| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of 1 Environmental Health Sciences, 2 Physiology, 3 Medicine, and 4 Anesthesiology/Critical Care Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Fitzgerald, Robert S., Machiko Shirahata, and Tohru Ide.
Further cholinergic aspects of carotid body chemotransduction of
hypoxia in cats. J. Appl. Physiol.
82(3): 819-827, 1997.
From the 1930s into the 1970s, the role of
acetylcholine (ACh) in the carotid body's chemotransduction of hypoxia
was debated. Since the late 1970s, the issue has been pursued only
intermittently or not at all. The purpose of this study was to test
again with a new preparation the hypothesis that ACh is an excitatory
neurotransmitter in the cat carotid body's chemotransduction of
hypoxia. We tested the effect of the specific nicotinic blocker
mecamylamine and the muscarinic blocker of all five muscarinic
receptors, atropine. We further tested the effects of
M1 and
M2 muscarinic-receptor blockers.
The carotid body region was selectively perfused with hypoxic
Krebs-Ringer bicarbonate (KRB) solutions that were blocker free or
contained varying doses of the blockers. Both mecamylamine and atropine
reduced the response to hypoxic KRB in a dose-related manner. The
M2 muscarinic-receptor blockers
gallamine and AFDX 116 increased the response to hypoxic KRB, whereas
the M1 muscarinic-receptor blocker
pirenzepine reduced the response to hypoxic KRB. These data are
consistent with an excitatory role for ACh in the carotid body
chemotransduction of hypoxia in the cat.
acetylcholine; mecamylamine; atropine; gallamine; pirenzepine; AFDX
116
INTRODUCTION THE IDENTITY of the excitatory neurotransmitter(s) in
the carotid body's response to hypoxia has been a matter of
controversy over several decades. Exogenously applied acetylcholine
(ACh) clearly stimulates the cat carotid body. And several early
investigators (11, 30, 31), using techniques such as
acetylcholinesterase blockers, generated data supportive of an
excitatory role for endogenously released ACh in the carotid body of
the cat during hypoxia. In the 1970s, Eyzaguirre and colleagues
(12-17) published several papers that provided strong evidence
that, in the cat, ACh was an excitatory neurotransmitter in the carotid
body's chemotransduction of several stimuli. However, data
unsupportive of an excitatory role for ACh during hypoxic
chemotransduction have also been presented by several investigators (9,
10, 25, 28, 35, 36, 40). The controversy has remained unresolved and
only intermittently examined since the late 1970s.
The present study represents a continuation of our efforts to provide
some new insight into answering the question "Does ACh play an
excitatory role in the chemotransduction of hypoxia in the cat carotid
body?" In previous studies it has been shown
(20-22) that when the isolated carotid body in situ
was transiently and selectively perfused with a hypoxic Krebs-Ringer-
bicarbonate solution (KRB) containing a combination of muscarinic and
nicotinic blockers, the neural response, compared with the response to
the blocker-free hypoxic KRB, was reduced in a dose-related way. The study reported herein describes carotid body neural responses to
perfusions of hypoxic KRB containing either the nicotinic blocker alone
or the muscarinic blocker alone. We also report the neural response to
a perfusion of hypoxic KRB that contained either an M1 or an
M2 muscarinic-receptor blocker.
METHODS Preparation
1 · h1).
Body temperature was maintained at 37-38°C with a heating
blanket (model K-20, American Hamilton Aquamatic Module). A tracheal
cannula was inserted. A femoral arterial catheter was inserted for the measuring of blood pressure and for sampling of blood to determine PCO2,
PO2, and pH, and a femoral venous
catheter was inserted for the administration of drugs and fluids. Blood samples were measured with a Radiometer BMS 3Mk2 Blood MicroSystem. During the experiment, the animals' physiological condition, as judged
from arterial pH, PCO2 and
PO2 values, and arterial blood
pressure, was maintained best if they were ventilated on
oxygen-enriched room air and were kept hydrated with 3-5
ml · kg1 · h1
of Ringer solution to which 3.1 mg/ml of sodium lactate were added. If
necessary, arterial pH was adjusted with an infusion (1 ml/min) of 1 M
NaHCO3 in a 2.5% glucose saline
solution until pH returned to normal.
A perfusion loop was inserted into the common carotid artery, and the remaining vasculature in the area was ligated except for the lingual, the external carotid artery, and the venous outflow of the carotid body. The whole carotid sinus nerve was used for recording chemoreceptor activity after the baroreceptor input was removed by gently crushing the carotid sinus and gently and briefly applying heat from an ophthalmological cautery to the sinus. The nerve was put under warm mineral oil. Neural activity was recorded from bipolar platinum-iridium electrodes connected to a Grass P15 preamplifier. The amplified neural activity was displayed on an oscilloscope (Hitachi V-302 F) and integrated (Grass wide-band AC preamplifier and integrator model 7P3C). This unit was a capacity-coupled differential preamplifier with frequency-response characterisitics enabling it to record neural activity. Full-wave rectification circuits ensured amplitude linearity of integration. The integrated trace displayed on the polygraph (Grass 79E) was proportional to the average level of the ongoing signal generated by the activity in the carotid sinus nerve. Femoral blood pressure, amplified raw nerve activity, and blood pressure in the common carotid artery loop were also recorded on the polygraph.
Except for 10-12 perfusions of KRB for 120-180 s each, the animal perfused its own carotid body with its own arterial blood throughout the experiment. When a perfusion was to be made, the snares around common and external carotid arteries were drawn tightly, the stopcock was turned, and KRB was perfused into the loop. The composition of KRB was (mM) 120 NaCl; 3.5 KCl; 1.8 CaCl2; 0.6 MgCl2; 20 glucose; 0.56 NaH2PO4; and 22 NaHCO3. Pressure in the loop during the KRB perfusion replicated the preceding arterial pressure, showing systolic and diastolic components generated by the perfusion pump. The perfusion escaped the carotid body area by the carotid body venous outflow and the lingual artery, which was fitted with a variable resistance. KRB was perfused at 5-8 ml/min via a water-jacketed catheter that preserved the perfusate's temperature at 37-38°C.
Two types of perfusions were made. In the first, while the animal was
being ventilated on oxygen-enriched room air, a perfusion of hypoxic,
blocker-free or hypoxic, blocker-containing KRB was made into the
common carotid artery. In the second, the animal was again ventilated
on oxygen-enriched room air. A perfusion of blocker-free, hypoxic KRB
was made for 1 min from the first syringe. At the 1-min mark, this
syringe was switched off, and immediately a second, containing equally
hypoxic KRB with or without the blocker, was turned on. The two-syringe
perfusion technique allowed each animal to serve as its own control
without any intervening time period and possible change in neural
output from the carotid body. Figure 1
presents an example of syringes switched repeatedly after perfusions of
20-60 s.
Drugs used in the experiments were AFDX 116 (a generous gift from Dr. Allyson Fryer), atropine (free base; Sigma Chemical), gallamine triethiodide (Sigma Chemical), mecamylamine hydrochloride (N,2,3,3-tetramethylbicyclo[2.2.1]heptan-2-amine hydrochloride; Sigma Chemical), and pirenzepine dihydrochloride (Sigma Chemical). These were dissolved in KRB.
Mean values ±SE are presented. Statistical analysis was made by using analysis of variance (ANOVA) with Duncan's new multiple-range test for determining significant differences among the means. Student's paired or unpaired t-tests were also used. P values were judged to be significant when they were at the 0.05 or better level.
RESULTS
Mecamylamine
Figure 2 presents the responses of the carotid body to a perfusion of hypoxic KRB that was either free of or contained increasing doses of mecamylamine. Statistical comparison of the data extended from 30 to 120 s and showed the response values at each time point during the 134 and 670 µM mecamylamine perfusions to be significantly different from the corresponding time points of the other two perfusions. The 67 µM perfusion differed from the control perfusion at all time points except at 100 and 120 s.
Atropine
The carotid body's response to hypoxic KRB was reduced in a dose-related manner by inclusion of atropine in the perfusate. The mean values at the various time points (Fig. 3) were significantly different from each other.
By way of a summary presentation, Fig. 4
shows the response to the different concentrations of mecamylamine as
whole treatments (i.e., the response during the entire perfusion
period). The histograms are significantly different from each other at
the 0.05 level except for the difference between the control and 67 µM mecamylamine histograms, which narrowly misses the
P < 0.05 level
(P = 0.073). This probably reflects
the influence of time points 100 and 120 s in Fig. 2.
In two experiments, a lower dose of atropine was used with a different
technique (Fig. 5). For 60 s the first
syringe infused hypoxic KRB, followed by a second syringe perfusing
hypoxic KRB that either contained or was free from one of the two doses
of atropine. Interestingly, ANOVA testing of the data in only two experiments indicated that there was a significant difference between
the time points from 30-s mark of the second perfusion onward.
AFDX 116, Gallamine, and Pirenzepine
Because atropine blocks all known muscarinic receptors, further pharmacological testing was done in an effort to determine whether M1 and M2 muscarinic-receptor activity could be detected. AFDX 116, an M2 muscarinic-receptor blocker, was tested in four experiments. Figure 6 presents mean values from five perfusions in three animals in which one syringe with blocker-free or blocker-containing hypoxic KRB was used in normoxic cats. All time points are significantly different, except at 10 s.
To test the impact of gallamine or pirenzepine on increased neural
activity produced by hypoxic KRB, double perfusion-type experiments
were performed (Fig. 7). Hypoxic KRB was
delivered from the first syringe for 60 s followed by a 70-s perfusion
from the second syringe containing equally hypoxic KRB that was blocker free like the first or contained either gallamine (a second
M2 muscarinic-receptor blocker;
450 µM) or pirenzepine (an M1
muscarinic-receptor blocker; 400 µM). The second perfusate was
blocker free or gallamine containing in seven experiments and
pirenzepine containing in four of those same seven experiments. ANOVA
demonstrated significant differences between the hypoxic KRB and the
hypoxic+gallamine KRB perfusions at each of the 10-s time intervals. An
unpaired t-test analysis showed
significant differences between the control and pirenzepine perfusions
starting at the 40-s mark. We assume that the differences between
gallamine and pirenzepine are also significant at each of the time
points.
In five subsequent experiments in the normoxic cat (Fig.
8), a single-perfusion technique was used
to confirm our observation that, with this technique, pirenzepine acted
more slowly than gallamine. Paired
t-test comparisons showed that there
were significant differences from the 30-s mark onward.
DISCUSSION
Summary
Using a perfusion technique that restricts the cholinergic blockers to the area of the carotid body, we have observed in vivo a dose-related depression of the carotid body neural response to a 2-min perfusion of hypoxic KRB containing either mecamylamine (67-670 µM) or atropine (78-1,570 µM). We have also observed that when either of two M2 muscarinic-receptor blockers (gallamine or AFDX 116) was included in the hypoxic KRB perfusate, the carotid body neural response was greater than that to the blocker-free hypoxic KRB. Also, when pirenzepine, an M1 muscarinic-receptor blocker was included, the response to hypoxic KRB was less than that to the blocker-free hypoxic KRB. These data support an excitatory role for ACh in the chemotransduction of hypoxia in the cat carotid body. Furthermore, the data suggest the presence of M1 and M2 muscarinic receptors in the carotid body of the cat that function during a hypoxic challenge.Critique of Methods
Preparation. The overall integrity of the preparation appeared to remain good throughout the experiment. Examination of the carotid body after perfusions showed no structural damage, at least at the level of light microscopy, but there was a mild infiltration of inflammatory cells. This is not surprising given the 6- to 8-h duration of the experiment and the fact that sterile conditions were not maintained. The application of the stimulus was local, transient, and reversible. This seems to have contributed to the overall viability of the preparation, and it provided exact information as to the concentration of the blockers delivered to the carotid body vasculature. However, the responses of the carotid bodies to these perfusates were probably not the equilibrium responses. We have chosen to use a whole nerve recording as opposed to the few-fiber recording for practical reasons but with experimental justification. Single- or few-fiber preparations are difficult to maintain in the face of drawing up on snares (especially on the external carotid artery), adjusting the variable resistor on the lingual arterial catheter (to ensure proper pressure in the carotid body area), and the turning of stopcocks. However, previous studies (18, 19) have compared the neural output of a whole nerve recording minus the baroreceptor component (removed by thermal and mechanical treatment of the carotid sinus area) in response to hypercapnia at different levels of hypoxia with the single-/few-fiber preparation's response to the same stimulus protocol. The results were qualitatively identical. Protocol. In the application of a pharmacological agent to the vasculature of the carotid body to evaluate its ability to be excited or inhibited, a first consideration might be the passage of the agents from the vasculature to the putative structures involved in chemotransduction. The vasculature of the carotid body is extensive, and the passage of simple molecules like O2 and CO2 through membranes is rapid. However, Ross (39), studying the cat carotid body with the electron microscope, reported an impressive number of layers between the endothelium of the capillaries and the synaptic cleft of the type I cell/apposed nerve fiber. According to Ross, no very intimate contact exists between the type I cells and the blood channels. However, there seems to be little question that a large molecule can reach into the synaptic cleft. Woods (45) showed that horseradish peroxidase (HRP; molecular wt ~40,000) penetrated to all intercellular spaces in the carotid bodies of 100-g rats. What is unclear is how the HRP got to these spaces. Woods describes his observation as a "paradox" in that the HRP could be observed in the intercellular clefts after only 30 s, whereas it had not left normal capillaries by 10 min. Hence, although the HRP molecule can occupy the spaces, it is impossible, as Woods himself states, "simply from these experiments to make calculations of the minimum time it might take some other substance . . . to pass from the blood to the receptor." Hence, it seems that the responses of the carotid body to blocker-containing hypoxic perfusates over the first 2 min should be considered nonequilibrium responses.Agreement/Disagreement With Other Studies
The results reported in this study, although consistent with some reports (11-17, 30, 31), fail to be consistent with others (9, 10, 25, 28, 35, 36, 40). Presently, it is difficult to account completely for these differences. Sampson (40) was unable to find that intravenous injections of mecamylamine reduced the excitation of the carotid bodies due to arterial injections of NaCN, acid, and hypoxic blood. In a more quantitative approach, McQueen (35) reported results virtually identical to those of Sampson (40). On the other hand, the studies of Eyzaguirre and Zapata (15-17) showed mecamylamine to be effective in reducing several forms of excitation of the carotid body. Sampson (40) suggested that perhaps part of the difference was due to the in vivo preparation with normal circulation vs. the superfused in vitro preparation of Eyzaguirre and Zapata (15-17). McQueen's (35) preparation was also in vivo, as is the one in this study, in which, although many vessels in the area of the carotid sinus have been ligated, the major blood supply to the carotid body is untouched. Hence it would seem that circulation to and within the carotid body itself was no less normal than the circulation in the studies of Sampson (40) and McQueen (35). However, here the results are consistent with the data of Eyzaguirre and Zapata (15-17).Perhaps the concentrations in the studies of Sampson (40) and McQueen (35) were too low. In McQueen's study, the mean weight of the cats was 2.9 kg. A reasonable estimate of extracellular fluid would be 0.580 liter. A homogeneous distribution of the 250 µg/kg to 6 mg/kg would create a concentration in the extracellular fluid of 6.1 and 147 µM, respectively. It is puzzling, therefore, that neither Sampson (40) nor McQueen (35) saw any impact on either resting or stimulated neural activity when they used concentrations of mecamylamine in the range of 150 µM in extracellular fluid. Some of the perfusates used in the study reported here that were in this concentration range (67 and 134 µM) did reduce the response to hypoxic KRB consistently. Perhaps the assumption of a homogeneous distribution of mecamylamine is incorrect or a significant fraction of mecamylamine became attached to plasma protein. Kurz and his colleagues (29) reported that ~39% of a total dose of atropine was bound to plasma protein in adult human subjects. Hence in the preparations of Sampson (40) and McQueen (35) the synaptic receptors in the carotid body may never have been exposed to the concentrations calculated above. We know the concentrations delivered to the carotid body vasculature in our studies, although we, too, do not know the tissue concentrations of the blockers. With the high concentrations delivered, we saw the effect in a matter of seconds. This is consistent with the concept that a high concentration of cholinergic blockers focused on the appropriate site may be required to produce the depression (23).
Antagonist Concentration
A major concern, however, is the possibility that the high micromolar concentration of antagonists used in this study may have generated their effects simply by way of a nonspecific inhibition. Clear descriptions of nonspecific inhibition or depression are not plentiful, making the addressing of this possibility somewhat difficult. But Taylor and Insel (41) give the following description: ". . . a depression of all cellular excitability by affecting the energy charge, physicochemical state of the membrane, or capacity to generate second messengers. . . ." In an earlier study (21) data were reported pertinent to the possibility of nonspecific inhibition. First, the carotid body area was perfused with hypoxic KRB containing both 402 µM mecamylamine and 942 µM atropine. Integrated baroreceptor activity, recorded from the carotid sinus nerve, retained the same amplitude and pattern it had shown when the carotid body was perfused with blocker-free hypoxic KRB. Integrated chemoreceptor activity, however, was markedly reduced. Then the preparation was perfused with hypoxic KRB containing only 300 µM lidocaine. Lidocaine inhibited both baroreceptor and chemoreceptor activities in the whole nerve recording in 25 s. These results suggested that atropine and mecamylamine did not exercise an anesthetic effect, which we presumed was the explanation for the effect of the lidocaine.We have no information suggesting that any of the agents used as blockers of cholinergic receptors exert an effect on the energy charge of glomus cells or neurons, nor is there any information addressing the issue of whether they impede the generation of second messengers.
Finally, if the effect of the antagonists was due to a nonspecific inhibition or depression in the carotid body, it would be difficult to explain why including either of the two M2-receptor antagonists gallamine and AFDX 116 in the hypoxic KRB generated a larger response than the blocker-free hypoxic KRB, whereas including the M1 muscarinic-receptor antagonist pirenzepine generated a smaller response than the blocker-free hypoxic KRB.
Receptor Type and Location
Considerations such as those mentioned above warrant searching for other explanations that are based on the presence of cholinergic receptors in the cat carotid body. Data support this presence. In the cat, Dinger and colleagues (6, 7) have reported the presence of
-bungarotoxin (-Bgt)-binding sites on type I cells, type II
cells, and, by inference, on sympathetic nerves supplying the
vasculature of the carotid body. They were not visible on the
chemoreceptor afferent fibers of the carotid sinus nerve in the carotid
body. However, their presence has been reported (20) on an
extra-carotid body segment of whole carotid sinus nerve, appearing in
somewhat heavier concentration on each side of a ligature that had been
in place for 10 h.
However, it is important to point out that, in the species studied,
neuronal nicotinic (nACh) receptors are pentameres made up of -and
-subunits. To date, at least eight (2- to
9-)- and three (2-to
4-)-subunits have been cloned
from chick, rat, and human neuronal tissues. The -Bgt-binding site
contains 7-subunits
(8- and
9-subunits are also sensitive
to -Bgt). It is now well accepted, as was acknowledged by Dinger et
al. in 1981 (6) and by Hirano et al. in 1992 (26), that this nACh
receptor is not involved in synaptic neuronal transmission; it is
extrasynaptic and frequently presynaptic (33). It could function as a
cation channel (46). Indeed, in
Xenopus oocytes nACh receptors made up
of five 7-subunits have a
Ca2+/Na+
permeability ratio of 20:1 (34). The most frequently studied nACh
receptors involved in neuronal transmission in the central and
peripheral nervous systems are composed of three
2-,
3-, or
4-subunits with two
2- or
4-subunits. The permeability
coefficient (KD),
inhibitory constant
(Ki), 50%
effective concentration, and 50% inhibitory concentration
(IC50) values, expressive of the affinity of these receptors for agonists and antagonists, vary greatly.
-Subunits seem to codetermine the sensitivity of rat nACh receptors
to some antagonsists (1).
Dinger et al. (7) also reported that in the in vitro cat carotid body
50 nM -Bgt reduced by 50% the hypoxia- or nicotine-generated release of
[3H]dopamine. It also
reduced significantly the stimulus-evoked discharges recorded from the
carotid sinus nerve. Gonzalez and colleagues (24) described the
inhibitory effects on catecholamine release and on discharge in their
earlier study (7) as being "incomplete." Using nicotine-evoked
[3H]catecholamine
release, they compared the inhibitory effects of 50 µM mecamylamine
and 100 nM -Bgt. Mecamylamine reduced the release by 92%, whereas
-Bgt reduced the release by only 56%. They concluded that nACh
receptors and -Bgt-binding sites are not "completely
identical." They felt their results "call into question earlier
interpretations based solely on -Bgt binding in the organ, and
rekindle uncertainty regarding the localization of nACh receptors on
type I cells versus CSN afferent terminals." Recently, we have
reported data strongly suggesting the presence of the
4-subunit of the nACh receptor
on the type I cell and possibly on cell bodies and some fibers in the
petrosal ganglion (27). However, there may be a variety of nACh
receptors in the carotid body. This is currently being investigated.
As mentioned above, the affinity characteristics
(KD,
IC50, and so on) of the nACh
receptors for agonists and antagonists vary greatly. Dinger and
colleagues (7) reported a
KD of 5.7 nM for
the carotid body -Bgt-binding site. However, Zorumski et al. (47),
using whole cell voltage-clamp techniques in cultured postnatal rat
hippocampal neurons, found that 100 µM mecamylamine reduced the fast
current generated by 1 mM ACh by 69%, and 1 mM reduced it by 89%.
Mulle et al. (37) compared the electrophysiological and pharmacological
characteristics of nACh receptors located at pre- and postsynaptic
sites within the medial habenula and interpeduncular nucleus system in
the rat brain slice preparation. The
IC50 values for presynaptic nACh
receptors on afferent axons to the interpedunular nucleus (50 µM
nicotine stimulus) were (in µM) 2 mecamylamine, 4 hexamethonium, 250 dihydro--erythroidine, and 200 curare. Whiting and Lindstrom (44),
characterizing nACh receptors in the human brain, found the
Ki for
mecamylamine to inhibit the binding of
DL-[3H]nicotine
was >103 M. Finally, it
seems important to remember that more than one type of nicotinic
receptor can be found in a given location. Vernallis and her colleagues
(43) found in chick ciliary ganglia a class of nACh receptors,
predominantly synaptic, which contained receptors having at least three
types of subunits; they were encoded by the
3-,
4-, and
5-ACh receptor genes. They also
showed a class of extrasynaptic ACh receptors on the same neurons; it
contained the 7-subunits but
lacked the 3-,
4-, and
5-subunits.
In summary, across several species there is a variety of nACh receptors that have widely varying affinity/inhibitory characteristics (4, 32, 34). In any one location, the nACh receptors are not necessarily homogeneous. We are only beginning to look for these nACh receptors in the carotid body of the cat. On the basis of the intrinsic characteristics of the nACh receptor briefly described above, it seems quite premature to assign the inhibition generated by cholinergic antagonists in the present study only to nonspecific effects.
With regard to muscarinic receptors, Dinger et al. (5, 8) and Hirano et al. (26) have reported [3H]quinuclidinyl benzilate-binding sites in both the rabbit and cat carotid bodies. In the cat they describe the failure of the carotid body to respond to the muscarinic agonist pilocarpine. Our use of gallamine, AFDX 116, and pirenzepine confirms the presence of M1 and M2 muscarinic receptors in the cat carotid body. Our data suggest that whereas the M2 receptor seems to inhibit the response to hypoxic KRB, the M1 receptor seems to enhance the response to hypoxic KRB. [3H]quinuclidinyl benzilate-binding sites are reported to be plentiful in the cat carotid body (26). If, in the cat carotid body, the combination of characteristics that determine the neural output due to the muscarinic M1 (e.g., number, distribution, affinity, access by exogenously delivered agents) matched the combination for the M2 muscarinic receptor, then the failure of pilocarpine to produce any response would be understandable.
A final consideration regarding the use of micromolar concentrations of
cholinergic antagonists to block either nicotinic or muscarinic
receptors seems pertinent. Several studies have reported that the
concentrations of neurotransmitters in the synaptic cleft can reach
millimolar levels. Udgaonkar and Hess (42) reported that in aplysia ACh
released from the presynaptic cell within 0.5-1 ms of the arrival
of an action potential can reach a concentration at the postsynaptic
membrane of ~300 µM within a few microseconds. Nishi et al. (38)
found that the concentration of liberated ACh per impulse in an
eserinized lumbar sympathetic ganglion of the toad could reach 840 µM. Clements et al. (3) found that glutamate peaked at 1.1 mM in the
clefts of cultured hippocampal synapses from rats. Clements (2) has
recently reviewed several classes of central synapses to find broad
agreement that the average concentration of transmitter peaks in the
range 1-5 mM with a biphasic clearance, having time constants of
~100 µs and 2 ms. The pulse of transmitter, although very brief,
can prolong the time course of some synaptic currents and is enough to
saturate certain postsynaptic receptors (e.g., 
-aminobutyric acid,
glycine, or
N-methyl-D-aspartate).
Hence, if there are cholinergic receptors in the synaptic cleft between
the type I cell and the apposed nerve fiber and if this synapse
functions like so many others in the central and peripheral nervous
systems, it is quite reasonable to postulate a need for a high level of
antagonist to block the effects of endogenously released agonists.
Conclusion
At this point, every model has its shortcomings in trying to incorporate all the available data into an explanation of hypoxic chemotransduction in the carotid body of any species. What has been shown here are data consistent with an excitatory role for ACh during the chemotransduction of hypoxia in the cat carotid body. If, on the basis of these pharmacological studies, one speculates that both the nACh receptors involved in neural transmission and the M1 and M2 muscarinic receptors are present both pre- and postsynaptically, several different models could be generated. However, further study is needed before any of them could claim a preferential position.ACKNOWLEDGEMENTS
The authors gratefully acknowledge Dr. Chung Long Chou for assistance with computer graphics.
FOOTNOTES
Address for reprint requests: R. S. Fitzgerald, EHS/SHPH/JHU, 615 N. Wolfe St., Baltimore, MD 21205.
Received 21 August 1995; accepted in final form 27 November 1996.
REFERENCES
| 1. |
Cachelin, A.,
and
G. Rust.
-Subunits co-determine the sensitivity of rat neuronal nicotinic receptors to antagonists.
Pflügers Arch.
429:
449-451,
1995.
[Medline]
|
| 2. | Clements, J. Transmitter time course in the synaptic cleft: its role in central synaptic function. Trends Neurosci. 19: 163-172, 1996. [Medline] |
| 3. |
Clements, J.,
R. Lester,
G. Tong,
C. Jahr,
and
G. Westbrook.
The time course of glutamate in the synaptic cleft.
Science
258:
1498-1501,
1992.
|
| 4. | Deneris, E., J. Connolly, S. Rogers, and R. Duvoisin. Pharmacological and functional diversity of neuronal nicotinic acetylcholine receptors. Trends Pharmacol. Sci. 121: 34-40, 1991. |
| 5. | Dinger, B., L. Almaraz, T. Hirano, K. Yoshizaki, C. Gonzalez, A. Gomez-Nino, and S. Fidone. Muscarinic receptor localization and function in rabbit carotid body. Brain Res. 562: 190-198, 1991. [Medline] |
| 6. | Dinger, B., C. Gonzalez, K. Yoshizaki, and S. Fidone. Alpha-bungarotoxin binding in cat carotid body. Brain Res. 205: 187-193, 1981. [Medline] |
| 7. | Dinger, B., C. Gonzalez, K. Yoshizaki, and S. Fidone. Localization and function of cat carotid body nicotnic receptors. Brain Res. 339: 295-304, 1985. [Medline] |
| 8. | Dinger, B., T. Hirano, and S. J. Fidone. Autoradiographic localization of muscarinic receptors in rabbit carotid body. Brain Res. 367: 328-331, 1986. [Medline] |
| 9. | Douglas, W. The effect of a ganglion-blocking drug, hexamethonium, on the response of the cat's carotid body to various stimuli. J. Physiol. (Lond.) 118: 373-383, 1952. |
| 10. |
Douglas, W.
Is there chemical transmission at chemoreceptors?
Pharmacol. Rev.
6:
81-83,
1954.
|
| 11. | Euler, U. von, G. Liljestrand, and Y. Zotterman. The excitation mechanism of the chemoreceptors of the carotid body. Scand. Arch. Physiol. 83: 132-152, 1939. |
| 12. |
Eyzaguirre, C.,
and
H. Koyano.
Effects of some pharmacological agents on chemoreceptor discharges.
J. Physiol. (Lond.)
178:
410-437,
1965.
|
| 13. |
Eyzaguirre, C.,
and
H. Koyano.
Effects of electrical stimulation on the frequency of chemoreceptor discharges.
J. Physiol. (Lond.)
178:
438-462,
1965.
|
| 14. |
Eyzaguirre, C.,
H. Koyano,
and
J. R. Taylor.
Presence of acetylcholine and transmitter release from carotid body chemoreceptors.
J. Physiol. (Lond.)
178:
463-476,
1965.
|
| 15. | Eyzaguirre, C., and P. Zapata. A discussion of possible transmitter or generator substances in carotid body chemoreceptors. In: Arterial Chemoreceptors, edited by R. W. Torrance. Oxford, UK: Blackwell, 1968, p. 213-252. |
| 16. |
Eyzaguirre, C.,
and
P. Zapata.
Pharmacology of pH effects on carotid body chemoreceptors in vitro.
J. Physiol. (Lond.)
195:
557-588,
1968.
|
| 17. |
Eyzaguirre, C.,
and
P. Zapata.
The release of acetylcholine from carotid body tissues. Further study on the effects of acetylcholine and cholinergic blocking agents on the chemosensory discharge.
J. Physiol. (Lond.)
195:
589-607,
1968.
|
| 18. |
Fitzgerald, R. S.,
and
G. Dehghani.
Neural responses of the cat carotid and aortic bodies to hypercapnia and hypoxia.
J. Appl. Physiol.
52:
596-601,
1982.
|
| 19. | Fitzgerald, R. S., and D. Parks. Effect of hypoxia on carotid chemoreceptor response to carbon dioxide in cats. Respir. Physiol. 12: 218-229, 1971. [Medline] |
| 20. | Fitzgerald, R. S., and M. Shirahata. The role of acetylcholine in the chemoreception of hypoxia by the carotid body. In: Arterial Chemoreception, edited by C. Eyzaguirre, S. J. Fidone, R. S. Fitzgerald, S. Lahiri, and D. M. McDonald. New York: Springer-Verlag, 1990, p. 124-130. |
| 21. |
Fitzgerald, R. S.,
and
M. Shirahata.
Acetylcholine and carotid body excitation during hypoxia in the cat.
J. Appl. Physiol.
76:
1566-1574,
1994.
|
| 22. | Fitzgerald, R. S., M. Shirahata, and T. Ide. Cholinergic aspects of carotid body chemotransduction. In: Arterial Chemoreceptors: Cell to System, edited by R. G. O'Regan, P. Nolan, D. S. McQueen, and D. J. Paterson. New York: Plenum, 1994, p. 213-215. |
| 23. | Gilman, A. G., L. S. Goodman, T. W. Rall, and F. Murad (Editors). The Pharmacological Basis of Therapeutics (7th ed.). New York: Macmillan, 1985, p. 132. |
| 24. | Gonzalez, C., A. Obeso, B. Dinger, and S. Fidone. Non-identity of nicotinic receptors and alpha-bungarotoxin binding sites in cat carotid body (Abstract). Soc. Neurosci. Abstr. 12: 236, 1986. |
| 25. | Heymans, C., and E. Neil. Reflexogenic Areas of the Cardiovascular System. London: Churchill, 1958, p. 191. |
| 26. | Hirano, T., B. Dinger, K. Yoshizaki, C. Gonzalez, and S. Fidone. Nicotinic versus muscarinic binding sites in cat and rabbit carotid bodies. Biol. Signals 1: 143-149, 1992. [Medline] |
| 27. | Ishizawa,Y., R. S. Fitzgerald, M. Shirahata, and B. Schofield. Localization of nicotinic acetylcholine receptors in cat carotid body and petrosal ganglion. In: Frontiers in Arterial Chemoreception, edited by P. Zapata, C. Larrain, C. Eyzaguirre, and R. Torrance. New York: Plenum. In press. |
| 28. | Joels, N., and E. Neil. The idea of a sensory transmitter. In: Arterial Chemoreceptors, edited by R. W. Torrance. Oxford, UK: Blackwell, 1968, p. 153-178. |
| 29. | Kurz, H., A. Mauser-Ganshorn, and H. H. Stickel. Differences in the binding of drugs to plasma proteins from newborn and adult man. I. Eur. J. Clin. Pharmacol. 11: 463-467, 1977. [Medline] |
| 30. | Landgren, S., G. Liljestrand, and Y. Zotterman. The effect of certain autonomic drugs on the action potentials of the sinus nerve. Acta Physiol. Scand. 26: 264-290, 1952. [Medline] |
| 31. |
Liljestrand, G.
The problem of transmission at chemoreceptors.
Pharmacol. Rev.
6:
73-76,
1954.
|
| 32. | Lindstrom, J., R. Schoepfer, W. Conroy, and P. Whiting. Structural and functional heterogeneity of nicotinic receptors. In: The Biology of Nicotinic Dependence. Ciba Foundation Symposium. New York: Wiley Chichester, 1990, p. 23-52. |
| 33. |
McGehee, D.,
M. Heath,
S. Gelber,
P. Devay,
and
L. Role.
Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors.
Science
269:
1692-1696,
1995.
|
| 34. | McGehee, D., and L. Role. Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu. Rev. Physiol. 57: 521-546, 1995. [Medline] |
| 35. |
McQueen, D.
A quantitative study of the effects of cholinergic drugs on carotid chemoreceptors in the cat.
J. Physiol. (Lond.)
273:
515-532,
1977.
|
| 36. | Moe, G., L. Capo, and B. Peralta. Action of tetraethyl ammonium on chemoreceptor and stretch receptor mechanisms. Am. J. Physiol. 153: 601-605, 1948. |
| 37. | Mulle, C., C. Vidal, P. Benoit, and J.-P. Changeux. Existence of different subtypes of nicotinic acetylcholine receptors in the rat habenulo-interpeduncular system. J. Neurosci. 11: 2588-2597, 1991. [Abstract] |
| 38. | Nishi, S., H. Soeda, and K. Koketsu. Release of acetylcholine from sympathetic preganglionic nerve terminals. J. Neurophysiol. 30: 114-134, 1967. |
| 39. |
Ross, L.
Electron microscopic observations of the carotid body of the cat.
J. Biophys. Biochem. Cytol.
6:
253-262,
1959.
|
| 40. |
Sampson, S. R.
Effects of mecamylamine on responses of carotid body chemoreceptors in vivo to physiological and pharmacological stimuli.
J. Physiol. (Lond.)
212:
655-666,
1971.
|
| 41. | Taylor, P., and P. Insel. Molecular basis of pharmacologic selectivity. In: Principles of Drug Action, edited by W. Pratt, and P. Taylor. New York: Churchill Livingstone, 1990, p. 61. |
| 42. | Udgaonkar, J., and G. Hess. Acetylcholine receptor kinetics: chemical kinetics. J. Membr. Biol. 93: 93-109, 1986. [Medline] |
| 43. | Vernallis, A., W. Conroy, and D. Berg. Neurons assemble acetylcholine receptors with as many as three kinds of subunits while maintaining subunit segregation among receptor subtypes. Neuron 10: 451-464, 1993. [Medline] |
| 44. | Whiting, P., and J. Lindstrom. Characterization of bovine and human neuronal nicotinic acetylcholine receptors using monoclonal antibodies. J. Neurosci. 8: 3395-3404, 1988. [Abstract] |
| 45. | Woods, R. I. Penetration of horseradish peroxidase between all elements of the carotid body. In: The Peripheral Arterial Chemoreceptors, edited by M. Purves. London: Cambridge Univ. Press, 1975, p. 195-205. |
| 46. |
Zhang, Z.-W.,
S. Vijayaraghavan,
and
D. K. Berg.
Neuronal acetylcholine receptors that bind -bungarotoxin with high affinity function as ligand-gated ion channels.
Neuron
12:
167-177,
1994.
[Medline]
|
| 47. | Zorumski, C., L.-L. Thio, K. Isenberg, and D. Clifford. Nicotinic acetylcholine currents in cultrued postnatal rat hippocampal neurons. Mol. Pharmacol. 41: 931-936, 1992. [Abstract] |
This article has been cited by other articles:
|
|
 |
|
  D. F. Donnelly Spontaneous action potential generation due to persistent sodium channel currents in simulated carotid body afferent fibers J Appl Physiol, May 1, 2008; 104(5): 1394 - 1401. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. H Vidruk, E B. Olson Jr, L. Ling, and G. S Mitchell Responses of single-unit carotid body chemoreceptors in adult rats J. Physiol., February 15, 2001; 531(1): 165 - 170. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Zhang, H. Zhong, C. Vollmer, and C. A Nurse Co-release of ATP and ACh mediates hypoxic signalling at rat carotid body chemoreceptors J. Physiol., May 15, 2000; 525(1): 143 - 158. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bairam, H. Neji, and F. Marchal Cholinergic dopamine release from the in vitro rabbit carotid body J Appl Physiol, May 1, 2000; 88(5): 1737 - 1742. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
