Differential effects of apamin on neuronal excitability in the nucleus tractus solitarii of rats studied in vitro.

We demonstrated previously that microinjection of the calcium-dependent potassium channel antagonist, apamin, into the nucleus tractus solitarius (NTS) in vivo potentiated the baroreceptor reflex mediated bradycardia but attenuated the cardiopulmonary reflex. The latter result was surprising since, intuitively, potassium channel blockade would be expected to increase neuronal excitability leading to reflex potentiation. The aim of this in vitro study was to investigate possible neuronal mechanisms to explain our in vivo observations. Transverse brainstem slices of rat were cut at the level of area postrema and recordings were made from 36 NTS neurones in whole-cell mode. The neurones were classified into three groups, based on their response to apamin (10 nM). Each group had a similar resting membrane potential (RMP; -55 +/- 1 mV; n = 36) and input resistance (404 +/- 20 M omega; n = 36). (1) In 15/36 neurones, apamin decreased the number of spikes evoked during injection of positive current by 37 +/- 6%; this was associated with a concomitant fall in input resistance of 12 +/- 2% (P < 0.05). Stimulation of the ipsilateral tractus solitarius evoked EPSP-IPSP complexes in nine of the 12 neurones tested; the inhibitory components were increased in amplitude, at a holding potential of -46 mV, from -1.7 +/- 0.4 to -3.2 +/- 0.6 mV (P < 0.01) in the presence of apamin, while the EPSPs were unaffected. All three of these effects were bicuculline (10 microM) sensitive. (2) In 8/36 neurones, apamin increased the number of spikes evoked during injection of positive current by 27 +/- 8%, but affected neither RMP nor input resistance. Only one of five neurones tested demonstrated a synaptically evoked EPSP-IPSP complex. The remaining four neurones displayed a single evoked EPSP, the amplitudes of which were unaffected by apamin. (3) In the remaining neurones (13/36), apamin affected neither responsiveness to positive current injection, RMP, nor input resistance. Six of 12 neurones demonstrated synaptically evoked EPSP-IPSP complexes; at a holding potential of -46 mV, apamin increased the IPSP component from -2.6 +/- 1 to -3.6 +/- 0.8 mV (P < 0.05), while the EPSPs were unaffected. In conclusion, apamin can both increase and decrease NTS neuronal excitability: the former reflecting blockade of channels on the recorded neurone; the latter may possibly result from an increase in GABA release by interneurones impinging onto the recorded neurone. The possibility of a differential distribution of apamin-sensitive channels in sub-populations of NTS neurones subserving different reflexes is discussed.

Design and synthesis of potent, selective, and orally bioavailable tetrasubstituted imidazole inhibitors of p38 mitogen-activated protein kinase.

Novel potent and selective diarylimidazole inhibitors of p38 MAP (mitogen-activated protein) kinase are described which have activity in both cell-based assays of tumor necrosis factor-alpha (TNF-alpha) release and an animal model of rheumatoid arthritis. The SAR leading to the development of selectivity against c-Raf and JNK2alpha1 kinases is presented, with key features being substitution of the 4-aryl ring with m-trifluoromethyl and substitution of the 5-heteroaryl ring with a 2-amino substituent. Cell-based activity was significantly enhanced by incorporation of a 4-piperidinyl moiety at the 2-position of the imidazole which also enhanced aqueous solubility. In general, oral bioavailability of this class of compounds was found to be poor unless the imidazole was methylated on nitrogen. This work led to identification of 48, a potent (p38 MAP kinase inhibition IC50 0.24 nM) and selective p38 MAP kinase inhibitor which inhibits lipopolysaccharide-stimulated release of TNF-alpha from human blood with an IC50 2.2 nM, shows good oral bioavailability in rat and rhesus monkey, and demonstrates significant improvement in measures of disease progression in a rat adjuvant-induced arthritis model.

Comparison of cardiorespiratory reflexes in NK1 receptor knockout, heterozygous and wild-type mice in vivo.

Neurokinin-1 receptors (NK1) are present within the nucleus of the solitary tract, a nucleus which plays a vital role in cardiovascular and respiratory homeostasis. We compared the efficacy of the baroreceptor and pulmonary chemoreflexes between NK1 knockout, heterozygous and wild-type urethane-anaesthetised mice. The magnitude of the baroreceptor reflex mediated bradycardia, induced by a phenylephrine induced pressor response, was significantly greater in NK1 knockout mice (P < 0.001) compared to heterozygous and wild-type animals. In comparison, administration of an NK1 antagonist, CP-99,994 (1.5 mg/kg i.v.) to wild-type animals, had no significant effect on baroreceptor reflex performance. In contrast to the baroreceptor reflex, there were no significant differences in the magnitude of the reflex evoked falls in heart rate, arterial pressure, or respiratory depression between the three groups of mice when the pulmonary chemoreflex was evoked with right atrial injections of phenylbiguanide. It is concluded that the baroreceptor reflex pathway over-compensates for the lack of NK1 receptors in knockout mice. Plausible mechanisms accounting for the enhanced baroreceptor reflex responsiveness in NK1 knockout animals are discussed.

K+ channel blockade in the NTS alters efficacy of two cardiorespiratory reflexes in vivo.

We investigated the role of potassium conductances in the nucleus of the solitary tract (NTS) in determining the efficacy of the baroreceptor and cardiopulmonary reflexes in anesthetized rats. The baroreceptor reflex was elicited with an intravenous injection of phenylephrine to evoke a reflex bradycardia, and the cardiopulmonary reflex was evoked with a right atrial injection of phenylbiguanide. Microinjection of two Ca-dependent potassium channel antagonists (apamin and charybdotoxin) into the NTS potentiated the baroreceptor reflex bradycardia. This may reflect the increased neuronal excitability observed previously in vitro with these blockers. In contrast, the Ca-dependent potassium channel antagonists attenuated the cardiopulmonary reflex, whereas voltage-dependent potassium channel antagonists (4-aminopyridine and dendrotoxin) attenuated both the baro- and cardiopulmonary reflexes when microinjected into the NTS. The possibility that the reflex attenuation observed indicates a predominant distribution of certain potassium channels on gamma-aminobutyric acid interneurons is discussed.

Cardiorespiratory reflexes in mice.

The various transgenic strains of mice make this species an attractive experimental model. We compared qualitatively some cardiorespiratory reflexes in two different preparations of mouse: in vivo urethane anaesthetised and a working heart-brainstem preparation (WHBP). Cardiorespiratory reflexes were evoked by stimulating baroreceptors, pulmonary vagal C fibres and cardiac receptors in both preparations, while peripheral chemoreceptors were also stimulated in the WHBP. In anaesthetised mice, activation of baroreceptors, pulmonary C fibres and cardiac receptors evoked an atropine-sensitive bradycardia (range: 21-414 bts/min) and depressed ventilation. A reflex fall in arterial pressure was also observed during pulmonary C fibre and cardiac receptor stimulation. Similar reflex bradycardia (range 81-164 bts/min) and respiratory responses were observed in the WHBP following stimulation of baro-, pulmonary C fibre and cardiac receptors. Additionally, sodium cyanide stimulation of peripheral chemoreceptors in the WHBP produced an atropine-sensitive bradycardia and increased respiratory frequency and amplitude. Thus, the cardiorespiratory reflex responses elicited in the mouse are similar to those reported in other species. It is concluded that the qualitatively similar reflex performances between the in vivo anaesthetised mouse and the WHBP make the latter an adequate model for studying central mechanisms controlling the cardiorespiratory system.

Angiotensin II receptors within the nucleus of the solitary tract mediate the developmental attenuation of the baroreceptor vagal reflex in pre-weaned rats.

This study tested the hypothesis that baroreceptor vagal reflex (BVR) attenuation in developing rats, which occurs between postnatal ages (P) of 10 to 20 days old, is due to a central action of angiotensin II (Ang II). In urethane or halothane anaesthetised mature (P > 45) or pre-weaned rats (P14-18), BVR sensitivity was estimated as the ratio between the fall in heart rate and the increase in arterial pressure induced by i.v. phenylephrine. An Ang II AT1 receptor antagonist, losartan, was administered intra-venously (i.v.) or microinjected into brainstem structures. In pre-weaned rats BVR sensitivity was increased significantly by losartan (5 mg/kg; urethane anaesthesia: p < 0.01; halothane anaesthesia: p < 0.05) while a larger dose (10 mg/kg) was ineffective in mature animals. In pre-weaned rats, microinjection of losartan (500 pmol) into the nucleus tractus solitarii (NTS) but neither area postrema nor subjacent nuclei, reversibly increased the sensitivity of BVR (+89 +/- 19%; p < 0.01, n = 12). Microinjection of losartan (500 or 1500 pmol) into the NTS of mature rats did not change the BVR. An AT2-antagonist, PD123-319 did not restore the BVR sensitivity in pre-weaned rats. Thus, AT1 receptors located within the NTS play a pivotal role in the developmental attenuation of the BVR in pre-weaned rats.

Inhibition of human leukocyte elastase. 1. Inhibition by C-7-substituted cephalosporin tert-butyl esters.

Time-dependent inhibitors of the enzyme human leukocyte elastase have been developed based on the cephem nucleus. A series of cephalosporin tert-butyl esters has been examined, and the activity of these compounds has been found to be very sensitive to C-7 substituents, with small, alpha-oriented, electron-withdrawing groups showing greatest activity. Additionally, the oxidation state of the sulfur atom has been found to play a role in potency, with sulfones showing considerably greater activity than the corresponding sulfides or beta-sulfoxides. The alpha-sulfoxides were inactive.