Ryanodine receptor-mediated [Ca(2+)](i) release in glomus cells is independent of natural stimuli and does not participate in the chemosensory responses of the rat carotid body.

The hypothesis that intracellular calcium ([Ca(2+)](i)) release in glomus cells via ryanodine receptor (RyR) activation by caffeine may be independent of natural stimuli and chemosensory discharge was tested in the rat carotid body (CB). CB type I cells were isolated, plated and preloaded with calcium-sensitive fluorescent probe, Indo-1AM. With the increase of caffeine dose (0-50 mM) cytosolic calcium ([Ca(2+)](c)) increased from 85+/-15 nM to 1933+/-190 nM (n=6) at normoxia (PO(2)=125-130 Torr, PCO(2)=25-30 Torr, pH 7.30-7.35). Hypoxia (PO(2)=10-15 Torr) increased and hypocapnia (PCO(2)=7-9 Torr) decreased the cytoplasmic calcium [Ca(2+)](c) levels, independent of caffeine. Caffeine-related [Ca(2+)](c) increase was the same in the presence and the absence of extracellular calcium ([Ca(2+)](o)), indicating the source of Ca(2+) ions is the cellular store. Permeabilization of the cell membrane with saponin (25 microg/ml) retained the caffeine response. Additional treatment of the cells with 50 microM ryanodine (an inhibitor of the caffeine-activated RyR site) abolished caffeine-stimulated response. In vitro CB chemosensory (carotid sinus nerve, CSN) responses to hypoxia (PO(2)=35-40 Torr) were not altered by caffeine. These results suggest that [Ca(2+)](i) stores in CB cells, mobilized by RyR activation, do not participate in the CSN responses to natural stimuli.

Regulation of oxygen sensing in peripheral arterial chemoreceptors.

The carotid bodies are a small pair of highly vascularized and well perfused organs located at each carotid artery bifurcation, strategically situated to sense oxygen in arterial blood as it leaves the heart. Carotid body glomus cells are identified as the primary oxygen sensors, which respond to changes in blood P(O(2)) within milliseconds. Acute hypoxia causes a rapid increase in carotid sinus nerve (CSN) activity, providing afferent signals to the respiratory center in the brainstem. Glomus cells secrete numerous neurotransmitters that modulate CSN firing rates. This review will discuss major hypotheses that have emerged regarding acute oxygen sensing by glomus cells. In contrast, chronic responses to hypoxia are much slower, involving cytosolic reactions that take place over several minutes and nuclear reactions which occur over several hours. Converging concepts from different areas of research in oxygen sensing cells and tissues (including the carotid body) have been combined to describe molecular and biochemical changes that take place in the carotid body with chronic hypoxia. These include oxygen dependent proteolytic processes in the cytosol and gene transcription in the nucleus. In addition, cellular and nuclear responses to chronic hypoxia will be discussed.

Barium-stimulated chemosensory activity may not reflect inhibition of background voltage-insensitive K+ channels in the rat carotid body.

To test the hypothesis that the voltage-insensitive background leak K+ channel is responsible for the oxygen-sensitive properties of glomus cells in the rat carotid body (CB) we used Ba2+, a non-specific inhibitor of K+ currents. In vitro changes in cytosolic calcium ([Ca2+]c) and chemosensory discharge were studied to measure the effect of Ba2+. In normal Tyrode buffer, Ba2+ (3 and 5 mM) significantly increased carotid sinus nerve (CSN) discharge over baseline firing rates under normoxia (PO2 approximately 120 Torr) from approximately 150 to approximately 600 imp/0.5 s. However, addition of 200 microM Cd2+ which completely blocked increase in CSN activity stimulated by hypoxia (PO2 approximately 30 Torr), hypercapnia (PCO2 approximately 60 Torr, PO2 approximately 120 Torr) and high CO (PCO approximately 550 Torr, PO2 approximately 120 Torr) did not significantly inhibit Ba2+-stimulated CSN discharge. The response to hypoxia is abolished with Ca2+-free tyrode buffer containing 10 mM EGTA. Yet, in the same buffer, Ba2+ increased CSN discharge from approximately 2 to approximately 180 imp/0.5 s. With 200 microM Cd2+ and 10 mM EGTA, Ba2+ still increased CSN discharge from approximately 2 to approximately 150 imp/0.5 s. Oligomycin (2 microg) abolished the hypoxic response. However, in the presence of oligomycin CSN response to Ba2+ was significant. Since Ba2+ increased neural discharge under conditions where hypoxia stimulated CSN discharge is completely abolished, we suggest that the effect of Ba2+ on CSN discharge may not have anything to do with the oxygen sensing mechanism in the CB.

Reduced glutathione, dithiothreitol and cytochrome P-450 inhibitors do not influence hypoxic chemosensory responses in the rat carotid body.

Glomus cells and carotid sinus afferents are anatomically connected, and the chemical events in the glomus cells are expected to be conveyed reflexly as afferent signals. Accordingly, K(+) channel inhibition of the glomus cell membrane is expected to be followed by excitation of the afferents. In order to test the redox inhibition of K(+) channels of glomus cells by reduced glutathione (GSH), dithiothreitol (DTT) and by cytochrome P-450 inhibitors (clotrimazole and miconazole), we measured the carotid sinus nerve (CSN) discharge using an in vitro perfused adult rat carotid body (CB) in the presence and absence of these chemicals which are expected to excite the afferents. Our findings were that these agents did not stimulate the CSN activities during normoxia and kept the hypoxic responses intact. These results led us to conclude that the redox modulation of glomus cells was not conveyed to the afferents, and this functional disconnection did not support the redox hypothesis of O(2) chemoreception in the whole carotid body.

Mice lacking in gp91 phox subunit of NAD(P)H oxidase showed glomus cell [Ca(2+)](i) and respiratory responses to hypoxia.

The hypothesis that NAD(P)H oxidase may serve as an oxygen sensor was tested using the mice deficient (knock-out) in gp91phox subunit of NAD(P)H oxidase enzyme complex and compared with wild-type (C57BL/6J) strain measuring the ventilatory and glomus cell intracellular calcium ([Ca(2+)](i)) responses of carotid body to hypoxia. The hypoxic ventilatory responses as well as the [Ca(2+)](i) were preserved in the NAD(P)H oxidase knock-out mice. NAD(P)H oxidase, though a major source of oxygen radical production, is not the oxygen sensor in mice carotid body.

P(O(2))-P(CO(2)) stimulus interaction in [Ca(2+)](i) and CSN activity in the adult rat carotid body.

Since glomus cell intracellular calcium ([Ca(2+)](i)) plays a key role in generating carotid sinus nerve (CSN) discharge, we hypothesized that glomus cell [Ca(2+)](i) would correspond to CSN discharge rates during P(O(2))-P(CO(2)) stimulus interaction in adult rat carotid body (CB). Accordingly, we measured steady state P(O(2))-P(CO(2)) interaction in CSN discharge rates during hypocapnia (P(CO(2))=8-10 Torr), normocapnia (P(CO(2))=33-35 Torr) and hypercapnia (P(CO(2))=68-70 Torr) in normoxia (P(O(2)) approximately 130 Torr) and hypoxia (P(O(2)) approximately 36 Torr). The results showed P(O(2))-P(CO(2)) stimulus interaction in CSN responses. [Ca(2+)](i) levels were measured in isolated type I cells (2-3 cells/field), using Ca(2+) sensitive fluoroprobe indo-1AM. The [Ca(2+)](i) responses increased with increasing P(CO(2)) in normoxia. In hypoxia, [Ca(2+)](i) did not increase during hypocapnia but increased during normocapnia, showing P(O(2))-P(CO(2)) interaction. However, CSN response during hypoxia was far greater than that for [Ca(2+)](i) response, particularly during hypocapnic hypoxia. Thus, the [Ca(2+)](i) interaction cannot account for the whole CSN interaction. The origin of this CSN P(O(2)-)P(CO(2)) interaction must have occurred in part beyond cellular [Ca(2+)](i) interaction. Interactions at both sites (glomus cell membrane and sinus nerve endings) are reminiscent of reversible O(2)-heme protein reaction with a Bohr effect.

The metabolic hypothesis revisited.

High levels of CO are used to mimic the stimulatory response of the CSN initiated by hypoxia. Using light of different wavelengths we show that the stimulatory effects of high CO can be pinpointed to the cytochrome c oxidase in the mitochondrial respiratory chain. This supports the metabolic theory of oxygen sensing in the mitochondria.

Effects of 2,4-dinitrophenol (DNP) on the relationship between the chemosensory activities of the rat carotid body and the intracellular calcium of glomus cells.

To test the hypothesis that the uncoupler 2,4-dinitrophenol (DNP) increases [Ca2+]i equally well, independent of pHi, we studied the effects of 250 microM DNP on [Ca2+]i and carotid sinus nerve (CSN) activity of rat carotid body (CB). CSN activity was measured in CB perfused and superfused with hypocapnic (pHo 7.80) and normocapnic (pHo 7.42) Tyrode solutions. [Ca2+]i of glomus (type I cells) was assessed by superfusion techniques under identical conditions as for CSN recording experiments. The results indicate that 250 microM DNP increased [Ca2+]i of type I cells as well as CSN activity at both pHos, although alkalosis diminished these responses. Given that pHi will change with pHo, DNP did not make any additional pHi change, although [Ca2+]i changed. We conclude that DNP effects were due to [Ca2+]i change alone, and the relationship between [Ca2+]i and CSN activity are internally consistent.

Altered structure and function of the carotid body at high altitude and associated chemoreflexes.

The ventilatory response to hypoxia is complex. First contact with hypoxia causes an increase in ventilation within seconds that reaches full intensity within minutes because of an increase in carotid sinus nerve (CSN) input to the brain stem. With continued exposure, ventilation increases further over days (ventilatory acclimatization). Initially, it was hypothesized that ventilatory acclimatization arose from a central nervous system (CNS) mechanism. Compensation for alkalosis in the brain and restoration of pH in the vicinity of central chemoreceptors was believed to cause the secondary increase in ventilation. However, when this hypothesis could not be substantiated, attention was turned to the peripheral chemoreceptors. With the lowering of arterial PO2 at high altitude, there is an immediate increase in firing of afferents from chemoreceptors in the carotid body. After peaking over the next few minutes, the firing rate of afferents begins to rise again within hours until a steady state is reached. This secondary increase occurs along with increase in neurotransmitter synthesis and release and altered gene expression followed by hypertrophy of carotid body glomus cells. Further exposure to hypoxia eventually leads to blunting of the CSN output and ventilatory response in some species. This mini review is about the altered structure and function of the carotid body at high altitude and the associated blunting of the chemoreceptor and ventilatory responses observed in some species.

Evidence that 3,3',5-triiodothyronine is concentrated in and delivered from the locus coeruleus to its noradrenergic targets via anterograde axonal transport.

Recent immunohistochemical studies of rat brain triiodothyronine reveal heaviest localization in locus coeruleus perikarya. The cellular distribution is similar to that observed in concomitant studies of tyrosine hydroxylase immunohistochemistry: heavy clumps of immunoreactive triiodothyronine are distributed within locus coeruleus cytosol and in cell processes, leaving cell nuclei unstained. At the same time, in locus coeruleus targets, cell nuclei as well as surrounding neuropil are prominently triiodothyronine labeled. These observations, combined with diverse evidence linking thyroid hormone with norepinephrine at many levels of physiological and pathophysiological function, led to the hypothesis that the locus coeruleus binds and accumulates triiodothyronine and delivers the hormone via anterograde axonal transport to postsynaptic locus coeruleus targets, where nuclear triiodothyronine receptors are densely concentrated. Furthermore, the hypothesis predicts that destruction of locus coeruleus nerve terminals would interrupt this neural route of triiodothyronine delivery and prevent or reduce triiodothyronine labeling of nuclear receptors in noradrenergic target cells. To test this formulation, we gave the specific locus coeruleus lesioning agent, N-(2-chloroethyl)-N-2-bromobenzylamine hydrochloride (DSP-4), to adult male rats and examined their brains three, five and seven days thereafter by triiodothyronine and, in alternate sections, tyrosine hydroxylase immunohistochemistry. Treatment with DSP-4 resulted in specific and selective reduction in tyrosine hydroxylase and triiodothyronine immunohistochemical labeling in cell nuclei and in nerve cell processes within the neuropil of the hippocampus and cerebral cortex at all time periods examined. The results demonstrate that full occupancy of locus coeruleus target cells by triiodothyronine requires the presence of intact locus coeruleus projections and supports the proposal that, like norepinephrine, triiodothyronine delivery to noradrenergic targets occurs through delivery by locus coeruleus terminals. These findings provide strong support for earlier proposals that triiodothyronine functions as a co-transmitter with norepinephrine in addition to or as part of its genomic role in the cells receiving noradrenergic innervation.

Chemosensory response to high pCO is blocked by cadmium, a voltage-sensitive calcium channel blocker.

In the dark, during normocapnic (pCO2=35 Torr, pHo=7.4) normoxia (pO2=100 Torr), high pCO (>300 Torr) causes Ca2+-dependent photolabile excitation of chemosensors in the carotid body (CB). We previously proposed that the source of this Ca2+ was the [Ca2+]i stores because CO would react only intracellularly. However, influx of extracellular Ca2+ was not excluded. Now, using perfused rat CB (n=6) in the presence of normal extracellular [Ca2+] we show that chemosensory response to CO (pCO approximately 550 Torr) in normoxic (pO2 approximately 100 Torr) normocapnia (pCO2 approximately 30 Torr, pH approximately 7.4) is completely but reversibly inhibited by Cd2+ (200 microM), a voltage-gated Ca2+ channel blocker. Thus, extracellular Ca2+ is necessary for excitatory chemosensory response to high pCO. Cd2+ block occurs in spite of an enhanced [Ca2+]i rise. This shows that Ca2+ rise alone is unable to release neurotransmitter and to elicit a chemosensory response. Therefore, as a corollary, we conclude that Cd2+ blocks the Ca2+ flux that is needed for vesicle-membrane fusion for neurotransmitter release and neural discharge.

High PCO does not alter pHi, but raises [Ca2+]i in cultured rat carotid body glomus cells in the absence and presence of CdC12.

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.

Inhibition of dopamine release with simultaneous chemosensory excitation by hypercapnia with and without [Ca2+]0 in the cat carotid body.

The hypothesis that dopamine (DA) overflow corresponds to carotid sinus nerve (CSN) discharge during hypercapnia and is dependent on [Ca2+]0 was tested. We simultaneously measured the time course of DA overflow and CSN discharge of the cat carotid body, perfused/superfused in vitro at 37 degrees C at decreasing [Ca2+]0, during transition from normocapnia (PCO2 approximately 30-35 Torr) to hypercapnia (PCO2 approximately 60-65 Torr). In the presence of normal [Ca2+]0, hypercapnia instantaneously increased nerve discharge to peak levels followed by a decrease to steady states which were above the basal rate of activity. CSN discharge rate did not differ at decreasing [Ca2+]0 between 2.2 and 1.0 mM, and it began to decline at 0.1 mM [Ca2+]0, culminating to zero level in most cases, at zero [Ca2+]0. DA overflow increased slightly during hypercapnic peak CSN activity. Thereafter it declined to steady state levels below those of normocapnic conditions. Decreases in steady state DA levels were significantly less at 0 mM [Ca2+]0 compared to the higher calcium concentrations (0.1, 1.0 and 2.2 mM). Overall, steady state CSN activity and DA overflow were inversely related. Thus, DA release cannot have excitatory implications for carotid chemoreceptors during hypercapnia in the cat.

K+-current modulated by PO2 in type I cells in rat carotid body is not a chemosensor.

According to the membrane channel hypothesis of carotid body O2 chemoreception, hypoxia suppresses K+ currents leading to cell depolarization, [Ca2+]i rise, neurosecretion, increased neural discharge from the carotid body. We show here that tetraethylammonium (TEA) plus 4-aminopyridine (4-AP) which suppressed the Ca2+ sensitive and other K+ currents in rat carotid body type I cells, with and without low [Ca2+]o plus high [Mg2+]o, did not essentially influence low PO2 effects on [Ca2+]i and chemosensory discharge. Thus, hypoxia may suppress the K+ currents in glomus cells but K+ current suppression of itself does not lead to chemosensory excitation. Therefore, the hypothesis that K+-O2 current is linked to events in chemoreception is not substantiated. K+-O2 current is an epiphemenon which is not directly linked with O2 chemoreception.

Suppression of glomus cell K+ conductance by 4-aminopyridine is not related to [Ca2+]i, dopamine release and chemosensory discharge from carotid body.

The hypothesis that suppression of O2-sensitive K+ current is the initial event in hypoxic chemotransduction in the carotid body glomus cells was tested by using 4-aminopyridine (4-AP), a known suppressant of K+ current, on intracellular [Ca2+]i, dopamine secretion and chemosensory discharge in cat carotid body (CB). In vitro experiments were performed with superfused-perfused cat CBs, measuring chemosensory discharge, monitoring dopamine release by microsensors without and with 4-AP (0.2, 1.0 and 2.0 mM in CO2-HCO3- buffer) and recording [Ca2+]i by ratio fluorometry in isolated cat and rat glomus cells. 4-AP decreased the chemosensory activities in normoxia but remained the same in hypoxia and in flow interruption. It decreased the tissue dopamine release in normoxia, and showed an additional inhibition with hypoxia. Also, 4-AP did not evoke any rise in [Ca2+]i in glomus cells either during normoxia and hypoxia, although hypoxia stimulated it. Thus, the lack of stimulatory effect on chemosensory discharge, inhibition of dopamine release and unaltered [Ca2+]i by 4-AP are not consistent with the implied meaning of the suppressant effect on K+ current of glomus cells.