Role of glutathione redox state in oxygen sensing by carotid body chemoreceptor cells.
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Little is known about the cells or mechanisms of O2 chemoreception in vertebrates other than mammals. The purpose of this study, therefore, was to identify O2-sensitive chemoreceptors in a fish. Putative O2-sensitive chemoreceptors were dissociated from the gills of channel catfish, Ictalurus punctatus, and cultured. A population of cells was identified with morphology and a histochemical profile similar to mammalian carotid body Type I (glomus) cells and pulmonary neuroepithelial cells. These cells stain with neutral red and appear to be the branchial neuroepithelial cells. Immunocytochemical staining showed that these cells contain neuron-specific enolase (NSE), tyrosine hydroxylase (TH) and 5-hydroxytryptamine (5HT). Patch-clamp experiments showed that these cells have a O2-sensitive, voltage-dependent outward K+ current like mammalian O2 sensors. Two kinds of electrophysiological responses to hypoxia (P(O2) < 10 Torr) were observed. Some cells showed inhibition of outward current in response to hypoxia, whereas other cells showed potentiation. Neurochemical content and electrophysiological responses to hypoxia indicate that these cells are piscine O2-sensitive chemoreceptors.
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The effects of dopamine (DA) were studied on type I cells of the rat carotid body. DA (0.35 to 8.4 x 10(-4) M) elicited a long-lasting depolarization and decreased input resistance (Ro) in 75% of the cells, in a dose-dependent manner. Hyperpolarization and increased Ro were observed in 8% of cells. Membrane potential (Em) and Ro changes were highly correlated (-0.62, P < 0.001). The reversal potential (Erev) of the DA effect was +2.16 +/- 4.75 (mV +/- S.E.M.).
The present study shows that smell (antennular) receptor cells are as narrowly tuned to single compounds as taste (leg) receptor cells in the lobster. Antennular receptors responded best to hydroxy-L-proline (57% of the 30 cells sampled) and taurine (24%). In the presence of 14 other compounds in equimolar concentrations, the hydroxy-L-proline and taurine receptors showed a suppressed response to their best stimulus. Other cells had best responses to ammonium chloride, betaine, L-glutamate or L-proline. The results have implications for molecular receptor processes and for the neural basis of feeding behavior.
The ionic currents of carotid body type I cells and their possible involvement in the detection of oxygen tension (Po2) in arterial blood are unknown. The electrical properties of these cells were studied with the whole-cell patch clamp technique, and the hypothesis that ionic conductances can be altered by changes in PO2 was tested. The results show that type I cells have voltage-dependent sodium, calcium, and potassium channels. Sodium and calcium currents were unaffected by a decrease in PO2 from 150 to 10 millimeters of mercury, whereas, with the same experimental protocol, potassium currents were reversibly reduced by 25 to 50 percent. The effect of hypoxia was independent of internal adenosine triphosphate and calcium. Thus, ionic conductances, and particularly the O2-sensitive potassium current, play a key role in the transduction mechanism of arterial chemoreceptors.
The hypothesis that changes in environmental O2 tension (pO2) could affect the ionic conductances of dissociated type I cells of the carotid body was tested. Cells were subjected to whole-cell patch clamp and ionic currents were recorded in a control solution with normal pO2 (pO2 = 150 mmHg) and 3-5 min after exposure to the same solution with a lower pO2. Na and Ca currents were unaffected by lowering pO2 to 10 mmHg, however, in all cells studied (n = 42) exposure to hypoxia produced a reversible reduction of the K current. In 14 cells exposed to a pO2 of 10 mmHg peak K current amplitude decreased to 35 +/- 8% of the control value. The effect of low pO2 was independent of the internal Ca2+ concentration and was observed in the absence of internal exogenous nucleotides. Inhibition of K channel activity by hypoxia is a graded phenomenon and in the range between 70 and 120 mmHg, which includes normal pO2 values in arterial blood, it is directly correlated with pO2 levels. Low pO2 appeared to slow down the activation time course of the K current but deactivation kinetics seemed to be unaltered. Type I cells subjected to current clamp generate large Na- and Ca-dependent action potentials repetitively. Exposure to low pO2 produces a 4-10 mV increase in the action potential amplitude and a faster depolarization rate of pacemaker potentials, which leads to an increase in the firing frequency. Repolarization rate of individual action potentials is, however, unaffected, or slightly increased. The selective inhibition of K channel activity by low pO2 is a phenomenon without precedents in the literature that explains the chemoreceptive properties of type I cells. The nature of the interaction of molecular O2 with the K channel protein is unknown, however, it is argued that a hemoglobin-like O2 sensor, perhaps coupled to a G protein, could be involved.
Ticks are ectoparasites of vertebrates and utilize a variety of infochemicals for host finding and acceptance as well as for intraspecific aggregation and mating responses. Individual male and female Ixodes ricinus. the vector of Lyme disease in Europe. readily arrest on filter paper strips contaminated with their own faeces. I. ricinus also responds, but to a lesser degree, to faeces-contaminated papers enclosed in metal mesh envelopes. i.e. without directly contacting the faeces, suggesting a role for volatiles in the arrestment response. The faccal constituents guanine. xanthine, uric acid and 8-azaguanine (a bacterial breakdown product of guanine) also caused arrestment of individual I. ricinus males and females. However, mixtures of these products induced arrestment of I. ricinus at doses one hundred fold lower than the lowest active dose of any of them tested singly. Saline extracts of faeces activated receptor cells in terminal pore sensilla on the first leg tarsi of I. ricinus. One cell in these sensilla responded in a similar dose dependent manner to guanine and 8-azaguanine, whereas a second cell was more sensitive to lower doses of 8-azaguanine. The response threshold approached 100 fM for both cells. These findings suggest that faeces and faecal breakdown products are implicated in aggregation responses of I. ricinus. This may account for the clumped distribution of this ectoparasite on the ground and contribute to the high proportion of mated individuals recorded prior to host colonization.
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Membrane potential in oxygen-sensitive type I cells in carotid body is controlled by diverse sets of voltage-dependent and -independent K(+) channels. Coupling of Po(2) to the open-closed state of channels may involve production of reactive oxygen species (ROS) by NADPH oxidase. One hypothesis suggests that ROS are produced in proportion to the prevailing Po(2) and a subset of K(+) channels closes as ROS levels decrease. We evaluated ROS levels in normal and p47(phox) gene-deleted [NADPH oxidase knockout (KO)] type I cells using the ROS-sensitive dye dihydroethidium (DHE). In normal cells, hypoxia elicited an increase in ROS, which was blocked by the specific NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF, 3 mM). KO type I cells did not respond to hypoxia, but the mitochondrial uncoupler azide (5 microM) elicited increased fluorescence in both normal and KO cells. Hypoxia had no effect on ROS production in sensory and sympathetic neurons. Methodological control experiments showed that stimulation of neutrophils with a cocktail containing the chemotactic peptide N-formyl-Met-Leu-Phe (1 microM), arachidonic acid (10 microM), and cytochalasin B (5 microg/ml) elicited a rapid increase in DHE fluorescence. This response was blocked by the NADPH oxidase inhibitor diphenyleneiodonium (10 microM). KO neutrophils did not respond; however, azide (5 microM) elicited a rapid increase in fluorescence. Physiological studies in type I cells demonstrated that hypoxia evoked an enhanced depression of K+ current and increased intracellular Ca2+ levels in KO vs. normal cells. Moreover, AEBSF potentiated hypoxia-induced increases in intracellular Ca2+ and enhanced the depression of K+ current in low O(2). Our findings suggest that local compartmental increases in oxidase activity and ROS production inhibit the activity of type I cells by facilitating K+ channel activity in hypoxia.
The stimulus integration time of lobster olfactory receptor cells in situ was determined using extracellularly recorded spiking responses from receptor cells and on-line high-resolution measurement of odor square pulses. At a fixed odor concentration, odor steps of 200 ms duration elicited maximum responses; shorter odor steps did not drive the cells to their maximum response and longer odor steps added spikes but did not result in higher firing rates. Excitatory processes peaked within 220 ms of stimulus onset. At 160­300 ms, stimulus intensity discrimination was most evident. Adaptation processes reduced response magnitude to near-zero levels within 1000 ms of stimulus onset. Olfactory receptor cells thus resolve odor peak onsets within the first few hundred milliseconds: this time window corresponds to the 4­5 Hz frequency of olfactory sampling (i.e. 'sniffing') as well as the rapid fluctuations in odor concentration that are common in natural odor plumes. The stimulus integration time of 200 ms may play a role in the filtering of information used by lobsters to orient to distant odor sources.
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