The NHE3 inhibitor AVE1599 stimulates phrenic nerve activity in the rat.

The effect of AVE1599, an inhibitor of the sodium/proton exchanger type 3 (NHE3), on phrenic nerve (PN) activity was investigated using the working heart brainstem preparation (WHBP). Hypercapnia (Delta pH: -0.1) applied for 10 min reversibly increased PN frequency (f) by 66.0 +/- 19.5% and decreased burst duration by 23.3 +/- 3% (mean +/- SE, n = 21). Similarly, AVE1599 (0.3 microM) increased f after 10 and 30 min by 75.1 +/- 13.2 and 176 +/- 36.2% (n = 10), respectively, and reduced duration of PN bursts by 24.9 +/- 10.8%. Hypercapnia-induced increases of f were attenuated by AVE1599. An elevated concentration of AVE1599 (0.9 microM) had no significant effect on PN. As AVE1599 accumulates in brain tissue and might interfere with the less affine NHE1, we furthermore tested the NHE1-inhibitor HOE642. In fact, HOE642 (0.9 microM) diminished f by 88.5 +/- 9.2 and 58.6 +/- 10.0% after 10 and 30 min (n = 6), respectively, but did not abolish hypercapnic responses. We conclude that AVE1599 augments central respiratory drive in the WHBP via NHE3 but not NHE1 inhibition.

Pharmacological impact on loop gain properties to prevent irregular breathing.

Theory predicts respiratory instabilities at elevated system loop gain (G), determined by such factors as ventilatory CO(2) sensitivity, set-point PCO(2), and metabolic rate. In anesthetized rabbits, the effects on G of carbonic anhydrase (CA) inhibitors and of different sodium/proton exchanger type 3 (NHE3) inhibitors were studied. Acetazolamide significantly reduced G by 42.0 +/- 9.3% and methazolamide by 35.0 +/- 9.5% (each n = 7, P<0.01). Irrespective of the substance, NHE3 inhibition reduced G by 33.0 +/- 7.8% (n = 10, P<0.01) at 35.5 +/- 1.6 mmHg PaCO(2) (mean +/-SE), but not at lower arterial CO(2) levels (n=5). Since high baseline PCO(2) coincides with elevated brainstem NHE3 mRNA expression, this may also account for a higher risk of sleep apnea (or even occurrence of sudden infant death). Therefore, NHE3 inhibitors may gain similar therapeutic importance in the treatment of irregular breathing as CA inhibitors. Generally, effective treatment should aim at a low system loop gain, by reducing respiratory chemosensitivity, improving blood gases and preventing low metabolic rates.

Patterns of intrathoracic gas volume and airway resistance in children with asthma.

Airway resistance (Raw) decreases with an increase of lung volume (ITGV) during body-growth. In asthmatic subjects, an increase of Raw may be modified by hyperinflation. In this study thirty five asthmatic children underwent histamine challenges with the monitoring of changes of Raw and ITGV. Control measurements (after withhold of spasmolytic medication) showed increased values of ITGV and Raw with high interindividual variability. Histamine challenge resulted in a further increase of both ITGV and Raw in 18 patients (normal pattern). Twelve patients showed an increase predominantly of Raw (obstructive pattern), 5 patients an increase predominantly of ITGV (hyperinflatory pattern). On provocation and after bronchospasmolysis, all subjects presented the expected inverse relation between ITGV and Raw. The variability of ITGV-Raw patterns in children with asthma may agree with the concomitantly established role for vagal reflex mechanisms in prolonged inspiratory diaphragmatic innervation during experimentally induced bronchoconstriction in animals. In children with asthma the ITGV-Raw pattern may point to different risk profiles.

A novel inhibitor of the Na+/H+ exchanger type 3 activates the central respiratory CO2 response and lowers the apneic threshold.

Cultured CO2-sensitive neurons from the ventrolateral medulla of newborn rats enhanced their bioelectric activity upon intracellular acidification induced by inhibition of the Na+/H+ exchanger type 3 (NHE3). Now we detected NHE3 also in the medulla oblongata of adult rabbits. Therefore, this animal model was employed to determine whether NHE3 inhibition also affects central respiratory chemosensitivity in vivo. Seven anesthetized (pentobarbital), vagotomized, paralyzed rabbits were artificially ventilated with O2-enriched air. From the phrenic nerve compound discharge, integrated burst amplitude (IPNA), respiratory rate (fR), and phrenic minute activity (IPNA. fR) were taken as measures of central respiratory rhythm and drive. Effects of potent NHE3 inhibition with the novel brain permeant substance S8218 were studied by comparing respiratory characteristics before and after up to 9.2 +/- 1.1 mg/kg cumulative drug application, yielding average plasma concentrations of 0.9 +/- 0.2 microg/ml. In response to S8218, the baseline level of IPNA. fR was significantly enhanced by an average of 51.0 +/- 6.4% (n = 27, p < 0.0001). The influence of NHE3 inhibition on the respiratory CO2 response was studied at plasma concentrations of S8218 maintained in the range of 0.3 microg/ml (10(-6) M). Although the metabolic acid-base status thereby remained widely unchanged, the group mean apneic threshold PaCO2 was significantly lowered by 0.45 +/- 0.11 kPa (n = 7, p < 0.01), whereby in four of seven animals even strong hyperventilation failed to suppress phrenic nerve rhythmicity completely. Likewise, S8218 significantly augmented IPNA. fR, in the range of PaCO2 between 1 and 6 kPa above threshold, by an average of 38.0 +/- 8.5% (n = 35, p < 0.0001). These in vivo results are compatible with the effects of NHE3 inhibition on chemosensitive brainstem neurons in vitro. Moreover, rhythmogenesis is supported through NHE3 inhibition by lowering the threshold PCO2 for central apnea.

Low-dose acetazolamide does affect respiratory muscle function in spontaneously breathing anesthetized rabbits.

Patients with chronic obstructive pulmonary diseases (COPD) and/or central sleep apnea are sometimes treated with the carbonic anhydrase inhibitor acteazolamide to improve blood gas values. Studies have shown that this agent may have a complicated effect on lung ventilation, because carbonic anhydrase has a widespread distribution within the body, particularly in tissues involved in the control of breathing. To investigate whether acetazolamide may have (neuro)muscular effects on respiration, we measured the responses of ventilation, phrenic nerve activity, and transpulmonary pressure to changes in arterial PCO2 before and after intravenous administration of a low-dose (4.6 +/- 0.2 mg x kg(-1), mean +/- SEM) of this inhibitor in anesthetized spontaneously breathing rabbits. The agent decreased the mean resting end-tidal PCO2 by 1 kPa and increased ventilation from 258 +/- 15 to 292 +/- 14 ml x min(-1) x kg(-1) (p < or = 0.05). The ventilatory and tidal volume responses to CO2 were reduced, and the response curves were shifted to lower PCO2 values. At the level of phrenic activity, however, the response was shifted leftward without altering CO2 sensitivity. With an unchanged lung compliance, the slopes of the relationships between tidal volume and phrenic activity and that between the tidal change in transpulmonary pressure and phrenic amplitude were both reduced by about 40%, indicating an action of acetazolamide on (neuro)muscular level. The results raise the suggestion that treatment of some hypercapnic COPD patients with acetazolamide may have undesired clinical implications, particularly in those with already weakened respiratory muscles.

Minimal-invasive approach to study pulmonary, metabolic and renal responses to alimentary acid-base changes in conscious rabbits.

BACKGROUND: Systemic acid-base balance is maintained by the complex interplay of renal and pulmonary control functions and metabolic adaptations, whereby intake and mineral composition of feed are important factors. AIM OF THE STUDY: It was intended to explore the role of alimentary acid-base load and carbonic anhydrase activity for regulatory responses of renal, pulmonary or metabolic origin in rabbits as typical herbivores. METHODS: Sixty-eight conscious male rabbits (about 3.5 kg) were kept in a metabolic cage, to determine daily water intake, urine excretion and food consumption. Different groups were fed either alkali-rich rabbit standard pellets, or modified rabbit chow with low Ca++-content, or a special diet with very low alkali content, or standard food together with a low oral dose (about 20 mg x kg(-1) x d(-1)) acetazolamide. Samples from the central ear artery were analyzed for blood gases (PaO2, PaCO2), pHa, base excess (BE) and actual bicarbonate (HCO3a-). The metabolic CO2 production (VCO2 STPD) was determined, to calculate alveolar ventilation (VA BTPS). Anaerobically collected urine was analyzed for pHu and for concentrations of bicarbonate/carbonate (HCO3-/CO3--), ammonium (NH4+), and phosphate. RESULTS: 1) Systemic BE was not affected by alimentary alkali load, either varied spontaneously by standard food intake or by the low-Ca++ diet, and decreased only slightly on the low-alkali diet, but distinctly upon carbonic anhydrase inhibition. 2) Under all conditions of alimentation, PaCO2 was closely correlated with BE without a detectable set-point, the normal-range variability of BE being sufficient to elicit corresponding changes in VA. In contrast, acetazolamide led to much lower values of PaCO2 than predicted by the reference PCO2/BE relationship, being primarily caused by significant reductions in VCO2 (> 20%). 3) Prior to other systems, renal base excretion, normally being high on species-adapted standard chow, closely followed any variation of alimentary alkali load and approached zero upon the low-alkali diet. It was, however, not significantly influenced by carbonic anhydrase (CA) inhibition on alkalirich alimentation. CONCLUSIONS: Blood acid-base balance in rabbits is maintained over a wide range of alimentary alkali load by effective adaptation of renal base excretion, independent of CA activity. Ventilatory pH control is perpetuated even in the normal range of BE, provided metabolic rate is not impaired, e. g., by CA inhibition. These results may help one understand the different manifestations of acid-base disorders in body fluids under clinical conditions.

The Haldane effect under different acid-base conditions in premature and adult humans.

The Haldane effect (HE) was investigated in human adults and prematures under normal metabolic acid-base conditions but at different levels of PCO2. Venous blood samples were equilibrated with low and high PCO2 in either O2 or N2. The change in plasma pH of oxygenated blood by deoxygenation did not differ between both groups. Thus, ontogenetic differences of human hemoglobin structure do not influence the net proton Haldane effect measured in terms of whole blood pH-changes. Since the present data quantitatively agree with those we reported earlier for rabbits, cats and dogs (Kiwull-Schöne et al., 1992), phylogenetic differences in hemoglobin structure of these mammalian species do not either seem to play a role in this respect. The influence of the Haldane effect on plasma pH has to be considered in blood-gas and acid-base analysis of samples with incomplete oxygenation. This is important for the indirect determination of PCO2 through pH by the equilibration method (Astrup and Schrøder, 1956), serving as reference method for determination of metabolic acid-base status and CO2 buffering capacity. Likewise, HE-correction is important for indirect estimation of metabolic acid-base status (BE and HCO-3st) from clinical routine PCO2- and pH-measurement. In spite of the vaste amount of literature on the Haldane effect in human blood, quantitative data for practical purpose are less available and still equivocal. By the present study, a strong inverse linear correlation between the HE-induced delta pH and 1g[HCO3-] could be shown over a wide range of acid-base changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoxia and the "reaction theory" of central respiratory chemosensitivity.

In peripherally chemodenervated and vagotomized cats and rabbits, either spontaneously breathing or artificially ventilated, we studied the reaction of the respiratory control system to changes in the extracellular fluid (ECF) pH at the ventral surface of the medulla oblongata. The brainstem ECF-pH was varied either by alternating periods of hypoxia and hyperoxia or by intravenous infusion of lactic acid to achieve endogenous or exogenous lactacidosis, respectively. Additionally, the arterial PCO2 was changed by varying the inspiratory CO2-fraction or the respirator's pumping rate. When pulmonary ventilation or central respiratory drive (in terms of phrenic nerve activity) was related to brainstem ECF-pH, no unique function resulted for respiratory (CO2-induced) and metabolic (lactic acid induced) acid-base changes, thus contradicting the "reaction theory" for central respiratory chemosensitivity. Under steady state conditions, there was no ventilatory reaction to endogenous or exogenous metabolic brainstem acidosis at all. However, the apneic threshold was shifted towards the acid range, although the sensitivity of the respiratory system to CO2 remained nearly unchanged, no matter whether CO2 was inhaled or increased by acetazolamide. This points to a dominating role of CO2 or at least carbonic acid over fixed acids for the central chemosensitive control of pulmonary ventilation.

The Haldane effect of rabbit blood under different acid-base conditions.

The Haldane effect (HE), i.e. the difference in plasma pH of oxygenated and deoxygenated blood, was investigated in rabbits over a wide range of respiratory (PCO2 2.7 to 8.0 kPa) and metabolic (BE +5 to -15 mM) acid-base conditions and compared to cats, dogs and humans. Even under standard conditions (PCO2 = 5.3 kPa, BE = O mM) and normalized to the same Hb-concentration, the HE-induced pH-difference was distinctly greater in rabbits, cats and dogs (about 0.045) than in humans (0.034 - 0.039). During respiratory and metabolic acid-base changes, the HE-induced delta pH was inversely related to PCO2 and BE. The dependency of the Haldane effect on the acid-base status can be estimated by means of a linear inverse relationship between delta pH and lgHCO3-, as originally proposed for human blood by v. Mengden et al., 1969. Correspondingly, the regression analysis of the present experimental Haldane effect data of rabbits, cats and dogs, yielded highly linear correlations as well as characteristic species-related regression coefficients. Additionally considering the influence of the Hb-concentration, a most useful tool for quantitative estimation of the Haldane effect is thus available. This is of practical importance, e.g. when determining the arterial PCO2 indirectly by the Astrup method. However, Haldane corrections based only on human blood data available so far, would lead to an underestimation of the Haldane effect in common laboratory animals. If the appropriate Haldane shift of pH for a species is not considered, the resulting PCO2 may be erroneous by up to several 100 Pa, particularly in the range of metabolic acidosis.

The effects of CO2 and fixed acid on the O2-Hb affinity of rabbit and cat blood.

The action of respiratory and metabolic acid-base disturbances on the O2-Hb affinity was studied in rabbits and cats. Blood samples of both species were exposed to in vitro pH-changes, which were either achieved by variation of PCO2 (2.8-8.3 kPa) at constant lactic acid concentration, or by addition of lactic acid (5-14 mmol X 1(-1) at constant PCO2. The PO2 at halfsaturation (P50) and the Hill's n were determined from O2-Hb dissociation curves (ODC) in a range between 20 and 80% SO2. Under standard conditions (T = 311 K, PCO2 = 5.33 kPa, pH = 7.4), the average P50 value was 4.66 +/- 0.05 kPa in rabbits, that is slightly higher than reported by others, and 5.17 +/- 0.03 kPa in cats. The average values of Hill's n were 2.91 +/- 0.02 and 2.95 +/- 0.03 for rabbits and cats, respectively. When plasma pH was varied by CO2, the resulting classical CO2 Bohr factor phi CO2 = delta lgP50/delta pH50 was distinctly higher in cats (-0.560 +/- 0.006, n = 25) than in rabbits (-0.504 +/- 0.014, n = 22), although in the latter species being even higher than reported elsewhere. Concomitant metabolic acidosis did not significantly affect phi CO2, but shifted the P50 at a given plasma pH to lower values. Substitution of lactic acid with equimolar amounts of sodium lactate left both phi CO2 and P50 unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

On the seemingly diminished CO2-Bohr effect in hypoxic chemodenervated rabbits.

In hypoxic rabbits, different levels of Pco2 before and after carotid chemodenervation were applied in order to get information about the acid-base status and the position of the O2-Hb dissociation curve (ODC). A CO2-induced change in pH caused a smaller change in the half-saturation pressure (P50) than was to be expected from the CO2-Bohr effect alone. Considering both, the numerically different CO2- and fixed acid-Bohr factors as well as the corresponding respiratory or metabolic pH changes, a method is presented to calculate the position of the ODC with high accuracy. From these considerations it can be derived that the seemingly diminished CO2-Bohr effect in hypoxic rabbits in vivo, especially after chemodenervation , is due more or less to accumulation of lactid acid. This leads to an increasing error if only the CO2-Bohr factor and the actual pH change are taken into account.

The involvement of expiratory termination in the vagally mediated facilitation of ventilatory CO2 responsiveness during hyperoxia.

In anaesthetized rabbits the influence of differential vagal cold blockade on the ventilatory response to inhaled CO2 during hyperoxia was investigated. Following total inactivation, the relationship between ventilation (V) and arterial PCO2 (PaCO2) was shifted to the left and steepened slightly over a range of modest hypercapnia, but was progressively flattened as hypercapnia intensified. The latter effect, suggestive of a vagally mediated facilitation of ventilatory CO2 responsiveness, was studied further. Differential vagal cold blockade to a temperature (5-11 degrees C) which abolished the Breuer-Hering inflation reflex (end-inspiratory tracheal occlusion no longer eliciting a prolongation of expiratory duration, TE) had no effect on V either during normocapnia or at a substantial level of hypercapnia. Only with further vagal cooling to 0 degrees C did the ventilatory depression during hypercapnia emerge, largely because TE failed to shorten in response to the hypercapnic stimulus. It is concluded that the integrity of expiratory-terminating mechanisms is crucial for the manifestation of the vagally mediated facilitation of V and its CO2 responsiveness which is evident during hyperoxic hypercapnia. A possible role is suggested for lung epithelial irritant receptors or for the tonic late-expiratory activity from pulmonary stretch receptors.

The role of the vagus nerves in the ventilatory response to lowered PaO2 with intact and eliminated carotid chemoreflexes.

In anaesthetized rabbits the influence of vagal cold-block on the ventilatory response to lowered arterial oxygen pressure was investigated. With intact carotid chemoreflexes, lowered PaO2 caused hyperventilation, which was progressively intensified with the degree of hypoxia, regardless of whether the alveolar PCO2 was uncontrolled or kept constant at the hyperoxic control. The V-PaO2 response was to a greater extent due to an increase of respiratory rate than to one of tidal volume. During hyperoxia, vagal cold-block caused a distinct increase in ventilation provided the alveolar PCO2 was not allowed to decrease. During moderate hypoxia, vagal block caused only a slight increase in ventilation, when PACO2 was not controlled, but a distinct decrease in ventilation, when PACO2 was maintained at the hyperoxic level. Without carotid chemoreflexes, lowered PaO2 did not change ventilation at any level, provided the vagus nerves were left intact. This was due to a substantial increase in respiratory rate counteracting a corresponding decrease in tidal volume. Then vagal block led to a ventilatory depression depending on the degree of hypoxia, which was due to a simultaneous decline in respiratory rate and tidal volume. It is concluded that during hypocapnic hypoxia the vagal stretch reflex primarily inhibits the carotid chemoreflex drive of ventilation. During normocapnic hypoxia, however, the mode of interaction between the peripheral and the central chemical drive has to be considered, which without vagal feed-back is occlusive. This occlusion appears to be counteracted by a vagal mechanism sensitive to CO2 in the airways--and possibly also to a lack of O2--, mainly shortening respiratory cycle duration.