The biophysical and pharmacological properties of an oxygen‐sensitive background K+ current in rat carotid body type‐I cells were investigated and compared with those of recently cloned two pore domain K+ channels. Under symmetrical K+ conditions the oxygen‐sensitive whole cell K+ current had a linear dependence on voltage indicating a lack of intrinsic voltage sensitivity. Single channel recordings identified a K+ channel, open at resting membrane potentials, that was inhibited by hypoxia. This channel had a single channel conductance of 14 pS, flickery kinetics and showed little voltage sensitivity except at extreme positive potentials. Oxygen‐sensitive current was inhibited by 10 mM barium (57 % inhibition), 200 μM zinc (53 % inhibition), 200 μM bupivacaine (55 % inhibition) and 1 mM quinidine (105 % inhibition). The general anaesthetic halothane (1.5 %) increased the oxygen‐sensitive K+ current (by 176 %). Halothane (3 mM) also stimulated single channel activity in inside‐out patches (by 240 %). Chloroform had no effect on background K+ channel activity. Acidosis (pH 6.4) inhibited the oxygen‐sensitive background K+ current (by 56 %) and depolarised type‐I cells. The pharmacological and biophysical properties of the background K+ channel are, therefore, analogous to those of the cloned channel TASK‐1. Using in situ hybridisation TASK‐1 mRNA was found to be expressed in type‐I cells. We conclude that the oxygen‐ and acid‐sensitive background K+ channel of carotid body type‐I cells is likely to be an endogenous TASK‐1‐like channel.

oseph priestley, one of the three scientists credited with the discovery of oxygen, described the death of mice that were deprived of oxygen. However, he was also well aware of the toxicity of too much oxygen, stating, "For as a candle burns much faster in dephlogisticated [oxygen-enriched] than in common air, so we might live out too fast, and the animal powers be too soon exhausted in this pure kind of air. A moralist, at least, may say, that the air which nature has provided for us is as good as we deserve." 1In this review we examine the remarkable mechanisms by which different organs detect and respond to acute changes in oxygen tension. Specialized tissues that sense the local oxygen tension include glomus cells of the carotid body, neuroepithelial bodies in the lungs, chromaffin cells of the fetal adrenal medulla, and smooth-muscle cells of the resistance pulmonary arteries, fetoplacental arteries, systemic arteries, and the ductus arteriosus. Together, they constitute a specialized homeostatic oxygen-sensing system. Although all tissues are sensitive to severe hypoxia, these specialized tissues respond rapidly to moderate changes in oxygen tension within the physiologic range (roughly 40 to 100 mm Hg in an adult and 20 to 40 mm Hg in a fetus) (Fig. 1).In fetal life, the pulmonary vascular bed has a high resistance to blood flow. Consequently, oxygenated blood returning from the placenta is diverted from the unventilated lungs and across the foramen ovale and ductus arteriosus. At birth, when air breathing begins, the lungs expand and oxygen levels rise. With reversal of fetal hypoxic pulmonary vasoconstriction, the pulmonary vessels dilate and the ductus arteriosus constricts, thereby establishing the transition from the fetal to the neonatal circulation.After birth, hypoxic pulmonary vasoconstriction remains important, because it reduces perfusion of poorly ventilated areas of lung, and in so doing it decreases the shunting of desaturated, mixed venous blood to the systemic circulation. Inhibition of hypoxic pulmonary vasoconstriction reduces the systemic arterial oxygen tension, particularly in small-airway disease. 2 Moreover, as was first demonstrated in humans in 1947, 3 the intensity of hypoxic pulmonary vasoconstriction depends on the severity and duration of alveolar hypoxia. 4,5 The endothelium produces vasodilators, such as nitric oxide and prostacyclin, and vasoconstrictors, such as endothelin and thromboxane A 2 ; these molecules from endothelial cells modulate hypoxic pulmonary vasoconstriction, but the ability of small pulmonary vessels to contract in response to hypoxia resides in their smooth-muscle cells. 6 Three sites in these cells are involved in the mechanism of hypoxic pulmonary vasoconstriction: the membrane, the sarcoplasmic reticulum, and the contractile apparatus.

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1. We have studied the effects of hypoxia on membrane potential and [Ca2+]i in enzymically isolated type I cells of the neonatal rat carotid body (the principal respiratory 02 chemosensor). Isolated cells were maintained in short term culture (3-36 h) before use.[Ca2+]j was measured using the Ca2+-sensitive fluoroprobe indo-1. Indo-1 was loaded into cells using the esterified form indo-1 AM. Membrane potential was measured (and clamped) in single isolated type I cells using the perforated-patch (amphotericin B) whole-cell recording technique.

SUMMARY1. Intracellular pH (pHi) was recorded ratiometrically in isolated guinea-pig ventricular myocytes using the pH-sensitive fluoroprobe, carboxy-SNARF-1 (carboxy-seminaphthorhodafluor).2. Following an intracellular acid load (10 mm NH4Cl removal), pHi recovery in HEPES-buffered Tyrode solution was inhibited by 1-5 mm amiloride (Na+-H+ antiport blocker). In the presence of amiloride, switching from HEPES buffer to HCO3J/CO2 (pH0 of both solutions = 7 4) stimulated a pHi recovery towards more alkaline levels.3. Amiloride-resistant, HCO3--dependent pHi recovery was inhibited by removal of external Na+ (replaced by N-methyl-D-glucamine), whereas removal of external Cl-(replaced by glucuronate, leading to depletion of internal Cl-), removal of external K', or decreasing external Ca2+ by tenfold had no inhibitory effect. These results suggest that the amiloride-resistant recovery is due to a Na+-HCO3 cotransport into the cell.4. The stilbene derivative DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, 500 /tM) slowed Na+-HCO3--dependent pHi recovery. Intracellular pH increased in Cl--free solution and this increase still occurred inNa+-free solution indicating that it is not caused via Na+-HCO3-symport and is more likely to be due to Cl-efflux in exchange for HC03-influx on a sarcolemmal Cl--HC03-exchanger. The lack of any significant pHi recovery from intracellular acidosis in Na+-free solution suggests that this exchanger does not contribute to acidequivalent extrusion.6. Possible voltage sensitivity and electrogenicity of the co-transport were examined by using the whole-cell patch clamp technique in combination with SNARF-1 recordings of pHi. Stepping the holding potential from -110 to -40 mV did not affect amiloride-resistant pHi recovery from acidosis. Moreover, following an intracellular acid load, the activation of Na+--HCO3-co-influx (by switching from HEPES to HCO3J/CO2 buffer) produced no detectable outward current (outward current would be expected if the coupling of HC03-with Na+ were > 1-0). 8. Comparison of acid-equivalent efflux via Na+-HCO3-symport and Na+-H+ antiport showed that, following an intracellular acidosis, the symport accounts for about 40 % of total acid efflux, the other 60 % being carried by the antiport. Over the pHi range 7-1-6-9, the activity of both acid extrusion systems increases similarly as pHi falls below 7K1.9. Application of 100 /M acetazolamide (a carbonic anhydrase inhibitor) slowed and attenuated intracellular acid loads imposed by introduction of C02-buffered Tyrode solution and slowed subsequent pH, recovery. This suggests that carbonic anhydrase is present in mammalian myocardium.10. In conclusion, acid-equivalent extrusion from the guinea-pig ventricular myocyte in a HCO3-/CO2-buffered medium is achieved by both Na+-H+ antiport and a voltage-insensitive Na+-HCO3-symport. The efficiency of the latter system is dependent upon carbonic anhydrase activity.

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We report the use of a new pH-sensitive dual-emission fluoroprobe, carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1) for ratiometric recording of intracellular pH (pHi) in small isolated cells. The method is illustrated with pHi measurement in single type-1 cells (cell diameter approximately 10 microns) isolated from the carotid body of the neonatal rat. Carboxy-SNARF-1 is loaded using bath application of the acetoxymethyl ester. When excited at 540 nm, the fluoroprobe gives strong, inversely related emission signals at 590 nm and 640 nm. Stable ratiometric recordings of pHi can be achieved from a single cell (pHi 8.5-6.5) for up to 50 min. Photo-bleaching of the probe is minimised by illuminating at relatively low light intensity (50 W xenon lamp with 0.2% transmission neutral density filter). The probe can be calibrated in situ using the nigericin technique and this is in good quantitative agreement with the independent null-point technique (extracellular weak acid/weak base application) of Eisner et al. (1989).(ABSTRACT TRUNCATED AT 250 WORDS)

1. An acid-induced rise in the intracellular calcium concentration ([Ca2"]