Shinobu Osanai

The primary oxygen sensor of the cat carotid body is cytochrome a3 of the mitochondrial respiratory chain

Carbon monoxide was shown to be competitive with O2 in oxygen sensing by perfused carotid bodies isolated from cats, afferent electrical activity increasing with either decreasing O2 or increasing CO. The CO‐induced increase in afferent activity was fully reversed by bright light. At submaximal light intensities the extent of reversal, after correcting to equal light intensity of light quanta at each wavelength, was maximal for light of 432 ± 2 and 590 ± 2 nm, with a ratio (432/590) of approximately 6. This spectrum is characteristic of the CO compound of mitochondrial cytochrome a 3. The photo‐reversible inhibition of oxygen sensing activity by CO accounts for at least 80% of the oxygen chemosensory activity of the carotid body.

Reciprocal photolabile O2 consumption and chemoreceptor excitation by carbon monoxide in the cat carotid body: evidence for cytochrome a3 as the primary O2 sensor

High carbon monoxide (CO) gas tensions (> 500 Torr) at normoxic PO2 (125-140 Torr) stimulates carotid chemosensory discharge in the perfused carotid body (CB) in the absence but not in the presence of light. According to a metabolic hypothesis of O2 chemoreception, the increased chemosensory discharge should correspond to a photoreversible decrease of O2 consumption, unlike a non-respiratory hypothesis. We tested the respiratory vs. non-respiratory hypotheses of O2 chemoreception in the cat CB by measuring the effect of high CO. Experiments were conducted using CBs perfused and superfused in vitro with high CO in normoxic, normocapnic cell-free CO2-HCO3- buffer solution at 37 degrees C. Simultaneous measurements of the rate of O2 disappearance with recessed PO2 microelectrodes and chemosensory discharge were made after flow interruption with and without CO in the perfusate. The control O2 disappearance rate without CO was -3.66 +/- 0.43 (S.E.) Torr/s (100 measurements in 12 cat CBs). In the dark, high CO reduced the O2 disappearance rate to -2.35 +/- 0.33 Torr/s, or 64.2 +/- 9.0% of control (P < 0.005, 34 measurements). High CO was excitatory in the dark, with an increase in baseline neural discharge from 129.2 +/- 47.0 to 399.3 +/- 49.1 impulses per s (P < 0.0001), and maximum discharge rate of 659 +/- 76 impulses/s (N.S. compared to control) during flow interruption. During perfusion with high CO in the light, there were no significant differences in baseline neural discharge or in the maximum neural discharge after flow interruption, and little effect on O2 metabolism (88.8 +/- 11.5% of control, N.S., 29 measurements).(ABSTRACT TRUNCATED AT 250 WORDS)

Cat carotid body chemosensory discharge (in vitro) is insensitive to charybdotoxin

Charybdotoxin (ChTX), a venom protein, suppresses Ca2+-activated K+ (K+(Ca)) currents in the glomus cell of neonatal rat carotid body. If it works similarly for cat carotid body chemoreceptors, charybdotoxin is expected to stimulate the chemosensory discharge during normoxia, and particularly hypoxia and hypercapnia. We studied the effects of charybdotoxin (20-40 nM) in vitro (perfused/superfused) on the cat carotid chemosensory discharge, and simultaneously tissue PO2 (PtiO2), as a measure of positive control. ChTX (20 nM) only increased PtiO2 and decreased carotid chemosensory discharge during hypoxia, indicating vasodilation. We conclude that K+(Ca) channels do not appear to play a significant role in chemotransduction in the cat carotid body.

High PCO does not alter pHi, but raises [Ca2+]i in cultured rat carotid body glomus cells in the absence and presence of CdCl2

We measured the effect of high PCO (500-550 Torr) on the pHi and [Ca2+]i in cultured glomus cells of adult rat carotid body (CB) as a test of the two models currently proposed for the mechanism of CB chemoreception. The metabolic model postulates that the rise in glomus cell [Ca2+]i, the initiating reaction in the signalling pathway leading to chemosensory neural discharge, is due to [Ca2+] release from intracellular Ca2+ stores. The membrane potential model postulates that the rise in [Ca2+]i comes from influx of extracellular Ca2+ through voltage-dependent Ca2+ channels (VDCC) of the L-type. High PCO did not change pHi at PO2 of 120-135 Torr, showing that CO-induced changes in [Ca2+]i are not due to changes in pHi. High PCO caused a highly significant rise in [Ca2+]i from 90+/-12 nM to 675+/-65 nM, both in the absence and in the presence of 200 microM CdCl2, a potent blocker of L-type VDCCs. This result is fully consistent with release of Ca2+ from glomus cell intracellular stores according to metabolic model, but inconsistent with influx of extracellular Ca2+ through VDCCs according to the membrane potential model.

Dopamine increases in cat carotid body during excitation by carbon monoxide: Implications for a chromophore theory of chemoreception

Studies of dopamine (DA) release were conducted with 10 perfused/superfused cat carotid bodies using shallow recessed Nafion polymer-coated microsensors (tips approximately 5 microns). Simultaneous measurements of tissue DA and neuronal discharge (ND) from the sinus nerve were made after switching from normoxic, normocapnic control perfusate (20% O2, 5% CO2, balance N2) to a normoxic, normocapnic perfusate equilibrated with a high tension (> 550 Torr) of carbon monoxide (CO). When high PCO perfusate was delivered in the dark, ND increased from a baseline of 89 +/- 24 (SE) impulses/s, to a peak excitation of 374 +/- 44 impulses/s within 15-30 s. Excitation then diminished to a plateau of 281 +/- 36 impulses/s within 1-2 min. Both peak and plateau ND were significantly above baseline (P < 0.05). Average tissue DA values increased above basal levels by +7.2 +/- 1.0 and +5.6 +/- 0.6 microns, respectively, during the peak and plateau ND phases (P < 0.05). Bright light restored the chemosensory activity to baseline, but had no effect on DA. Both chemosensory excitation and tissue DA responses to high CO in the dark were diminished in 3 carotid bodies perfused with Ca(2+)-free solutions. Responses were reduced even further with Ca2+ chelator (EGTA) in the perfusate. The results suggest that the effect of high PCO on DA release and chemosensory excitation are dependent on Ca2+ in the media, but the two events are not coupled.

Potential role of H2O2 in chemoreception in the cat carotid body

The hypothesis that H2O2 plays a critical role in hypoxic chemoreception in the cat carotid body (CB) was tested using a perfused-superfused preparation in vitro, measuring chemosensory discharge and CB tissue PO2 (PtiO2). According to the hypothesis NADPH mediated, PO2 dependent increase in H2O2 production would hyperpolarize the glomus cell, decreasing the chemosensory discharge. Thus, lactate and aminotriazole which would increase H2O2 concentration, would decrease the chemosensory discharge during hypoxia. However, 2.5-5.0 mM lactate and 25 mM aminotriazole did not diminish the hypoxic response. But, 2.5 mM lactate decreased the chemosensory discharge during normoxia which can be explained by an increase of CB PtiO2. Diethyldithiocarbamic acid (5 mM), which blocks the conversion of superoxide to H2O2, also diminished the chemosensory discharge, presumably due to an increased CB PtiO2. Menadione (increasing H2O2) and t-butyl hydroperoxide irreversibly decreased the chemosensory discharge, and the data are not useful. H2O2 increased the PO2 of the perfusate, and therefore could not be tested against PO2. Thus, perturbation of endogenous or exogenous H2O2 did not provide any evidence for its critical role in O2 chemoreception.

Thapsigargin enhances carotid body chemosensory discharge in response to hypoxia in zero [Ca2+]e: evidence for intracellular Ca2+ release.

To test the hypothesis that Ca2+ is released from intracellular store in the carotid body glomus cells during hypoxia, we stimultaneously measured chemosensory discharge and tissue PO2 of perfused-superfused cat carotid body before and during flow interruption in the presence and absence of extracellular [Ca2+] with and without thapsigargin (1-10 microM). Ca(2+)-free solution increased the latency of sensory response, and decreased the rate of rise and peak activity but thapsigargin significantly influenced these responses, without affecting oxygen consumption. Since thapsigargin depletes the intracellular Ca2+ store, and since Ca2+ is needed for the sensory discharge, these results suggest that intracellular release and influx of Ca2+ occur during hypoxia.

Stimulus interaction between CO and CO2 in the cat carotid body chemoreception

Since high PCO in the dark works like hypoxia in the carotid body chemoreceptors and since hypoxia shows a stimulus interaction with CO2, it is hypothesized that high PCO will show a similar interaction with PCO2 in the chemosensory excitation in the dark. We tested the hypothesis using cat carotid body perfused and superfused in vitro with Po2 of about 100 Torr. In one series, the chemosensory discharges were tested at three levels of PCO2 at high PCO of 500 Torr in the absence and presence of light. In the dark, normocapnia (PCO2 approximately 30 Torr) with high PCO promptly stimulated the sensory discharges to a peak, subsiding to a lower level. In hypocapnia (PCO2 approximately 18 Torr) with high PCO, all phases of activities were significantly lower than those of normocapnia, showing stimulus interaction. Hypercapnia saturated the activity with high PCO and seems to preclude a clear demonstration of stimulus interaction. In another series, an intermediate level of PCO (approximately 150 Torr), which showed a half-maximal activity in normoxia, showed a clear interaction with hypercapnia in the dark. With high PCO, bright light promptly reduced the activity to baseline at all PCO2 levels. This then increased somewhat to a steady-state. Withdrawal of the light was followed by a sharp rise in the activity to a peak which then fell to a somewhat lower level of steady-state. The peak discharge rate in the presence of light did not differ significantly from those of PCO2 alone.

Effect of CO on VO2 of carotid body and chemoreception with and without Ca2

This study was done using high PCO (> 500 Torr at PO2 of 120 Torr) in the carotid body perfusate in vitro, and recording simultaneously the activity of the whole carotid sinus nerve (CSN) and VO2 of the carotid body. In the cascade of excitation of CSN by high PCO in the dark [light eliminated the excitation; S. Lahiri, News Physiol. Sci. 9 (1992) 161-165], Ca2+ effects occur at the level of neurosecretion after the level of oxygen consumption, according to the following scheme: CO-hypoxia-->VO2 decrease-->K+ conductance decrease-->cell depolarization-->cytosolic Ca2+ rise-->neurosecretion-->neural discharge. Thus, a part of the hypothesis was that [Ca2+] decrease, being a downstream event, may not affect VO2 of the carotid body. Also, to determine to what extent the intracellular calcium stores contribute to cystolic [Ca2+] and chemosensory discharge with high PCO, we tested the effect of interruption of perfusate flow with medium nominally free of [Ca2+] on CSN excitation and VO2 of the carotid body with and without high PCO. High PCO in the dark decreased carotid body VO2, independent of [Ca2+]o. CSN excitation was always enhanced by high PCO, and its sensitivity to perfusate flow interruption. Also, nominally Ca(2+)-free solution increased the latency and decreased the rate of rise and peak activity of CSN during interruption of perfusate flow, but CO augmented the responses. This reversal effect by CO suggests that Ca2+ is released from intracellular stores, because CO has no other way to excite the chemoreceptors than by acting on the intracellular stores.

CO interact with intracellular [H+] with and without CO2–HCO3− in the cat carotid chemosensory discharge

To test the hypothesis whether CO2-HCO3- buffer is essential for the expression of chemoreception and to distinguish between pHi and pHo interaction with pCO in the carotid chemosensory response, we superfused-perfused in vitro cat carotid bodies using HEPES-Tyrodes solution with and without CO2-HCO3-, and compared the responses at the same pHo in the absence and presence of light. In the absence of light, pCO (> 138 Torr) stimulated the carotid body chemoreceptors in CO2-HCO3- buffer at pHo of 7.40, whereas pCO (69-550 Torr) did not stimulate the neural discharge in HEPES buffer at the pHo of 7.4-7.1 but did so below pHo 7.1. In the presence of light, all the responses were diminished proportionately.