The anti-emetic effects of CP-99,994 in the ferret and the dog: role of the NK1 receptor.

1. The selective NK1 receptor antagonist, CP-99,994, produced dose-related (0.1-1.0 mg kg-1, s.c.) inhibition of vomiting and retching in ferrets challenged with central (loperamide and apomorphine), peripheral (CuSO4) and mixed central and peripheral (ipecac, cisplatin) emetic stimuli. 2. Parallel studies with the enantiomer, CP-100,263 (1 mg kg-1, s.c.), which is > 1,000 fold less potent as a NK1 antagonist, indicated that it was without significant effect against CuSO4, loperamide, cisplatin and apomorphine-induced emesis. Against ipecac, it inhibited both retching and vomiting, expressing approximately 1/10th the potency of CP-99,994. 3. The 5-HT3 receptor antagonist, tropisetron (1 mg kg-1, s.c.) inhibited retching and vomiting to cisplatin and ipecac, but not CuSO4 or loperamide. 4. CP-99,994 (1 mg kg-1, i.v.) blocked retching induced by electrical stimulation of the ventral abdominal vagus without affecting the cardiovascular response, the apnoeic response to central vagal stimulation or the guarding and hypertensive response to stimulation of the greater splanchnic nerves. CP-99,994 (1 mg kg-1, i.v.) did not alter baseline cardiovascular and respiratory parameters and it failed to block the characteristic heart rate, blood pressure and respiratory rate/depth changes in response to i.v. 2-methyl-5-HT challenge (von Bezold-Jarisch reflex). 5. Using in vitro autoradiography, [3H]-substance P was shown to bind to several regions of the ferret brainstem with the density of binding in the nucleus tractus solitarius being much greater than in the area postrema. This binding was displaced by CP-99,994 in a concentration-related manner. 6. In dogs, CP-99,994 (40 micrograms kg-1 bolus and 300 micrograms kg-1 h-1, i.v.) produced statistically significant reductions in vomiting to CuSO4 and apomorphine as well as retching to CuSO4. 7. Together, these studies support the hypothesis that the NK1 receptor antagonist properties of CP-99,994 are responsible for its broad spectrum anti-emetic effects. They also suggest that CP-99,994 acts within the brainstem, most probably within the nucleus tractus solitarius although the involvement of the area postrema could not be excluded.

CP-96,345 antagonism of NK1 receptors and smoke-induced protein extravasation in relation to its cardiovascular effects.

The effects of the non-peptide NK1 receptor antagonist, CP-96,345, on cardiovascular homeostasis were investigated in conscious and anaesthetized rats in vivo and on heart function and muscle tonicity of vessels in vitro. CP-96,345 and its enantiomer, CP-96,344, which does not exhibit NK1 receptor-blocking activity when tested at a concentration of 1 microM, significantly decreased blood pressure in conscious rats at a dose of 0.32 mg/kg i.v. CP-96,345 and CP-96,344 additionally reduced heart rate at doses of 1 and 3.2 mg/kg, respectively. Studies in anaesthetized rats showed that ganglionic blockade did not modify the decreases in blood pressure and heart rate elicited by CP-96,345. In the isolated guinea-pig heart, CP-96,345 and CP-96,344 exerted negative chronotropic effects at 10(-7) M; negative inotropic effects were observed at 10(-6) M. At 10(-5) M, both CP-96,345 and CP-96,344 decreased the amplitude of contraction of the rat portal vein, whereas at 10(-4) M, both compounds increased the frequency of contraction of this vessel. CP-96,345, at 5 x 10(-8) M, caused relaxation of precontracted pig coronary arteries. Since both CP-96,345 and CP-96,344 produced similar changes in haemodynamics and in the contractility of vascular and cardiac tissue, the cardiovascular effects of CP-96,345 are probably not related to NK1 receptor antagonism. As only the enantiomer with NK1 antagonistic activity inhibited cigarette smoke-induced plasma protein extravasation in rat trachea, CP-96,345 remains a useful tool for elucidating NK1 receptor-mediated responses, provided CP-96,344 is included as control.

Early toxicity of recombinant interleukin-2 in cats.

Studies were conducted in cats to determine whether this species could serve as an animal model for the therapeutic toxicity of recombinant interleukin-2 in humans, and to establish the role of the area postrema in the causation of recombinant interleukin-2-induced vomiting. Injections of recombinant interleukin-2 (3.6 x 10(6) IU/kg, i.m.), given once every 24 hr for one to three days, evoked repeated vomiting in 4 out of 6 area postrema intact cats and in 3 out of 3 area postrema-ablated cats. These results suggest that the area postrema is not essential for the emetic action of recombinant interleukin-2. In anesthetized intact cats, no remarkable changes in ventilation, blood pressure, heart rate or blood pH were observed over 4.5 to 54 hr of continuous physiological recording after i.v. injections of recombinant interleukin-2 to a total dose as high as 27 x 10(6) IU/kg. Cat lymphocytes responded appropriately to the cytokinetic action of human recombinant interleukin-2.

Chemosensitivity of medullary neurons in explant tissue cultures.

To determine whether cultured medulla contains chemosensitive neurons which are excited by CO2 and fixed acid and whether this function is specific to the ventral medulla, tissue explants of ventral and dorsal medulla were prepared from neonatal rats and incubated for two to three weeks. Cultures were superfused with artificial cerebrospinal fluid, maintained at 37 degrees C, and pH of the superfusate was varied either with PCO2 (14-71 Torr) at constant HCO3- (22 mM) or HCO3- (10-30 mM) at constant PCO2 (35 Torr). Spontaneous action potentials were recorded extracellularly in 51 ventral and 23 dorsal medullary neurons. Ventral medullary neurons exhibited a steady baseline firing frequency of 4 +/- 0.8 Hz. In contrast, dorsal medullary neurons exhibited two different patterns of spontaneous activity: 11 fired continuously (7.2 +/- 1.4 Hz) while 12 fired with a bursting pattern. Burst duration was 0.80 +/- 0.14 min and cycle time was 1.74 +/- 0.43 min. Decreasing pH with CO2 caused an increase in the activity of 10 of 27 ventral medullary neurons and two of six dorsal medullary neurons with a mean response of 7.5 Hz/pH unit. Varying pH by changing HCO3- had no effect on firing frequency. These results demonstrate that: (i) chemosensitive neurons are present in both ventral and dorsal medullary explant cultures; (ii) these cells only respond to changes in pH induced with CO2; and (iii) about half of the dorsal medullary neurons fire spontaneously with a regular bursting pattern of activity.

Anticonvulsant activity of the glial GABA uptake inhibitor, THAO, in chemical seizures.

The antiseizure activity of the glia-selective GABA uptake inhibitor, 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-c]azepin-3-ol (THAO), was evaluated in rats in models of acute chemoconvulsion. In these experiments, intracerebroventricular administration of the drug 30 min prior to testing in doses between 100-750 micrograms provided protection against maximal pentylenetetrazol seizures and increased the latency to isonicotinic acid hydrazide seizures. Pentylenetetrazol seizure thresholds, in contrast, were not significantly elevated. The ability of THAO to suppress tonic but not generalized minor seizures suggests that it may block seizure spread.

Anticonvulsant activity of intracerebroventricularly administered glial GABA uptake inhibitors and other GABAmimetics in chemical seizure models.

The antiseizure activities of glial or neuronal GABA uptake inhibitors and GABA agonists were compared following intracerebroventricular administration in 2 acute models of chemoconvulsion in rats. The glia-selective GABA uptake inhibitor, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO), given at doses of 100-750 micrograms, i.c.v., protected against maximal pentylenetetrazol (PTZ) seizures and increased the latency to isonicotinic acid hydrazide (INH) seizures for at least 1 h following central administration. THPO failed to increase PTZ seizure thresholds. In contrast, the more potent partly glia-selective GABA uptake inhibitor, cis-4-hydroxynipecotic acid (30-300 micrograms), which is also a substrate for neuronal and glial transport systems, protected only 33% of rats against PTZ-induced tonic extension and had no effect on INH seizure latency. The neuron-selective uptake inhibitor L-2,4-diaminobutyric acid (DABA) at 1500 micrograms exhibited anti-PTZ activity initially and then, after a delay, produced proconvulsant behavior and spontaneous myoclonus in some animals. Intracerebroventricular injection of the GABA receptor agonist, muscimol, at toxic doses, gave rise to mixed anticonvulsant (INH seizures) and proconvulsant (PTZ seizure thresholds) effects. The results suggest that THPO, of the 4 compounds tested, possesses significant anticonvulsant activity. Its ability to suppress tonic but not generalized minor seizures suggests that it may block seizure spread.

Persistent reversal of tolerance to anticonvulsant effects and GABAergic subsensitivity by a single exposure to benzodiazepine antagonist during chronic benzodiazepine administration.

The persistence of benzodiazepine antagonists in reversing neuronal and behavioral tolerance during chronic diazepam exposure was examined in rodents by investigating the time course for antagonist-induced alterations in iontophoretic sensitivity to gamma-aminobutyric acid on dorsal raphe neurons and the re-emergence of anticonvulsant efficacy to bicuculline-induced seizures. In these studies, exposure to Ro15-1788 resulted in the persistent reversal of GABAergic subsensitivity and restoration of anticonvulsant actions of diazepam despite the continued presence of diazepam in the rats. Reversal of tolerance appears to persist for up to 7 days after a single exposure to benzodiazepine antagonists.

Effects of pentobarbital and flurazepam on respiratory neurons in undrugged cats.

Two hypnotic drugs known to enhance GABAergic transmission, a barbiturate (pentobarbital) and a benzodiazepine (flurazepam), were applied locally to respiratory-related neurons (RN) located in the ventral respiratory area in the medulla of non-anaesthetized cats which were either decerebrated or chronically implanted. Pentobarbital applied iontophoretically depressed the spontaneous discharge rate of most RN tested as well as the increase in firing of RN discharge induced by iontophoretic application of glutamate; pentobarbital also potentiated inhibition induced by iontophoresed GABA. Flurazepam applied by iontophoresis or pressure pulses depressed a minority of RN tested and did not enhance GABA-induced inhibition. These results suggest: that inhibition of RN activity through GABAergic mechanisms can be affected by drugs which act at the chloride ionophore but not those acting indirectly through the benzodiazepine binding site, and the effects of the agents on medullary neuronal activity are independent of an effect on the states of consciousness or on structures rostral to the medulla.

Time course for development of anticonvulsant tolerance and GABAergic subsensitivity after chronic diazepam.

The time courses for development of neuronal and behavioral tolerance to diazepam (DZ) were estimated in rats continuously exposed to low levels of DZ for 3, 7, 14 or 21 days. Microiontophoretic sensitivity of dorsal raphe neurons to gamma-aminobutyric acid (GABA) was initially facilitated after short-term exposure to DZ released from implanted capsules for up to 3 days but returned to control levels by 7 days postimplantation and continued to decrease thereafter. GABAergic sensitivity remained depressed for a minimum of 5 days following removal of DZ capsules. To obtain a behavioral measure of tolerance, the anticonvulsant activity of DZ against bicuculline-induced seizures was also assessed. Rats studied 3 days after capsule implantation showed a significant elevation in seizure threshold. Seizure liability returned to control levels ca. 7 days after chronic treatment was initiated. These results indicate that tolerance to anticonvulsant efficacy against bicuculline seizures are temporally related to the onset of reduced GABA sensitivity on dorsal raphe neurons during prolonged exposure to DZ.

Excitability of 'silent' respiratory neurons during sleep-waking states: an iontophoretic study in undrugged chronic cats.

An iontophoretic study of respiratory-related neurons (RN) was conducted in the medullary ventral respiratory area of chronically implanted, undrugged cats during states of sleep and wakefulness. Most RN recorded were unaffected by sleep-wake states but a few RN decreased their firing rate during sleep (sleep sensitive cells). The excitability of RN was assessed in the different states by local application of L-glutamate. Glutamate iontophoresis revealed the presence of 5 cells which were silent during sleep and completely or mostly silent during undisturbed wakefulness but always discharged with a respiratory-modulated pattern of the expiratory type in response to glutamate application. Arousing stimuli induced spontaneous firing of these cells and REM sleep reduced glutamate effectiveness. It was concluded that silent RN and RN which become inactive during sleep permanently receive subthreshold respiratory-modulated inputs which are amplified or depressed by state-dependent tonic inputs.

Tolerance to anti-pentylenetetrazol effects following chronic diazepam.

Continuous release of diazepam from subcutaneously implanted silastic capsules provided significant protection against pentylenetetrazol seizures in rats for up to 3 weeks. However, the degree of protection seen after 3 weeks of exposure to diazepam was significantly less than after 1 h. These data suggest that continuous exposure to constant low levels of diazepam results in the development of partial tolerance which is not sufficient to eliminate significant anticonvulsant effects.

Continuous release of diazepam: electrophysiological, biochemical and behavioral consequences.

Neuronal GABAergic sensitivity was assessed using electrophysiological, biochemical and behavioral techniques following the continuous release and maintenance of relatively constant brain levels of diazepam for greater than or equal to 21 days. Our studies indicate that long-term exposure to diazepam results in: (1) a decrease in iontophoretic sensitivity to GABA in the dorsal raphe nucleus, (2) an increase in the affinity of the GABA recognition site in brain tissue and (3) an increase in susceptibility to bicuculline-induced seizures in the intact animal. Since the decrease in GABAergic responsiveness was observed in the presence of measurable levels of diazepam, it was concluded that this subsensitivity phenomenon is associated with tolerance and not with withdrawal effects of the benzodiazepines.

Spontaneous and RO 15-1788-induced reversal of subsensitivity to GABA following chronic benzodiazepines.

Chronic daily injections of diazepam (5 mg/kg i.p.) for 21-30 days have previously been shown to cause a selective subsensitivity to microiontophoretically applied GABA in serotonergic dorsal raphe neurons in the rat. Following termination of chronic benzodiazepine treatment, GABAergic sensitivity remained depressed for up to 96 h even though pharmacologically active concentrations of diazepam and its active metabolites could no longer be detected in brain tissue. In contrast, a single injection of the specific benzodiazepine antagonist, RO 15-1788, given 22 h prior to electrophysiological recording, restored GABAergic sensitivity to the control range. Sensitivity of dorsal raphe neurons to serotonin was not altered by either chronic treatment with or withdrawal from diazepam. These results indicate that (1) RO 15-1788 can accelerate the time course of withdrawal and (2) administration of this benzodiazepine antagonist can induce a persistent change in GABAergic responsiveness.

beta-Adrenoceptor blockade spares chemoreceptor responsiveness to hypoxia.

The effect of beta-adrenoceptor blockade on the carotid body chemoreceptor response to hypoxia was assessed in anesthetized and paralyzed cats. Propranolol, atenolol and ICI 118,551 each abolished the enhancement of chemoreceptor activity produced by i.v. infusion of exogenous isoproterenol; however, the blocking drugs did not significantly diminish the increase in chemoreceptor neural discharge induced by hypoxia. These results do not support the hypothesis that beta-adrenergic receptors play an essential role in the chemoreceptor response to oxygen deprivation.

Chronic benzodiazepine treatment decreases postsynaptic GABA sensitivity.

Benzodiazepines exert most of their pharmacological effects by a selective facilitation of the postsynaptic actions of GABA. Clinical, behavioural and electrophysiological studies have shown reduced drug response following chronic benzodiazepine administration. We present here electrophysiological evidence for decreased postsynaptic sensitivity to GABA following chronic benzodiazepine administration as measured by the direct iontophoretic application of GABA and serotonin onto serotonergic cells in the midbrain dorsal raphe nucleus (DRN), known to receive GABAergic input. The subsensitivity to GABA was found to be dose dependent and was seen when diazepam administration was three weeks or longer. Further, acute injection of the specific benzodiazepine antagonist, Ro15-1788, was found to reverse rapidly the decrease in GABA sensitivity observed in chronically diazepam-treated animals without altering GABA sensitivity in vehicle-treated rats. Decreased response to chronic benzodiazepines does not appear to be consistently related to alterations in the number or affinity of receptors for benzodiazepines. Our studies of radioligand-binding showed a decrease in the ability of GABA to enhance benzodiazepine binding in cerebral cortical membranes from chronic diazepam-treated animals without significant changes in benzodiazepine binding site density or affinity.

Apneustic respiration normalized by an atypical cholinergic drug action in cats.

Cats were anesthetized with a mixture of pentobarbital and barbital given intraperitoneally and vagotomized. All animals were pretreated with atropine i.v. which blocked conventional muscarinic receptors and at the same time revealed atropine-insensitive sites whose stimulation by the cholinergic drug, bethanechol, was earlier found to produce respiratory analepsis. Respiration was recorded by pneumotachograph and intrapleural cannula. Apneustic breathing was produced by placing electrolytic lesions in the pneumotaxic area or by injecting a local anesthetic agent into the cerebrospinal fluid. Experiments were performed under isocapnic conditions. Bethanechol, administered intracerebro-ventricularly, restored regular breathing and increased the tidal volume in a dose-related manner within minutes of its injection. In cats with lesions, the expiratory pause generally decreased first and was followed by shortening of the inspiratory plateau. In cats made apneustic by intracerebro-ventricular injection of procaine, bethanechol acted competitively to normalize the distorted waveshape. The central excitant drug pentylenetetrazol was given intravenously for comparison with bethanechol. Apneustic breathing was partially corrected by pentylenetetrazol through a facilitatory influence on the brain stem interpreted to be independent of its convulsant action. These experiments demonstrate correction of pathological breathing by two chemically unrelated agents which share the ability to increase central excitatory state.

Central respiratory and circulatory depression caused by intravascular saxitoxin.

1 In cats anaesthetized with pentobarbitone and vagotomized, observations were made on the phrenic nerve action potential and the diaphragm electromyogram (EMG) at constant end-tidal Pco(2). Arterial blood pressure was stabilized by intravenous infusions of noradrenaline.2 Intravenous administration of saxitoxin (STX) initially abolished respiratory activity in the EMG and caused a slowing of oscillation in the central phrenic neurogram. Additional STX produced apneustic phrenic discharges followed by a progressive loss of nerve action potentials.3 The inspiratory centre in the medulla oblongata was stimulated electrically to evoke a sustained phrenic nerve discharge. STX, given intravenously, resulted in the elimination of spontaneous nerve activity without interfering with the evoked response.4 The cephalic intravascular infusion of STX into a carotid or vertebral artery depressed spontaneous respiratory activity while sparing EMG activity evoked by electrical stimulation of the intact phrenic nerve.5 Spontaneous respiratory discharge in the phrenic nerve was eliminated by smaller doses of STX administered intra-arterially than were required intravenously. In addition, onset of and recovery from neural silence occurred faster following intra-arterial injection of STX.6 Depressant effects on arterial blood pressure coincided with those on respiration when STX was given intra-arterially.7 An electrophysiological assay on frog sartorius muscle was used to measure STX in the cerebrospinal fluid. Levels of STX detected were proportional to amounts of the toxin infused intra-arterially.8 It is concluded that STX exchanges rapidly between blood and brain to bring about central depression and this adds to its peripheral paralytic actions.

Dynamics of respiratory VT response to isocapnic pHa forcing in chemodenervated cats.

Arterial blood pH (pHa) was continuously monitored in decerebrate or pentobarbital-anesthetized cats with a rapidly responding hydrogen ion sensor inserted into the aorta. Alveolar carbon dioxide partial pressure and pHa were controlled independently during infusions of 1 N NaHCO3 or 0.5 N HCl into the inferior vena cava. Shifts in pHa up to 0.3 unit were effected isocapnically within 2.5-20 s over a working pHa range of 6.9-7.7. Before carotid sinus neurotomy, average onset latency of the tidal volume (VT) response to acid and alkaline pHa shifts was less than 5 S and the average VT response half time was less than 8.5 s regardless of whether the vagus nerves had been interrupted. After carotid sinus neurotomy, the deltaVT onset latency was approximately doubled, whereas the response half time was prolonged about eightfold on the average. Subsequent vagotomy tended further to increase the responding time lag. Nevertheless, the minimum response latency after peripheral chemodenervation was less than the deltapHa forcing rise time. It is concluded that the central chemoreceptors promptly sense pH change in the arterial blood and that neural processes adjust the time course of the respiratory response through the VT controller.