Endothelin modulates chemoreceptor cell function in mammalian carotid body.
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Adaptation, Physiological↗
Biomedical subjects
S Fidone
Publications and source records attributed to S Fidone.
Mechanisms underlying chemoreceptor inhibition induced by atrial natriuretic peptide in rabbit carotid body.
1. Previous studies in our laboratory revealed the presence of atrial natriuretic peptide (ANP) in preneural chemosensory type I cells of the cat carotid body, and demonstrated that submicromolar concentrations of the peptide inhibited carotid sinus nerve (CSN) activity evoked by hypoxia. In the present study, we have evaluated the role of the cyclic nucleotide second messenger, cyclic GMP (cGMP), and the involvement of type I cells in rabbit chemosensory inhibition. 2. Submicromolar concentrations of the potent ANP analogue, APIII, greatly elevated both the content and release of cGMP from the carotid body. Denervation experiments confirmed earlier immunocytochemical studies which suggested that APIII-induced cGMP production occurs almost exclusively in type I cells; these experiments also indicate that both the sympathetic and sensory innervation to the carotid body exert a trophic influence on the metabolism of this second messenger. 3. Submicromolar concentrations of APIII inhibited the CSN activity evoked by hypoxia (79.8 +/- 3.2% (mean +/- S.E.M.) inhibition with 100 nM APIII) and nicotine (74.5 +/- 3.6% inhibition with 100 nM APIII), but did not affect basal CSN activity established in 100% O2-equilibrated superfusion solutions. 4. The biologically inactive analogue of ANP, C-ANP, failed to produce CSN inhibition; however, the inhibitory effects of APIII were mimicked by cell-permeant analogues of cGMP (dibutyryl-cGMP and 8-bromo-cGMP, 2 mM), which likewise did not alter basal CSN activity. Because we found that unmodified cGMP was an ineffective inhibitor of CSN activity, our data suggest that APIII inhibition is mediated intracellularly by cGMP produced within the type I cells. 5. APIII does not inhibit the CSN activity produced by 20 mM K+ (in zero Ca2+ media), which very probably results from direct depolarization of the sensory nerve terminals. 6. Catecholamine release from the carotid body evoked by hypoxia is likewise not altered by APIII (100 nM). 7. The data are consistent with the notion that APIII and analogues of cGMP alter the release of excitatory and/or inhibitory transmitters from chemosensory type I cells in the carotid body.
Animals↗
Effect of low O2 on glucose uptake in rabbit carotid body.
Glucose consumption in the rabbit carotid body was studied in vitro by measuring phosphorylation rates of tracer concentrations of 2-[3H]deoxy-glucose. The rate of glucose consumption measured in 100% O2-equilibrated modified Tyrode medium was 61 nmol.g tissue-1 x min-1 and was linear for up to 30 min. Incubation of carotid bodies for 5 or 10 min in moderately hypoxic solution (20% O2-80% N2) resulted in a 44% increase in the rate of glucose consumption. The glucose consumption of the nodose ganglion was not affected during similar incubation with low-O2 medium. High-resolution autoradiography of freeze-dried tissues revealed that the type I parenchymal cells are the principal site of glucose consumption in both 100% O2- and 20% O2-incubated carotid bodies. This metabolic response of the carotid body to hypoxia was not secondary to neurotransmitter release, because similar elevations in glucose utilization were observed with low-O2 medium containing zero Ca2+, a condition in which the release of neurotransmitters from type I cells is inhibited. Lowering the pH of the incubation medium from 7.4 to 7 or 6.8 markedly reduced the rate of glucose utilization by both the carotid body and the nodose ganglion. Ouabain (2 x 10(-4) and 1 x 10(-3) M) reduced by 20% the glucose consumption of carotid bodies incubated in 100% O2-equilibrated solution and abolished the metabolic response produced by low-O2 medium. The results suggest that the utilization of metabolic energy is an integral component of the chemoreceptor response to hypoxia.
Animals↗
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)
Animals↗
The role of cyclic AMP in chemoreception in the rabbit carotid body.
The present study identified physiological factors which influence the generation (and degradation) of cyclic AMP (cAMP) in the arterial chemoreceptor tissue of the mammalian carotid body. Experiments established a 3-way correlation between cAMP generation, neurotransmitter release from chemoreceptor cells, and carotid sinus nerve (CSN) activity. Incubation of carotid bodies in vitro for 10 min in media equilibrated with different low O2 ('hypoxic') gas mixtures (5% O2 or 10% O2, balance N2) elevated basal cAMP levels (100% O2 media) in proportion to the stimulus intensity. Similar experiments using nodose sensory ganglia showed that low O2 stimulation did not alter cAMP levels in this non-chemosensory tissue. However, the adenylate cyclase (AC) activator, forskolin (10 microM), evoked large increases in the cyclic nucleotide content in both carotid bodies and nodose ganglia. After chronic (10 days) CSN denervation or sympathectomy, the basal levels of cAMP in the carotid body were elevated; the cAMP response to low O2 media (stimulus minus control) was increased after CSN denervation but remained unaltered after sympathectomy. The effects of zero Ca2+ media on cAMP generation was examined in order to assess whether feedback from released neurotransmitters acting on known (presynaptic) type I cell receptors could have contributed to the observed changes in cAMP. Basal levels of cAMP were increased 2.8-fold, and the response to hypoxic stimulation was elevated 5-fold, in the absence of extracellular Ca2+. Forskolin (10 microM) did not alter basal release of [3H]-catecholamines ([3H]CA; synthesized from [3H]tyrosine), or resting CSN discharge; however, stimulus-evoked [3H]CA release and CSN discharge were potentiated in the presence of forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)
3,4-Dihydroxyphenylacetic Acid↗
Metabolic activation of carotid body glomus cells by hypoxia.
The effects of low O2 on glucose consumption in the rabbit carotid body were studied using the in vitro 2-deoxyglucose technique. Metabolically active structures within the tissue were localized autoradiographically after freeze-drying and vacuum fixation/embedding of selected incubated tissue samples. In 100% O2-equilibrated media, the mean basal glucose consumption calculated from the rate of 2-[1,2-3H]deoxy-D-glucose phosphorylation and its specific activity in the incubation media was 61 nmol.g tissue-1.min-1 in the carotid body and 42 nmol.g tissue-1.min-1 in parallel experiments with nodose ganglia. Low PO2 (20% O2-equilibrated media in vitro) increased glucose consumption in the carotid body by 44% but did not alter glucose metabolism of nodose ganglia. Autoradiographic data showed that preneural type I parenchymal cells are the principal site of glucose consumption in carotid chemosensory tissue. The mechanisms responsible for the hypoxia-induced increase in glucose consumption by the type I cells are discussed in relation to sensory transduction by the carotid body chemoreceptors.
Animals↗
Carotid sinus nerve C-fibers release catecholamines from the cat carotid body.
Previous studies have demonstrated that unmyelinated fibers in the cat carotid sinus nerve (CSN) are capable of inhibiting chemoreceptor activity from the carotid body. While the mechanism(s) underlying this phenomenon are unknown, it has been postulated that carotid body catecholamines may be involved in mediating this inhibitory effect. In this study, the in vitro cat carotid body-CSN preparation was used to investigate the effects of CSN stimulation on the release of [3H]catecholamines from the carotid body. The results showed that labeled catecholamines were released from this organ only when C-fibers were recruited by nerve stimulation.
Action Potentials↗
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)
Animals↗
Effects of hypoxia on neuropeptide levels in the rabbit superior cervical ganglion.
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Animals↗
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.
Animals↗
Effects of low pH on synthesis and release of catecholamines in the cat carotid body in vitro.
The rates of dopamine and noradrenaline synthesis in the cat carotid body (c.b.) are 5.9 +/- 0.58 pmol/c.b./2 h and 0.3 +/- 0.02 pmol/c.b./2 h, respectively. The synthesis is doubled when the organs are incubated at pH 7. Similarly, low pH induces a release of dopamine from the c.b. which is proportional to increased activity in the carotid sinus nerve.
3,4-Dihydroxyphenylacetic Acid↗
Cytospecific properties of arterial chemosensory neurons.
The carotid body of the cat was reinnervated either by the carotid branch or by the glossal branch of the IXth nerve and evaluated histologically and neurophysiologically. Regenerating foreign fibers re-established 90% fewer specialized terminals on glomus cells and displayed a greatly diminished chemosensory response, compared to axons of the regenerating carotid branch. Regenerating sensory neurons appear to develop chemosensitivity as a consequence of contact with glomus cells.
Animals↗
Catecholamine synthesis in rabbit carotid body in vitro.
1. Catecholamine synthesis in rabbit carotid body was studied in vitro using [(3)H]DOPA and [(3)H]tyrosine as precursors. The effects of sympathectomy and transection of the carotid sinus nerve on [(3)H]dopamine ([(3)H]DA) and [(3)H]noradrenaline ([(3)H]NA) synthesis were investigated in chronically denervated carotid bodies.2. When [(3)H]DOPA was used as precursor, the synthesis of [(3)H]DA was linear for more than 6 hr. The carotid body synthesized larger amounts of [(3)H]catecholamines than when [(3)H]tyrosine was used as precursor, but most of this excess was liberated into the incubation media. Using 10 muM-[(3)H]DOPA as precursor, the synthesis rates were 6.76 and 1.51 n-mole/g per hr for [(3)H]DA and [(3)H]NA, respectively; with 40 muM-[(3)H]DOPA, these values increased to 19.22 and 3.23 n-mole/g per hr, respectively.3. The relationship between [(3)H]DOPA concentration and [(3)H]DA synthesis was linear throughout the range 5-40 muM-[(3)H]DOPA.4. Sympathectomy reduced the synthesis of [(3)H]NA by 90% and [(3)H]DA by 37% when [(3)H]DOPA was used as precursor.5. When [(3)H]tyrosine (40 muM) was used as precursor, synthesis of [(3)H]catecholamines was linear for at least 4 hr, with rates of 12.10 and 0.85 n-mole/g per hr for [(3)H]DA and [(3)H]NA, respectively.6. [(3)H]DA and [(3)H]NA synthesis from [(3)H]tyrosine exhibited the characteristics of saturable processes, with K(m) values of 16.8 and 17.6 muM, respectively.7. 6-methyltetrahydropterine (6-MPH(4), 100 muM), a synthetic analogue of the natural co-factor for tyrosine hydroxylase, increased [(3)H]DA and [(3)H]NA synthesis from [(3) H]tyrosine in both the carotid body and superior cervical ganglion, with the greatest effect seen in the carotid body.8. When [(3)H]tyrosine was used as precursor, sympathectomy of the carotid body reduced [(3)H]NA synthesis by 80%, but did not alter [(3)H]DA or [(3)H]tyrosine levels in the tissue. Transection of the carotid sinus nerve had no effect on [(3)H]catecholamine synthesis in the carotid body.
Animals↗
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% O(2) in N(2)) or a hyperoxic gas mixture (50% O(2) in N(2)). The carotid bodies were then removed and incubated for 3 hr in modified Tyrode media equilibrated with 100% O(2) and containing either [(3)H]tyrosine or [(3)H]DOPA. The contents of [(3)H]DA and [(3)H]NA in the tissue were determined as described in the preceding paper.2. When [(3)H]DOPA was used as precursor, neither labelled dopamine (DA) or noradrenaline (NA) synthesis was increased in carotid bodies from rabbits exposed to 10% O(2) in N(2). Following exposure to 10% O(2) in N(2) and incubation with [(3)H]tyrosine, however, [(3)H]DA synthesis was increased by 72% above control (air) values while [(3)H]NA synthesis was unchanged. Less severe hypoxia, 14% O(2) in N(2), resulted in a smaller increase in [(3)H]DA synthesis, i.e. 53% above control value. Again, [(3)H]NA synthesis was unchanged. Similar experiments with the superior cervical ganglion involving exposure of the animals to either 10% or 14% O(2) in N(2) did not produce any change in the amounts of [(3)H]DA or [(3)H]NA synthesized from [(3)H]tyrosine when compared to control animals breathing air.3. Sympathectomy of the carotid body or transection of the carotid sinus nerve 12-15 days prior to hypoxic exposure (10% O(2) in N(2)) did not alter the increase in [(3)H]DA synthesis compared to normally innervated carotid bodies.4. Carotid bodies incubated with [(3)H]tyrosine for 2 hr in an alternating O(2)/N(2) sequence (5 min in media equilibrated with 100% O(2) followed by 3 min in media equilibrated with 100% N(2)) synthesized 37% more [(3)H]DA than control carotid bodies similarly exposed to an alternating O(2)/O(2) sequence. [(3)H]NA synthesis was unchanged. However, tissue levels of non-metabolized [(3)H]tyrosine were reduced by 19% in the carotid bodies exposed to the O(2)/N(2) sequence.5. Exposure of rabbits for 3 hr to 50% O(2) in N(2), followed by incubation of their carotid bodies in [(3)H]tyrosine, resulted in a 19% decrease in the absolute value for [(3)H]DA synthesis compared to control carotid bodies, but this difference was not significant (P > 0.05). However, [(3)H]NA synthesis was significantly reduced (51%; P < 0.05) in the hyperoxic carotid bodies. Similar experiments with the superior cervical ganglion showed that [(3)H]DA and [(3)H]NA synthesis were unchanged under control vs. hyperoxic conditions.6. Carotid bodies incubated with [(3)H]tyrosine for 3 hr, then transferred for 1 hr to unlabelled media equilibrated with 10% O(2) in N(2), released 81% more [(3)H]DA, and contained 38% less [(3)H]DA, than similarly treated carotid bodies exposed to 100% O(2). [(3)H]NA was not detectable in the media, and tissue levels of [(3)H]NA were the same in both hypoxic and control carotid bodies.
Animals↗
Effects of low oxygen on the release of dopamine from the rabbit carotid body in vitro.
1. Rabbit carotid bodies were pre-loaded with [(3)H]dopamine (DA) synthesized from [(3)H]tyrosine and then mounted in a vertical drop-type superfusion chamber which permitted simultaneous collection of released [(3)H]DA and recording of chemoreceptor discharge from the carotid sinus nerve.2. The time course of the spontaneous release of [(3)H]DA (superfusion with media equilibrated with 100% O(2)) in the presence of monoamine oxidase inhibitors exhibited two linear components, an initial steep phase followed after 3-4 hr by a later slower phase of release.3. When a 5 min low O(2) stimulus was delivered during the initial steep linear component of resting [(3)H]DA release, there was an abrupt increase in release, the magnitude of which was stimulus-dependent.4. The efflux of total radioactivity from the preparation declined exponentially with time; under resting conditions it was principally non-metabolized [(3)H]tyrosine. During stimulation, however the efflux increased, and 60-80% of the radioactivity could be attributed to [(3)H]DA.5. For a given low O(2) stimulus, the ratio of [(3)H]DA release during the stimulus period over that in the preceding control period remained approximately the same throughout a single experiment. Ratios for different low O(2) stimuli (50, 40, 30, 20, 10 and 0% O(2) in N(2)) yielded a parabolic relationship when plotted against stimulus intensity.6. Transection of the carotid sinus nerve or removal of the superior cervical ganglion 12-15 days prior to the experiment did not affect the release of [(3)H]DA at moderate stimulus intensities (superfusion with media equilibrated with 30% or 10% O(2) in N(2)) but both procedures significantly depressed release at the highest stimulus intensity (100% N(2)).7. Chemoreceptor discharge and [(3)H]DA release were simultaneously monitored in experiments using superfusion media free of monoamine oxidase inhibitors. In these experiments, the efflux of [(3)H]dihydroxyphenyl acetic acid (DOPAC) was also measured. The increase in peak chemosensory discharge was closely correlated with the increase in total release ([(3)H]DA + [(3)H]DOPAC) during stimulation with a series of low O(2) stimuli.8. Release of [(3)H]DA was almost completely abolished during superfusion with Ca(2+)-free, high Mg(2+) (2.1 mM) media, and the stimulus-related efflux of [(3)H]DOPAC was significantly reduced. However, chemoreceptor discharge was diminished by only 55%. These data are discussed with respect to their implications for DA as a chemosensory transmitter in rabbit carotid body.
Action Potentials↗
Taste buds reinnervated by carotid sinus nerve fibers.
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Animals↗
Alpha-bungarotoxin binding in cat carotid body.
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Acetylcholine↗
[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.
Animals↗