K. Mückenhoff

Theophylline and hypoxic ventilatory response in the rat isolated brainstem-spinal cord.

We have used the isolated brainstem-spinal cord preparation of the neonatal rat to study the effects of theophylline on the ventilatory response to hypoxia. The brainstem-spinal cord was isolated from neonatal rats (0-4 days) and superfused with mock cerebrospinal fluid (CSF), equilibrated with a gas mixture (FO2, 0.90; FCO2, 0.02; FN2, 0.08; control CSF) at 27 degrees C. We recorded phrenic nerve discharge from C4 roots, using suction electrodes, and measured respiratory frequency (fR) and the amplitude of the integrated phrenic neurogram (integral of phr). We examined how theophylline and the specific adenosine antagonist, 8-p-sulfophenyltheophylline (SPT), modify the ventilatory response to hypoxia. The response during superfusion with hypoxic CSF (FO2, 0.06) consisted of a marked decrease in fR (to 60% of control) and a slight decline in integral of phr (to 85% of control). By contrast, in the presence of theophylline (30 mg/L = 165 microM) and SPT (5 mg/L = 15 microM) in the superfusate hypoxia reduced fR only moderately (to 87% of control) and exerted virtually no effect on integral of phr (105% of control). Theophylline and SPT attenuated the rate of decrease in fR and completely blocked the decrease in integral of phr. There was no difference between the effects of theophylline and those of SPT. The results suggest that theophylline attenuates hypoxic respiratory depression, and that this effect is mediated by the blockade of adenosine.

Role of the pons in hypoxic respiratory depression in the neonatal rat

The main purpose of this study was to evaluate the role of the pons in hypoxic respiratory depression (HRD) of the neonatal rat. Experiments were conducted using the isolated brainstem-spinal cord preparation of the neonatal rat (1-3 days old). The brainstem was transected at various levels. We found that ablation of the diencephalon decreased respiratory frequency (fR), and conversely, that ablation of the midbrain or pons increased fR. In the preparation with the pons intact (without the midbrain), hypoxia (superfusate PO2 = 56 mmHg) caused strong depression of respiratory activity, which was characterized by a steady decrease in fR and in integrated inspiratory burst amplitude (integral of Phr). In the preparation with the intact ventral pons (without midbrain and dorsal pons) we observed similar, though weaker, HRD. When the entire pons was ablated, integral of Phr was little depressed by hypoxia and thus, HRD was further attenuated. We conclude that the pons contributes importantly to the induction of hypoxic respiratory depression in the neonatal rat. Both the ventral and dorsal portions of the pons are involved in the control of hypoxic respiratory depression. In addition, we show that the respiratory modulatory functions of the diencephalon (facilitating) and midbrain (inhibitory) are already expressed at the time of birth.

Locus coeruleus neurones in vitro: pH-sensitive oscillations of membrane potential in an electrically coupled network

The response to hypercapnic acidosis (2-8% CO2, bath pH 7.8-7.2) was examined in whole cell recordings from neonatal (P1 to P5) rat Locus coeruleus (LC) neurones in the in vitro brainstem-spinal cord preparation exposed to low Ca2+ (0.2 mM)-high Mg2+ (5 mM). This medium suppressed chemical synaptic transmission and resulted in a pattern of subthreshold oscillations of membrane potential and rhythmic burst discharge which was synchronized throughout the network. The oscillation was suppressed, and the discharge of individual neurones desynchronized, by the gap junction uncoupler, carbenoxolone, indicating that in low Ca2+-high Mg2+ LC neurones form an electrically coupled network. Switching from 2 to 8% CO2 decreased the oscillation amplitude and increased its frequency. The oscillation was suppressed by external Cd2+ and by TTX. but persisted during injection into the cell soma of QX-314. We conclude that in LC neurones acidosis increases the frequency of a Ca2+- and Na+-dependent dendritic oscillator which is synchronized by gap junction coupling throughout the network. This coupling is retained during acidosis.

Thrombin stimulates Na+-H+ exchange across the human platelet plasma membrane.

We have investigated the release of protons from thrombin‐stimulated platelets. Addition of thrombin to suspensions of washed platelets resulted in fast liberation of H+. In the presence of 0.1 mM amiloride, a potent inhibitor of the Na+/H+ transport system, the amount of protons liberated was decreased by about 50%, and was further reduced to about 15% by 1 mM amiloride. Similar inhibition of H+ release was observed after Na+ in the incubating medium had been replaced by choline. We conclude that one of the earliest events in thrombin‐stimulated platelets consists of the activation of an Na+/H+ countertransport, which leads to an increase in intracellular pH.

Rhythms, synchrony and electrical coupling in the Locus coeruleus

Electrical coupling of neurones is believed to promote synchronized activity. It may, however, also be a requirement for the maintenance of endogenous rhythmic activity in some systems. In en bloc isolated brainstem-spinal cord of the neonatal rat simultaneous whole cell recordings from pairs of LC neurones (n = 47 pairs) disclosed for the most part strongly synchronized activity which could take the form of tonic spiking or phasic bursts. Simultaneous whole cell recording from LC neurones and glia also revealed synchronized waves of depolarization in 7 of 17 pairs. This synchrony was partly due to respiratory-phased synaptic input and partly due to mechanisms, which were not dependent on chemical synapses. The gap junction uncoupler carbenoxolone suppressed non-synaptic rhythmic activity in LC neurones, but did not suppress either respiratory-phased synaptic input to these neurones or their excitatory response to increased CO(2). We give preliminary direct evidence for the existence of a current pathway between LC neurones, which is inhibited by carbenoxolone. Within the LC nucleus carbenoxolone-sensitive electrical coupling, which may involve neurone-glia as well as neurone-neurone interactions, may be required not just for synchronization, but also for the maintenance of rhythm.

Synchronized rhythms in chemosensitive neurons of the locus coeruleus in the absence of chemical synaptic transmission

The activity of locus coeruleus (LC) neurons was examined in the en bloc isolated brainstem-spinal cord of the neonatal rat using paired whole cell or whole cell plus extracellular recording. In artificial cerebrospinal fluid (ACSF) LC neurons were synchronized by their respiratory innervation and in some neurons showing tonic or burst patterns of discharge these patterns of discharge could also be synchronized. Replacing ACSF with low Ca(2+)-high Mg(2+) generated synchronized rhythmic bursts which remained synchronized at high CO(2) (up to 20%). This rhythm was suppressed by TTX. Substitution of Ba(2+) for Ca(2+) in ACSF generated a synchronized rhythm which was TTX-insensitive. The frequency of this rhythm increased by 31+/-16% on raising CO(2) concentration from 2 to 10%. We conclude that the capacity of chemosensitive LC neurons to generate a synchronized rhythm depends on their electrical coupling, but not on chemical synaptic transmission.

Evidence for A role of Na+H+ exchange in platelets activated with calcium-ionophore A 23187

We have investigated the release of protons from human platelets and platelet aggregation induced by the calcium ionophore, A 23187. Addition of the ionophore to suspensions of washed platelets resulted in fast liberation of H+. In the presence of 0.2 mM amiloride, a potent inhibitor of Na+/H+ countertransport, the amount of protons liberated was decreased by 50% and was further reduced to about 10% by 1 mM amiloride. Similar inhibition of H+-release was observed after decreasing Na+ in the incubation medium. Both results suggest that increasing internal Ca2+ by the ionophore induces Na+/H+ exchange in human platelets. Platelet aggregation could be induced by adding the ionophore to the platelet suspension. This aggregation was inhibited by amiloride, at least when induced by low ionophore concentrations. The results suggest that stimulation of Na+/H+ exchange, and the concomitant increase in intraplatelet pH, are important mechanisms in platelet activation.

Measurements of lactate in exhaled breath condensate at rest and after maximal exercise in young and healthy subjects

Arterial lactate concentrations, taken as indicators of physical fitness, in athletes as well as in patients with cardio-respiratory or metabolic diseases, are measured invasively from arterialized ear lobe blood. Currently developed micro enzyme detectors permit a non-invasive measurement of hypoxia-related metabolites such as lactate in exhaled breath condensate (EBC). The aim of our study is to prove whether this technology will replace the traditional measurement of lactate in arterialized blood. Therefore, we determined the functional relation between lactate release in EBC and lactate concentration in blood in young and healthy subjects at rest and after exhausting bicycle exercise. During resting conditions as well as after exhausting bicycle exercise, 100 L of exhaled air along with blood samples from the ear lobe was collected after stationary load conditions in 16 healthy subjects. EBC was obtained by cooling the expired air volume with an ECoScreen I (FILT GmbH, Berlin) condenser. The analysis was performed within 90 min using an ECoCheck ampere meter (FILT GmbH, Berlin). Lactate measurements were performed using a bi-enzyme sensor after lactate oxidase-induced oxidation of lactate to pyruvate and H2O2. The rates of lactate release via the exhaled air were calculated from the lactate concentration, the volume and the collection time of the EBC. The functional relation of lactate release in exhaled air and lactate concentration of arterial blood was computed. At rest, the mean lactate concentration in arterialized blood was 0.93 ± 0.30 mmol L(-1). At a resting ventilation of 11.5 ± 3.4 L min(-1), the collection time for 100 L of exhaled air, Ts, was 8.4 ± 2.9 min, and 1.68 ± 0.40 mL EBC was obtained. In EBC, the lactate concentration was 21.4 ± 7.7 µmol L(-1), and the rate of lactate release rate in collected EBC was 4.5 ± 1.7 nmol min(-1). After maximal exercise load (220 ± 20 W), the blood lactate concentration increased to 10.9 ± 1.8 mmol L(-1) and the ventilation increased to 111.6 ± 21.4 L min(-1). The EBC collection time decreased to 3.9 ± 1.9 min, and 1.20 ± 0.44 mL EBC were obtained in the recovery period after termination of exercise. The lactate concentration in EBC increased to 40.3 ± 23.0 µmol L(-1), and the lactate release in EBC increased to 13.6 ± 8.6 nmol min(-1) (p < 0.01). Assuming a volume of 4.3 mL water in 100 L of exhaled air (saturated with water at 37 °C), we calculated a lactate release at rest of 11.5 ± 4.3 nmol min(-1) and 48.6 ± 30.7 nmol min(-1) (p < 0.01) after exhausting exercise. Detectable releases of lactate in exhaled breath condensate were found already under resting conditions. During exhausting external load on a bicycle spiroergometer, an increase in the lactate concentration was found in arterialized blood along with an increased lactate release in EBC. The correlation between expiratory lactate release via EBC and lactate concentration in arterialized blood is studied in pursuing investigations.

Fast measurement of the CO2 partial pressure in gases and fluids.

AbstractA CO2-electrode system consisting of a membrane covered pH electrode, an electronic antilog modul and a special electronic analog circuit is described. Since the electrode output signal is a logarithmic function of the CO2 partial pressure the output signal of the antilog module is proportional to the CO2 partial pressure. The time course of the electrode signal has been analyzed after a step change of