Robert S. Fitzgerald

Oxygen and carotid body chemotransduction: the cholinergic hypothesis — a brief history and new evaluation

Oxygen can be said to be the most fundamentally necessary substrate for life. In those organisms having a cardiopulmonary system for delivering it in blood to the tissues the carotid body functions as the principal detector of decreases in arterial oxygen. Such a decrease stimulates an increase in neural output from the carotid body to the nucleus tractus solitarii, and this can precipitate a wide array of systemic reflex responses. The neural mechanisms involved in the genesis of increased signal from the carotid body remain unclear. But a current model of carotid body chemotransduction postulates that transmitter-laden glomus cells initiate the neural activity by being depolarized by hypoxemia and releasing an excitatory transmitter which binds to postsynaptic receptors of the adjacent sensory afferent fibers as well as to presynaptic glomus cell autoreceptors. This Frontiers Review evaluates anew the data supporting the hypothesis that acetylcholine (ACh) is an (the) essential excitatory transmitter in this process by examining AChs fulfillment of criteria required to establish a substance as a synaptic transmitter. All eight criteria are fulfilled in the case of ACh. Indeed, additional data further support the Cholinergic Hypothesis.

Cerebral circulatory responses to arterial hypoxia in normal and chemodenervated dogs

Cerebral hemodynamic responses to arterial hypoxia were studied in 13 normal and 9 chemodenervated anesthetized, paralyzed dogs. Arterial O2 content was lowered from control (18.0 vol%) to 14.0, 8.0, and 4.0 vol%, respectively, by either decreasing arterial Po2 (hypoxic hypoxia) or increasing carboxyhemoglobin saturation (CO hypoxia) at normal Po2. Both hypoxic hypoxia and CO hypoxia at each value of the lowered arterial O2 content resulted in progressive significant increases in cerebral blood flow (134, 169, 276, and 146, 206, 244% of control, respectively). Before chemoreceptor denervation, arterial blood pressure increased with hypoxic hypoxia but decreased with CO hypoxia. After chemodenervation, hypoxic hypoxia and CO hypoxia at each value of lowered arterial O2 content resulted in similar significant increases in cerebral blood flow. These increases were not significantly different from those observed prior to chemodenervation. After chemodenervation, hypoxic hypoxia and CO hypoxia both resulted in similar decreases in arterial blood pressure and cerebral vascular resistance, whereas, before chemodenervation, cerebral vascular resistance decreased more with CO hypoxia than with hypoxic hypoxia. These data show that cerebral vasodilation induced by both forms of hypoxia in chemodenervated dogs resembles that in animals with CO hypoxia and intact chemoreceptors in which Pao2 is high and the carotid chemoreceptors may not be activated. We also have shown that the transient responses to both types of hypoxia are not altered by carotid chemodenervation, and conclude that the carotid chemoreceptors do not play a role in the mechanism by which cerebral blood flow increases during decreased blood O2 content.

Role of acetylcholine in neurotransmission of the carotid body

Acetylcholine (ACh) has been considered an important excitatory neurotransmitter in the carotid body (CB). Its physiological and pharmacological effects, metabolism, release, and receptors have been well documented in several species. Various nicotinic and muscarinic ACh receptors are present in both afferent nerve endings and glomus cells. Therefore, ACh can depolarize or hyperpolarize the cell membrane depending on the available receptor type in the vicinity. Binding of ACh to its receptor can create a wide variety of cellular responses including opening cation channels (nicotinic ACh receptor activation), releasing Ca(2+) from intracellular storage sites (via muscarinic ACh receptors), and modulating activities of K(+) and Ca(2+) channels. Interactions between ACh and other neurotransmitters (dopamine, adenosine, nitric oxide) have been known, and they may induce complicated responses. Cholinergic biology in the CB differs among species and even within the same species due to different genetic composition. Development and environment influence cholinergic biology. We discuss these issues in light of current knowledge of neuroscience.

Presence of nicotinic acetylcholine receptors in cat carotid body afferent system.

With immunocytochemical techniques using a monoclonal antibody for alpha7 subunits of neuronal nicotinic acetylcholine receptors, we have found these subunits to be exclusively expressed in nerve fibers in the carotid body. Double-immunostaining showed that alpha7 subunit-positive nerve endings enveloped tyrosine hydroxylase-positive glomus cells. Some carotid sinus nerve fibers and tyrosine hydroxylase-positive petrosal ganglion neurons also expressed alpha7 subunits. These data support a role for acetylcholine in carotid body neurotransmission.

Acetylcholine release from cat carotid bodies.

Hypoxia, hypercapnia and acidosis stimulate the carotid body (CB) sending increased neural activity via a branch of the glossopharyngeal nerve to nucleus tractus solitarius; this precipitates an impressive array of cardiopulmonary, endocrine and renal reflex responses. However, the cellular mechanisms by which these stimuli generate the increased CB neural output are only poorly understood. Central to the understanding of these mechanisms is the determination of which agents are released within the CB in response to hypoxia, and serve as the stimulating transmitter(s) for chemosensory nerve endings. Acetylcholine (ACh) has been proposed as such an agent from the outset, but this proposal has been, and remains, controversial. The present study tests two hypotheses: (1) The CB releases ACh under normoxic/normocapnic conditions; and (2) The amount released increases during hypoxia and other conditions known to increase neural output from the CB. These hypotheses were tested in 12 experiments in which both CBs were removed from the anesthetized cat and incubated at 37 degrees C in a physiological salt solution while the solution was bubbled with four different concentrations of oxygen and carbon dioxide. The incubation medium was exchanged at 10 min intervals for 30 min (three periods of incubation). The medium was analyzed with high performance liquid chromatography-electrochemical detection for ACh content. Normoxic/normocapnic conditions (21% O2/6% CO2) produced a total of 0.639 +/- 0.106 pmol/150 microl (mean +/- S.E.M.; n = 12). All stimulating conditions produced larger total outputs: 4% O2/2% CO2 produced 1.773 +/- 0.46 pmol/150 microl; 0% O2/5% CO2, 0.868 +/- 0.13 pmol/150 microl; 4% O2/10% CO2, 1.077 +/- 0.21 pmol/150 microl. These three amounts were significantly greater than the normoxic/normocapnic condition, but indistinguishable among themselves. Further, the amount of ACh released did not diminish over the 30 min of stimulation. These data support the concept that during hypoxia ACh functions as a stimulating transmitter in the CB, and are consistent with the earlier reports of cholinergic enzymes and receptors found in the CB.

The effect of hyperoxia, hypoxia and hypercapnia on FRC and occlusion pressure in human subjects.

The measurement of pressure in the mouth 0.1 sec after the initiation of an occluded inspiratory effort (P0.1) has been proposed as an index of activity of medullary inspiratory neurons. If changes in FRC can be interpreted as important changes in the length-tension curve of the diaphragm or the total respiratory musculature, then changes in FRC from one occlusion pressure measurement to another can complicate such an interpretation of the P0.1 measurement. Forty-five subjects divided into three different groups were seated in a variable volume body plethysmograph. They had their FRC, P0.1, VT and VE measured while breathing air, 100% oxygen, 11% oxygen balance nitrogen, and 4% carbon dioxide in 20% oxygen balance nigrogen. All 45 showed a decrease in FRC during hyperoxia (-12%); 40 of 43 showed increases in FRC during hypoxia (14%); 42 of 43 showed an increased FRC during hypercapnia (15%). Changes in VE were small as were changes in P0.1 values. These latter changes generally followed the same pattern of changes as FRC though the magnitude of the changes showed more variability. We were unable to demonstrate a significant correlation between changes in FRC and changes in P0.1 under the conditions of our experiments.

Carotid chemoreceptor response to intermittent or sustained stimulation in the cat

Abstract In order to determine how closely the carotid chemoreceptor system follows a repeated transient stimulus, intermittent infusions of hypercapnic-hypoxic, hypercapnic, and hypoxic blood were made into the common carotid arteries of anesthetized, artificially ventilated cats. The system demonstrated an ability to follow blood-home stimuli presented at frequencies up to about 70 min −1 though at decreasing amplitude. In response to sustained stimulations, the carotid body showed a slow adaptation to hypercapnic stimuli and perhaps to hypoxic stimuli also. A transient undershoot occurred at the cessation of stimulation when the animal was breathing air, an event generally not seen if the animal was breathing pure oxygen.

EFFECTS OF A CONTINUOUS INFUSION OF DOPAMINE ON THE VENTILATORY AND CAROTID BODY RESPONSES TO HYPOXIA IN CATS

1. We investigated how a continuous infusion of dopamine (DA; 5μg/kg per min), which is often used clinically, would affect the ventilation and carotid chemoreceptor neural activity in anaesthetized cats.

The presence of CO2/HCO3- is essential for hypoxic chemotransduction in the in vivo perfused carotid body

Carotid chemoreceptor activity was increased by the perfusion of the carotid body in vivo with hypoxic HEPES-buffered solution (HBS) containing CO2/HCO3- (HBA+), but not with hypoxic HBS without CO2/HCO3- (HBS-). When the perfusate was switched to hypoxic HBS+ during hypoxic HBS-perfusions, chemoreceptor activity increased immediately. Thus, CO2/HCO3- played a critical role in the hypoxic chemotransduction of the in vivo perfused carotid body.

The impact of hypoxia and low glucose on the release of acetylcholine and ATP from the incubated cat carotid body

The carotid body (CB) is a polymodal sensor which increases its neural output to the nucleus tractus solitarii with a subsequent activation of several reflex cardiopulmonary responses. Current reports identify acetylcholine (ACh) and adenosine triphosphate (ATP) as two essential excitatory neurotransmitters in the cat and rat CBs. This study explored the impact of hypoxia, low glucose, and the two together on the release of both ACh and ATP from two incubated cat CBs. The CBs were prepared with standard procedures in accordance with the policies and regulations of the Institutional Animal Care and Use Committee. When normalized to their controls, a significant increase of ACh in the incubation medium was measured in response to hypoxia, low glucose, and the combined stimuli. When normalized to their controls, a significant increase in ATP in the incubation medium was measured in response to hypoxia and to the combined stimuli. Low glucose generated an increase in ATP which was not statistically significant (P>0.05). Second, normalizing the initial 3-4 or 2-3 min Time Segment of the challenge Stage to the final 3-4 or 2-3 min Time Segment of the control Stage for both ACh and ATP generated significant increases in response to hypoxia, low glucose (ACh only), and the combined stimuli. The data suggested the possibility that in the cat the increased CB neural output in response to low glucose might be due primarily to ACh.