Role of IP3 Receptors in Shaping the Carotid Chemoreceptor Response to Hypoxia But Not to Hypercapnia in the Rat Carotid Body: An Evidence Review.

This article addresses the disparity in the transduction pathways for hypoxic and hypercapnic stimuli in carotid body glomus cells. We investigated and reviewed the experimental evidence showing that the response to hypoxia, but not to hypercapnia, is mediated by 1,4,5-inositol triphosphate receptors (IP3R/s) regulating the intracellular calcium content [Ca2+]c in glomus cells. The rationale was based on the past observations that inhibition of oxidative phosphorylation leads to the explicit inhibition of the hypoxic chemoreflex. [Ca2+]c changes were measured using cellular Ca2+-sensitive fluorescent probes, and carotid sinus nerve (CSN) sensory discharge was recorded with bipolar electrodes in in vitro perfused-superfused rat carotid body preparations. The cell-permeant, 2-amino-ethoxy-diphenyl-borate (2-APB; 100 µM) and curcumin (50 µM) were used as the inhibitors of IP3R/s. These agents suppressed the [Ca2+]c, and CSN discharge increases in hypoxia but not in hypercapnia, leading to the conclusion that only the hypoxic effects were mediated via modulation of IP3R/s. The ATP-induced Ca2+ release from intracellular stores in a Ca2+-free medium was blocked with 2-APB, supporting this conclusion.

Effects of iron-chelators on ion-channels and HIF-1alpha in the carotid body.

Acute hypoxia instantaneously increases the chemosensory discharge from the carotid body, increasing ventilation mostly by inhibiting the oxygen sensitive ion channels and exciting the mitochondrial functions in the glomus cells. On the other hand, Fe2+-chelation mimics hypoxia by inhibiting the prolyl hydroxylases and the degradation of HIF-1alpha in non-excitable cells. Whether Fe2+-chelation can inhibit the ion channels giving rise to the sensory responses in excitable cells was the question. We characterized the responses to Fe2+-chelators on excitable glomus cells of the rat, and found that they instantaneously blocked the ion-channels, exciting the chemosensory discharge, and later causing a gradual accumulation of HIF-1alpha. Although initiated by the same stimuli, the two effects (on ion channels and cytosolic HIF-1alpha) possibly occurred by two different mechanisms.

Activation of HIF-1alpha mRNA by hypoxia and iron chelator in isolated rat carotid body.

The hypoxia inducible factor-1alpha (HIF-1alpha) protein level is increased by hypoxia and iron chelator (ciclopirox olamine) in isolated rat carotid body (CB) and glomus cells. Reverse transcription and polymerase chain reaction (RT-PCR) are performed to test whether this increase is caused, at least in part, by increased HIF-1alpha gene transcription. HIF-1alpha mRNA levels dose-dependently increased and decreased in the rat CBs incubated for 1 h in a medium saturated with O(2) levels that were varied around nominally normoxic level of 21% in the 0-95% range. The iron chelator, ciclopirox olamine (5 microM), stimulated HIF-1alpha mRNA production under normoxic condition. Thus, in the CB, the main systemic O(2)-sensing organ, HIF-1alpha transcription is regulated by O(2) supply around the normoxic level; this may contribute to cellular and organismal adaptations to chronic changes in ambient O(2).

Effects of hypoxia and intracellular iron chelation on hypoxia-inducible factor-1alpha and -1beta in the rat carotid body and glomus cells.

The present investigation provides for the first time, unambiguous information on the occurrence of hypoxia-inducible factors (HIF-1alpha and HIF-1beta proteins) in normoxia (Nx) and their interaction with hypoxia (Hx) and intracellular Fe(2+) chelation in the rat carotid body (CB) glomus cells. HIF-1alpha bound to HIF-1beta translocated into the nucleus is identified on the basis of immunohistochemistry and immunofluorescence. In Nx, a weak expression of HIF-1alpha was observed in CB glomus cells. However, exposure of CB and glomus cells to Hx (Po(2) approximately 7 Torr) and Nx with ciclopirox olamine (CPX, 5 microM) for 1 h showed a significant ( P<0.001) increase in HIF-1alpha protein. The CBs and glomus cells exposed to Nx, Hx, and Nx with CPX showed a constant level of HIF-1beta protein expression. HIF-1alpha subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. Hydroxylation of HIF-1alpha by prolyl hydroxylase for proteasomal degradation was dependent on iron, 2-oxoglutarate, and oxygen concentration. The intracellular iron that acts as a cofactor for prolyl hydroxylase activity belongs to the labile iron pool and can be easily chelated. Thus, chelation of intracellular labile iron by CPX in Nx significantly increased HIF-1alpha in CB glomus cells. Thus, the results are consistent with the hypothesis that HIF-1alpha which is present in the glomus cells translocates to the nucleus during exposure to Hx and to CPX in Nx.

Extra- and intracellular free iron and the carotid body responses.

The hypothesis that chelation of free iron, by decreasing reactive oxygen species (ROS), might mimic hypoxia and stimulate the carotid body was tested. We used the iron chelators, desferrioxamine (DFO, 200-400 microM) initially, and later ciclopirox olamine (CPX, 2.5-5.0 microM), on rat carotid body in vitro and measured chemosensory activity and [Ca2+]i in isolated cultured glomus cell clusters during normoxia and hypoxia. Although acute treatment of DFO might not penetrate the cell, and extracellular DFO would not influence these activities whereas CPX significantly increased chemosensory activities as well as increased [Ca2+]i in normoxia. We concluded that chelation of extracellular free iron did not alter ROS formation and oxygen sensing. Chelation of intracellular free iron and, therefore, a decrease in intracellular ROS appears to influence oxygen sensing in the carotid body.

Effect of acute hypoxia on glomus cell Em and psi m as measured by fluorescence imaging.

We have reinvestigated the hypothesis of the relative importance of glomus cell plasma and mitochondrial membrane potentials (E(m) and psi(m), respectively) in acute hypoxia by a noninvasive fluorescence microimaging technique using the voltage-sensitive dyes bis-oxonol and JC-1, respectively. Short-term (24 h)-cultured rat glomus cells and cultured PC-12 cells were used for the study. Glomus cell E(m) depolarization was indirectly confirmed by an increase in bis-oxonol (an anionic probe) fluorescence due to a graded increase in extracellular K(+). Fluorescence responses of glomus cell E(m) to acute hypoxia (approximately 10 Torr Po(2)) indicated depolarization in 20%, no response in 45%, and hyperpolarization in 35% of the cells tested, whereas all PC-12 cells consistently depolarized in response to hypoxia. Furthermore, glomus cell E(m) hyperpolarization was confirmed with high CO (approximately 500 Torr). Glomus cell psi(m) depolarization was indirectly assessed by a decrease in JC-1 (a cationic probe) fluorescence. Accordingly, 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (an uncoupler of oxidative phosphorylation), high CO (a metabolic inhibitor), and acute hypoxia (approximately 10 Torr Po(2)) consistently depolarized the mitochondria in all glomus cells tested. Likewise, all PC-12 cell mitochondria depolarized in response to FCCP and hypoxia. Thus, although bis-oxonol could not show glomus cell depolarization consistently, JC-1 monitored glomus cell mitochondrial depolarization as an inevitable phenomenon in hypoxia. Overall, these responses supported our "metabomembrane hypothesis" of chemoreception.

CO-induced K(+) currents in rat glomus cells are insensitive to light unlike carotid body neural discharge and Vo(O(2)).

The hypothesis that the light sensitive properties of CO-induced chemosensory nerve (CSN) discharge and oxygen consumption of the carotid body (CB) were shared by the pre-synaptic glomus cells was tested. The light effect on K(+) currents were measured before and during perfusion of the isolated rat glomus cells with high P(CO) of 550 Torr during nomoxia (P(O(2)approximately equal 100 Torr) at extra-cellular pH 7.0 and intracellular pH 6.8 with HEPES buffer. CO increased the K(+) currents with a left ward shift of the reversal potential, which showed no light effect. Thus the K(+) permeability of the glomus cell membrane were not shared by the light-sensitive CSN discharge of the CB and oxygen consumption in the presence of high P(CO.)