Carotid Body AT(4) Receptor Expression and its Upregulation in Chronic Hypoxia

Abstract

Hypoxia regulates the local expression of angiotensin-generating system in the rat carotid body and the me-tabolite angiotensin IV (Ang IV) may be involved in the modulation of carotid body function. We tested the hypothesis that Ang IV-binding angiotensin AT(4) receptors play a role in the adaptive change of the carotid body in hypoxia. The expression and localization of Ang IV-binding sites and AT(4) receptors in the rat carotid bodies were studied with histochemistry. Specific fluorescein-labeled Ang IV binding sites and positive staining of AT(4) immunoreactivity were mainly found in lobules in the carotid body. Double-labeling study showed the AT(4) receptor was localized in glomus cells containing tyrosine hydroxylase, suggesting the expression in the chemosensitive cells. Intriguingly, the Ang IV-binding and AT(4) immunoreactivity were more intense in the carotid body of chronically hypoxic (CH) rats (breathing 10% oxygen for 4 weeks) than the normoxic (Nx) control. Also, the protein level of AT(4) receptor was doubled in the CH comparing with the Nx group, supporting an upregulation of the expression in hypoxia. To examine if Ang IV induces intracellular Ca(2+) response in the carotid body, cytosolic calcium ([Ca(2+)](i)) was measured by spectrofluorimetry in fura-2-loaded glomus cells dissociated from CH and Nx carotid bodies. Exogenous Ang IV elevated [Ca(2+)](i) in the glomus cells and the Ang IV response was significantly greater in the CH than the Nx group. Hence, hypoxia induces an upregulation of the expression of AT(4) receptors in the glomus cells of the carotid body with an increase in the Ang IV-induced [Ca(2+)]i elevation. This may be an additional pathway enhancing the Ang II action for the activation of chemoreflex in the hypoxic response during chronic hypoxia.

Keywords: Angiotensin IV; angiotensin IV receptor; chemoreceptor; type-I cells.

Fig. (1)

Angiotensin IV (Ang IV)-binding sites in normoxic (Nx) and chronically hypoxic (CH) rat carotid body. Panels on top show FITC-coupled Ang IV binding (A), + Ang IV (B), + Sarile (C) in Nx group. Panels in middle show FITC-coupled Ang IV binding (D), + Ang IV (E), + Sarile (F) in CH group. Panels at bottom show FITC-coupled Ang IV binding (G), + Ang IV (H), + Sarile (I) in rat kidney, which was used as the positive control. Concentration of FITC-coupled Ang IV, unlabeled Ang IV and Sarile were 0.2, 10, 1 μM, respectively. Calibration bar is 20 μm.

Fig. (2)

The expression and localization of AT₄ receptor-immunoreactivity in (A) Nx and (B) CH rat carotid body. Note the intensity of staining is at a high level in the CH comparing with that of the Nx group. Sections with tyrosine hydroxylase (TH) for the Nx and CH carotid bodies are shown in C and D. E and F are overlays of the AT₄ receptor- and TH-stainings. Calibration bars are 20 and 50 μm, respectively, for low (A, C, E) and high magnification (B, D, F).

Fig. (3)

Protein expression of AT₄ receptors in Nx and CH rat carotid body. Note the level of protein expression increased significantly in the CH group. The expression in the hippocampus serves as the positive control. Densitometric analysis depicts the relative expression of AT₄ receptor protein in the CH group of carotid bodies and the expression is normalized as percent of the Nx group. Statistical significance (n=5, p<0.05, unpaired-t test) is shown in asterisk when comparing with the Nx group.

Fig. (4)

The [Ca²⁺]_i response to exogenous Ang IV in dissociated glomus cells of the rat carotid body from Nx control (A) and CH group (B). The records were made in cluster of 8-10 cells loaded with fura-2. Ang IV-induced [Ca²⁺]_i elevation was more significant in the CH glomus cells than that of the Nx group. Note that the transient [Ca²⁺]_i increase following the injection of Ang IV (0.01-10 μM) into the bath. In addition, histotoxic hypoxia with sodium cyanide (NaCN, 1 mM) increased the [Ca²⁺]i of the cells. Results are summarized in (C). On average, the [Ca²⁺]_i elevation (ΔR (340/380)) induced by 10 μM Ang IV in the CH group (n=9) was significantly greater than that of the Nx control (n=7). Statistical significance (p<0.05, unpaired-t test) is shown in asterisk when comparing with the other groups.