Rabbit Carotid Body Research Articles

Hypoxia inhibits the synthesis of phosphoinositides in the rabbit carotid body.

Hypoxic transduction in the carotid body (CB) is regulated by several systems of second messengers, but the role of the phospholipase C system has not been studied. The aim of the present study was to characterize the turnover rate of inositol phosphates (InsPs) and phosphoinositides (PIs) and their modifications by hypoxia in the rabbit CB in vitro. In CBs, in which the PIs had been labelled previously with 3H-myo-inositol, hypoxia in the presence of LiCl did not modify the accumulation of 3H-InsPs, whilst exposure to hypoxia during the loading period in the presence of LiCl reduced the accumulation of 3H-InsPs by more than 50%. Endogenous levels of inositol 1,4,5-trisphosphate were unaltered by hypoxia. Synthesis of 3H-PIs from 3H-myo-inositol was markedly inhibited by hypoxia in the CB, but not in the rat superior cervical ganglion used as control tissue. Levels of 3H-phosphatidylinositol (3H-PtdIns), 3H-phosphatidylinositol 4-monophosphate and 3H-phosphatidylinositol 4,5-bisphosphate were similarly decreased, indicating that inhibition occurs at a step prior to PtdIns synthesis. It is concluded that the phospholipase C system of second messengers does not play a significant role in the short-term regulation of hypoxic transduction cascade. It can be speculated that the decrease in PI availability produced by hypoxia might be involved in the functional changes observed in the CB on chronic hypoxic exposure.

Nitric oxide inhibits L-type Ca2+ current in glomus cells of the rabbit carotid body via a cGMP-independent mechanism.

Previous studies have shown that nitric oxide (NO) inhibits carotid body sensory activity. To begin to understand the cellular mechanisms associated with the actions of NO in the carotid body, we monitored the effects of NO donors on the macroscopic Ca2+ current in glomus cells isolated from rabbit carotid bodies. Experiments were performed on freshly dissociated glomus cells from adult rabbit carotid bodies using the whole cell configuration of the patch-clamp technique. The NO donors sodium nitroprusside (SNP; 600 microM, n = 7) and spermine nitric oxide (SNO; 100 microM, n = 7) inhibited the Ca2+ current in glomus cells in a voltage-independent manner. These effects of NO donors were rapid in onset and peaked within 1 or 2 min. In contrast, the outward K+ current was unaffected by SNP (600 microM, n = 6), indicating that the inhibition by SNP was not a nonspecific membrane effect. 2-(4-carboxyphenyl)-4,4,5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO; 500 microM), an NO scavenger, prevented inhibition of the Ca2+ current by SNP (n = 7), whereas neither superoxide dismutase (SOD; 2,000 U/ml, n = 4), a superoxide scavenger, nor sodium hydrosulfite (SHS; 1 mM, n = 7), a reducing agent, prevented inhibition of the Ca2+ current by SNP. However, SNP inhibition of the Ca2+ current was reversible in the presence of either SOD or SHS. These results suggest that NO itself inhibits Ca2+ current in a reversible manner and that subsequent formation of peroxynitrites results in irreversible inhibition. SNP inhibition of the Ca2+ current was not affected by 30 microM LY 83, 583 (n = 7) nor was it mimicked by 600 microM 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP; n = 6), suggesting that the effects of NO on the Ca2+ current are mediated, in part, via a cGMP-independent mechanism. N-ethylmaleimide (NEM; 2.5 mM, n = 6) prevented the inhibition of the Ca2+ current by SNP, indicating that SNP is acting via a modification of sulfhydryl groups on Ca2+ channel proteins. Norepinephrine (NE; 10 microM) further inhibited the Ca2+ current in the presence of NEM (n = 7), implying that NEM did not nonspecifically eliminate Ca2+ current modulation. Nisoldipine, an L-type Ca2+ channel blocker (2 microM, n = 6), prevented the inhibition of Ca2+ current by SNP, whereas omega-conotoxin GVIA, an N-type Ca2+ channel blocker (1 microM, n = 9), did not prevent the inhibition of Ca2+ current by SNP. These results demonstrate that NO inhibits L-type Ca2+ channels in adult rabbit glomus cells, in part, due to a modification of calcium channel proteins. The inhibition might provide one plausible mechanism for efferent inhibition of carotid body activity by NO.

Adenosine inhibits L-type Ca2+ current and catecholamine release in the rabbit carotid body chemoreceptor cells.

In an in vitro preparation of the intact carotid body (CB) of the rabbit, adenosine (100 microM) inhibited hypoxia-induced catecholamine release by 25%. The specific A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 microM) prevented the inhibition and increased the response to hypoxia further. In isolated chemoreceptor cells from the same species, adenosine inhibited voltage-dependent Ca2+ currents by 29% at 1 microM (concentration producing half-maximal inhibition, IC50 = 50 nM). This inhibition was mimicked by R(-)N6-(2-phenylisopropyl)-adenosine and 2-chloroadenosine (1 microM), two purinergic agonists poorly active at the intracellular ('P') site, and persisted in the presence of dipyridamole (a blocker of adenosine uptake; 1 microM) and was fully inhibited by 8-phenyltheophylline (10 microM). The A1 antagonists DPCPX (10 microM) and 8-cyclopentyl-1,3-dimethylxantine (0.1 microM) inhibited the effect of adenosine by 93% (IC50 = 0.14 microM) and 59%, respectively. The inhibition of the Ca2+ current (I(Ca)) was reduced by nisoldipine (an L-type Ca2+ channel antagonist) by nearly 50%, and was unaltered by omega-conotoxin GVIA, a blocker of N-type Ca2+ channels. Adenosine did not affect the voltage-dependent Na+ current (I(Na)) or K+ current (I(K)). We conclude that adenosine A1 receptors are located in chemoreceptor cells and mediate the inhibition of L-type Ca2+ channels and thereby the release of catecholamines produced by hypoxia. The data also indicate that endogenous adenosine acts as a physiological negative modulator of the chemoreceptor cell function. The previously reported excitatory action of adenosine on the activity of the sensory nerve of the CB is discussed in terms of a balance between the inhibition mediated by A1 receptors and the excitation mediated by A2 receptors.

Norepinephrine inhibits a toxin resistant Ca2+ current in carotid body glomus cells: evidence for a direct G protein mechanism.

Previous studies have demonstrated that endogenous norepinephrine (NE) inhibits carotid body (CB) sensory discharge, and the cellular actions of NE have been associated with inhibition of Ca2+ current in glomus cells. The purpose of the present study was to elucidate the characteristics and mechanism of NE inhibition of whole cell Ca2+ current isolated from rabbit CB glomus cells and to determine the type(s) of Ca2+ channel involved. NE (10 microM) inhibited 24 +/- 2% (SE) of the macroscopic Ca2+ current measured at the end of a 25 ms pulse to 0 mV and slowed activation of the current. The alpha2 adrenergic receptor antagonist, SK&F 86466, attenuated these effects. Inhibition by NE was fast and voltage-dependent i.e., maximal at -10 mV and then diminished with stronger depolarizations. This is characteristic of G protein betagamma subunit interaction with the alpha1 subunit of certain Ca2+ channels, which can be relieved by depolarizing steps. A depolarizing step (30 ms to +80 mV) significantly increased (14 +/- 1%) current in the presence of NE, whereas it had no effect before application of NE (1 +/- 1%). To further test for the involvement of G proteins, NE was applied to cells where intracellular GTP was replaced by GDP-betaS. NE had little or no effect on Ca2+ current in cells dialyzed with GDP-betaS. To determine whether NE was inhibiting N- and/or P/Q-type channels, we applied NE in the presence of omega-conotoxin MVIIC (MVIIC). In the presence of 2.5 microM MVIIC, NE was equally potent at inhibiting the Ca2+ current (23 +/- 4% vs. 23 +/- 4% in control), suggesting that NE was not exclusively inhibiting N- or P/Q-type channels. NE was also equally potent (30 +/- 2% vs. 26 +/- 4% in control) at inhibiting the Ca2+ current in the presence of 2 microM nisoldipine, suggesting that NE was not inhibiting L-type channels. Further, NE inhibited a significantly larger proportion (47 +/- 6%) of the resistant Ca2+ current remaining in the presence of NISO and MVIIC. These results suggest that NE inhibition of Ca2+ current in rabbit CB glomus cells is mediated in most part by effects on the resistant, non L-, N-, or P/Q-type channel and involves a direct G protein betagamma interaction with this channel.

Carotid body I 1-imidazoline receptors: Binding, visualization and modulatory function

The carotid body is influenced by many neurotransmitter receptors. A novel receptor specific for imidazolines has been implicated in cardiorespiratory regulation in the brain. To test for both I 1-imidazoline and α 2-adrenergic receptors, which also recognize imidazolines, specific [ 125I] p-iodoclonidine binding to carotid body membranes was characterized. The specific α 2-agents epinephrine (100 μM) or SK&F 86466 (10 μM) inhibited only a portion of specific [ 125I] p-iodoclonidine binding in both cat and rabbit carotid bodies, indicating the presence of I 1-imidazoline as well as α 2-adrenergic sites. The distribution of [ 125I] p-iodoclonidine binding sites was visualized autoradiographically. The cat carotid body was intensely labeled by [ 125I] p-iodoclonidine, with both I 1-imidazoline and α 2-adrenergic sites expressed. The relevance of I 1-imidazoline receptors in modulation of chemosensory discharge was determined in seven cats after α 2-adrenergic blockade. Clonidine (100 μg/kg) facilitated chemosensory activity particularly under hypoxia. We conclude that I 1-imidazoline receptors are expressed within the carotid body and may potentiate chemosensory discharge, in contrast to the inhibitory action of α 2-adrenergic receptors.

Ca2+ current in rabbit carotid body glomus cells is conducted by multiple types of high-voltage-activated Ca2+ channels.

Ca2+ current in rabbit carotid body glomus cells is conducted by multiple types of high-voltage-activated Ca2+ channels. J. Neurophysiol. 78: 2467-2474, 1997. Carotid bodies are sensory organs that detect changes in arterial oxygen. Glomus cells are presumed to be the initial sites for sensory transduction, and Ca2+-dependent neurotransmitter release from glomus cells is believed to be an obligatory step in this response. Some information exists on the Ca2+ channels in rat glomus cells. However, relatively little is known about the types of Ca2+ channels present in rabbit glomus cells, the species in which most of the neurotransmitter release studies have been performed. Therefore we tested the effect of specific Ca2+ channel blockers on current recorded from freshly dissociated, adult rabbit carotid body glomus cells using the whole cell configuration of the patch-clamp technique. Macroscopic Ba2+ current elicited from a holding potential of -80 mV activated at a Vm of approximately -30 mV, peaked between 0 and +10 mV and did not inactivate during 25-ms steps to positive test potentials. Prolonged ( approximately 2 min) depolarized holding potentials inactivated the current with a V1/2 of -47 mV. There was no evidence for T-type channels. On steps to 0 mV, 6 mM Co2+ decreased peak inward current by 97 +/- 1% (mean +/- SE). Nisoldipine (2 mu M), 1 mu M omega-conotoxin GVIA, and 100 nM omega-agatoxin IVa each blocked a portion of the macroscopic Ca2+ current (30 +/- 5, 33 +/- 5, and 19 +/- 3% after rundown correction, respectively). Simultaneous application of these blockers revealed a resistant current that was not affected by 1 mu M omega-conotoxin MVIIC. This resistant current constituted 27 +/- 5% of the total macroscopic Ca2+ current. Each blocker had an effect in every cell so tested. However, the relative proportion of current blocked varied from cell to cell. These results suggest that L, N, P, and resistant channel types each conduct a significant proportion of the macroscopic Ca2+ current in rabbit glomus cells. Hypoxia-induced neurotransmitter release from glomus cells may involve one or more of these channels.

CAMP production in rabbit carotid body: role of adenosine.

In the present study, we have investigated the possible role of adenosine in the hypoxia-mediated increase in adenosine 3',5'-cyclic monophosphate (cAMP) in the carotid body. cAMP levels in rabbit carotid bodies superfused in vitro for 10 min were increased in the presence of adenosine (100 microM and 1.0 mM; maximum increase = 127%, P < 0.01). These effects were reduced by the nonspecific adenosine-receptor antagonist 1,3-dipropyl-8[p-sulfophenyl]xanthine (DPSPX; 10 microM). The specific A2-receptor agonist 2-[4'(2-carboxymethyl)phenylethylamino]-5'-N-ethylcarboxamido adenosine (CGS-21680; 100 nM) also elevated carotid body cAMP levels, an effect that was blocked by the specific A2-antagonist 3,7-dimethyl-L-propargyl-xanthine (DMPX; 50 microM). Hypoxia-evoked elevations in cAMP were potentiated in the presence of the adenosine-uptake inhibitor dipyridamole (100 nM) and blocked by exposure to adenosine-receptor antagonists. Our data suggest that the rabbit carotid body contains specific adenosine receptors (A2 subtype) that are positively coupled to adenylate cyclase and that increases in cAMP associated with hypoxia are mediated by the release of endogenous adenosine.

Expression of dopamine D2 receptor mRNA isoforms in the carotid body of rat, cat and rabbit

Using the Reverse transcription-Polymerase chain reaction, we detected dopamine D2 receptor mRNA short and long isoforms in the adult carotid body of rats, cats, and rabbits. For these animals, the relative short/long ratios were 0.60, 0.65 and 0.57, respectively. Our results suggest that the variety of dopamine effects on carotid chemoreceptor activity, that has been related to species differences, may not be dependent on the expression levels of the dopamine D2 receptor mRNA isoforms in the studied species.

Properties of ionic currents from isolated adult rat carotid body chemoreceptor cells: effect of hypoxia.

1. The electrical properties of chemoreceptor cells from neonatal rat and adult rabbit carotid bodies (CBs) are strikingly different. These differences have been suggested to be developmental and/or species related. To distinguish between the two possibilities, the whole-cell configuration of the patch-clamp technique was used to characterize the ionic currents present in isolated chemoreceptor cells from adult rat CBs. Since hypoxia-induced inhibition of O2-sensitive K+ currents is considered a crucial step in O2 chemoreception, the effect of hypoxia on the adult rat chemoreceptor cell currents was also studied. 2. Outward currents were carried mainly by K+, and two different components could be distinguished: a Ca(2+)-dependent K+ current (IK(Ca)) sensitive to Cd2+ and charybdotoxin (CTX), and a Ca(2+)-insensitive, voltage-dependent K+ current (IK(V)). IK(V) showed a slow voltage-dependent activation (time constant (tau) of 87.4 ms at -20 mV and 8.8 ms at +60 mV) and a very slow inactivation, described by the sum of two exponentials (tau 1 = 684 +/- 150 ms and tau 2 = 4.96 +/- 0.76 s at + 30 mV), that was almost voltage insensitive. The kinetic and pharmacological properties of IK(V) are typical of a delayed rectifier K+ channel. 3. Voltage-dependent Ca2+ currents (ICa) were present in nineteen of twenty-seven cells. TTX-sensitive Na+ currents were also observed in about 10% of the cells. 4. Low PO2 (< 10 mmHg) reduced the whole outward current amplitude by 22.17 +/- 1.96% (n = 27) at +20 mV. This effect was absent in the presence of Cd2+. Since low PO2 did not affect ICa, we conclude that hypoxia selectively blocks IK(Ca). 5. The properties of the currents recorded in adult rat chemoreceptor cells, including the specific inhibition of IK(Ca) by hypoxia, are similar to those reported in neonatal rat CB cells, implying that the differences between rat and rabbit chemoreceptor cells are species related.

Peptidases of the peripheral chemoreceptors: biochemical, immunological, in vitro hydrolytic studies and electron microscopic analysis of neutral endopeptidase-like activity of the carotid body

The purposes of the present study are to identify and characterize the major peptidase(s) that may be involved in the inactivation of neuropeptides in the mammalian carotid body. Measurements of a number of peptidase activities in the cell-free extract of the cat carotid body using specific substrates and inhibitors indicated that the previously identified neutral endopeptidase (NEP)-like activity [Kumar et al., Brain Res., 517 (1990) 341–343] is the major peptidase in the chemoreceptor tissue. The NEP-like activity of the carotid body was further characterized using a monoclonal antibody to human neutral endopeptidase, EC 3.4.24.11. Immune blot analysis indicated strong immunoreactivity toward the cat and calf carotid bodies but a weak cross-reactivity with the rabbit carotid body. Furthermore, western blot analysis of the cat carotid body extract revealed the presence of a major 97-kDa protein and a minor 200-kDa protein. The 97-kDa NEP form of the carotid body was comparable to EC 3.4.24.11 and was consistent with its reported molecular weight suggesting NEP-like activity of the carotid body is structurally similar to the neutral endopeptidase, EC 3.4.24.11. In order to assess whether NEP is the primary peptide degrading activity in the cat carotid body in vitro hydrolysis studies using substance P (SP) as a model peptide were performed. HPLC analysis showed that SP is hydrolyzed maximally at pH 7.0 by carotid body peptidases with the formation of SP(1–7) and SP(1–8) as stable intermediates. Inhibitors specific to NEP also inhibited the SP-hydrolyzing activity of the carotid body. Analyses of the cell-free extracts showed the occurrence of both NEP and SP-hydrolyzing activities in the rabbit and rat carotid bodies although at 2- and 4-fold lower levels respectively than that observed in the cat carotid body. Immunoelectron microscopy showed that NEP-specific immunoreactivity is associated with the intercellular region between the type I cells and cell clusters of the carotid body. Taken together, the results from this investigation demonstrate that neutral endopeptidase (EC 3.4.24.11) is one of the major endopeptidases which mediates the degradation and inactivation of neuropeptides in the carotid body.

Mechanisms of alpha2-adrenoceptor-mediated inhibition in rabbit carotid body.

We have used the in vitro preparation of the intact carotid body (CB) and isolated chemoreceptor cells to elucidate the distribution and function of alpha2-adrenoreceptors. The significance of the study lies in the fact that norepinephrine (NE), being the neurotransmitter of the sympathetic innervation to the CB, is also abundant in chemoreceptor cells. In intact CB whose catecholamine (CA) deposits had been labeled by prior incubation with the CA precursor [3H]tyrosine, the alpha2-antagonist yohimbine (10 microM) potentiated the low-PO2 (33 and 60 mmHg)-induced release of [3H]CA by 100 and 53%, respectively. Yohimbine (10 microM) and SKF-86466 (50 microM; another alpha2-antagonist) reversed the inhibition of the release of [3H]CA produced by the alpha2-receptor agonists clonidine and UK-14304 (10 microM). The increase in adenosine 3',5'-cyclic monophosphate produced by low PO2 was further augmented by yohimbine and nearly halved by UK-14304 and clonidine. In isolated chemoreceptor cells, UK-14304 and NE inhibited voltage-dependent Ca2+ currents by 28 and 32%, respectively. These results indicate that alpha2-receptors are present in chemoreceptor cells, where they reduce the release of [3H]CA. Inhibition of adenylate cyclase(s) and Ca2+ channels may be involved in this effect. Using intact CB from normal and chronically sympathectomized animals, we demonstrated a specific accumulation of [3H]NE in intraglomic sympathetic endings. Hypoxia (PO2 approximately 33 mmHg) did not elicit release of [3H]NE from the sympathetic endings, but high extracellular K+ (K+(e)) induced a release of [3H]NE that was inhibited by alpha2-agonists and augmented by alpha2-antagonists. These findings demonstrate that alpha2-receptors are also present in the sympathetic endings of the CB, where they modulate the release of NE. As a whole, this work provides a more detailed understanding of the role of the sympathetic innervation in the control of the CB chemoreceptor function, including the cellular mechanisms of the action of NE.

Cholera and pertussis toxins reveal multiple regulation of cAMP levels in the rabbit carotid body.

It is known that hypoxia (PO2 approximately equal to 66-18 mm Hg), acting via unknown receptors, increases carotid body cAMP levels in Ca(2+)-free solutions, indicating that low PO2 activates adenylate cyclases independently of the action of the released neurotransmitters. The aim of the present work was to investigate the involvement of G proteins in the genesis of the basal level of cAMP and on the increase in cAMP induced by low PO2. In carotid body homogenates, cholera toxin- and pertussis toxin-induced [32P]ADP-ribosylation of two protein bands of approximately equal to 42 and 45 kDa, and approximately equal to 39 and 40 kDa respectively; in both cases, prior incubation of the carotid bodies with the toxins reduced [32P]ADP-ribosylation by > 90%. In intact carotid bodies, cholera toxin treatment increased cAMP levels more in normoxic than in hypoxic organs, indicating that hypoxia releases neurotransmitters acting on receptors negatively coupled to adenylate cyclases. Cholera toxin-treated carotid bodies incubated in Ca(2+)-free solution had identical cAMP levels in normoxia and in hypoxia. In pertussis toxin-treated normoxic carotid bodies the cAMP level was close to control, but in pertussis toxin-treated hypoxic carotid bodies cAMP rose to a level similar to those seen in normoxic cholera toxin-treated organs, indicating that low PO2 releases neurotransmitters acting on receptors positively coupled to adenylate cyclases. Pertussis toxin-treated carotid bodies incubated in Ca(2+)-free solution lost their capacity to increase cAMP in response to hypoxia, indicating that a G protein sensitive to pertussis toxin is needed for this response. This implies that the carotid bodies express a pertussis toxin-sensitive G protein positively coupled to adenylate cyclases, or that a Gs protein requiring the cooperative action of Go/Gi donated beta gamma subunits mediates the increase in cAMP level produced by hypoxia.

Dopamine efflux from the carotid body during hypoxic stimulation.

The carotid bodies of cats are known to contain high levels of dopamine (DA), persisting even after complete denervation of these organs (Zapata et al, 1969; Mir et al, 1982; Fitzgerald et al, 1983). Thus DA appears to be concentrated in glomus cells, characterized by their chromaffin reaction, abundance in dense-core granules and formaldehydeinduced fluorescence (see Eyzaguirre & Zapata, 1984). Since the DA content and the number of dense-core granules are reduced in direct proportion to the severity and duration of hypoxia in rats (Hanbauer & Hellström, 1978; Hansen, 1981), and radiolabeled DA effluxes from rabbit and cat carotid bodies —in parallel with the frequency of carotid chemosensory discharges (f x)— are also proportional to the intensity of hypoxic superfusions (Fidone et al, 1982; Rigual et al, 1986), the idea that DA serves as the glomus cell to sensory nerve endings transmitter during hypoxia is highly attractive (see Gonzalez et al, 1994).KeywordsCarotid BodyGlomus CellDopamine EffluxHypoxic ChallengeHypoxic StimulationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Intracellular Ca2+ deposits and catecholamine secretion by chemoreceptor cells of the rabbit carotid body.

The pivotal role of intracellular free [Ca2+] fluctuations in the control of cellular functions such as contraction and secretion, including the release of neurotransmitters, was recognized many decades ago (see Rubin, 1982). More recently, the list of cellular functions triggered or modulated by the levels of Ca2+ i has grown enormously. Additional functions regulated by [Ca2+]i include neuronal excitability, synaptic plasticity, gene expression, cellular metabolism, cell division and differentiation, and programmed cell dead (Miller, 1991; Clapham, 1995). Paralleling the growth in this list of Ca2+-controlled functions, a multiplicity of cellular mechanisms aimed at maintaining resting free [Ca2+]i in the range of 100 nM for most cells has been described, allowing increases in Ca2 i levels that are specific in their magnitude, time course and spatial distribution, according to the cell function activated (Toescu, 1995).KeywordsCarotid BodyCatecholamine SecretionCarotid Sinus NerveArterial ChemoreceptorChemoreceptor CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Modulation of voltage-gated Ca2+ channels by O2 tension. Significance for arterial oxygen chemoreception.

Physiological adaptations to changes in oxygen availability are expressed in several tissues, which facilitate survival in situations of oxygen rarity, or hypoxia. These physiological responses to hypoxia are mediated by the arterial chemoreceptors which have classically been treated as synonymous with the carotid bodies (for a review see González et al, 1992) but which, in a broader sense, should also include the arterial smooth muscle cell layer (Sparks, 1980; Wadsworth, 1994). Among the acute responses to hypoxia are hyperventilation and active changes in vascular resistance controlling local circulation. Combined these physiological responses to hypoxia act to increase the uptake and ventilation of oxygen within the lungs, as well as, to better irrigate hypoxic regions of the body. Hyperventilation ensues from the activity of peripheral chemoreceptors located in the carotid and aortic bodies which transduce changes in blood oxygen content into neurosecretory activity. Chemotransduction in the carotid body has been attributed, among other factors, to the existence in glomus cells of K+ channels regulated by blood PO2 (López-Barneo et al, 1988; Delpiano & Hescheler, 1989). Thus, similar O2-modulated ion channels may also be present in other tissues responsive to alterations in PO2. We have recently demonstrated the existence of O2-modulated Ca2+ channels in vascular smooth muscle cells where they may play a key role in the vasomotor responses to hypoxia (Franco-Obregón et al, 1995; Franco-Obregón & López-Barneo, 1996). A similar effect of PO2 on the voltage-gated Ca2+ channels of the chemoreceptive cells of the rabbit carotid body has also been recently found (Montoro et al, 1996). Since in both preparations (glomus cells and vascular smooth muscle) the transduction of the hypoxic stimulus requires the modulation of cytosolic Ca2+ levels, the O2-sensitive Ca2+ channels described here are well suited to confer chemoreceptive properties to these tissues.

Developmental changes in intracellular Ca2+ response of carotid chemoreceptor cells to hypoxia

The carotid chemoreceptor response to hypoxia is weak just after birth and increases during postnatal development. The mechanisms underlying chemoreceptor maturation are unknown. We tested the hypothesis that carotid chemoreceptor maturation occurs at the glomus cell level by measuring intracellular calcium ([Ca2+]i) mobilization in response to hypoxia, anoxia, and NaCN in freshly dissociated cells from newborn vs. adult rabbit carotid bodies. Cells were loaded with fura 2 and superfused at 37 degrees C with balanced salt solution equilibrated with 5% CO2. [Ca2+]i mobilization in response to 3-min challenges of hypoxia (PO2 approximately 15 mmHg), anoxia (PO2 approximately 0 mmHg), and NaCN (1 mM) was measured using a digital imaging microscope. The fluorescence intensity ratio was used to calculate [Ca2+]i. Peak [Ca2+]i responses to all three challenges were three- to fivefold greater in glomus cells from adult compared with newborn carotid chemoreceptors. In addition, the average normoxic [Ca2+]i baseline was approximately threefold higher in the adult glomus cells. These results suggest that carotid chemoreceptor glomus cell sensitivity to natural stimuli, as reflected by the [Ca2+]i response, depends on the level of postnatal maturity.

Localization of dopamine D2 receptor mRNA in glomus cells of the rabbit carotid body by in situ hybridization.

The localization of mRNA coding for the dopamine D2 receptor was studied in the rabbit carotid body using in situ hybridization with synthetic 35S-labelled oligodeoxynucleotides. Using autoradiography on cryostat or semi-thin sections, labelling was observed over the cytoplasm of glomus cells, but not over sustentacular cells. A quantitative study showed that labelling intensity (silver grain density) was increased by haloperidol treatment. These results suggest that glomus cells express the dopamine D2 receptor gene and that this expression is regulated.

Hypoxia induces voltage-dependent Ca2+ entry and quantal dopamine secretion in carotid body glomus cells.

We have investigated the changes of cytosolic [Ca2+] and the secretory activity in single glomus cells dispersed from rabbit carotid bodies during exposure to solutions with variable O2 tension (Po2). In normoxic conditions (Po2 = 145 mmHg; 1 mmHg = 133 Pa), intracellular [Ca2+] was 58 +/- 29 nM, and switching to low Po2 (between 10 and 60 mmHg) led to a reversible increase of [Ca2+] up to 800 nM. The response to hypoxia completely disappeared after removal of external Ca2+ or with the addition of 0.2 mM Cd2+ to the external solution. These same solutions also abolished both the Ca2+ current of the cells and the increase of internal [Ca2+] elicited by high external K+. Elevations of cytosolic [Ca2+] in response to hypoxia or to direct membrane depolarization elicited the release of dopamine, which was detected by amperometric techniques. Dopamine secretion occurred in episodes of spike-like activity that appear to represent the release from single secretory vesicles. From the mean charge of well-resolved secretory events, we estimated the average number of dopamine molecules per vesicle to be approximately 140,000, a value about 15 times smaller than a previous estimate in chromaffin granules of adrenomedullary cells. These results directly demonstrate in a single-cell preparation the secretory response of glomus cells to hypoxia. The data indicate that the enhancement of cellular excitability upon exposure to low Po2 results in Ca2+ entry through voltage-gated channels, which leads to an increase in intracellular [Ca2+] and exocytotic transmitter release.

Response of cytosolic Ca 2+ to hypercapnic acidosis in cultured glomus cells of the adult rabbit carotid body

The characteristics underlying the response of cytosolic Ca 2+ ([Ca 2++] i ) to hypercapnic acidosis in clusters of cultured glomus cells of the adult rabbit carotid body were evaluated using fura-2 microscopic fluorometry. Application of CO 2 by bubbling through both superfusions of bicarbonate-buffered saline and HEPES-buffered saline produced a rapid and sustained increase in [Ca 2+] i. The [Ca 2+] i response increased correspondingly with a rise in concentrations of CO 2 to 20% but at a point between 20 and 50% adapted to CO 2 and it decreased its linear assent. The hypercapnic acidosis-induced increased of [Ca 2+] i was diminished by removing external Ca 2+. Also the [Ca 2] i response was reduced in dose-dependent fashion by the addition of the voltage-gated Ca 2+ channel blocker D600. An additional response of [Ca 2+] i to acetate was also diminished by Ca 2+ removal. These results suggest that the [Ca 2+] i response to hypercapnic acidosis involves an influx of external Ca 2+ through voltage-gated Ca 2+ channels.

Participation of Na+ channels in the response of carotid body chemoreceptor cells to hypoxia.

The role played by Na+ channels of carotid body (CB) chemoreceptor cells was investigated by studying the effects of tetrodotoxin (TTX) on the release of 3H-labeled catecholamines ([3H]CA) by adult rabbit CBs previously incubated with the precursor [3H]tyrosine. TTX inhibited partially the release of [3H]CA elicited by mild hypoxia (10 or 7% O2) or by depolarizing incubation medium containing 20 or 30 mM KCl, but the response to more intense hypoxia (5 or 2% O2) or to higher KCl concentration (40 or 50 mM) was not significantly affected. The release of [3H]CA elicited by acidic stimuli, either 20% CO2 (pH 6.6) or the protonophore dinitrophenol (100 microM), although comparable in magnitude to that elicited by mild hypoxia, was not modified by TTX. These results provide evidence for the first time that Na+ channels of chemoreceptor cells participate in the transduction of hypoxic stimuli into the neurotransmitter release response of these cells and suggest that Na+ current operates as an amplifying device that enhances the initial cell depolarization mediated by the closure of the O2-sensitive K+ channels. Sympathetic denervation of CBs was followed by a marked reduction in the release of [3H]CA elicited by veratridine or by 20 mM KCl, suggesting that the number of Na+ channels in chemoreceptor cells decreases after denervation.