AMP-activated protein kinase and the regulation of Ca2+ signalling in O2-sensing cells

All cells respond to metabolic stress. However, a variety of specialized cells, commonly referred to as O₂-sensing cells, are acutely sensitive to relatively small changes in   P _O2  . Within a variety of organisms such O₂-sensing cells have evolved as vital homeostatic mechanisms that monitor O₂ supply and alter respiratory and circulatory function, as well as the capacity of the blood to transport O₂. Thereby, arterial   P _O2  may be maintained within physiological limits. In mammals, for example, two key tissues that contribute to this process are the pulmonary arteries and the carotid bodies. Constriction of pulmonary arteries by hypoxia optimizes ventilation–perfusion matching in the lung, whilst carotid body excitation by hypoxia initiates corrective changes in breathing patterns via increased sensory afferent discharge to the brain stem. Despite extensive investigation, the precise mechanism(s) by which hypoxia mediates these responses has remained elusive. It is clear, however, that hypoxia inhibits mitochondrial function in O₂-sensing cells over a range of   P _O2  that has no such effect on other cell types. This raised the possibility that AMP-activated protein kinase might function to couple mitochondrial oxidative phosphorylation to Ca²⁺ signalling mechanisms in O₂-sensing cells and thereby underpin pulmonary artery constriction and carotid body excitation by hypoxia. Our recent investigations have provided significant evidence in support of this view.     

Reduced mitochondrial coupling in vivo alters cellular energetics in aged mouse skeletal muscle

The mitochondrial theory of ageing proposes that the accumulation of oxidative damage to mitochondria leads to mitochondrial dysfunction and tissue degeneration with age. However, no consensus has emerged regarding the effects of ageing on mitochondrial function, particularly for mitochondrial coupling (P/O). One of the main barriers to a better understanding of the effects of ageing on coupling has been the lack of in vivo approaches to measure P/O. We use optical and magnetic resonance spectroscopy to independently quantify mitochondrial ATP synthesis and O₂ uptake to determine in vivo P/O. Resting ATP demand (equal to ATP synthesis) was lower in the skeletal muscle of 30-month-old C57Bl/6 mice compared to 7-month-old controls (21.9 ± 1.5 versus 13.6 ± 1.7 nmol ATP (g tissue)⁻¹ s⁻¹, P = 0.01). In contrast, there was no difference in the resting rates of O₂ uptake between the groups (5.4 ± 0.6 versus 8.4 ± 1.6 nmol O₂ (g tissue)⁻¹ s⁻¹). These results indicate a nearly 50% reduction in the mitochondrial P/O in the aged animals (2.05 ± 0.07 versus 1.05 ± 0.36, P = 0.02). The higher resting ADP (30.8 ± 6.8 versus 58.0 ± 9.5 μmol g⁻¹, P = 0.05) and decreased energy charge (ATP/ADP) (274 ± 70 versus 84 ± 16, P = 0.03) in the aged mice is consistent with an impairment of oxidative ATP synthesis. Despite the reduced P/O, uncoupling protein 3 protein levels were not different in the muscles of the two groups. These results demonstrate reduced mitochondrial coupling in aged skeletal muscle that alters cellular metabolism and energetics.     

Ozone inhalation induces exacerbation of eosinophilic airway inflammation and hyperresponsiveness in allergen-sensitized mice

Background:  Ozone (O₃) exposure evokes asthma exacerbations by mechanisms that are poorly understood. We used a murine model to characterize the effects of O₃ on allergic airway inflammation and hyperresponsiveness and to identify factors that might contribute to the O₃-induced exacerbation of asthma.
Methods:  BALB/c mice were sensitized and challenged with Aspergillus fumigatus ( Af ). A group of sensitized and challenged mice was exposed to 3.0 ppm of O₃ for 2 h and studied 12 h later (96 h after Af challenge). Naive mice and mice exposed to O₃ alone were used as controls. Bronchoalveolar lavage (BAL) cellular and cytokine content, lung function [enhanced pause (P_enh)], isometric force generation by tracheal rings and gene and protein expression of Fas and FasL were assessed. Apoptosis of eosinophils was quantified by FACS.
Results:  In sensitized mice allergen challenge induced a significant increase of P_enh and contractile force in tracheal rings that peaked 24 h after challenge and resolved by 96 h. O₃ inhalation induced an exacerbation of airway hyperresponsiveness accompanied by recurrence of neutrophils and enhancement of eosinophils 96 h after allergen challenge. The combination of allergen and O₃ exposure inhibited Fas and FasL gene and protein expression and eosinophil apoptosis and increased interleukin-5 (IL-5), granulocyte-macrophage-colony stimulating factor (GM-CSF) and G-CSF protein levels.
Conclusions:  O₃ affects airway responsiveness of allergen-primed airways indirectly by increasing viability of eosinophils and eosinophil-mediated pathological changes.     

Does nitric oxide allow endothelial cells to sense hypoxia and mediate hypoxic vasodilatation?in vivo and in vitro studies

Hypoxia-evoked vasodilatation is a fundamental regulatory mechanism that is often attributed to adenosine. The identity of the O₂ sensor is unknown. Nitric oxide (NO) inhibits endothelial mitochondrial respiration and ATP generation by competing with O₂ for its binding site on cytochrome oxidase. We proposed that in vivo this interaction allows endothelial cells to release adenosine when O₂ tension falls or NO concentration increases. Using anaesthetised rats, we confirmed that the increase in femoral vascular conductance (FVC, hindlimb vasodilatation) evoked by systemic hypoxia is attenuated by NO synthesis blockade with l -NAME, but restored when baseline FVC is restored by infusion of NO donor. This 'restored' hypoxic response, like the control hypoxic response, is inhibited by the adenosine A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Similarly, the FVC increase evoked by adenosine infusion was attenuated by l -NAME but restored by infusion of NO donor. However, when baseline FVC was restored after l -NAME with 8-bromo-cGMP, the FVC increase evoked by adenosine infusion was restored, but not in response to systemic hypoxia, suggesting that adenosine was no longer released by hypoxia. Infusion of NO donor at a given rate after treatment with l -NAME evoked a greater FVC increase during systemic hypoxia than during normoxia, both responses being reduced by 8-cyclopentyl-1,3-dipropylxanthine. Finally, both bradykinin and NO donor released adenosine from superfused endothelial cells in vitro ; l -NAME attenuated only the former response. We propose that in vivo , shear-released NO increases the apparent K _m of endothelial cytochrome oxidase for O₂, allowing the endothelium to act as an O₂ sensor, releasing adenosine in response to moderate falls in O₂.     

Effects of hypoxic and hyperoxic incubation on the reactivity of the chicken embryo (Gallus gallus) ductus arteriosi in response to catecholamines and oxygen

Development in chronic hypoxia has been shown to have a significant negative impact on the developing cardiovascular system. The developing chicken embryo has two ductus arteriosi (DA) that shunt blood away from the lungs to the systemic circuit and constrict during hatching in response to an increase in arterial partial pressure of O₂. The goal of this study was to determine the influence of O₂ levels during incubation on the vascular reactivity of the O₂-sensitive DA using the chicken as a novel model system. In addition, we measured blood gas and air cell O₂ during these developmental stages. Chicken embryos were incubated in hypoxia (15% O₂), normoxia (21% O₂) or hyperoxia (30% O₂) and examined on incubation days 16 and 18 and as internally pipped and externally pipped embryos. The vasoreactivity of the DA was measured in response to an increase in O₂ and during stepwise increases in noradrenaline (NA) and phenylephrine (PE). The DA from embryos incubated in hypoxic conditions contracted in response to O₂ at a later hatching stage than the DA from embryos incubated in normoxic or hyperoxic conditions. The DA from day 18 embryos incubated in hypoxic conditions had a significantly weaker contractile response to NA and PE when compared with the DA from embryos incubated in normoxic or hyperoxic conditions. Blood gas and air cell O₂ were lowest for embryos incubated in hypoxic conditions and highest for embryos incubated in hyperoxic conditions. Incubation in hypoxia significantly delays the maturation of DA, while incubation in hyperoxia accelerates development.     

Hypoxia reduces the hypothalamic thermogenic threshold and thermosensitivity

Hypoxia is well known to reduce the body temperature ( T _b) of mammals, although the neural origins of this response remain uncertain. Short-term hypoxic exposure causes a reduction in the lower critical temperature of the thermal neutral zone and a reduction in whole body thermal conductance of rodents, providing indirect support that hypoxia lowers T _b in a regulated manner. In this study, we examined directly the potential for changes in central thermosensitivity to evoke the hypoxic metabolic response by heating and cooling the preoptic area of the hypothalamus (the area which integrates thermoreceptor input and regulates thermoeffector outputs) using chronic, indwelling thermodes in ground squirrels during normoxia and hypoxia (7, 10 and 12% O₂). We found that the threshold hypothalamic temperature for the metabolic response to cooling ( T _th) of ∼38°C in normoxia was proportionately reduced in hypoxia (down to 28–31°C at 7% O₂) and that the metabolic thermosensitivity (α; the change in metabolic rate for any given change in hypothalamic temperature below the lower critical temperature) was comparatively reduced by 5 to 9 times. This provides strong support for the hypothesis that the fall in temperature that occurs during hypoxia is the result of a reduction in the activation of thermogenic mechanisms. The decrease in the central thermosensitivity in hypoxia, however, appears to be a critical factor in the alteration of mammalian T _b. We suggest, therefore, that an altered central thermosensitivity may provide a proximate explanation of how low oxygen and similar stressors reduce normal fluctuations in T _b (i.e. circadian), in addition to the depression in regulated T _b.     

Low glucose effects on rat carotid body chemoreceptor cells' secretory responses and action potential frequency in the carotid sinus nerve

Glucose deprivation (hypoglycaemia) is counterbalanced by a neuroendocrine response in order to induce fast delivery of glucose to blood. Some central neurons can sense glucose, but nevertheless the most important glucose sensors/glycaemia regulators are located outside the brain. Some recent experimental evidence obtained in carotid body (CB) slices and isolated chemoreceptor cells in culture supports a role for the CB in glucose sensing and presumably glucose homeostasis, but this role has been questioned on the basis of a lack of effect of low glucose on the carotid sinus nerve activity. This work was performed in an attempt to clarify if low glucose is or is not a stimulus for the rat CB chemoreceptors. Using freshly isolated intact CB preparations we have monitored the release of catecholamines (CAs) and ATP from chemoreceptor cells in response to several concentrations of glucose, as indices of chemoreceptor cell sensitivity to glycaemia, and the electrical activity in the carotid sinus nerve (CSN), as an index of reflex-triggering output of the CB. We have observed that basal (20% O₂) and hypoxia (7 and 10% O₂)-evoked release of CAs was identical in the presence of normal (5.55 m m ) and low (3, 1 and 0 m m ) glucose concentrations. 0 m m glucose did not activate the release of ATP from the CB, while hypoxia (5% O₂) did. Basal and hypoxia (5% O₂)-induced CSN action potential frequency was identical with 5.55 and 1 m m glucose. Our results indicate that low glucose is not a direct stimulus for the rat carotid body chemoreceptors.     

Mutant mice deficient in NOS-1 exhibit attenuated long-term facilitation and short-term potentiation in breathing

The objective of the present study is to examine the potential role of nitric oxide (NO) in short-term potentiation (STP) and long-term facilitation (LTF) of breathing. Experiments were performed in wild-type (WT) and mutant mice deficient in nitric oxide synthase-1 (NOS-1), as well as in WT mice administered the NOS-1 inhibitor 7-nitroindazole (7-NI; 50 mg kg⁻¹; i.p. ). Respiratory responses following either single or recurrent episodes of hypoxia (7 % O₂, balance N₂) were analysed in unanaesthetised animals by body plethysmography along with rate of O₂ consumption (_O2) and CO₂ production (_CO2). After a single hypoxic challenge, respiration in WT mice remained elevated for 5 min, suggesting STP in ventilation. Following termination of three consecutive hypoxic challenges, respiration remained elevated during normoxia for as long as 30 min, indicating LTF in breathing under awake conditions. STP and LTF were significantly attenuated or absent in WT mice after 7-NI. A similar attenuation or absence of STP and LTF was also seen in NOS-1 mutant mice. Changes in _O2 and _CO2 were comparable among mice during the post-hypoxic period, suggesting that the absence of STP and LTF was not due to alterations in body metabolism. These results suggest endogenous NO is an important physiological modulator of ventilatory STP and LTF.     

A novel O2-sensing mechanism in rat glossopharyngeal neurones mediated by a halothane-inhibitable background K+ conductance

Modulation of K⁺ channels by hypoxia is a common O₂-sensing mechanism in specialised cells. More recently, acid-sensitive TASK-like background K⁺ channels, which play a key role in setting the resting membrane potential, have been implicated in O₂-sensing in certain cell types. Here, we report a novel O₂ sensitivity mediated by a weakly pH-sensitive background K⁺ conductance in nitric oxide synthase (NOS)-positive neurones of the glossopharyngeal nerve (GPN). This conductance was insensitive to 30 m m TEA, 5 m m 4-aminopyridine (4-AP) and 200 μ m Cd²⁺, but was reversibly inhibited by hypoxia (O₂ tension ( P _O2) = 15 mmHg), 2–5 m m halothane, 10 m m barium and 1 m m quinidine. Notably, the presence of halothane occluded the inhibitory effect of hypoxia. Under current clamp, these agents depolarised GPN neurones. In contrast, arachidonic acid (5–10 μ m ) caused membrane hyperpolarisation and potentiation of the background K⁺ current. This pharmacological profile suggests the O₂-sensitive conductance in GPN neurones is mediated by a class of background K⁺ channels different from the TASK family; it appears more closely related to the THIK (tandem pore domain halothane-inhibited K⁺) subfamily, or may represent a new member of the background K⁺ family. Since GPN neurones are thought to provide NO-mediated efferent inhibition of the carotid body (CB), these channels may contribute to the regulation of breathing during hypoxia via negative feedback control of CB function, as well as to the inhibitory effect of volatile anaesthetics (e.g. halothane) on respiration.     

Functional analysis of neurotransmission at β2-laminin deficient terminals

Carotid body glomus cells release transmitters in response to hypoxia due to the increase of excitability resulting from inhibition of O₂ -regulated K⁺ channels. However, the mechanisms involved in the detection of changes of O₂ tension are unknown. We have studied the interaction between glomus cell O₂ sensitivity and inhibition of the mitochondrial electron transport chain (ETC) in a carotid body thin slice preparation in which catecholamine release from intact single glomus cells can be monitored by amperometry. Inhibition of the mitochondrial ETC at proximal and distal complexes induces external Ca²⁺-dependent catecholamine secretion. At saturating concentration of the ETC inhibitors, the cellular response to hypoxia is maintained. However, rotenone, a complex I blocker, selectively occludes the responsiveness to hypoxia of glomus cells in a dose-dependent manner. The effect of rotenone is mimicked by 1-methyl-4-phenylpyridinium ion (MPP⁺), an agent that binds to the same site as rotenone, but not by complex I inhibitors acting on different sites. In addition, the effect of rotenone is not prevented by incubation of the cells with succinate, a substrate of complex II. These data strongly suggest that sensitivity to hypoxia of carotid body glomus cells is not linked in a simple way to mitochondrial electron flow and that a rotenone (and MPP⁺)-sensitive molecule critically participates in acute oxygen sensing in the carotid body.     

Fetal cerebral blood flow, electrocorticographic activity, and oxygenation: responses to acute hypoxia

Arterial blood gases are critical in regulation of cerebral blood flow (CBF) and cerebral metabolic rate for O₂ (CMRO₂). However, the relation of these variables to cortical tissue     (t     ), and electrocorticographic (ECoG) activity (high voltage low frequency, HVLF, versus low voltage high frequency, LVHF), are not well defined. In the fetus, we tested the hypothesis that ECoG pattern is associated closely with cerebral oxygenation. In fetal sheep ( n = 8) with laser Doppler flowmeter, fluorescent O₂ probe and ECoG electrodes, we measured laser Doppler CBF (LD-CBF), t     , ECoG and spectral edge frequency-90 (SEF₉₀) in response to 40 min isocapnic hypoxia. In the normoxic fetus, LD-CBF and CMRO₂ correlated highly with ECoG state. With a shift from HVLF to LVHF, t     decreased followed by increased LD-CBF (18%) and CMRO₂ (13%). With acute hypoxia (     = 12 ± 1 Torr), t     decreased to ∼3 Torr, LD-CBF increased 48 ± 10%, ECoG shifted to chiefly the HVLF state, SEF₉₀ decreased ∼15%, and CMRO₂ decreased ∼20% ( P < 0.05 for each). For the normoxic fetus, CBF was closely related to ECoG state, but this association was less evident during acute hypoxia. We speculate that, in the otherwise stressed fetus, acute hypoxia may further compromise cerebral oxygenation.     

Adaptive responses of vertebrate neurons to anoxia--matching supply to demand

Oxygen depleted environments are relatively common on earth and represent both a challenge and an opportunity to organisms that survive there. A commonly observed survival strategy to this kind of stress is a lowering of metabolic rate or metabolic depression. Whether metabolic rate is at a normal or a depressed level the supply of ATP (glycolysis and oxidative phosphorylation) must match the cellular demand for ATP (protein synthesis and ion pumping), a condition that must of course be met for long-term survival in hypoxic and anoxic environments. Underlying a decrease in metabolic rate is a corresponding decrease in both ATP supply and ATP demand pathways setting a new lower level for ATP turnover. Both sides of this equation can be actively regulated by second messenger pathways but it is less clear if they are regulated differentially or even sequentially with the onset of anoxia. The vertebrate brain is extremely sensitive to low oxygen levels yet some species can survive in oxygen depleted environments for extended periods and offer a working model of brain survival without oxygen. Hypoxia tolerant vertebrate brain will be the primary focus of this review; however, we will draw upon research involving hypoxia/ischemia tolerance mechanisms in liver and heart to offer clues to how brain can tolerate anoxia. The issue of regulating ATP supply or demand pathways will also be addressed with a focus on ion channel arrest being a significant mechanism to reduce ATP demand and therefore metabolic rate. Furthermore, mitochondria are ideally situated to serve as cellular oxygen sensors and mediator of protective mechanisms such as ion channel arrest. Therefore, we will also describe a mitochondria based mechanism of ion channel arrest involving ATP-sensitive mitochondrial K(+) channels, cytosolic calcium and reaction oxygen species concentrations.     

Dissociation of the store-operated calcium current ICRAC and the Mg-nucleotide-regulated metal ion current MagNuM

Rat basophilic leukaemia cells (RBL-2H3-M1) were used to study the characteristics of the store-operated Ca²⁺ release-activated Ca²⁺ current ( I _CRAC) and the magnesium-nucleotide-regulated metal cation current (MagNuM) (which is conducted by the LTRPC7 channel). Pipette solutions containing 10 m m BAPTA and no added ATP induced both currents in the same cell, but the time to half-maximal activation for MagNuM was about two to three times slower than that of I _CRAC. Differential suppression of I _CRAC was achieved by buffering free [Ca²⁺]_i to 90 n m and selective inhibition of MagNuM was accomplished by intracellular solutions containing 6 m m Mg.ATP, 1.2 m m free [Mg²⁺]_i or 100 μ m GTP-γ-S, allowing investigations on these currents in relative isolation. Removal of extracellular Ca²⁺ and Mg²⁺ caused both currents to be carried significantly by monovalent ions. In the absence or presence of free [Mg²⁺]_i, I _CRAC carried by monovalent ions inactivated more rapidly and more completely than MagNuM carried by monovalent ions. Since several studies have used divalent-free solutions on either side of the membrane to study selectivity and single-channel behaviour of I _CRAC, these experimental conditions would have favoured the contribution of MagNuM to monovalent conductance and call for caution in interpreting results where both I _CRAC and MagNuM are activated.     

The early effects of chronic hypoxia on the cardiovascular system in the rat: role of nitric oxide

Experiments were performed under Saffan anaesthesia on normoxic (N) rats and on chronically hypoxic rats exposed to 12% O₂ for 1, 3 or 7 days (1, 3 or 7CH rats): N rats routinely breathed 21% O₂ and CH rats 12% O₂. The 1, 3 and 7CH rats showed resting hyperventilation relative to N rats, but baseline heart rate (HR) was unchanged and arterial blood pressure (ABP) was lowered. Femoral vascular conductance (FVC) was increased in 1 and 3CH rats, but not 7CH rats. When 1–7CH rats were acutely switched to breathing 21% O₂ for 5 min, ABP increased and FVC decreased, consistent with removal of a hypoxic dilator stimulus that is waning in 7CH rats. We propose that this is because the increase in haematocrit and vascular remodelling in skeletal muscle help restore the O₂ supply. The increases in FVC evoked by acute hypoxia (8% O₂ for 5 min) and by infusion for 5 min of α-calcitonin gene-related peptide (α-CGRP), which are NO-dependent, were particularly accentuated in 1CH, relative to N rats. The NO synthesis inhibitor l -NAME increased ABP, decreased HR and greatly reduced FVC, and attenuated increases in FVC evoked by acute hypoxia and α-CGRP, such that baselines and responses were similar in N and 1–7CH rats. We propose that in the first few days of chronic hypoxia there is tonic NO-dependent vasodilatation in skeletal muscle that is associated with accentuated dilator responsiveness to acute hypoxia and dilator substances that are NO -dependent.     

The intent to exercise influences the cerebral O2/carbohydrate uptake ratio in humans

During and after maximal exercise there is a 15–30 % decrease in the metabolic uptake ratio (O₂/[glucose + 1/2 lactate]) and a net lactate uptake by the human brain. This study evaluated if this cerebral metabolic uptake ratio is influenced by the intent to exercise, and whether a change could be explained by substrates other than glucose and lactate. The arterial-internal jugular venous differences (a-v difference) for O₂, glucose and lactate as well as for glutamate, glutamine, alanine, glycerol and free fatty acids were evaluated in 10 healthy human subjects in response to cycling. However, the a-v difference for the amino acids and glycerol did not change significantly, and there was only a minimal increase in the a-v difference for free fatty acids after maximal exercise. After maximal exercise the metabolic uptake ratio of the brain decreased from 6.1 ± 0.5 (mean ± s.e.m. ) at rest to 3.7 ± 0.2 in the first minutes of the recovery (   P < 0.01  ). Submaximal exercise did not change the uptake ratio significantly. Yet, in a second experiment, when submaximal exercise required a maximal effort due to partial neuromuscular blockade, the ratio decreased and remained low (4.9 ± 0.2) in the early recovery (   n = 10  ;   P < 0.05  ). The results indicate that glucose and lactate uptake by the brain are increased out of proportion to O₂ when the brain is activated by exhaustive exercise, and that such metabolic changes are influenced by the will to exercise. We speculate that the uptake ratio for the brain may serve as a metabolic indicator of 'central fatigue'.     

Target-specific PIP2 signalling: how might it work?

Phosphatidylinositol 4,5-bisphosphate (PIP₂)-mediated signalling is a new and rapidly developing area in the field of cellular signal transduction. With the extensive and growing list of PIP₂-sensitive membrane proteins (many of which are ion channels and transporters) and multiple signals affecting plasma membrane PIP₂ levels, the question arises as to the cellular mechanisms that confer specificity to PIP₂-mediated signalling. In this review we critically consider two major hypotheses for such possible mechanisms: (i) clustering of PIP₂ in membrane microdomains with restricted lateral diffusion, a hypothesis providing a mechanism for spatial segregation of PIP₂ signals and (ii) receptor-specific buffering of the global plasma membrane PIP₂ pool via Ca²⁺-mediated stimulation of PIP₂ synthesis or release, a concept allowing for receptor-specific signalling with free lateral diffusion of PIP₂. We also discuss several other technical and conceptual intricacies of PIP₂-mediated signalling.     

Non-selective β-adrenergic blockade prevents reduction of the cerebral metabolic ratio during exhaustive exercise in humans

Intense exercise decreases the cerebral metabolic ratio of oxygen to carbohydrates [O₂/(glucose +½lactate)], but whether this ratio is influenced by adrenergic stimulation is not known. In eight males, incremental cycle ergometry increased arterial lactate to 15.3 ± 4.2 m m (mean ± s.d. ) and the arterial–jugular venous (a–v) difference from −0.02 ± 0.03 m m at rest to 1.0 ± 0.5 m m ( P < 0.05). The a–v difference for glucose increased from 0.7 ± 0.3 to 0.9 ± 0.1 m m ( P < 0.05) at exhaustion and the cerebral metabolic ratio decreased from 5.5 ± 1.4 to 3.0 ± 0.3 ( P < 0.01). Administration of a non-selective β-adrenergic (β₁+β₂) receptor antagonist (propranolol) reduced heart rate (69 ± 8 to 58 ± 6 beats min⁻¹) and exercise capacity (239 ± 42 to 209 ± 31 W; P < 0.05) with arterial lactate reaching 9.4 ± 3.6 m m . During exercise with propranolol, the increase in a–v lactate difference (to 0.5 ± 0.5 m m ; P < 0.05) was attenuated and the a–v glucose difference and the cerebral metabolic ratio remained at levels similar to those at rest. Together with the previous finding that the cerebral metabolic ratio is unaffected during exercise with administration of the β₁-receptor antagonist metropolol, the present results suggest that the cerebral metabolic ratio decreases in response to a β₂-receptor mechanism.     

Antioxidants reverse depression of the hypoxic ventilatory response by acetazolamide in man

The carbonic anhydrase inhibitor acetazolamide may have both inhibitory and stimulatory effects on breathing. In this placebo-controlled double-blind study we measured the effect of an intravenous dose (4 mg kg⁻¹) of this agent on the acute isocapnic hypoxic ventilatory response in 16 healthy volunteers (haemoglobin oxygen saturation 83–85%) and examined whether its inhibitory effects on this response could be reversed by antioxidants (1 g ascorbic acid i.v. and 200 mg α-tocopherol p.o. ). The subjects were randomly divided into an antioxidant (Aox) and placebo group. In the Aox group, acetazolamide reduced the mean normocapnic and hypercapnic hypoxic responses by 37% ( P < 0.01) and 55% ( P < 0.01), respectively, and abolished the O₂–CO₂ interaction, i.e. the increase in O₂ sensitivity with rising P _CO2. Antioxidants completely reversed this inhibiting effect on the normocapnic hypoxic response, while in hypercapnia the reversal was partial. In the placebo group, acetazolamide reduced the normo- and hypercapnic hypoxic responses by 33 and 47%, respectively ( P < 0.01 versus control in both cases), and also abolished the O₂–CO₂ interaction. Placebo failed to reverse these inhibitory effects of acetazolamide in this group. We hypothesize that either an isoform of carbonic anhydrase may be involved in the regulation of the redox state in the carotid bodies or that acetazolamide and antioxidants exert independent effects on oxygen-sensing cells, in which both carbonic anhydrase and potassium channels may be involved. The novel findings of this study may have clinical implications, for example with regard to a combined use of acetazolamide and antioxidants at high altitude.     

pH-dependent and -independent effects inhibit Ca2+-induced Ca2+ release during metabolic blockade in rat ventricular myocytes

We have investigated the role of changes of intracellular pH (pH_i) in the effects of metabolic blockade (cyanide plus 2-deoxyglucose) on Ca²⁺ release from the sarcoplasmic reticulum (SR) in rat ventricular myocytes. pH_i and cell length were measured simultaneously. Metabolic blockade decreased the frequency of Ca²⁺ waves, an effect previously shown to be due to inhibition of Ca²⁺ release from the SR. This was accompanied by an intracellular acidification. Intracellular acidification was produced in the absence of metabolic inhibition by application of sodium butyrate. A maintained intracellular acidosis produced a decrease of wave frequency. A hysteresis between pH_i and wave frequency was observed such that during the onset of the acidification the wave frequency decreased more than in the steady state. Comparison of the steady state relationship between pH_i and wave frequency showed that the decrease of wave frequency produced by metabolic blockade was greater than could be accounted for simply by the accompanying decrease of pH_i. In other experiments the buffering power of the solution was increased. Under these conditions, metabolic blockade produced no change of pH_i but the decrease of wave frequency persisted. We conclude that, although intracellular acidification occurs during metabolic blockade, it is not responsible for most of the inhibition of Ca²⁺ release from the SR.