B. Dinger

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.

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.

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.

Cellular mechanisms involved in rabbit carotid body excitation elicited by endothelin peptides

The present study evaluated the effects of endothelin (ET) peptides on carotid sinus nerve (CSN) activity, catecholamine (CA) release, and second messenger signaling pathways in rabbit carotid bodies superfused in vitro, and in dissociated chemosensory type I cells. ET-1 (1.0 microM) and ET-3 (1.0 microM) did not alter basal CSN activity and CA release, but they potentiated nerve activity (P<0. 05) and CA release (P<0.05) evoked by hypoxia. Under basal conditions, ET-1 and ET-3 (1.0 microM each) elevated tissue cyclic AMP (cAMP) levels nearly 3-fold (P<0.001, ET-1; P<0.05, ET-3) and inositol phosphate (IP(n)) levels nearly 4-fold (P<0.01, ET-1). Hypoxia evoked an increase in carotid body cAMP, and this response was also potentiated in the presence of 1.0 microM ET-1 (P<0.01) or 1.0 microM ET-3 (P<0.001). Patch-clamp studies of isolated type I cells showed that 100 nM ET-1 elevated the peak amplitude of voltage-sensitive (L-type) Ca(2+)-currents by an average of 37.6% (P<0.001). Fluorescent Ca(2+)-imaging revealed that 100 nM ET-1 did not alter [Ca(2+)](i) under basal conditions, but that [Ca(2+)](i)-responses evoked by hypoxia were potentiated by 87% (P<0. 01). Our data indicate that ET augments chemoreceptor responses by activating second messenger signaling pathways which promote the phosphorylation of Ca(2+)-channel protein, thereby enhancing stimulus-evoked intracellular Ca(2+) levels.

The role of cyclic AMP in chemoreception in the rabbit caroid 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 (CNS) 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 intesity. 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 μM), evoked large increases in the cyclic nucleotide content in both carotid bodies and nodose ganglia. After chronic (10 days) CSN denervation or synpathectomy, 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 μM) 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. This increased release was primarily due to enhanced efflux of dopamine (DA). At increasing stimulus strengths, however, the relative effect of forskolin on [3H]CA release was diminished. The data suggest that the chemoreceptor type I cells in the carotid body generate cAMP in their transductive response to hypoxia, but that the net levels of cAMP in the tissue are also regulated by both feedback actions of released neurotransmitters and by the sympathetic and sensory innervation to the organ. The effects of forskolin on [3H]CA release and CSN activity, combined with the finding that hypoxia increases the cAMP content of the carotid body, suggest the immediate invlovement of this classical second messenger in chemotransduction and chemotransmission of natural carotid body stimuli.

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-

The Role of Endogenous NADPH Oxidases in Airway and Pulmonary Vascular Smooth Muscle Function

Reactive oxygen species generated from NADPH oxidase(s) in airway smooth muscle cells and pulmonary artery smooth muscle cells are important signaling intermediates. Nox4 appears to be the predominant gp91 homologue in these cells. However, expression of NADPH oxidase components is dependent on phenotype, and different homologues may be expressed during different functional states of the cell. NADPH oxidase(s) appear to be important not only for mitogenesis by these cells, but also for O(2) sensing. The regulation of NADPH oxidase(s) in airway and pulmonary artery smooth muscle cells has important implications for the pathobiochemistry of asthma and pulmonary vascular diseases.