S. Fidone

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.

The role of dihydropyridine-sensitive Ca2+ channels in stimulus-evoked catecholamine release from chemoreceptor cells of the carotid body

The present study utilized an in vitro preparation of the rabbit carotid body, with tissue catecholamine stores labeled by incubation with 3H-tyrosine. The goal was to characterize pharmacologically the voltage-dependent Ca2+ channels present in the type I (glomus) cells of this arterial chemoreceptor organ, and to elucidate their role as pathways for Ca2+ entry. We found that release of 3H-dopamine induced by high external potassium was over 95% dependent on external calcium concentration and that this release was 90-100% inhibited by the dihydropyridine antagonists, nisoldipine and nitrendipine, and was potentiated by the dihydropyridine agonist, BayK 8644. Therefore, any stimulus-induced, calcium-dependent release of 3H-dopamine that was inhibited by nisoldipine and potentiated by BayK 8644, was considered to be supported by Ca2+ entry into the cells via voltage-dependent Ca2+ channels. Significant differences were observed in the release of 3H-dopamine induced by 75 vs 25 mM K+. On prolonged stimulation, release induced by 75 mM K+ was large and transient, whilst that induced by 25 mM K+, although more moderate, was sustained. The release elicited by 75 mM K+ was inhibited approximately 90% by 1.5 mM Co2+ or 625 nM nisoldipine, while release by 25 mM K+ was completely blocked by 0.6 mM Co2+ or 125 nM nisoldipine. Low PO2-induced release of 3H-dopamine was 95% dependent on Ca2+, and was inhibited by nisoldipine (625 nM) in a manner inversely proportional to the intensity of hypoxic stimulation, i.e. 79% inhibition at a PO2 of 49 Torr, and 20% inhibition at PO2 of 0 Torr. BayK 8644 potentiated the release induced by moderate hypoxic stimuli. Release elicited by high PCO2/low pH, or by Na(+)-propionate or dinitrophenol-containing solutions, was approximately 80% Ca(2+)-dependent, and the dihydropyridines failed to modify this release. It is concluded that type I cells possess voltage-dependent Ca2+ channels sensitive to the dihydropyridines, which in agreement with previous electrophysiological data should be defined as L-type Ca2+ channels. Calcium entry which supports the release of 3H-dopamine elicited by moderate hypoxia should occur mainly through these channels while the release induced by strong hypoxic stimuli will be served by Ca2+ entry which occurs in part via voltage-dependent Ca2+ channels, and in part through an additional pathway, probably a Na+/Ca2+ exchanger.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Efferent inhibition and antidromic depression of chemoreceptor A-fibers from the cat carotid body

Single unit chemoreceptor A-fiber activity was isolated in very fine filaments peeled back from the peripheral cut end of the cat carotid nervein vivo. Electrical stimulation of the remainder of the carotid nerve produces two types of suppression of chemoreceptor discharge. The first type arises from activation of an efferent inhibitory pathway mediated by carotid nerve C-fibers, while the second type results from antidromic depression of chemoreceptor discharge consequent to the direct stimulation of the sensory fiber. The shortness of the carotid nerve gives rise to the problem of stimulus spread which can confuse the nature of the type of discharge suppresion observed. A technique based on the relative refractory period of carotid nerve A-fibers is described which monitored the stimulus spread and the direct excitation of the sensory fiber. Utilizing this technique, it was possible to separate the effects of antidromic depression from efferent inhibition. The possible functional significance of efferent inhibition and antidromic depression is discussed.

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.

Adaptation to chronic hypoxia involves immune cell invasion and increased expression of inflammatory cytokines in rat carotid body

Exposure to chronic hypoxia (CH; 3-28 days at 380 Torr) induces adaptation in mammalian carotid body such that following CH an acute hypoxic challenge elicits an abnormally large increase in carotid sinus nerve impulse activity. The current study examines the hypothesis that CH initiates an immune response in the carotid body and that chemoreceptor hyperexcitability is dependent on the expression and action of inflammatory cytokines. CH resulted in a robust invasion of ED1(+) macrophages, which peaked on day 3 of exposure. Gene expression of proinflammatory cytokines, IL-1beta, TNFalpha, and the chemokine, monocyte chemoattractant protein-1, was increased >2-fold after 1 day of hypoxia followed by a >2-fold increase in IL-6 on day 3. After 28 days of CH, IL-6 remained elevated >5-fold, whereas expression of other cytokines recovered to normal levels. Cytokine expression was not restricted to immune cells. Studies of cultured type I cells harvested following 1 day of in vivo hypoxia showed elevated transcript levels of inflammatory cytokines. In situ hybridization studies confirmed expression of IL-6 in type I cells and also showed that CH induces IL-6 expression in supporting type II cells. Concurrent treatment of CH rats with anti-inflammatory drugs (ibuprofen or dexamethasone) blocked immune cell invasion and severely reduced CH-induced cytokine expression in carotid body. Drug treatment also blocked the development of chemoreceptor hypersensitivity in CH animals. Our findings indicate that chemoreceptor adaptation involves novel neuroimmune mechanisms, which may alter the functional phenotypes of type I cells and chemoafferent neurons.

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 cyclic nucleotide formation in rabbit carotid body in vitro

The present experiments measured cAMP and cGMP in the arterial chemosensory tissue of the rabbit carotid body exposed for 10 min in vitro to normoxic or hypoxic conditions, or to specific activators of adenylate cyclase (forskolin) and guanylate cyclase (sodium nitroprusside). The enzyme activators elevated the basal levels of cAMP (48 x) and cGMP (3.7 x), respectively. Hypoxic media increased cAMP in the carotid body by 3.6-fold, but the levels of cGMP were reduced by 33% in media equilibrated with low O2. The data are consistent with the notion that cyclic nucleotides are involved in the transduction of natural stimuli and/or the neurotransmitter feedback modulation of chemosensory type I cells.

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.