Irene Chang

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.

The impact of adenosine on the release of acetylcholine, dopamine, and norepinephrine from the cat carotid body.

Exogenously administered adenosine provokes an increase in respiration in both animal models and in man. Administered near the carotid body adenosine increases neural output from the carotid body in rats and cats. Hypoxia has the same effect. Hypoxia also provokes a release of acetylcholine (ACh), dopamine (DA), and norepinephrine (NE) from the carotid body. The present study aimed to determine the effect of exogenous adenosine on the release of ACh, DA, and NE from the carotid bodies of cats. After a recovery period (from surgery) carotid bodies were first incubated for 10 (DA, NE) or 15 (ACh) min in Eppendorf tubes containing 85 microL of a physiological salt solution equilibrated with 40% O2/5% CO2 at 37 degrees C (hyperoxia). At the end of the incubation period the medium was drawn off, and measured for ACh, DA, and NE using HPLC-ECD methods. Next 85 microL of the medium and the tubes were equilibrated with a hypoxic gas mixture (4% O2/5% CO2) and the carotid bodies were incubated for 10 (DA, NE) or 15 (ACh) min, at the end of which the medium was drawn off and measured for ACh, DA, and NE. In the ACh studies there followed a post-hypoxic hyperoxic exposure (40% O2/5% CO2). ACh tubes were then made 100 microM with respect to adenosine, and the hyperoxic, hypoxic, and post-hypoxic hyperoxic challenges were repeated. One of the two DA, NE tubes had the 100 microM adenosine from the start. Adenosine significantly increased the release of ACh, but significantly decreased the hypoxia-induced release of DA. Potential mechanisms for these changes are reviewed.

The effect of a nitric oxide donor, sodium nitroprusside, on the release of acetylcholine from the in vitro cat carotid body

The purpose of the present study was to determine the impact of a nitric oxide (NO) donor, sodium nitroprusside (SNP), on the release of acetylcholine (ACh), an essential excitatory neurotransmitter, from the in vitro cat carotid body (CB). Bilateral CBs were harvested from five deeply anesthetized cats according to the regulations contained in the policies of the Johns Hopkins Animal Care and Use Committee. After recovering from the surgical procedures for extraction and cleaning, the CBs were taken through a 15-step protocol in which they were exposed to a hyperoxic gas mixture (40% O2/5% CO2; 20 min), then a hypoxic gas mixture (6% O2/5% CO2; 20 min), and a final 10 min hyperoxic mixture. This sequence was applied twice, followed by the same sequence in the presence, first, of 5 microM SNP, and secondly in the presence of 10 microM SNP. After washing and a recovery period the CBs were again exposed to the gases as in the first two non-SNP trials. The SNP exposures significantly reduced the overall release of ACh by about 20% (P=0.039). Further, SNP significantly reduced the hypoxia-induced increase in ACh release (without SNP: 82.4+/-19.1 fmol/20 microL versus with SNP: 49.7+/-15.0 fmol/20 microL; mean+/-S.E.M.; P=0.032). Trials #1 and #2 which preceded the application of SNP and Trial #3 which followed SNP were statistically indistinguishable. The CBs had recovered their original status. The data support the hypothesis that the frequently reported NO-induced reduction in CB neural output during hypoxia is at least in part due to the reduction in ACh release. The results are consistent with a previous report in which l-arginine, an NO precursor, had the same reducing effect. Possible mechanisms are discussed.

The impact of adenosine and an A2A adenosine receptor agonist on the ACh-induced increase in intracellular calcium of the glomus cells of the cat carotid body.

The carotid body (CB) is a polymodal chemosensor of arterial blood located next to the internal carotid artery. The basic chemosensing unit is composed of the neurotransmitter (NT)-containing glomus cells (GCs) and the sensory afferent fibers synapsing onto the GCs. Nicotinic and muscarinic receptors have been found on both the sensory afferent fibers and on the GCs. Neural output from the CB (CBNO) increases when arterial blood perfusing it is hypoxic, hypoglycemic, hypercapnic, or acidic. The increased CBNO due to GC release of excitatory NTs must be preceded by an entrance of calcium into the GCs. With repeated release of ACh from the GCs, cholinergic receptors could become desensitized, particularly nicotinic receptors which function as calcium channels. The purpose of the present study was to see if adenosine (ADO), known to alter receptor sensitivities, could attenuate or eliminate any desensitization of the nicotinic receptors occurring during the repeated application of ACh. Cat CBs were harvested with techniques approved by the Universitys Animal Care/Use Committee. The GCs were cultured and prepared for detecting [Ca(++)](i) with standard techniques. Repeated application of ACh produced a progressively decreasing increase in [Ca(++)](i). With the use of ADO or an A2(A) ADO receptor agonist the decrease was avoided. Though ADO also increased GC [Ca(++)](i), the sum of ADO increase and ACh increase, when superfused separately, was less than the increase when they were both included in the same superfusion. This suggested the possible involvement of a new path in the action. Potential mechanisms to explain the phenomena are discussed.

The impact of PCO2 and H+ on the release of acetylcholine from the cat carotid body

The carotid body (CB) is a sensor of oxygen, carbon dioxide, hydrogen ion, and glucose in the arterial blood. Many studies of the CBs responses to low oxygen (hypoxia) have been reported. Recently attention has been increasingly focused on its responses to elevated CO2 (hypercapnia). An increase in ventilation or carotid body neural output (CBNO) can result from stimulating the CB with blood or perfusion fluids having an elevated CO2 or H+. The increase in ventilation seen with a hypoxic stimulus is accompanied with an increase in CBNO and an increased release of both acetylcholine (ACh) and ATP from the CB. The present in vitro study using both CBs harvested from six cats was undertaken to determine if hypercapnia also provoked an increased release of ACh from the incubated CBs. The anesthetizing, handling, and euthanizing of the animals were according to the guidelines of the Johns Hopkins Animal Care and Use Committee which are totally consonant with those of the NIH. CBs, once harvested and prepared for the experimental protocol, were subjected to the following steps each lasting 10 min: (1) control; (2) stress; (3) recovery. The stresses were respiratory acidosis (RAC; acidic hypercapnia), compensated respiratory acidosis (CRAC; isohydric hypercapnia), and metabolic acidosis (MtAC). The first and last forms of acidosis generated small but significant increases in the release of ACh from the CBs; the second generated a very small and insignificant increase in ACh release. Since it is generally accepted that ACh is a key excitatory neurotransmitter in the CB along with ATP, these data are consistent with other studies measuring the increase in ventilation in response to a small increase in CO2 and those studies recording CBNO in response to hypercapnia. In five of the six animals the responses to RAC and MtAC were compared to the responses to hypoxia. The latter were statistically indistinguishable from the former two.

Hypoxic modulation of the cholinergic system in the cat carotid glomus cell.

The carotid body is a primary sensory organ for arterial hypoxia. Chemosensory glomus cells in the carotid body release neurotransmitters, including ACh, in response to hypoxia. The release of neurotransmitters from the glomus cell, a putative chemoreceptor cell, appears to be triggered by an influx of calcium and subsequent increase in intracellular calcium ([Ca]i). Several reports indicate that L-type and some other types of voltage-gated

Modulators of Cat Carotid Body Chemotransduction

The Carotid Body (CB) senses hypoxia, hypercapnia, and acidosis in the arterial blood. The resulting increase in CB neural output (CBNO) to the nucleus tractus solitarius in the medulla promotes reflex responses in the respiratory, circulatory, renal, and endocrine systems. Increases in CBNO are commonly thought to be due to the release of neurotransmitters from glomus cells in the CB. Additional to the action of these released transmitters on the postsynaptic afferent neurons which abut on the glomus cells the transmitters act presynaptically on glomus cell autoreceptors. Among the several transmitters contained in the glomus cells there now exists considerable evidence supporting excitatory roles for both acetylcholine (ACh) and ATP and an inhibitory role for dopamine (DA) and norepinephrine (NE) (Fitzgerald, 2000). The release of ACh (Fitzgerald et al., 1999; Kim et al., 2004) and catecholamines (Wang and Fitzgerald, 2002) appears to be influenced by modulators. The present study investigated the action of adenosine (ADO) on the release of ACh, DA, and NE since it has been reported that ADO influences CBNO (McQueen and Ribeiro, 1981) and CB-mediated increases in ventilation (Monteiro and Ribeiro, 1987). The study further investigated the action of nitric oxide (NO) on the release of ACh since NO has been reported to reduce the hypoxia-induced increase in CBNO (Wang, et al., 1994).

Hydrogen sulfide acting at the carotid body and elsewhere in the organism

H2S, the most recently explored gasotransmitter, has been found to have actions in at least three types of tissues.