Hypoxia-induced secretion of serotonin from intact pulmonary neuroepithelial bodies in neonatal rabbit

Abstract

We examined the effects of hypoxia on the release of serotonin (5-HT) from intact neuroepithelial body cells (NEB), presumed airway chemoreceptors, in rabbit lung slices, using amperometry with carbon fibre microelectrodes. Under normoxia (P(O2) ~155 mmHg; 1 mmHg approximately 133 Pa), most NEB cells did not exhibit detectable secretory activity; however, hypoxia elicited a dose-dependent (P(O2) range 95-18 mmHg), tetrodotoxin (TTX)-sensitive stimulation of spike-like exocytotic events, indicative of vesicular amine release. High extracellular K(+) (50 mM) induced a secretory response similar to that elicited by severe hypoxia. Exocytosis was stimulated in normoxic NEB cells after exposure to tetraethylammonium (20 mM) or 4-aminopyridine (2 mM). Hypoxia-induced secretion was abolished by the non-specific Ca(2+) channel blocker Cd(2+) (100 microM). Secretion was also largely inhibited by the L-type Ca(2+) channel blocker nifedipine (2 microM), but not by the N-type Ca(2+) channel blocker omega-conotoxin GVIA (1 microM). The 5-HT(3) receptor blocker ICS 205 930 also inhibited secretion from NEB cells under hypoxia. These results suggest that hypoxia stimulates 5-HT secretion from intact NEBs via inhibition of K(+) channels, augmentation of Na(+)-dependent action potentials and calcium entry through L-type Ca(2+) channels, as well as by positive feedback activation of 5-HT(3) autoreceptors.

Figure 1. Low magnification view of neonatal rabbit lung slice immunostained for 5-HT

A small bronchiole (BR) with NEB cell cluster stongly positive for 5-HT located at airway bifurcation(arrow). PA, pulmonary artery. (Immunoperoxidase method for 5-HT; scale bar represents 100 μm). Inset, higher magnification of airway epithelium with two 5-HT immunoreactive NEBs facing each other with their apical surfaces exposed to airway lumen (arrowhead). (Immunoperoxidase method for 5-HT; scale bar represents 5 μm.)

Figure 2. Amperometric recording from individual NEB cells

A, example of exocytosis induced from NEB cells by perfusion with a hypoxia solution (P_O2 = 18 mmHg). At the point indicated by the arrow, the perfusate was changed to hypoxia solution. A, bottom left panel, a large spike-like exocytotic event is shown on an expanded time base. Scale bars apply to traces A, C and D. A, bottom right panel, frequency distribution of the charge of secretory events evoked by hypoxia in six different cells. B, effects of 4 different levels of partial pressures of oxygen in solution on secretory response from NEB cells. Each point represents the mean values of between 8 and 18 cells. The vertical bars show the mean ± s.e.m. * P < 0.05. C, effect of TTX on hypoxia-induced secretory response. Note that exocytosis was abolished by hypoxia plus 1 μm TTX; all recordings are from one cell. D, secretory response of an NEB cell to high extracellular potassium (50 mm). E, bar graph showing mean frequency of exocytosis induced by hypoxia with 5 mm K⁺ and normoxia with 50 mm K⁺ extracelluar solution. Each bar represents mean ± s.e.m. determined from the number of cells indicated in parentheses.

Figure 3. Secretory response of NEB cells to TEA and 4-AP

A, example of an amperometric recording showing excytosis in normoxic solution (5 mm K⁺) containing 20 mm TEA. B, example recording showing exocytosis in normoxic solution (5 mm K⁺) containing 2 mm 4-AP. C, example recording illustrating lack of potentiation of the effect of hypoxia by TEA. D, bar graph showing mean frequency of exocytosis evoked by TEA and 4-AP in the absence or presence of hypoxia (P_O2 = 18 mmHg). Each bar represents mean ± s.e.m. determined from the number of cells indicated in parentheses.

Figure 4. Effects of Ca²⁺ channels blockers and 5-HT₃-R blocker on hypoxia-induced secretory reponses

A, example of an amperometric recording of exocytosis evoked from an NEB cell following exposure to hypoxia (P_O2 = 18 mmHg). Excytosis was abolished by switching the solution to one of the same P_O2, containing 200 μm Cd²⁺. B, example of amperometric recording of exocytosis evoked from NEB cell following exposure to L-type Ca²⁺ blocker, 1 μm ω-CgTx. C, example of an amperometric recording of exocytosis evoked from an NEB cell following exposure to the 5-HT₃-R blocker, 50 μm ICS 205 930. D, bar graph showing the mean frequency of exocytosis evoked by hypoxia (no blockers) in the presence of different selective blockers of voltage-gated Ca²⁺ channels and 5-HT₃-R blocker. Cells were exposed to Cd²⁺ (200 μm), nifedipine (2 μm), ω-CgTx (1 μm), or ICS 205 930 (50 μm), as indicated under each bar. Each bar represents mean ± s.e.m. determined from the number of cells indicated in parentheses. * P < 0.05.