C. Gonzalez

Effects of low oxygen on the release of dopamine from the rabbit carotid body in vitro.

1. Rabbit carotid bodies were pre‐loaded with [3H]dopamine (DA) synthesized from [3H]tyrosine and then mounted in a vertical drop‐type superfusion chamber which permitted simultaneous collection of released [3H]DA and recording of chemoreceptor discharge from the carotid sinus nerve.

Catecholamine synthesis in rabbit carotid body in vitro

1. Catecholamine synthesis in rabbit carotid body was studied in vitro using [3H]DOPA and [3H]tyrosine as precursors. The effects of sympathectomy and transection of the carotid sinus nerve on [3H]dopamine ([3H]DA) and [3H]noradrenaline ([3H]NA) synthesis were investigated in chronically denervated carotid bodies.

3H)SPIROPERIDOL BINDING IN NORMAL AND DENERVATED CAROTID BODIES

Specific dopamine receptors were studied in freshly dissected, unhomogenized rabbit carotid bodies incubated in [3H]spiroperidol. Total binding and non-specific binding were determined in the absence and presence of 0.2 microM (+)-butaclamol, respectively. Specific binding in normal carotid bodies incubated at near saturating concentrations (0.38 nM) was 1.63 +/- 0.58 pmol/g of tissue. Chronic section of the carotid sinus nerve (14 days) resulted in a 64% reduction (P less than 0.05) in specific binding. We conclude that the majority of specific dopaminergic receptors are located on carotid sinus nerve afferent terminals.

Localization and function of cat carotid body nicotinic receptors

Acetylcholine and nicotinic agents excite cat carotid body chemoreceptors and modify their response to natural stimuli. The present experiments utilized [125I]alpha-bungarotoxin [( 125I]alpha-BGT) to localize within the chemosensory tissue the possible sites of action of exogenous and endogenous nicotinic cholinergic substances. In vitro equilibrium binding studies of intact carotid bodies determined a Kd of 5.57 nM and a Bmax of 9.21 pmol/g of tissue. Chronic section (12-15 days) of the carotid sinus nerve (CSN) did not change the amount of displaceable toxin binding. In contrast, the specific binding was reduced by 46% following removal of the superior cervical ganglion. Light microscope autoradiography of normal, CSN-denervated and sympathectomized carotid bodies revealed displaceable binding sites concentrated in lobules of type I and type II cells. Treatment of carotid bodies with 50 nM alpha-BGT in vitro reduced by 50% the release of [3H]dopamine (synthesized from [3H]tyrosine) caused by hypoxia or nicotine, and also significantly reduced the stimulus-evoked discharges recorded from the CSN. The data suggest an absence of alpha-BGT binding sites on the afferent terminals of the CSN and that nicotinic receptors located with parenchymal cell lobules may modulate the release of catecholamines from these cells.

Differential stimulus coupling to dopamine and norepinephrine stores in rabbit carotid body type I cells

Recent studies suggest that preneural type I (glomus) cells in the arterial chemoreceptor tissue of the carotid body act as primary transducer elements which respond to natural stimuli (low O2, pH or increased CO2) by releasing chemical transmitter agents capable of exciting the closely apposed afferent nerve terminals. These type I cells contain multiple putative transmitters, but the identity of the natural excitatory agents remains an unresolved problem in carotid body physiology. Characterization of putative transmitter involvement in the response to natural and pharmacological stimuli has therefore become fundamental to further understanding of chemotransmission in this organ. The present study demonstrates that a natural stimulus (hypoxia) evokes the release of dopamine (DA) and norepinephrine (NE) in approximate proportion to their unequal stores in rabbit carotid body (DA release/NE release = 8.2). In contrast, nicotine (100 microM), a cholinomimetic agent thought to act on the nicotinic receptors present on the type I cells, evokes the preferential release of NE (DA release/NE release = 0.17). These findings suggest that distinct mechanisms are involved in a differential mobilization of these two catecholamines from the rabbit carotid body.

EFFECTS OF HYPOXIA ON TYROSINE HYDROXYLASE ACTIVITY IN RAT CAROTID BODY

Abstract— Tyrosine hydroxylase (TH) activity was measured in the carotid body. superior cervical ganglion and adrenal glands of the rat under normal conditions and at 48 h following exposure of the animals for 1‐3 h in a low O2 atmosphere. Basal TH levels were 5‐6 nmol/h/mg tissue for both the carotid body and the ganglion. Forty‐eight hours after hypoxia, there was an increase in enzyme activity in both tissues which paralleled the severity of the hypoxia but was greater in the carotid body than the superior cervical ganglion. Thus, following exposure to 5% O2 in N2 for two 30‐min periods (20‐min interim), TH activity had increased by 50% in the carotid body and 33% in the ganglion; after exposure to 10% O2 in N2 for 3 h (continuous), TH levels were increased by 37% in the carotid body and 12% in the ganglion. In the adrenal gland, basal TH activity was 3.42 ± 1.87 nmol/h/mg tissue, and this value was unchanged following either level of hypoxia.

Effects of hypoxia on catecholamine synthesis in rabbit carotid body in vitro

1. Unanaesthetized, unrestrained rabbits were exposed for 3 hr in a chamber to either air, hypoxic gas mixtures (10% or 14% O2 in N2) or a hyperoxic gas mixture (50% O2 in N2). The carotid bodies were then removed and incubated for 3 hr in modified Tyrode media equilibrated with 100% O2 and containing either [3H]tyrosine or [3H]DOPA. The contents of [3H]DA and [3H]NA in the tissue were determined as described in the preceding paper.

Synthesis and release of catecholamines by the cat carotid body in vitro: Effects of hypoxic stimulation

The role of catecholamines (CAs) in cat carotid body chemoreception has been controversial. On the basis of pharmacological experiments, it would appear that endogenous dopamine (DA) may act either as an inhibitory or excitatory transmitter. Neurochemical studies on the effects of natural stimulation on the release of carotid body CAs in the cat have also been inconclusive. In the present study, we have characterized the synthesis and release of CAs in the in vitro cat carotid body preparation in response to different levels of hypoxic stimulation and have correlated these measures with the chemosensory activity of the carotid sinus nerve. The synthesis of [3H]DA and [3H]norepinephrine was linear for at least 4 h in carotid bodies incubated with their natural precursor [3H]tyrosine. Synthesis of both [3H]CAs plateaued when the [3H]tyrosine concentration in the media reached 40 microM, which is a concentration similar to that found in cat plasma. Exposure of the animals to an atmosphere of 10% O2 in N2 for 3 h prior to removal and incubation of the carotid bodies with [3H]tyrosine resulted in an approximately 100% increase in the rate of [3H]DA synthesis but no change in [3H]norepinephrine synthesis. This selective increase in [3H]DA synthesis was not detected when [3H]dihydroxyphenylalanine was used as precursor. Carotid bodies first incubated with [3H]tyrosine and later superfused with solutions equilibrated with different gas mixtures (0-100% O2 in N2) exhibited an increase in [3H]DA release and carotid sinus nerve discharge which were inversely related to the oxygen concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-bungarotoxin binding in cat carotid body

The carotid body is an arterial chemosensory organ which detects changes in blood gas tensions and pH, and reflexly contributes to the cardiorespiratory adjustments which occur during hypoxia, hypercapnia and acidosis. However, the sensory mechanisms involved in carotid chemoreception remain to be elucidated. Morphologically, the carotid body consists of an association of elemental units, or glomeruli, within a connective tissue stroma penetrated by a dense capillary net 5. The glomeruli are comprised of catecholamine-rich type I, or chief cells, which are enveloped by glial-like processes of type II, or sustentacular, cellsa,4,19. Sensory fibers from the carotid sinus nerve penetrate the glomeruli to terminate in synaptic-like apposition on type I cellst,18, 21. Schweitzer and Wright 25 first noted the stimulatory effects of acetylcholine (ACh) on carotid chemoreflexes in the cat, and suggested that this substance might be involved in the generation of chemosensory activity. Later experiments characterized in detail the excitatory potency of ACh and nicotinic agonists on the chemoreceptor discharge from the cat carotid body 7,9,10,24. They showed that cholinergic antagonists abolish the sensitivity to ACh and reduce the response to natural stimulation. More recently, it has been demonstrated that chemically identifiable ACh is present in the parenchymal tissue of the cat carotid body, rather than in the fibers or terminals of the carotid sinus nerve11,l~, 15. Although the site(s) of ACh storage in this tissue has not been firmly established, a high affinity component of choline uptake has been autoradiographically localized to the type I cells 12. Finally, there is evidence that ACh is released from the carotid body during natural stimulationS,L One interpretation of these findings is that ACh is a sensory transmitter in the cat carotid body, and that as such, this substance is released from the type I cells by natural stimulation to activate nicotinic receptors on neighboring sensory nerve terminals, thereby leading to the initiation of chemosensory impulses in the carotid sinus nerve 1°. Other recent studies have shown, however, that ACh directly depolarizes the type I cells in both normal and de-

Increased release of [3H]dopamine during low O2 stimulation of rabbit carotid body in vitro

SUMMARY Rabbit carotid bodies synthesized [3H]dopamine (DA) during a 3-h incubation period in modified Tyrodes solution containing 40 uM [3H]tyrosine. Following tbis loading period, the carotid bodies were exposed for one additional hour to unlabelled Tyrodes solution equilibrated with either 10% oxygen in nitrogen or with 100% oxygen. The carotid bodies exposed to low O2 released 81% more [3H]dopamine during this one-hour period than the carotid bodies exposed only to pure oxygen. These data suggest that hypoxia induces release of DA from the carotid body. It is now well documented that the mammalian carotid body contains the biogenic amines dopamine, norepinephrine and 5-hydroxytryptamine [9,12,29], and that of these, dopamine (DA) is present in the tissue in the highest concentration [12,15]. ~ere is also convincing evidence that these substances are contained primarily within the glomus, or Type I, cells of the carotid body [6,7]. Electrophysiological and pharmacological studies in the cat [23,24,28], dog [4] and rabbit (Monti-Bloch and Eyzaguirre, pets. comm.) have shown that exogenous DA alters chemoreceptor activity recorded from the carotid nerve, and it has been suggested that during natural stimulation of the carotid body release of endogenous DA from the glomus cells may function to modulate the activity of chemoreceptor nerve fibers which terminate upon these cells [2,14,28]. However, previous studies of the effects of natural stimulation on the DA stores of the carotid body have produced conflicting results, and there are no ~tudies which have attempted to directly demonstrate either increased release or tunlover of carotid body DA during natttral stimu]a