Sustained exposure to cytokines and hypoxia enhances excitability of oxygen-sensitive type I cells in rat carotid body: correlation with the expression of HIF-1α protein and adrenomedullin.

Recent studies in our laboratory demonstrated that chronic hypoxia (CH) induces a localized inflammatory response in rat carotid body that is characterized by macrophage invasion and increased expression of inflammatory cytokines. Moreover, CH-induced increased hypoxic sensitivity is blocked by concurrent treatment with the common anti-inflammatory drugs, ibuprofen and dexamethasone. The present study examines the hypothesis that selected cytokines enhance the excitability of oxygen-sensitive type I cells in the carotid body, and that downstream effects of cytokines involve upregulation of the transcription factor, hypoxia inducible factor-1α (HIF-1α). Cultured type I cells were exposed for 24 h to hypoxia and/or a cocktail of cytokines consisting of interleukin-1ß, interleukin-6, and tumor necrosis factor-α. Subsequent evaluation of hypoxia-evoked intracellular Ca(2+)-responses showed that previous exposure to cytokines plus hypoxia resulted in a 110% (p<0.001) increase in cell excitability, whereas exposure to cytokines or hypoxia alone elicited smaller increases of 22% (not significant) and 35% (p<0.01), respectively. These changes were correlated with increased immunostaining for HIF-1α in similarly treated type I cells, where exposure to cytokines plus hypoxia promoted the nuclear translocation of the transcription factor. Moreover, treatment with cytokines and/or hypoxia elevated the expression of the HIF-1-regulated gene, adrenomedullin. These in vitro results are supported by studies which show that elevated type I cell sensitivity following in vivo CH is blocked by concurrent treatment with ibuprofen. The data suggest that CH-induced adaptation in arterial chemoreceptors may in part be mediated by cytokine/hypoxia-induced upregulation of HIF-1α, and consequent enhanced expression of specific hypoxia-sensitive genes in type I cells.

Effect of endothelin receptor antagonist bosentan on chronic hypoxia-induced inflammation and chemoafferent neuron adaptation in rat carotid body.

Chronic hypoxia (CH) induces an inflammatory response in rat carotid body that is characterized by immune cell invasion and the expression of pro-inflammatory cytokines. In the present study, we have investigated the role of type-A endothelin (ET-A) receptors in the development of CH-induced inflammation. After 7 days of CH (380 Torr), double-label immunofluorescence studies demonstrated elevated levels of ET-A receptor and tyrosine hydroxylase (TH) in O(2)-sensitive type I cells. Following CH, ET-A receptors were also expressed on resident and invasive CD45+ immune cells distributed in tissue surrounding chemosensory cell lobules. Immnofluorescence and quantitative PCR studies showed that concurrent treatment with the ET-A/B receptor antagonist, bosentan (200 mg/kg/day), blocked CH-induced ED-1+ macrophage invasion and the upregulation of cytokines, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and monocyte chemoattractant protein-1 (MCP-1). Moreover, bosentan treatment blocked the CH-induced increases in expression of acid-sensitive ion channels (ASICs) in chemoafferent neurons in the petrosal ganglion (PG). Our findings are consistent with the hypothesis that CH-induced inflammation involves the upregulation and release of ET-1 from type I cells. ET-1 may act in an autocrine/paracrine mechanism via ET-A receptors on chemosensory type I cells and immune cells to promote an inflammatory response.

Chronic hypoxia increases the gain of the hypoxic ventilatory response by a mechanism in the central nervous system.

We studied the effects of the ventilatory stimulant doxapram to test the hypothesis that chronic hypoxia increases the translation of carotid body afferent input into ventilatory motor efferent output by the central nervous system. Chronic hypoxia (inspired Po(2) = 70 Torr, 2 days) significantly increased the ventilatory response to an intravenous infusion of a high dose of doxapram in conscious, unrestrained rats breathing normoxic or hypoxic gas. The in vitro carotid body response to hypoxia increased with chronic hypoxia, but the response was not increased with a high dose of doxapram. Similarly, the phrenic nerve response to doxapram in anesthetized rats with carotid bodies denervated did not change with 7 days of chronic hypoxia. The results support the hypothesis that chronic hypoxia causes plasticity in the central component of the carotid chemoreceptor ventilatory reflex, which increases the hypoxic ventilatory response. We conclude that doxapram provides a promising tool to study the time course of changes in the central gain of the hypoxic ventilatory response during chronic hypoxia in awake animals and humans.

Effect of the endothelin receptor antagonist bosentan on chronic hypoxia-induced morphological and physiological changes in rat carotid body.

Previous experiments have repeatedly demonstrated that exposure to chronic hypoxia (CH) elicits remarkable structural changes and chemosensory hypersensitivity in the mammalian carotid body. Moreover, recent studies have shown that CH upregulates the neuroactive peptide, endothelin (ET), in oxygen-sensitive type I cells. The present study examines the possible involvement of ET in adaptation by concurrently exposing rats to hypobaric CH (B(P) = 380 Torr) and bosentan, a potent nonpeptide antagonist that blocks ET(A) and ET(B) receptors. Carotid body weight indicated that 14 days of CH induced organ enlargement, a response that was blunted in bosentan-treated rats (CH: 2.54 +/- 0.19-fold increase; CH plus bosentan: 1.92 +/- 0.14-fold increase; P < 0.05). Morphometric studies revealed that bosentan substantially eliminated CH-induced hyperplasia of chemosensory cell lobules as well as expansion of the connective tissue matrix. Vascular dilation associated with CH was not altered by the drug. In untreated animals exposed to 3 days of CH, expression of proliferating cell nuclear antigen (PCNA), a marker of mitosis, was increased in lobules of oxygen-sensitive type I cells and in extralobular vascular and connective tissue cells. The incidence of PCNA expression was significantly (P < 0.05) reduced in bosentan-treated animals. In vitro assessments of carotid sinus nerve (CSN) activity showed that enhancement of basal and hypoxia-evoked chemosensory activity following 9 days of CH was significantly (P < 0.001) blunted by concurrent treatment with bosentan. Collectively, our data are consistent with the hypothesis that CH-induced adaptation in the carotid body is at least partially mediated by signaling pathways involving ET receptors.

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.