Safety and efficacy of ketamine xylazine along with atropine anesthesia in BALB/c mice

Anesthetics are an indispensable prerequisite for surgical intervention and pharmacological animal studies. The objective of present study was to optimize the dose of ketamine (K) and xylazine (X) along with atropine sulfate (A) in order to achieve surgical tolerance in BALB/c mice. Several doses of ketamine (100, 150, 200 mg/kg) and xylazine (10, 15, 20 mg/kg) were mixed and combination of nine doses (K/X: 100/10, 100/15, 100/20, 150/10, 150/15, 150/20, 200/10,200/15,200/20) were evaluated (n=9 per combination). A constant dose of atropine (0.05 mg/kg) was also used to counter side effect. Time-related parameters were evaluated on the basis of reflexes. KX at dose 200/20 mg/kg produced surgical tolerance in all nine mice with duration 55.00±6.87 minutes. The induction time 0.97±0.09 minutes, sleeping time 90.67±5.81 minutes and immobilization time (102.23±6.83 minutes) were significantly higher than all combination. However, this combination was considered unsafe due to 11 % mortality. While, KX at dose 200/15 mg/kg results in none of the mortality, so was considered as safe. Moreover, this combination produces surgical tolerance in 89 % mice with duration (30.00±7.45 minutes). It was concluded that KX at dose 200/15 mg/kg along with atropine 0.05 mg/kg is safe for performing surgical interventions in BALB/c mice.

Aqueous extracts of Cordyceps militaris (Ascomycetes) lower the levels of plasma glucose by activating the cholinergic nerve in streptozotocin-induced diabetic rats.

In our previous research, Cordyceps militaris (CM) had a hypoglycemic effect in normal rats. In this study we wanted to elucidate whether CM also had an effect on diabetic rats. Twelve rats with streptozotocin-induced diabetes were separated randomly into 2 groups. First, aqueous extracts of CM 10 mg/kg (CM group) or saline (control group) was fed to the rats; then the plasma glucose levels were assayed. Second, the signaling proteins IRS-1 and GLUT-4 collected from the muscle were detected. Finally, another 2 groups of rats were injected with atropine 0.1 mg/kg intraperitoneally just before the CM/saline feeding, and the assays mentioned above were repeated. Blood glucose decreased 7.2% in the CM group but only 1.5% in the control group (P < 0.05). The IRS-1 signal was 2.9-fold higher than actin in the CM group but only 0.8-fold higher in the control group (P < 0.005). In GLUT-4 signal, the difference was 1.7- vs. 0.6-fold, respectively, compared with actin (P < 0.05). However, atropine injection made CM-induced hypoglycemia or elevation of IRS-1 and GLUT-4 not significant. In conclusion, CM had a hypoglycemic effect in diabetic rats and atropine blocked it. Therefore, the cholinergic activation also was considered to be involved in the hypoglycemic effect of CM in rats with streptozotocin-induced diabetes.

Prefrontal cortex and reversion of atropine-induced disruption of the degraded contingency effect by antipsychotic agents and N-desmethylclozapine in rats.

Interactive context processing is a cognitive ability that is altered in psychotic states, including schizophrenia. This deficit has been linked to prefrontal cortical dysfunction in humans. The degraded contingency effect (DCE) is a simple form of interactive context processing by which contextual information interferes with a target conditioned stimulus for control over conditioned responding. We have previously shown that the DCE was disrupted by the muscarinic receptor antagonist atropine and that this disruption was specifically restored by cholinergic drugs displaying an antipsychotic-like profile, such as physostigmine or xanomeline. The DCE was selectively associated with an increase in Fos immunoreactivity in the medial prefrontal cortex (mPFC), an increase that was not observed in the presence of atropine. Here, we set out to test the actions of typical, atypical and potential antipsychotics on atropine-induced disruption of the DCE and the related mPFC Fos-immunoreactivity profile. Low doses of haloperidol, olanzapine, clozapine and N-desmethylclozapine reversed atropine-induced disruption of the DCE, but with different dose-dependent curves (linear shapes for haloperidol and N-desmethylclozapine, inverted U shapes for olanzapine and clozapine). The level of Fos within the mPFC paralleled the pharmacological profile of the different drugs. Compared to contingent control groups, an increased level of Fos immunoreactivity within the mPFC was observed only with doses that reversed atropine-induced disruption of the DCE. These results suggest that the deficit of interactive context processing, which is a hallmark of psychotic states, might originate from a mere deficit of fundamental associative processes. This deficit might result from a cholinergic blockade of the PFC.

Identification of atropine- and P2X1 receptor antagonist-resistant, neurogenic contractions of the urinary bladder.

Acetylcholine and ATP are excitatory cotransmitters in parasympathetic nerves. We used P2X1 receptor antagonists to further characterize the purinergic component of neurotransmission in isolated detrusor muscle of guinea pig urinary bladder. In the presence of atropine (1 microM) and prazosin (100 nM), pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (0.1-100 microM) and suramin (1-300 microM) inhibited contractions evoked by 4 Hz nerve stimulation in a concentration-dependent manner (IC50 of 6.9 and 13.4 microM, respectively). Maximum inhibition was 50-60%, which was unaffected by coadministration of the ectonucleotidase inhibitor ARL67156 (6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP) (100 microM). The remaining responses were abolished by tetrodotoxin (1 microM). PPADS and suramin also reduced contractions to exogenous ATP (300 microM) by 40-50%, but abolished those to the P2X1 agonist alpha,beta-methyleneATP (alpha,beta-meATP) (1 microM). The P2X1 antagonists reactive blue 2, NF279 (8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)] bis-1,3,5-naphthalenetrisulfonic acid), MRS2159 (pyridoxal-alpha5-phosphate-6-phenylazo-4'-carboxylic acid) (100 microM), and NF449 [4,4',4,4-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid] (3 microM) abolished contractions to alpha,beta-meATP (1 microM; n = 4-5), but only reduced contractions evoked by 4 Hz nerve stimulation by approximately 40-60% (n = 4-6) and ATP by 30-60% (n = 4-7). However, prolonged exposure to alpha,beta-meATP (50 microM) abolished contractions evoked by all three stimuli (n = 5-12). PPADS (100 microM) and suramin (300 microM) reduced the peak neurogenic contraction of the mouse urinary bladder to 30-40% of control. At the same concentrations, the P2X1 antagonists abolished the nonadrenergic, purinergic component of neurogenic contractions in the guinea pig vas deferens (n = 4-5). Thus, P2X1 receptor antagonists inhibit, but do not abolish, the noncholinergic component of neurogenic contractions of guinea pig and mouse urinary bladder, indicating a second mode of action of neuronally released ATP. This has important implications for treatment of dysfunctional urinary bladder, for which this atropine- and P2X1 antagonist-resistant site represents a novel therapeutic target.

Studies on cardio-suppressant, vasodilator and tracheal relaxant effects of Sarcococca saligna.

Sarcococca saligna is a shrub that is traditionally used for its medicinal properties in Pakistan. In this study we report the cardio-suppressant, vasodilator and tracheal relaxant activities of the aqueous-methanolic extract (Ss.Cr) of the plant. Ss.Cr, that tested positive for the presence of saponins, flavonoids, tannins, phenols, and alkaloids, exhibited a dose-dependent (0.3-5 mg/mL) negative inotropic and chronotropic effect on the isolated guinea-pig atrium which was resistant to atropine (1 microM) and aminophylline (10 microM) pretreatment. In rabbit thoracic aorta, Ss.Cr dose-dependently (0.1-3 mg/mL) relaxed the high K+ (80 mM) and phenylephrine (PE, 1 microM)-induced contractions, indicating a possible Ca++ channel blocking (CCB) effect. When tested against PE (1 microM) control peaks in normal Ca++ and Ca++-free Kreb's solution, Ss.Cr exhibited dose-dependent (0.1-3 mg/mL) inhibition, being more potent in relaxing the PE responses in Ca++-free Kreb's solution, thus indicating specific blockade of Ca++ release from the intracellular stores. Ss.Cr also relaxed the agonist-induced contractions in: a) rat aorta irrespective of the presence of endothelium or nitric oxide synthase inhibitor L-NAME and b) rabbit and guinea-pig tracheal strips. The data shows that Ss.Cr possesses possible Ca++ channel blocking activity which might be responsible for its observed cardio-suppressant, vasodilator and tracheal relaxant effects though more tests are required to confirm this Ca++ channel blocking effect.

The early toxicology of physostigmine: a tale of beans, great men and egos.

Mid-19th century European visitors to Old Calabar, an eastern province of Nigeria, could not avoid becoming aware of native belief in the power of the seeds of a local plant to determine whether individuals were innocent or guilty of some serious misdemeanour. The seeds were those of a previously unknown legume and soon referred to as the ordeal bean of Old Calabar. Their administration was known locally as 'chop nut'. Missionaries who arrived in Calabar in 1846 estimated that chop nut caused some 120 deaths annually and documented the course of poisoning. The latter information and samples of the beans rapidly found their way to Scotland, the home of the missionaries' parent church, explaining why the early toxicology of physostigmine, quantitatively the most important of three active alkaloids in the beans, has such strong Scottish, predominantly Edinburgh, associations. However, it was 1855 before the first of many medical scientists, Robert Christison, a toxicologist of repute, investigated the effects of the beans to the extent of eating part of one himself and documenting the moderate, if not severe, consequences. A further 6 years were to pass before Balfour's comprehensive botanical description of the bean plant appeared. It was he who named it Physostigma venenosum. It was not so long until the next event, one that sparked more intensive and international interest in the beans. In 1863 a young Edinburgh ophthalmologist, Argyll Robertson, published a paper announcing the arrival of the first agent that constricted the pupil of the eye. The drug was an extract of Calabar beans and Argyll Robertson openly admitted that he had been alerted to its unusual property by his physician friend, Thomas Fraser. A minor flood of contributions on the ophthalmic uses of bean extracts followed in the medical press in the next few months; those on their systemic toxicity were fewer. Fraser's MD thesis, submitted to the University of Edinburgh in 1862 and clearly pre-dating Argyll Robertson's involvement with the beans, became generally available a few weeks after the appearance of Argyll Robertson's paper and was the first to address in detail the features of systemic administration of extracts of the beans. A major problem facing all early researchers of the beans was that of deciding how best to extract their active principle, a task made all the more difficult because bioassays were the only means of determining if the toxin was being tracked. The stability of extracts was an inevitable issue and the active principle finally became known as physostigma or physostigmine, after the botanical name of the parent plant. The features of physostigmine toxicity were soon exhaustively documented, both in animals and humans. How they were mediated was another matter altogether. Fraser maintained that muscular paralysis, the cardinal feature, was the result of depression of the spinal cord and was generally, but far from unanimously, supported. Of those who had reservations, Harley was the most prominent. He concluded that paralysis was secondary to effects on the motor nerve endings and, in so doing, came nearest to present-day knowledge at a time when acetylcholine, cholinesterases and cholinesterase inhibitors were not even imagined. Differences of opinion on the mode of action of the beans were to be expected and it is hardly surprising that they were not resolved. No standard formulation of physostigmine was available so the potency of those used would have varied from one investigator to another, the range of animals experimented upon was large while the number used by any researcher was commonly in single figures, more readily available cold-blooded creatures seemed less sensitive to physostigmine toxicity than warm-blooded ones and only Fraser determinedly pursued an answer; in general, the others made one foray into bean research then turned their attentions elsewhere. The same problems would beset other aspects of bean research. While Fraser did not get as close to the mode of action of physostigmine as Harley, he reigns supreme when it comes to antagonism between physostigmine and atropine. By this time, the 1870s had dawned and although the concept of antagonism between therapeutic agents was not new, it had little, if any, reliable scientific foundation. This was about to change; antagonism was becoming exciting and rational. Fraser's firm belief that physostigmine and atropine were mutually antagonistic at a physiological level was contrary to the conventional wisdom of his contemporaries. This alone would earn him a place in history but his contribution goes much, much further. Unlike any other at the time, he investigated it with scientific rigour, experimenting on only one species, ensuring as best he could the animals were the same weight, adjusting the doses of drugs he gave them for bodyweight, determining the minimum lethal dose of each drug before assessing their antagonistic effects, adopting a single, incontrovertible endpoint for efficacy and carrying out sufficient numbers of experiments to appear convincing in a later era where the statistical power of studies is all-important. To crown it all, he presented his results graphically. Fraser never claimed to have discovered the antagonism between physostigmine and atropine. Bartholow in 1873 did, based on work done in 1869. But his data hardly justify it. If anyone can reasonably claim this particular scientific crown it is an ophthalmologist, Niemetschek, working in Prague in 1864. His colleague in the same discipline, Kleinwächter, was faced with treating a young man with atropine intoxication. Knowing of the contrary actions of the two drugs on the pupil, Niemetschek suggested that Calabar bean extract might be useful. Kleinwächter had the courage to take the advice and his patient improved dramatically. Clearly, this evidence is nothing more than anecdotal, but the ophthalmologists were correct and, to the present day, physostigmine has had an intermittent role in the management of anticholinergic poisoning. The converse, giving atropine to treat poisoning with cholinesterase inhibitors, of which physostigmine was the first, has endured more consistently and remains standard practice today. It is salutary to realise that the doses and dosage frequency of atropine together with the endpoints that define they are adequate were formulated by Fraser and others a century and a half ago.

A long-form alpha-neurotoxin from cobra venom produces potent opioid-independent analgesia.

AIM: In light of the antinociceptive activity of the short-chain neurotoxin, cobrotoxin, and other acetylcholine antagonists, the antinociceptive activity and mechanisms of cobratoxin (CTX), a long-chain postsynaptic alpha-neurotoxin, was investigated in rodent pain models. METHODS: CTX was administered intraperitoneally (30, 45, 68 microg/kg), intra-cerebral ventricularly (4.5 microg/kg) or microinjected into periaqueductal gray (PAG; 4.5 microg/kg). The antinociceptive action was tested using the hot-plate and acetic acid writhing tests in mice and rats. The involvement of the cholinergic system and opioid system in CTX-induced analgesia was examined by pretreatment of animals with atropine (0.5 mg/kg, im; or 10 mg/kg, ip) or naloxone (1 and 5 mg/kg, ip). The effect of CTX on motor activity was tested using the Animex test. RESULTS: CTX exhibited a dose-dependent analgesic action in mice as determined by both the hot-plate and acetic acid writhing tests. The peak effect of analgesia was seen 3 h after administration. In the mouse acetic acid writhing test, the intra-cerebral ventricular administration of CTX at 4.5 microg/kg (1/12th of a systemic dose) produced marked analgesic effects. Microinjection of CTX (4.5 microg/kg) into the PAG region did not elicit an analgesic action in rats in the hot-plate test. Atropine at 0.5 mg/kg (im) and naloxone at 1 and 5 mg/kg (ip) both failed to block the analgesic effects of CTX, but atropine at 10 mg/kg (ip) did antagonize the analgesia mediated by CTX in the mouse acetic acid writhing test. Acetylsalicylic acid (300 mg/kg) did not enhance the analgesic effects of CTX. At the highest effective dose of 68 microg/kg the neurotoxin did not change the spontaneous mobility of mice. CONCLUSION: CTX has analgesic effects, which are mediated in the central nervous system though not through the PAG. The central cholinergic system but not opioid system appears to be involved in the antinociceptive action of CTX.

Cholinergic effects on fear conditioning I: the degraded contingency effect is disrupted by atropine but reinstated by physostigmine.

RATIONALE: The cholinergic system has been shown to modulate contextual fear conditioning. However, with the exception of trace conditioning studies, most of the available data have focussed on independent context, i.e., context that do not compete with the conditioned stimulus to control for the conditioned response (interactive context). OBJECTIVE: In the present series of experiments, the effects of the muscarinic antagonist, atropine, were assessed when contextual fear memory interacts with cued fear memory to regulate conditioned response, using a Pavlovian degraded contingency preparation in rats. This preparation not only afforded an insight into simple Pavlovian associations but also enabled us to test for the processes of competition that made use of these associations to make an appropriate response to a stimulus [degraded contingency effect (DCE)]. METHODS: In experiment 1, three doses of atropine [2.5, 5.0, and 10.0 mg/kg, intraperitoneally (i.p.)] were evaluated on male Sprague-Dawley rats. In experiment 2, physostigmine (0.037-0.3 mg/kg, i.p.) was injected after the administration of 5 mg/kg of atropine. RESULTS: Experiment 1A and its partial replication (experiment 1B) showed that at asymptotic level of training, atropine did not alter contextual and cued fear memories when the subjects were directly tested for them, whereas it suppressed the DCE for a 5 mg/kg dose. Indeed, atropine-induced suppression of the DCE was found to be an inverted U-shaped dose-response curve. Experiment 2 showed that physostigmine caused a dose-dependent reversal of the atropine-induced alleviation of the DCE, without altering the expression of simple cued and contextual fear memories. CONCLUSION: These results evidence at asymptotic level of training a cholinergic modulation of the processing of interactive context, but not of independent ones. They are discussed in the framework of the mechanisms that are involved in both types of contextual processing.

Cholinergic effects on fear conditioning II: nicotinic and muscarinic modulations of atropine-induced disruption of the degraded contingency effect.

RATIONALE: In a companion study (Carnicella et al., 2005), we showed that the muscarinic antagonist atropine, when administered after extensive training during both conditioning and testing, affected neither cued nor contextual fear memories when both of them did not compete for the control of the overt behaviour. In contrast, atropine altered the degraded contingency effect (DCE), that is, the processes by which contextual fear memory competes with the cued one for the control of the conditioned response. Atropine-induced disruption of the DCE was fully reversed by the administration of the anticholinesterase inhibitor physostigmine, which suggests a direct cholinergic implication. OBJECTIVE: The present series of experiments was conducted in order to define more precisely the involvement of the cholinergic system in such an effect. METHODS: Oxotremorine (0.0, 0.0075, 0.015, or 0.03 mg/kg), pilocarpine (0.0, 0.3, 1, or 3 mg/kg), xanomeline (0.0, 2.5, 5.0, 10.0 or 20.0 mg/kg) and nicotine (0.0, 0.1, 0.2, or 0.4 mg/kg) were tested for reversal of the atropine-induced alteration of the DCE. RESULTS: Oxotremorine and pilocarpine did not reverse the atropine-induced alteration of the DCE. In contrast, xanomeline and nicotine reversed the effect of atropine on the DCE. CONCLUSION: The present series of experiments reveals complex pharmacological interactions within the cholinergic system when cued and contextual fear memories interact. Results are discussed in this connection and with regard to the relation between the properties of cholinergic agonists and their therapeutic values.

Organophosphate-induced convulsions and prevention of neuropathological damages.

Such organophosphorus (OP) compounds as diisopropylfluorophosphate (DFP), sarin and soman are potent inhibitors of acetylcholinesterases (AChEs) and butyrylcholinesterases (BChEs). The acute toxicity of OPs is the result of their irreversible binding with AChEs in the central nervous system (CNS), which elevates acetylcholine (ACh) levels. The protective action of subcutaneously (SC) administered antidotes or their combinations in DFP (2.0 mg/kg BW) intoxication was studied in 9-10-weeks-old Han-Wistar male rats. The rats received AChE reactivator pralidoxime-2-chloride (2PAM) (30.0 mg/kg BW), anticonvulsant diazepam (2.0 mg/kg BW), A(1)-adenosine receptor agonist N(6)-cyclopentyl adenosine (CPA) (2.0 mg/kg BW), NMDA-receptor antagonist dizocilpine maleate (+-MK801 hydrogen maleate) (2.0 mg/kg BW) or their combinations with cholinolytic drug atropine sulfate (50.0 mg/kg BW) immediately or 30 min after the single SC injection of DFP. The control rats received atropine sulfate, but also saline and olive oil instead of other antidotes and DFP, respectively. All rats were terminated either 24 h or 3 weeks after the DFP injection. The rats treated with DFP-atropine showed severe typical OP-induced toxicity signs. When CPA, diazepam or 2PAM was given immediately after DFP-atropine, these treatments prevented, delayed or shortened the occurrence of serious signs of poisoning. Atropine-MK801 did not offer any additional protection against DFP toxicity. In conclusion, CPA, diazepam and 2PAM in combination with atropine prevented the occurrence of serious signs of poisoning and thus reduced the toxicity of DFP in rat.

Mechanism of atropine-resistant contraction induced by Dai-kenchu-to in guinea pig ileum.

To clarify the contractile mechanism of Dai-kenchu-to, the effects of hydroxy beta-sanshool (an ingredient of Zanthoxylum fruit), Zanthoxylum fruit (a constituent herb of Dai-kenchu-to) and Dai-kenchu-to were studied in mucosa-free longitudinal muscle of guinea pig ileum. Hydroxy beta-sanshool at 10(-7)-10(-5) g/ml induced dose-related contractions accompanied by autonomous contraction and produced an initial contraction at a concentration of 10(-4) g/ml or more. The contraction induced by hydroxy beta-sanshool (10(-5) g/ml) was significantly inhibited by tetrodotoxin or the capsaicin-receptor antagonist capsazepine. Although atropine or the substance P antagonist spantide tended to inhibit the contraction, a combination of atropine and spantide almost abolished the contraction by hydroxy beta-sanshool. The P2-purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid did not affect hydroxy beta-sanshool-induced contraction in the presence or absence of spantide. The tonic contractions by Zanthoxylum fruit (2 x 10(-4) g/ml) and Dai-kenchu-to (10(-3) g/ml) were significantly inhibited or tended to be inhibited by atropine, spantide, tetrodotoxin or capsazepine and were remarkably suppressed by the combination of atropine and spantide. These results suggested that acetylcholine release from intrinsic cholinergic nerves and tachykinins from sensory neurons are involved in the contractions induced by hydroxy beta-sanshool and that tachykinins may be involved in the atropine-resistant contraction by Dai-kenchu-to.

Learning deficits induced by 4 belladonna alkaloids are preferentially attenuated by tacrine.

AIM: To examine the antagonism of tacrine on the amnesic effects of scopolamine (Sco), anisodine (AT3), atropine (Atr), and anisodamine (Ani). METHODS: Cognitive functions and locomotor activities were determined using two sessions of step-through and open-field tests, respectively. Mice were injected with one of the belladonna alkaloids (0.05-50 mumol.kg-1, i.p.) and tacrine (50 mumol.kg-1, s.c.) 30 min before the first session. RESULTS: Tacrine completely blocked the avoidance-learning deficit caused by Sco 0.5 mumol.kg-1, AT3 and Atr 5 mumol.kg-1, or Ani 50 mumol.kg-1. But tacrine partly antagonized the learning deficit induced by Sco 5-50 mumol.kg-1 or Atr and AT3 50 mumol.kg-1. The avoidance-memory deficit caused by Sco 0.05-5 mumol.kg-1 or Atr 5 mumol.kg-1 was completely or partly attenuated by tacrine, which did not antagonize the memory deficit elicited by Sco and Atr 50 mumol.kg-1, AT3 5 and 50 mumol.kg-1, and Ani 50 mumol.kg-1. During the acquisition, the locomotor activity of the mice was inhibited by tacrine. This reduction was completely antagonized by Sco 0.5-50 mumol.kg-1, AT3 5-50 mumol.kg-1, Atr 5-50 mumol.kg-1, and only partly antagonized by AT3 and Atr 0.5 mumol.kg-1 or Ani 50 mumol.kg-1. CONCLUSION: Compared with the avoidance-memory deficit, the avoidance-learning deficit caused by belladonna alkaloids is more preferentially attenuated by tacrine.

The effects of novel, selective 5-hydroxytryptamine (5-HT)4 receptor ligands in rat spatial navigation.

Activation of central 5-hydroxytryptamine (5-HT4) receptors may enhance cognitive performance. In the present study, the effects of two novel, potent and selective 5-HT4 receptor agonists, RS 67333 (1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-n-burtl-4-piperidinyl)- 1-propanone) and RS 67506 (1-(4-amino- 5-chloro-2-methoxyphenyl)-3-[1-[2-[(methylsulfonyl)amino]ethyl]-4- piperidinyl]-1-propanone), were studied in a rat model of spatial learning and memory; the Morris water maze. RS 67333 (0.1, 10 and 1000 micrograms/kg, intraperitoneally (i.p.)), a highly potent, selective and hydrophobic 5-HT4 receptor agonist, reversed the decrements in cognitive performance induced by atropine (30 mg/kg, i.p.). By contrast, no effect was seen to RS 67506 (0.1, 10 and 1000 micrograms/kg, i.p.), a hydrophilic 5-HT4 receptor agonist, of equivalent potency and selectivity to RS 67333. This differential effect may reflect the enhanced ability of RS 67333 to enter the CNS, with respect to RS 67506. The ameliorative actions of RS 67333 on cognitive dysfunction were abolished by prior treatment with a selective 5-HT4 receptor antagonist, RS 67532 [1-(4-amino-5-chloro-2-(3, 5-dimethoxy benzyloxyphenyl)-5-(1-piperidinyl)-1-pentanone; 1 mg/kg, i.p.]. When given alone, or in naive rats, RS 67532 (0.1, 10 and 1000 micrograms/kg, i.p.), was without effect. None of the compounds tested affected the swim speed at any of the doses used. In separate locomotor studies, RS 67532 reduced activity at 1 and 10 mg/kg, i.p., although no effect was seen with RS 67333 or RS 67506 (0.01-10 mg/kg, i.p.). These data suggest that RS 67333 reversed the cognitive deficit induced by atropine and support a role of 5-HT4 receptors in rat spatial learning and memory.

Tachykinin receptors mediate atropine-resistant rat duodenal reflex contractions in vivo.

The study aimed to establish the possible role of tachykinins as mediators of atropine-resistant reflex contractions evoked by balloon distension in the proximal duodenum of urethane-anesthetized, guanethidine (34 mumol/kg s.c.)-pretreated rats. Distension of the balloon with a small amount (0.2-0.3 ml) of saline induced the appearance of phasic rhythmic contractions (about 11 mmHg in amplitude) which were promptly suppressed by either atropine (3 mumol/kg i.v.) or hexamethonium (28 mumol/kg i.v.). Despite the continuous i.v. infusion of atropine (2 mumol/h), low-amplitude rhythmic phasic contractions recovered, which were promptly suppressed by hexamethonium, to indicate the involvement of an atropine-resistant excitatory reflex. The amplitude of these atropine-resistant contractions was increased to about 4-5 mmHg by further distension of the balloon (0.4-0.6 ml) : under these conditions, the atropine-resistant contractions undergo a progressive fading. The fading was prevented by i.v. administration of the nitric oxide (NO) synthase inhibitor, L-nitroarginine methyl ester (L-NAME, 55 mumol/h), to provide a suitable baseline (amplitude of contractions was 7-8 mmHg) for studying the effect of tachykinin receptor antagonists. I.v. administration of the selective tachykinin NK2 receptor antagonists, MEN 10,627 (10-100 nmol/kg) and SR 48968 (100-300 nmol/kg) or of the selective NK1 antagonist SR 140333 (100 nmol/kg), at doses which do not affect the duodenal contractions induced by acetylcholine (5.5 mumol/kg i.v.), produced a prompt and long lasting suppression of the atropine-resistant reflex duodenal contractions produced by balloon distension in urethane-anesthetized rats, whilst SR-48965 (300 nmol/kg), the enantiomer of SR-48968 devoid, of NK2 receptor blocking activity, was without effect. I.v. administration of the selective NK1 receptor agonists [Sar9] substance P sulfone and septide or of the NK2 receptor selective agonist, [beta Ala8] neurokinin A(4-10) produced dose-dependent contractions of the duodenum. SR 140333 (100 nmol/kg i.v.) selectively antagonized the duodenal contractions produced by [Sar9] substance P sulfone and septide without affecting those produced by [beta Ala8] neurokinin A(4-10). On the other hand, MEN 10,627 (30-100 nmol/kg i.v.) and SR 48968 (100-300 nmol/kg i.v.) but not SR 48965 (300 nmol/kg i.v.) antagonized, at a comparable extent, duodenal contractions induced by both the selective NK2 and NK1 receptor agonists. We conclude that endogenous tachykinins are involved in mediating atropine-resistant reflex contractions evoked by distension of the rat duodenum in vivo: both NK1 and NK2 receptors are activated by endogenous ligands to produce NANC contractions of rat duodenum in vivo. However, the contractile response to i.v. administered NK1 receptor agonists, [Sar9] substance P sulfone and septide, may involve the release of mediators producing smooth muscle contraction via NK2 receptors.

Effects of acute and short-term repeated application of fullerene C60 on agonist-induced responses in various tissues of guinea pig and rat.

1. Effects of fullerene C60 in trachea, right atria, ileum and stomach (fundus) of guinea pig and vas deferens and uterus of rat were studied pharmacologically. 2. C60 (4 microM) had no direct effect in all tissues. In guinea pig trachea and heart, relaxation and positive inotropic and chronotropic actions of isoprenaline and in isolated rat vas deferens and uterus the responses on norepinephrine and oxytocin were not affected by the short-term repeated application of C60 30 mg/kg i.p. for 4 wk. 3. The pD2 values (potencies) of acetylcholine in ileum and its longitudinal muscle from guinea pig after the short-term repeated application of C60 were significantly smaller than those obtained without the application. The value of 5-hydroxytryptamine in rat stomach (fundus) also tended to be smaller than obtained without the application. 4. Atropine inhibited competitively the contractions for acetylcholine in the longitudinal muscles prepared from C60-treated and non-treated guinea pigs, and the pA2 values for atropine were not significantly different with each other. 5. These results suggest that C60 has no direct effects or antagonistic properties toward drug receptors, but sub-chronic exposure of C60 decreased responsiveness. This may be due to a change in post-receptor processes.

Glucose attenuation of atropine-induced deficits in paradoxical sleep and memory.

When administered systemically, glucose attenuates deficits in memory produced by several classes of drugs, including cholinergic antagonists and opiate agonists. Glucose also enhances memory in aged rats, mice, and humans. In addition, glucose ameliorates age-related reductions in paradoxical sleep. Because deficits in paradoxical sleep are most marked in those individual aged rats that also have deficits in memory, treatments which improve one of these functions may similarly improve the other. The present experiments show that glucose attenuates deficits in paradoxical sleep and memory after atropine administration, with similar dose-response curves for both actions. In the first experiment, rats received saline, atropine (1 mg/kg), glucose (100 mg/kg) or combinations of atropine + glucose (10, 100, 250, and 500 mg/kg) 30 min before assessment on a spontaneous alternation task. In the second experiment, 3-h EEGs were assessed for spontaneous daytime sleep in rats administered saline, atropine (1 mg/kg), glucose (100 mg/kg) or combinations of atropine + glucose (10, 100 and 250 mg/kg). In both experiments, glucose significantly attenuated deficits at an optimal dose of 100 mg/kg. A third experiment assessed blood glucose levels after injections of atropine + glucose (100 mg/kg) and determined that blood glucose levels were similar to those produced by other treatments which enhance memory. These results are consistent with the view that paradoxical sleep and at least one test of memory are similarly influenced by atropine and glucose.

Linopirdine (DuP 996) improves performance in several tests of learning and memory by modulation of cholinergic neurotransmission.

The actions of linopirdine (DuP 996; 3,3-bis[4-pyrindinylmethyl]-1-phenylindolin-2-one) were evaluated in rats and mice in several cognitive behavioral tests, and for its effects on hippocampal acetylcholine (ACh) overflow in rats. Using mice treated with the muscarinic receptor antagonist, scopolamine, we studied the effects of linopirdine on retention of a passive avoidance task. Linopirdine (0.1 and 1 mg/kg) ameliorated the scopolamine-induced deficit, but at doses ranging from 0.01-1 mg/kg, it did not affect passive avoidance retention in normal (untreated) mice. In a scopolamine-induced hyperactivity test, linopirdine (1 mg/kg) decreased the motoric stimulation associated with the cholinergic hypofunction, without affecting locomotor activity on its own. Using rats, we studied the effects of linopirdine on performance in the Morris water maze spatial memory task. Young rats treated with atropine (30 mg/kg), a muscarinic receptor antagonist, took significantly longer to locate the submerged platform across 12 trials. Linopirdine (0.01 and 0.1, but not 1 mg/kg) ameliorated the atropine deficit. In addition, linopirdine (0.1 mg/kg) ameliorated the deficit in cognition-impaired aged rats (23-24 mo), but did not affect unimpaired aged rats. In terms of neurochemical action, linopirdine (1, 10, and 100 microM) produced a concentration-dependent increase in K(+)-evoked ACh overflow from superfused rat hippocampal slices. Also, linopirdine (10 microM) similarly increased ACh release in young control rats and cognition-impaired and nonimpaired aged rats. Our results confirm and extend findings from other studies that demonstrate the cognition-enhancing action of linopirdine in rodent models.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide-like activity in guinea pig colon as determined by effector responses, bioassay and chemiluminescence analysis.

The role of nerve-induced release of nitric oxide (NO) as a modulator of neuroeffector transmission was studied in the longitudinal muscle of the guinea pig colon. The biological activity of a vascular relaxing factor released by nerve stimulation was examined in a bioassay cascade system. Furthermore, biochemical measurements of nerve-induced release of the NO metabolite nitrite (NO2-) were made with a chemiluminescence technique. Transmural nerve stimulation elicited contractile responses that were partly blocked by atropine and further inhibited after additional application of the tachykinin receptor antagonist CP-96, 345. The NO-synthase inhibitor N omega-nitro-L-arginine (NOARG) enhanced the nerve-induced contractions and concomitantly increased the basal degree of contraction ('tone'). The relaxations obtained by nerve stimulation after treatment with atropine and histamine were inhibited by NOARG. Electrical stimulation of the guinea pig colon released a non-adrenergic non-cholinergic (NANC) vascular relaxing factor into the tissue superfusate. The half-life of this factor down the cascade was the same as that observed with exogenous application of NO NOARG and tetrodotoxin (TTX) inhibited the release of the relaxing factor. During transmural nerve stimulation there was a significant increase in NO/NO2- release. This increase was inhibited by TTX and N omega-nitro-L-arginine methyl ester (L-NAME). In conclusion, pharmacological analysis as well as bioassay and biochemical measurements suggest that NO is released during nerve stimulation in the guinea pig colon, where it mediates smooth muscle relaxation.

[The effects of preanesthetic oral clonidine upon heart rate response to intravenous atropine in patients during general anesthesia].

In awake subjects the positive chronotropic effect of intravenously administered atropine 10 micrograms.kg-1 has been demonstrated to be blunted by preanesthetic medication of oral clonidine 5 micrograms.kg-1. The aim of the present study is to investigate whether general anesthesia could alter the clonidine-induced attenuation of positive chronotropic effect by atropine. Thirty-two patients were randomly assigned to one of the two groups; patients of the clonidine group received oral clonidine 5 micrograms.kg-1 (n = 12), whereas those of the control group received no clonidine. General anesthesia was induced with intravenous thiamylal 4-5 mg.kg-1, and was maintained with enflurane and nitrous oxide in oxygen after endotracheal intubation. Following the stable circulatory period of 10 min, hemodynamic measurements were made at 1 min intervals for 10 min after atropine 10 micrograms.kg-1 was administered intravenously as a bolus in both groups. A significant attenuation in heart rate response to intravenous atropine 10 micrograms.kg-1 was observed in patients receiving clonidine 5 micrograms.kg-1, as compared with that in the control group (P less than 0.01); maximal increases in heart rate were 15 +/- 8 and 22 +/- 6 beats.min-1 (mean +/- SD) in the clonidine and control groups, respectively. It is concluded that clonidine 5 micrograms.kg-1 blunts the heart rate response to intravenous atropine 10 micrograms.kg-1 in patients anesthetized with enflurane and nitrous oxide in oxygen.

Oral clonidine blunts the heart rate response to intravenous atropine in humans.

Clonidine, recently introduced into anesthesia practice, may cause bradycardia. Whether this bradycardia is reversible with atropine is not known. Accordingly, we studied heart rate (HR) responses to intravenous atropine in 80 patients assigned randomly to either a control group, who received no medication (n = 20), or a clonidine group, who received oral clonidine of approximately 1.2 micrograms.kg-1 (n = 20), 2.5 micrograms.kg-1 (n = 20), or 5 micrograms.kg-1 (n = 20). All patients received incremental doses of atropine, 2.5, 2.5, and 5 micrograms.kg-1, at 2-min intervals (total dose 10 micrograms.kg-1). Positive chronotropic response to the cumulative atropine dose of 10 micrograms.kg-1 was attenuated significantly only in patients given clonidine 5 micrograms.kg-1 (7 +/- 1 beats per min, mean +/- standard error) when compared with those given smaller doses of clonidine (15 +/- 2, 16 +/- 2 beats per min) or no clonidine (19 +/- 2 beats per min) (P less than 0.05). To determine whether HR hyporesponsiveness to atropine induced by clonidine can be overcome by a larger dose of atropine, the authors studied 30 additional patients given clonidine 5 micrograms.kg-1 or no medication. In all patients not receiving clonidine (n = 15), HR increased by more than 20 beats per min when atropine of 15 micrograms.kg-1 was administered, whereas in only 5 patients (33%) receiving clonidine did the HR increase by 20 beats per min after atropine 15 micrograms.kg-1 (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)