Chronic hypoxia-induced acid-sensitive ion channel expression in chemoafferent neurons contributes to chemoreceptor hypersensitivity.

Previously we demonstrated that chronic hypoxia (CH) induces an inflammatory condition characterized by immune cell invasion and increased expression of inflammatory cytokines in rat carotid body. It is well established that chronic inflammatory pain induces the expression of acid-sensitive ion channels (ASIC) in primary sensory neurons, where they contribute to hyperalgesia and allodynia. The present study examines the effect of CH on ASIC expression in petrosal ganglion (PG), which contains chemoafferent neurons that innervate oxygen-sensitive type I cells in the carotid body. Five isoforms of ASIC transcript were increased ∼1.5-2.5-fold in PG following exposure of rats to 1, 3, or 7 days of hypobaric hypoxia (380 Torr). ASIC transcript was not increased in the sympathetic superior cervical ganglion (SCG). In the PG, CH also increased the expression of channel-interacting PDZ domain protein, a scaffolding protein known to enhance the surface expression and the low pH-induced current density mediated by ASIC3. Western immunoblot analysis showed that CH elevated ASIC3 protein in PG, but not in SCG or the (sensory) nodose ganglion. ASIC3 transcript was likewise elevated in PG neurons cultured in the presence of inflammatory cytokines. Increased ASIC expression was blocked in CH rats concurrently treated with the nonsteroidal anti-inflammatory drug ibuprofen (4 mg·kg(-1)·day(-1)). Electrophysiological recording of carotid sinus nerve (CSN) activity in vitro showed that the specific ASIC antagonist A-317567 (100 µM) did not significantly alter hypoxia-evoked activity in normal preparations but blocked ∼50% of the hypoxic response following CH. Likewise, a high concentration of ibuprofen, which is known to block ASIC1a, reduced hypoxia-evoked CSN activity by ∼50% in CH preparations. Our findings indicate that CH induces inflammation-dependent phenotypic adjustments in chemoafferent neurons. Following CH, ASIC are important participants in chemotransmission between type I cells and chemoafferent nerve terminals, and these proton-gated channels appear to enhance chemoreceptor sensitivity.

Changes in tyrosine hydroxylase and substance P immunoreactivity in the cat carotid body following chronic hypoxia and denervation.

Long-term hypoxia elicits functional changes in the cat carotid body which are manifest as altered chemosensitivity in response to hypoxia. Previous studies have suggested that these functional adjustments may be mediated by changes in neurotransmitter levels in chemosensory type I cells. Neurotransmitter metabolism in the carotid body has also been shown to be regulated by the neural innervation to the organ. The present study using the cat carotid body demonstrates profound changes in the levels of immunoreactivity of the catecholamine-synthesizing enzyme, tyrosine hydroxylase, and the neuropeptide, substance P, in response to a two-week exposure to hypoxia (10% O2 in 90% N2). Furthermore, these changes were modulated both by sensory and sympathetic denervation of the organ. For TH, the intensity of immunostaining in type I cells was markedly increased by long-term hypoxia in both normal and chronic carotid sinus nerve-denervated carotid bodies, but this effect was blocked following chronic sympathectomy. Substance P immunoreactivity in type I cells was dramatically attenuated by hypoxia in both intact and chronic carotid sinus nerve-denervated preparations, but this effect was reduced following chronic sympathectomy. Tyrosine hydroxylase- and substance P-positive axon terminals were observed to innervate type I cells. These axons were also present in chronically sympathectomized preparations, but they disappeared following chronic carotid sinus nerve-denervation suggesting that they very likely arise from sensory neurons in the petrosal ganglion. Our data indicate that chronic chemoreceptor stimulation by hypoxia elicits multiple neurochemical adjustments in the cat carotid body. These changes suggest that catecholaminergic enzymes and neuropeptides play a significant role in the adaptive mechanisms of chemoreceptor function which occur in response to chronic physiological stimulation. Furthermore, the data suggest that neurotrophic mechanisms may influence neurotransmitter metabolism in chemosensory type I cells.

cAMP production in rabbit carotid body: role of adenosine.

In the present study, we have investigated the possible role of adenosine in the hypoxia-mediated increase in adenosine 3',5'-cyclic monophosphate (cAMP) in the carotid body. cAMP levels in rabbit carotid bodies superfused in vitro for 10 min were increased in the presence of adenosine (100 microM and 1.0 mM; maximum increase = 127%, P < 0.01). These effects were reduced by the nonspecific adenosine-receptor antagonist 1,3-dipropyl-8[p-sulfophenyl]xanthine (DPSPX; 10 microM). The specific A2-receptor agonist 2-[4'(2-carboxymethyl)phenylethylamino]-5'-N-ethylcarboxamido adenosine (CGS-21680; 100 nM) also elevated carotid body cAMP levels, an effect that was blocked by the specific A2-antagonist 3,7-dimethyl-L-propargyl-xanthine (DMPX; 50 microM). Hypoxia-evoked elevations in cAMP were potentiated in the presence of the adenosine-uptake inhibitor dipyridamole (100 nM) and blocked by exposure to adenosine-receptor antagonists. Our data suggest that the rabbit carotid body contains specific adenosine receptors (A2 subtype) that are positively coupled to adenylate cyclase and that increases in cAMP associated with hypoxia are mediated by the release of endogenous adenosine.

Second messenger regulation of tyrosine hydroxylase gene expression in rat carotid body.

Previous studies [Czyzyk-Krzeska et al.: J Neurochem 1992;58:1538] demonstrated the relationship between low O2 breathing and tyrosine hydroxylase (TH) gene expression in chemosensory type I cells of the carotid body. In the present study, we have exposed carotid bodies in vitro to hypoxic superfusion media, and subsequently used the reverse transcriptase-polymerase chain reaction technique to measure relative changes in the TH transcript in an effort to elucidate the cellular mechanisms which regulate TH gene expression. Carotid bodies and superior cervical ganglia (SCG) were exposed for 3 h to superfusion media equilibrated with either 10% O2 or 100% O2 and then rapidly frozen on dry ice prior to extraction of total RNA. Hypoxia elevated TH mRNA in the carotid body 3.63 +/- 0.84-fold (mean +/- SEM), while in contrast, these parameters were unchanged in SCG similarly exposed to hypoxic media. Incubation of carotid bodies in zero Ca2+ superfusates greatly attenuated the increase in TH mRNA evoked by hypoxia (1.39 +/- 0.34-fold increase; p < 0.025 compared to normal Ca2+ group). Likewise, exposure to the guanylate cyclase activator, atriopeptin III (100 nM), attenuated the TH mRNA hypoxic response (p < 0.005), while activation of adenylate cyclase with forskolin (10 microM) tended to elevate the response to low O2. Our data suggest that hypoxia, independent of circulating hormones, induces TH gene expression in the carotid body, and that multiple factors, including [Ca2+] and cyclic nucleotides, may be important components of the signal transduction pathway.

The role of nitric oxide in carotid chemoreception.

Immunocytochemical and histochemical studies of cat and rat carotid bodies have revealed a plexus of nitric oxide synthase (NOS)-positive nerve fibers associated with lobules of chemosensory type I cells as well as with the carotid body vasculature. NOS-positive fibers originate from (1) autonomic neurons located in the carotid body and distributed along the carotid sinus nerve (CNS) and IXth cranial nerve which terminate in the adventitial layer of carotid body blood vessels, and (2) from unipolar sensory neurons of the petrosal (IXth nerve) ganglion. Carotid bodies incubated with the NO precursor, 3H-arginine, yield 3H-citrulline, the detectable coproduct of NO synthesis. Furthermore, electrical stimulation of the CNS or exposure of carotid bodies to hypoxic incubation media elevates 3H-citrulline formation. Millimolar concentrations of L-arginine inhibit chemoreceptor activity evoked by hypoxia, an effect which is reversed by the specific NOS antagonist, L-NG-nitroarginine methylester (L-NAME, 0.1 mM). Electrical stimulation of CNS C fibers elevates cyclic GMP in the carotid body vasculature and lobules of type I cells. Cyclic GMP production is reduced during stimulation in the presence of L-NAME, a finding consistent with the known ability of NO to activate a soluble form of guanylate cyclase. Further studies showed that brief (< 1 min) stimulation of CNS C fibers inhibits basal chemoreceptor discharge in a perfused/superfused in vitro carotid body preparation, whereas prolonged (> 5 min) stimulation is required to inhibit the response to hypoxia. The inhibitory effect is reversed by L-NAME. Our combined anatomical, neuropharmacological and electrophysiological data suggest that NO plays a dual role in mediating CNS inhibition, one via its actions on the organ's vasculature and the other through direct effects on the chemosensory type I cells. The former pathway involves cholinergic/NOS presumptive parasympathetic autonomic neurons, while the latter may be mediated by axon reflex or primary affarent depolarization of chemosensory nerve terminals.

Nitric oxide mediates chemoreceptor inhibition in the cat carotid body.

Numerous studies have demonstrated that carotid sinus nerve fibers mediate a so-called "efferent" inhibition of carotid body chemoreceptors. However, the mechanism(s) underlying this phenomenon are not understood. Recently, it has been shown that an extensive plexus of nitric oxide synthase-containing carotid sinus nerve fibers innervate the carotid body, and that many fine, beaded fibers can be seen in close proximity to small blood vessels as well as lobules of parenchymal cells. The present study examined the effects of centrifugal neural activity in the carotid sinus nerve on the accumulation of [3H]citrulline synthesized from [3H]arginine in the cat carotid body, and the possible involvement of nitric oxide in mediating "efferent" chemoreceptor inhibition. Electrical stimulation of carotid sinus nerve C-fibers evoked an increase in [3H]citrulline accumulation in the carotid body, which was Ca(2+)-dependent and blocked by L-NG-nitroarginine methylester (0.1 mM), an inhibitor of nitric oxide synthase. Using a vascularly perfused in vitro carotid body preparation, chemoreceptor activity was recorded from thin nerve filaments split-off from the main trunk of the carotid sinus nerve. Electrical stimulation of the main nerve trunk at C-fiber intensities inhibited steady-state chemoreceptor discharge, and this effect was blocked by L-NG-nitroarginine methylester. However, when the organ preparation was switched to the superfuse-only mode, carotid sinus nerve stimulation failed to alter the steady-state discharge, but under these conditions, prolonged nerve stimulation (> 5 min) did attenuate the chemoreceptor response to hypoxia, an effect which was likewise blocked by L-NG-nitroarginine methylester. The present data, together with previous anatomical findings that nitric oxide synthase immunoreactivity is present in both sensory and autonomic ganglion cells innervating the carotid body, suggest that two neural mechanisms may be involved in the inhibitory neural regulation of carotid chemoreceptors. One mechanism appears to involve nitric oxide release from intralobular sensory C-fibers, which lie in close proximity to the chemoreceptor type I cells. The other mechanism involves release of nitric oxide from perivascular terminals of autonomic microganglia neurons, which control carotid body blood flow.

Localization and actions of nitric oxide in the cat carotid body.

An extensive plexus of nerve fibers capable of synthesizing nitric oxide was demonstrated in the cat carotid body by immunocytochemical and biochemical studies of nitric oxide synthase. Denervation experiments indicated that the axons originate from: (i) microganglial neurons located within the carotid body and along the glossopharyngeal and carotid sinus nerves, whose ramifications primarily innervate carotid body blood vessels; and (ii), sensory neurons in the petrosal ganglion, whose terminals end in association with lobules of type I cells. In the in vitro superfused cat carotid body, the nitric oxide synthase substrate, L-arginine, induced a dose-dependent inhibition of carotid sinus nerve discharge evoked by hypoxia. In contrast, the nitric oxide synthase inhibitor, L-NG-nitroarginine methylester, augmented the chemoreceptor response to hypoxia, and this effect was markedly enhanced when the preparation was both perfused and superfused in vitro. The nitric oxide donor, nitroglycerine, inhibited carotid sinus nerve discharge, and immunocytochemistry revealed that this drug stimulated the formation of cyclic 3',5'-guanosine monophosphate in both type I cells and blood vessels. Our data indicate that nitric oxide is an inhibitory neuronal messenger in the carotid body, which affects the process of chemoreceptor transduction/transmission via actions on both the receptor elements and their associated blood vessels.

Neurons synthesizing nitric oxide innervate the mammalian carotid body.

The carotid body is an arterial chemoreceptor organ sensitive to blood levels of O2, CO2 and pH. The present immunocytochemical and neurochemical study has demonstrated the presence of an extensive plexus of nitric oxide (NO)-synthesizing nerve fibers in this organ. These nitric oxide synthase (NOS)-containing axons are closely associated with parenchymal type I cells and with blood vessels in the carotid body. Denervation and retrograde tracing experiments have revealed that these fibers arise from NOS-immunoreactive and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive neuronal cell bodies located in the petrosal ganglion and the carotid body, and dispersed along the glossopharyngeal and carotid sinus nerves (CSN). Within the petrosal ganglion, these neurons are topographically segregated from the catecholaminergic cells, and they contain the neuropeptide, substance P. NOS-positive autonomic microganglial cells in the carotid body and CSN also exhibit choline acetyltransferase (ChAT) immunoreactivity. Our results suggest that nitric oxide may be a novel neuronal messenger in the mammalian carotid body involved in the modulation of chemosensory transduction and transmission in this organ.

Release of dopamine from carotid sinus nerve fibers innervating type I cells in the cat carotid body.

This study presents evidence that dopaminergic neurons innervate the cat carotid body. Immunocytochemical studies revealed many tyrosine hydroxylase (TH)-positive nerve fibers in the carotid body which establish extensive contacts with type I cells. All TH-positive intralobular profiles disappeared with chronic carotid sinus nerve (CSN) section, but survived sympathectomy following removal of the superior cervical ganglion. The level of endogenous dopamine (DA) in the CSN was higher than that for norepinephrine (NE). While both catecholamines were synthesized by the nerve at similar rates, NE synthesis was abolished by chronic sympathectomy, but DA synthesis remained largely unchanged following this procedure. Our data indicate that DA is not present in the CSN as a mere precursor of NE. Following a 3-hour incubation of carotid bodies with their attached nerves in media containing 20 microM 3H-tyrosine, electrical stimulation of CSN C-fibers in chronically sympathectomized preparations provoked the release of 3H-DA, but not 3H-NE.

Atrial natriuretic peptide increases cyclic guanosine monophosphate immunoreactivity in the carotid body.

The mammalian carotid body is a peripheral arterial chemoreceptor organ involved in the regulation of respiration, and in the modulation of blood pressure through reflex control of peripheral vascular resistance and cardiac output. In addition to its responsiveness to blood gases, the organ is also sensitive to hyperosmotic solutions, and we have recently shown that a systemic hormonal regulator of natriuresis and diuresis, atrial natriuretic peptide, is a potent inhibitor of chemoreceptor activity evoked by hypoxia in the cat carotid body. The present study demonstrates atrial natriuretic peptide immunoreactivity in type I cells of the carotid body, and shows further that a biologically active atrial natriuretic peptide fragment, atriopeptin III, increases cyclic guanosine monophosphate immunoreactivity in type I cells in a dose-dependent manner. Moreover, double-labeling techniques demonstrate co-existence of atrial natriuretic peptide immunoreactivity with the atriopeptin III-enhanced cyclic guanosine monophosphate reaction product. These findings indicate the probable existence of atrial natriuretic peptide receptors coupled to membrane-bound guanylate cyclase on the parenchymal type I cells. Our findings support the view that cyclic guanosine monophosphate functions as a second messenger in this organ, and may serve as a functional activity marker in identifying type I cells which respond to atrial natriuretic peptide.

The co-existence of biogenic amines and neuropeptides in the type I cells of the cat carotid body.

The mammalian carotid body consists of preneural type I (glomus) cells synaptically coupled to afferent axon terminals and enveloped by type II (sustentacular) cells. Recent studies indicate the presence of multiple putative neurotransmitters in this arterial chemoreceptor organ. A double-labeling immunocytochemical technique was utilized which allows simultaneous visualization of two neurochemicals in a single cell. The issue of transmitter co-occurrence in type I cells of the cat carotid body was addressed using specific antibodies for seven neurochemical agents: tyrosine hydroxylase, dopamine-beta-hydroxylase, choline acetyltransferase, serotonin, substance P, met-enkephalin and chromogranin. A high degree (greater than 70%) of co-localization was observed for most pairs of markers, indicating the co-existence of multiple neuroactive agents in type I cells of the cat carotid body. The intensity of staining varied greatly among cells but formed a pattern. Thus, for tyrosine hydroxylase and dopamine-beta-hydroxylase, the majority of double-labeled type I cells exhibited equivalently low or high levels of both, while for the neuropeptides unequal levels of the two markers predominated. Neuropeptides also co-existed in type I cells with catecholamine-synthesizing enzymes and with serotonin. The functional significance of such patterns of multiple co-existence involving biogenic amines and neuropeptides is discussed. Our results indicate a high degree of co-occurrence of reaction product for amine-synthesizing enzymes (tyrosine hydroxylase, dopamine-beta-hydroxylase and choline acetyltransferase), the indoleamine serotonin, and the neuropeptides substance P and met-enkephalin.

Muscarinic receptor localization and function in rabbit carotid body.

Acetylcholine and muscarinic agonists inhibit chemosensory activity in the rabbit carotid sinus nerve (CSN). Because the mechanism of this inhibition is poorly understood, we have investigated the kinetics and distribution of muscarinic receptors in the rabbit carotid body with the specific muscarinic antagonist [3H]quinuclidinylbenzilate ([3H]QNB). Equilibrium binding experiments identified displaceable binding sites (1 microM atropine) with a Kd = 71.46 pM and a Bmax = 9.23 pmol/g tissue. These binding parameters and the pharmacology of the displaceable [3H]QNB binding sites are similar to specific muscarinic receptors identified in numerous other nervous, muscular and glandular tissues. Comparisons of specific binding in normal and chronic CSN-denervated carotid bodies suggest that muscarinic receptors are absent on afferent terminals in the carotid body; however, nearly 50% of the specific [3H]QNB binding is lost following chronic sympathectomy, suggesting the presence of presynaptic muscarinic receptors on the sympathetic innervation supplying the carotid body vasculature. Autoradiographic studies have localized the remainder of [3H]QNB binding sites to lobules of type I and type II parenchymal cells. In separate experiments, the muscarinic agonists, oxotremorine (100 microM) stimulation of the in vitro carotid body. Our data suggest that muscarinic inhibition in the rabbit carotid body is mediated by receptors located on type I cells which are able to modulate the excitatory actions of acetylcholine at nicotinic sites.

Co-existence of tyrosine hydroxylase and dopamine beta-hydroxylase immunoreactivity in glomus cells of the cat carotid body.

Catecholamines are thought to play an important role in sensory transduction in the arterial chemoreceptors of the mammalian carotid body, and classical cytochemical techniques have demonstrated their presence in the type I (glomus) cells of this organ. However, it remains controversial whether dopamine (DA) and norepinephrine (NE) occur in the same or in different subtypes of glomus cells. In the present study, we have addressed this issue using immunocytochemistry to compare the localization of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (D beta H) in the cat carotid body. Both pre- and post-embedding double-labelling immunohistochemical techniques were employed. TH and D beta H were found to co-exist in over 90% of the glomus cells, and they were co-localized at equivalent levels in almost 80% of the cells; less than 5% contained only TH. The results suggest that DA and NE are synthesized and stored in a common cell population in the cat carotid body.

Localization and in vitro actions of atrial natriuretic peptide in the cat carotid body.

Previous studies of atrial natriuretic peptide (ANP) have indicated that its release from the heart and from discrete areas of the central nervous system evokes coordinated physiological and behavioral adjustments that mitigate the adverse hypertensive effects of volume overload and/or acute increases in sodium intake. Because the reflex activity initiated by arterial chemoreceptors of the carotid body directly contributes to the integrated regulation of systemic blood pressure, we have investigated the possibility that ANP has a significant role in the chemosensory process as well. Our immunocytochemical studies show that ANP-like immunoreactivity is present in the preneural chemosensitive type I cells in the cat carotid body. Furthermore we found that the biologically active ANP fragment atriopeptin III is a potent inhibitor of carotid sinus nerve activity evoked by hypoxia. Our findings suggest that circulating and/or endogenous ANP may modulate carotid body function as part of a coordinated response to changes in systemic volume and solute balance.

Immunocytochemical localization of cAMP and cGMP in cells of the rat carotid body following natural and pharmacological stimulation.

Although the chemoreceptive function of the carotid body has been known for many decades, the cellular mechanisms of sensory transduction in this organ remain obscure. Common elements in the transductive processes of many cells are the cyclic nucleotide second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Studies from our laboratory have revealed stimulus-induced changes in cyclic nucleotide levels in the carotid body as measured by RIA, but such changes in second messenger levels have not been localized to specific cellular elements in the organ. The present immunocytochemical study utilized the avidin-biotin-peroxidase method to investigate the distribution of cAMP and cGMP in the rat carotid body and to assess changes in the intensity of immunostaining following in vitro stimulation by hypoxia, forskolin, sodium nitroprusside, high potassium, and atrial natriuretic peptide. Both cAMP and cGMP immunoreactivity were localized to type I cells of organs maintained in vivo and fixed by perfusion. Organs exposed to 100% O2-equilibrated media in vitro produced low but visible levels of cAMP immunoreactivity in a majority of type I cells; hypoxia (5% O2-equilibrated media) for 10 min moderately increased the level of immunoreactivity; forskolin (10(-5) M), or forskolin combined with hypoxia, dramatically increased cAMP levels in virtually all cells. Moderate levels of cGMP immunoreactivity in control carotid bodies in vitro were strikingly reduced by hypoxia; a significant increase in cGMP levels occurred following incubation in high potassium (100 mM), and under these conditions, the decrease in cGMP immunoreactivity with hypoxia was much more pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)

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.