Mitochondrial Function and Responsiveness to Hypoxia of Glomus Cells

Despite the progress in the understanding of glomus cell electrophysiology and its responses to hypoxia, how 02 is sensed in the carotid body remains unknown. It has been reported that modulation of some K+ channels by Po2 in glomus cells is maintained in excised patches, thus suggesting that 02 sensing depends on membrane-delimited mechanisms (11, 18, 19, 35). On the other hand, mitochondria have traditionally been considered as the site for glomus cell 02 sensing by several investigators because these organelle consume most ofthe cellular 02 and similarly to hypoxia, inhibitors of the electron transport chain (ETC) or mitochondrial uncouplers increase the afferent activity of the carotid body sinus nerve (24, 27). Besides in the carotid body, mitochondria have also been postulated to participate in 02 sensing in other acutely responding systems, such as pulmonary vascular smooth muscle (1, 17, 41) or chromaffin cells (15, 25). We have investigated in our carotid body slice preparation whether sensitivity of intact glomus cells to hypoxia is altered by mitochondrial dysfunction (28). We have tested the effect of inhibition at complex I with rotenone, at complex II with thenoyltrifluoroacetone (TTFA), at complex III with myxothiazol or antimycin A (respectively proximal and distal inhibitors of this complex) and at complex IV with cyanide. These agents were used in a broad range of concentrations, however the lowest concentrations used were at least 5 to 10 times the reported K50 values (6, 40).

The typical secretory response to hypoxia (Po2 ~ 20 mmHg) of a glomus cell in the carotid body slice is illustrated in Figure 5A. As shown in previous figures (see also Refs. 30, 31), low Po2 induces spike-like quantal events corresponding to catecholamine release from individual vesicles. For these type of experiments we calculated the cumulative secretion signal (lower trace in Fig. 5A), a value of electric charge obtained by the sum of the time integral of successive amperometric events that is proportional to the number of catecholamine molecules oxidized. Thus, the secretion rate, in femtocoulombs per min (fC/min), is given by the amount of charge transferred to the recording electrode during 60 seconds once the solutions are equilibrated in the recording chamber. We have observed that the ETC inhibitors induce in one to three minutes an exocytotic response from glomus cells (Fig. 5B-F; see also Fig. 6). The difference in the secretagogue potency of applied ETC inhibitors is not very marked when they are used at concentrations above saturation. The mean area of individual quantal events (in fC, mean±sd) triggered by the ETC inhibitors (rotenone: 40±18, n=245 spikes in 5 cells; myxothiazol: 42±30, n=102 spikes in 7 cells; antimycin A: 40±22, n=132 spikes in 5 cells; cyanide: 48±33, n=116 spikes in 7 cells) is not significantly different (p>0.1) from the average values estimated with events evoked by hypoxia (43±26, n=576 spikes in 14 cells, see Fig. 4 above). These data indicate that hypoxia and the ETC inhibitors induce the release of a common type of catecholaminergic vesicle. Secretion evoked by all the ETC inhibitors can be completely abolished by blockade of membrane Ca2+ channels with (Fig. 5B-F). Only the secretory response induced by concentrations of cyanide in the millimolar range is partially maintained in the presence of 0.3 mM extracellular Cd2+, thus suggesting Ca2+ release from intracellular stores (2). These data indicate that, as described in cells exposed to hypoxia (Fig. 3) (5, 30, 39), activation of carotid body glomus cells by mitochondrial ETC inhibitors largely depends — ----

through channels of the plasma membrane (28).

on extracellular Ca + influx

Figure 5. Secretory responses of glomus cells to hypoxia and to the inhibition of the mitochondrial electron transport. A: Top. Amperometric signal showing catecholamine release from a glomus cell exposed to low Po2 («20 mmHg). Each spike represents an exocytotic event. Bottom. Cumulative secretion signal (in femtocoulombs) resulting from the time integral of the amperometric recording. B-F: Catecholamine release induced by exposure to several electron transport inhibitors. The concentrations are: rotenone (5 |iM), TTFA (0.3 |iM), myxothiazol (1 Hg/ml), antimycin A (1 ng/ml), and cyanide (100 |iM) (Modified from Ref. 28).

Figure 5. Secretory responses of glomus cells to hypoxia and to the inhibition of the mitochondrial electron transport. A: Top. Amperometric signal showing catecholamine release from a glomus cell exposed to low Po2 («20 mmHg). Each spike represents an exocytotic event. Bottom. Cumulative secretion signal (in femtocoulombs) resulting from the time integral of the amperometric recording. B-F: Catecholamine release induced by exposure to several electron transport inhibitors. The concentrations are: rotenone (5 |iM), TTFA (0.3 |iM), myxothiazol (1 Hg/ml), antimycin A (1 ng/ml), and cyanide (100 |iM) (Modified from Ref. 28).

The interaction between hypoxia and the mitochondrial electron flow has been studied in cells exposed to low Po2 before and during application of ETC inhibitors acting at either proximal or distal mitochondrial complexes. The rationale behind these experiments is that if hypoxia exerts its effect through alteration of the mitochondrial electron flow, preincubation with saturating concentrations of ETC blockers would prevent any further effect of low P02. In contrast, the effects of hypoxia and ETC inhibition would be additive, at least partially, if they were acting through separate pathways. Figure 6 (A and B) illustrates that when 02-responsive glomus cells are treated with cyanide or antimycin A, the concomitant exposure to low Po2 elicits further increase in the secretory activity. In each case the amperometric recordings are shown on the left and right panels are the cumulative secretion signals recorded during hypoxia in the presence of the ETC inhibitors. The secretion rates measured immediately before exposure to hypoxia are illustrated diagrammatically by the slopes of the cumulative secretion signals. The amperometric recordings show that hypoxic responsiveness was preserved in cells treated with the ETC inhibitors. In parallel, the cumulative secretion traces clearly illustrate that in the presence of ETC inhibitors hypoxia induces a reversible increase in the slope of the signals. Similar results have been obtained in experiments performed with myxothiazol, or TTFA (28).

Figure 6. Secretory responses of glomus cells exposed concomitantly to hypoxia (Po2,20 mmHg) and to blockade of the mitochondrial electron transport. A-C: Amperometric recordings (left panels). The concentration of drugs are: cyanide (100 fiM), antimycin A (1 jig/ml) and rotenone (5 nM). Right panels, Cumulative secretion signals before, during and after the exposure to hypoxia in the presence of the ETC inhibitors. The straight lines represent the slopes (secretion rates) of the cumulative secretion signals immediately before the exposure to hypoxia. D: Average secretion rate measured in cells in various experimental conditions. Secretion rate in the ordinate is expressed in fC/min (meaniSE). Experimental conditions: Control (Po2,150 mmHg, 75±15 fC/min, n=17 cells) and hypoxia (1710±65 fC/min, n=17 cells). Cyanide (CN, 0.1 nM, 1771±842 fC/min, n=4 cells) and CN plus hypoxia (3932±1339 fC/min, n=4 cells). Antimycin A (0.1-1 Hg/ml, 1910±151 fC/min, n=13 cells) and antimycin A plus hypoxia (4201±421 fC/min, n=7 cells). Myxothiazol (0.1-1 (xg/ml, 2167±199 fC/min, n=6 cells) and myxothiazol plus hypoxia (3188±240 fC/min, n=6 cells). TTFA (0.1-0.3 nM; 2093±488 fC/min, n=5 cells) and TTFA plus hypoxia (4134±587 fC/min, n=5 cells). Rotenone (0.1-5 nM, 2058±550 fC/min, n=14 cells), rotenone plus hypoxia (1915±552 fC/min, n=12 cells). Asterisks indicate statistically significant difference (P<0.05) between each pair of samples (Modified from Ref. 28).

An exception among the mitochondrial inhibitors tested is rotenone, a flavoprotein inhibitor that blocks mitochondrial complex I. Figure 6C shows that, as other ETC inhibitors, rotenone elicits secretion from the cells, however previous exposure to rotenone abolishes any further increase of secretion by hypoxia. In cells treated with rotenone the secretory response to depolarization with high potassium is unaltered (Fig. 6C). The average secretion rates measured in several cells exposed to hypoxia and the ETC inhibitors are given in Figure 6D. This summary plot shows that inhibition at various sites along the ETC with saturating concentrations ofmitochondrial inhibitors induces a secretory activity in glomus cells of a magnitude comparable to that evoked by low Po2 (=20 mmHg). With the exception of rotenone, the effects of hypoxia and ETC inhibitors are additive, thus suggesting that they might act through separate signaling pathways. Selective occlusion of hypoxia sensitivity by rotenone has been observed in all the cells studied with concentrations ofthe drug at 0.1 -5 jxM (28). The lowest concentration used in these experiments (0.1 (iM) can produce full blockade of complex I (6, 40) or saturation ofrotenone binding sites (14).

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