inhibitory effect was seen (Fig. 5C). Moreover, the disinhibitory effect of L-NAME was reversed by excess L-Arg (1 mM; Fig. 5D), demonstrating competitive inhibition of endogenous NO synthesis in the carotid body. Similar results were obtained from eight preparations in five cats, as summarized in Table 2. The stereospecificity of NOS inhibitors with respect to chemoreceptor inhibition was examined using L-N °-monomethylarginine (L-NMMA). 7A° This potent NOS inhibitor clearly blocked chemo-
Z.-Z. Wang et al.
222
receptor inhibition elicited by electrical stimulation of the CSN, whereas its inactive isomer, D-N ~monomethylarginine (D-NMMA), was ineffective (Fig. 6).
Chemoreceptor inhibition in the superfused carotid body Stimulation of the CSN at 6-10 V (10-40 Hz) in the superfused carotid body preparation failed to change the basal frequency of chemoreceptor discharge. This
was true even after the rate of discharge was adjusted with 100% O2-equilibrated superfusion medium to the level recorded from the vascularly perfused preparation (Fig. 7). Nerve stimulation was also without effect on the high frequency, steady-state discharge induced by hypoxia (40% and 20% 02; Fig. 7; but see below). These findings might suggest that chemoreceptor inhibition induced by C S N stimulation is a vascular phenomenon, as originally suggested by G o o d m a n 17 and by Belmonte and Eyzaguirre. 5 However, our experiments showed that high levels of
Control
~rg
i
S
"
Fig. 4. CSN stimulation induced cGMP formation in the cat carotid body and its reversal by an NOS inhibitor, cGMP immunoreactivity is not discernible in the unstimulated control (A) and in the A-fiber stimulated (15 min) carotid body (B). High levels ofcGMP appear in arterial smooth muscle (arrowhead) and a few type I cells (arrows) following 15 min of electrical stimulation of the CSN with stimulus intensities recruiting C-fibers maximally (C). The C-fiber activation-evoked cGMP production was blocked by 0.1 mM L-NAME (D). L-Arg (1 raM) restored cGMP immunoreactivity in L-NAME-treated carotid bodies (E), and D-Arg was ineffective (F).
Nitric oxide in the carotid body
A
15 m
~.
if)
CI.
E
a
Control 5 V, 20 Hz
10 s
60
120
15 ~
10
~ E
5
L-NAME (~o mmHg)
60
120
5V, :~0 Hz
12o
180
18o
Time (s)
T i m e (s)
°o
223
180
0
T i m e (s)
T i m e (s)
Fig. 5. L-NAME reverses chemoreceptor inhibition during CSN stimulation in vascularly perfused cat carotid body. (A) Single unit activity was attenuated during the nerve stimulation. (B) L-NAME (0.1 raM) elevates basal neural activity and it prevented the chemoreceptor inhibition. (C) Efferent inhibition was not elicited in the presence of L-NAME when the nerve discharge was adjusted to control levels by raising perfusion pressure. (D) L-Arg (1 mM) restored the chemoreceptor inhibition blocked by 0.1 mM L-NAME.
c G M P immunoreactivity are observed in type I cells following C S N stimulation, and it has been found that under these conditions c G M P inhibits the response to hypoxia while failing to alter basal discharge rates. 33 N o n e the less, we did find in these experiments that observable increases in tissue levels of c G M P required several minutes or more of continued CSN stimulation. Consequently, we examined whether the response to hypoxia in superfused carotid body preparations could be inhibited when C S N stimulation preceded the hypoxic stimulus. These experiments are illustrated in Fig. 8. As expected, when a short (5 min) hypoxic stimulus (20% O2-equilibrated superfusion medium) was delivered simultaneously with C S N stimulation, the chemosensory response was not markedly different
(Fig. 8B). However, the response to hypoxia was attenuated when nerve stimulation (10min) was begun 5 min prior to the start of the hypoxic episode (Fig. 8C). Maximal reduction of the chemoreceptor response occurred when C S N stimulation preceded the hypoxic episode by 10 min (Fig. 8D). This inhibition of the chemoreceptor response could be blocked by 0.1 m M L - N A M E (Fig. 8E), and the block was selectively reversed by 1 m M L-Arg (Fig. 8F). Similar results were obtained from multiple superfusion experiments, suggesting an important role for N O in chemoreceptor inhibition which is apparently independent of vascular effects (Table 3). As expected, inhibition of the hypoxic response could also be elicited in vascularly perfused preparations (Fig. 9); the magnitude and time course of the effect
Table 2. Carotid sinus nerve stimulation inhibits basal chemoreceptor activity in the perfused/superfused carotid body
CSN stimulation CSN stimulation plus 0.1 mM L-NAME CSN stimulation plus 0.1 mM L-NAME/1 mM L-Arg
Per cent of mean pre-stimulation frequency
n
61.9 + 5.9
8
97.8 4- 11.2"
6
43.2 4- 8.5
6
n represents the number of preparations; values are mean 4- S.E.M. *P < 0.05 vs CSN stimulation group.
Z.-Z. Wang et al.
224 A
15-
Control
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s-
60
1'~0
180
Time (s) S
is
DISCUSSION
I D-NMMA [o,1 raM)
s.sv, 2o Hz
_E 60
120
180
120
180
Time (s)
C
fused with 100% O2-equilibrated medium (Table 4). One hour of hypoxia (10% 02 in 90% N2) significantly increased [3H]citrulline levels in the carotid body. When expressed as a percentage of total radioactivity incorporated into the tissue (Table 4), the rate of [3H]citrulline formation was also elevated by hypoxia, indicating that hypoxia did not significantly change [3H]arginine uptake by the organ.
15
(,3 1
L-NMMA 0
-
5"
~
5.5 V, 20 Hz 60
Time (s)
Fig. 6. Stereospecific actions of NOS inhibitor on efferent chemoreceptor inhibition in the vascularly perfused/ superfused carotid body. Single unit discharge was reduced in control (A) and 0.1 mM D-NMMA-treated preparations during electrical stimulation of the CSN (B). L-NMMA (0.1 nM) blocked chemoreceptor inhibition (C). in perfused preparations differed little from those observed with the superfused preparation.
Effect of hypoxia on [3H]citrulline Jormation Experiments were conducted to determine the effects of hypoxia on NO synthesis. Moderate levels of [3H]citrulline were found in carotid bodies super-
The present study demonstrates that centrifugal neural activity in the CSN increases NO production in the carotid body. The elevated levels of [3H]citrulline which reflect this increase in NO synthesis occur only when CSN C-fibers are recruited by the stimulus, and only in the presence of extracellular Ca -,+ . These findings, together with our earlier immunocytochemical studies localizing NOS to unmyelinated axons in the carotid body, 34'35'37implicate CSN C-fibers as mediators of centrifugal, or "efferent," chemoreceptor inhibition. The present study further demonstrates that stimulation of CSN C-fibers induces cGMP formation in the carotid body. Our previous electrophysiological studies found that cGMP is a potent inhibitor of the hypoxia-evoked chemosensory response, 33 and that NO donors, including sodium nitroprusside and nitroglycerine, stimulate the production of cGMP in both type I cells and arterioles of the organ. 32'JT3s Taken together, these findings suggest that cGMP very likely acts as a second messenger mediator of chemoreceptor inhibition in the carotid body, triggered by the liberation of NO from NOS-positive CSN C-fibers. Stimulation of CSN C-fibers does not alter the resting chemosensory discharge of the superfused carotid body preparation, but does inhibit the chemoreceptor response to a hypoxic stimulus. Our results suggest that NOS-positive CSN sensory neurons, with cell bodies in the petrosal ganglia, mediate this non-vascular inhibitory effect through
1000
I Superfused carotid body
8OO
20% Oxygen O3 Ob ¢n C).
600 4.0%Oxygen 400-
E
2000
o
100% Oxygen
360
9 V, 20 Hz 660 960 T i m e (s)
12"0o
I 5oo
Fig. 7. The level of basal (100% 02) and hypoxic (40% and 20% 02) steady-state chemosensory discharge was unaffected by electrical stimulation of the CSN in the superfused carotid body.
Nitric oxide in the carotid body
225
Superfusion
A E
L-NAME(0.1mM)
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Fig. 8. In the superfused carotid body preparation, delayed inhibition by CSN stimulation of the dynamic chemoreceptor response to hypoxia followed CSN stimulation. The chemosensory response to a 5 min hypoxic stimulus (20% O2-equilibrated superfusion medium, black bar) was not markedly changed by concomitant nerve stimulation (open bar, B). However, the hypoxic response was attenuated when the nerve stimulation (10 min) was started 5 min prior to hypoxia (C). Maximal attenuation of the response occurred with a 15 rain CSN stimulation which was started 10 min preceding hypoxia (D). The inhibition was blocked by superfusion medium containing 0.1 mM L-NAME (E). L-Arg (1 mM) reversed the effect of L-NAME (F). Note the basal chemosensory discharge was not changed by the nerve stimulation.
226
Z.-Z. Wang et al.
Table 3. Carotid sinus nerve stimulation inhibits hypoxia (20% Oj-evoked chemoreceptor response in the superfused carotid body Time of CSN stimulation 5 min 10min 15min 15 min (plus 0.1 mM L-NAME) 15min (plus 0.1 mM L-NAME/I mM L-Arg)
Per cent of control hypoxic response
tz
100.7 _+ 5.2 74.5 + 7.1" 54.3_+3.8** 106.2 _+ I0.4
5 5 5 4
44.9 _+ 4.0**
3
n represents the number of preparations; values are mean _+ S.E.M. *P < 0.05, **P < 0.01 vs control.
regulation o f c G M P levels in type I cells. Several observations support this view. First, NOS-positive nerve fibers arising from small diameter petrosal sensory neurons innervate type I cell lobules in the carotid body; 34"37 these sensory neurons also contain
Table 4. Effects of hypoxia on nitric oxide synthesis in the in vitro cat carotid body [3H]Citrulline formation (d.p.m./h per mg tissue)
Per cent of total radioactivity
n
1196 _+ 200 1784_+399"
8.9 + 1.3 14.0_+ 1.8"
6 6
100% 02 10% O~
n represents the number of carotid bodies. Values are mean + S.E.M. *P < 0.05 vs 100% O_~ group. substance P, and represent a subset o f sensory neurons distinct from the tyrosine hydroxylasepositive petrosal sensory neurons which also innervate type I cell l o b u l e s Y 4 Second, electrical stimulation o f CSN C-fibers (but not A-fibers) increases c G M P levels in type I cells (Figs 3, 4). Third, c G M P is a potent inhibitor o f the chemosensory response to hypoxia, but fails to alter basal chemo-
Perfusion
A
B
L-NAME(~.1mM 1~:...=_16,,,,i,..u ,~..,.~ ..,JHypox~ll
.__~ . . . . .
8V, 20Hz
C
,
,,°
D
.........
m
5 mJn
is0
Fig. 9. Attenuation of the chemosensory response to hypoxia by electrical stimulation of the CSN in the vascularly perfused cat carotid body. Note the basal chemosensory discharge was immediately reduced following the onset of the nerve stimulation.
Nitric oxide in the carotid body receptor discharge. 33 Fourth, the required length of time that CSN stimulation must precede an hypoxic stimulus in order to inhibit the chemosensory response corresponds to the time required for an observable accumulation of cGMP in type I cells (Figs 3, 8) finally, both chemoreceptor inhibition and cGMP formation produced by CSN stimulation in the superfused carotid body preparation are blocked by the NOS inhibitor, L-NAME. The fact that CSN stimulation inhibits steady-state chemoreceptor discharge in the vascularly perfused/ superfused carotid body preparation, but is ineffective in the superfused-only preparation, could also suggest that chemoreceptor inhibition is at least partly mediated through vascular mechanisms. This interpretation would be consistent with results from an earlier study by Belmonte and Eyzaguirre showing that activation of a vasomotor neural pathway in the CSN increased carotid body blood flow and decreased chemosensory discharge. 5 On the other hand, a decrease in total blood flow through the organ following CSN stimulation was observed by Neff and O'Regan in their original demonstration of chemoreceptor inhibition]5 and this finding was later confirmed by Acker and O'Regan. L Such discrepant findings suggest complex vascular dynamics sensitive to variable experimental conditions, and underscore the need to clarify the anatomical locations and physiological actions of vasomotor autonomic neurons controlling regional carotid body blood flow and organ chemosensitivity. This need is further emphasized by studies which show that chemoreceptor inhibition survives transection of the CSN proximal to the petrosal ganglion, a "decentralization" procedure which should eliminate all vasomotor efferents projecting from the brain to the carotid body. 23 Our studies have shown that large NOS-containing autonomic neurons occur along the CSN and within the carotid body itself,34'37 and likely correspond to the isolated groups of ganglionic neurons described by de Castro many years ago as "microganglia.''H Our immunocytochemical experiments show that these neurons are distinguishable from sensory neurons, because they exhibit neuronal processes resembling dendrites and co-stain for choline acetyltransferase and NOS. Also, these neurons can be shown to give rise to axons which innervate nearby blood vessels. Thus, these microganglia neurons appear to constitute a population of local, post ganglionic vasomotor elements in the carotid body. 34'37 Our studies using the NOS inhibitor, L-NAME, in the vascularly perfused/superfused carotid body preparation suggest that NO is a principal mediator of chemoreceptor inhibition. In this regard, it should be noted that NO is the most potent vasodilator known, and has been shown to be involved in the non-adrenergic non-cholinergic inhibition of gastrointestinal motility, as well as relaxation of vascular smooth muscle in numerous other sites, including the penile corpus cavernosum and the cerebral cortex. 8 ~0
227
NO generated by the neural innervation to vascular elements stimulates guanylate cyclase and cGMP production, leading to relaxation of smooth muscle and vascular dilatation.4'8 Because blood vessels in the carotid body are extensively innervated by NOSpositive microganglial elements, activation of these neurons should increase cGMP production in vascular smooth muscle, leading to vasodilation, increased blood flow and depression of steady-state chemosensory discharge. Our observations that CSN stimulation rapidly induces high levels of cGMP in vascular smooth muscle of the carotid body, and that L-NAME blocks this effect in the perfused/superfused organ preparation, provides compelling evidence in favor of such an NO-mediated mechanism. The physiological mechanism(s) responsible for the activation of NOS-containing sensory C-fibers in the carotid body remains an unresolved issue. One possibility is that primary afferent depolarization of the central (brainstem) terminals of these petrosal sensory neurons leads to antidromic "backfiring" of action potentials into the carotid body. 23 Another, and perhaps simpler explanation, arises as a consequence of the profuse branching of individual CSN axons within the carotid body; 19 a centripetal nerve impulse initiated within one branch of a sensory fiber could by axon reflex invade other branches, resulting in a local cascade of increased NO production within a terminal axon arbor. Evidence favoring this latter mechanism is presented in Table 4, which shows that NO production ([3H]citrulline formation) is elevated in carotid bodies superfused in vitro with low 0 2 medium. During hypoxia, the net activity of the carotid chemoreceptors very likely results from a balance between stimulatory and inhibitory processes. In this regard, previous studies have shown that centrifugal impulse traffic in the cat CSN tonically inhibits the response to both acute 26'28 and chronic 2°'21 hypoxia. The present study suggests that excitation of the carotid chemoreceptors triggers an NO-mediated inhibitory co-response, which perhaps could provide for both acute dynamic control as well as chronic adaptive adjustments within the chemoreceptor. Furthermore, such chemoreceptor inhibitory mechanisms involving neuromodulators like NO (and perhaps atrial natriuetic peptide) 33 may be important determinants in the observed blunting of ventilatory sensitivity due to chronic hypoxic exposure at high altitudes. 31 CONCLUSIONS
Our combined anatomical, neuropharmacological and electrophysiological findings support the notion that NO plays two major roles in carotid body chemoreceptor inhibition. First, acting in an anterograde fashion as a transmitter released by efferent autonomic neurons, NO has potent actions on arterial vascular elements. Second, NO liberated by
228
Z.-Z. Wang et al.
Smooth muscle Autonomic efferents (Parasympathetic)
L-Arg
NOS
~
[
C
~
[~
cGMP'~
Vascular relaxation
Flow~crease
" ~ '-'-I~" ca2. Sensory C-fibers
,I Chemosensory
Type I cell
Fig. 10. Schematic diagram depicting the two NO-mediated efferent inhibitory neural pathways that control carotid body chemoreceptors. GC, guanylate cyclase. Chemosensory discharge generated in the afferent sensory A-fibers is influenced by two separate NO-synthesizing neural pathways. Centrifugal nerve activity activates NOS in nerve terminals in the carotid body via a Ca2+-dependent mechanism. NO released from parasympathetic vasomotor fibers stimulates cGMP production in postsynaptic vascular smooth muscle and triggers vasodilation. The resultant flow increase attenuates the basal steady-state chemosensory activity. NO generated by axonal terminals of the sensory C-fibers diffuses to adjacent type I cells and stimulates cGMP accumulation, which modulates the chemoreceptor response to hypoxia.
a n t i d r o m i c activity invading the terminals o f sensory fibers (resulting either from peripheral axon reflexes or central p r i m a r y afferent depolarization) increases c G M P in c h e m o r e c e p t o r type I cells, leading to c h e m o s e n s o r y inhibition. In c o m b i n a t i o n , the vasom o t o r n e u r o n a l p a t h w a y appears to m o d u l a t e steady-state c h e m o r e c e p t o r activity, while the sensory n e u r o n a l p a t h w a y inhibits the d y n a m i c response to a hypoxic stimulus (Fig. 10). These cellular m e c h a n i s m s therefore constitute two morphologically distinct, but
functionally c o m p l e m e n t a r y , neural pathways, which together may account for the k n o w n characteristics o f the so-called "efferent" inhibitory control o f the carotid chemoreceptors (Fig. 10). Acknowledgements--This work was supported by USPHS
grants NS12636 and NS07938. Z.-Z. Wang was supported in part by a graduate research fellowship from the University of Utah. The authors wish to thank Dr J. DeVente of the Free University, Amsterdam, The Netherlands, for supplying the antiserum to cyclic GMP.
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