Propoxur-induced acetylcholine esterase inhibition and impairment of cognitive function: attenuation by Withania somnifera.

Propoxur (2-isopropoxyphenyl N-methylcarbamate) is widely used as an acaricide in agriculture and public health programs. Studies have shown that sub-chronic exposure to propoxur can cause oxidative stress and immuno-suppression in rats. Carbamates are also known to exhibit inhibitory effect on cholinesterase activity, which is directly related to their cholinergic effects. In the present study, the effect of Withania somnifera (Ashwagandha), a widely used herbal drug possessing anti-stress and immunomodulatory properties was studied on propoxur-induced acetylcholine esterase inhibition and impairment of cognitive function in rats. Male Wistar rats were divided into four groups. Group I was treated with olive oil and served as control. Group II was administered orally with propoxur (10 mg/kg b.wt.) in olive oil, group III received a combination of propoxur (10 mg/kg b.wt.) and W. somnifera (100 mg/kg b.wt.) suspension and group IV W. somnifera (100 mg/kg b.wt.) only. All animals were treated for 30 days. Cognitive behaviour was assessed by transfer latency using elevated plus maze. Blood and brain acetylcholine esterase (AChE) activity was also assessed. Oral administration of propoxur (10 mg/kg b.wt.) resulted in a significant reduction of brain and blood AChE activity. A significant prolongation of the acquisition as well as retention transfer latency was observed in propoxur-treated rats. Oral treatment of W. somnifera exerts protective effect and attenuates AChE inhibition and cognitive impairment caused by sub-chronic exposure to propoxur.

Role of kappa- and delta-opioid receptors in the antinociceptive effect of oxytocin in formalin-induced pain response in mice.

Oxytocin has been implicated in the modulation of somatosensory transmission such as nociception and pain. The present study investigates the effect of oxytocin on formalin-induced pain response, a model of tonic continuous pain. The animals were injected with 0.1 ml of 1% formalin in the right hindpaw and the left hindpaw was injected with an equal volume of normal saline. The time spent by the animals licking or biting the injected paw during 0-5 min (early phase) and 20-25 min (late phase) was recorded separately. Oxytocin (25, 50, 100 microg/kg, i.p.) dose dependently decreased the licking/biting response, both in the early as well as the late phases. The antinociceptive effect of oxytocin (100 microg/kg, i.p.) was significantly attenuated in both the phases by a higher dose of the non-selective opioid receptor antagonist naloxone (5 mg/kg, i.p.), MR 2266 (0.1 mg/kg, i.p.), a selective kappa-opioid receptor antagonist and naltrindole (0.5 mg/kg, i.p.), a selective delta-opioid receptor antagonist but not by a lower dose of naloxone (1 mg/kg, i.p.) or beta-funaltrexamine (2.5 microg/mouse, i.c.v.), a selective mu-opioid receptor antagonist. Nimodipine, a calcium channel blocker (1 and 5 mg/kg, i.p.) produced a dose-dependent analgesic effect. The antinociceptive effect of oxytocin was significantly enhanced by the lower dose of nimodipine (1 mg/kg, i.p.) in both the phases. Chronic treatment with oxytocin (100 microg/kg/day, i.p. daily for 7 days) did not produce tolerance in both the phases of formalin-induced pain response. The results thus indicate that oxytocin displays an important analgesic response in formalin test; both kappa- and delta-opioid receptors as well as voltage-gated calcium channels seem to be involved in the oxytocin-induced antinociception.

Protective effect of melatonin against propoxur-induced oxidative stress and suppression of humoral immune response in rats.

Effect of melatonin in attenuation of propoxur induced oxidative stress and suppression of humoral immune response was studied in rats. Oral administration of propoxur (10 mg/kg) increased lipid peroxidation in serum after 28 days treatment. Superoxide dismutase, catalase and glutathione were also altered following propoxur exposure. In addition propoxur exposure markedly suppressed humoral immune response as assessed by antibody titre and plaque forming cell assay. Simultaneous treatment with melatonin (5 mg/kg, ip) markedly attenuated the effect of propoxur on (a) lipid peroxidation, (b) oxidative stress parameters and (c) immunotoxicity. Results have been discussed in the light of possible immunopotentiating and antioxidant effects of melatonin to understand the influence of oxidative stress on propoxur induced immunomodulation.

Modulation by naltrexone of stress-induced changes in humoral immune responsiveness and gastric mucosal integrity in rats.

The effects of restraint stress (RS) and the opioid antagonist, naltrexone, were evaluated on humoral immune responsiveness and gastric mucosal integrity in rats. RS for 24 h, but not 6 h, attenuated both the primary (PAR) and secondary antibody response (SAR) to sheep red blood cells (SRBC) after a single exposure to the stressor. Naltrexone (1 or 5 mg/kg) dose-dependently aggravated the effects of RS on anti-SRBC antibody titre in both PAR and SAR studies. Further, in rats sensitized with SRBC, RS (24 h), in addition to lowering the humoral antibody response, also induced gastric mucosal lesions. Both these responses were further aggravated with naltrexone pretreatment. These results are discussed in light of interactions between immune and visceral responses, and their regulation by endogenous opioids, during RS.

Evaluation of immunomodulatory potential of Ocimum sanctum seed oil and its possible mechanism of action.

The present study investigates the effect of Ocimum sanctum seed oil (OSSO) on some immunological parameters in both non-stressed and stressed animals. An attempt has also been made to explore the possible mechanism of immunomodulatory activity. OSSO (3 ml/kg, ip) produced a significant increase in anti-sheep red blood cells (SRBC) antibody titre and a decrease in percentage histamine release from peritoneal mast cells of sensitized rats (humoral immune responses), and decrease in footpad thickness and percentage leucocyte migration inhibition (LMI) (cell-mediated immune responses). Restraint stress (RS) produced a significant reduction in the anti-SRBC antibody titre, foot pad thickness and percentage LMI (% LMI). The effects of RS on humoral as well as cell-mediated immune responses were effectively attenuated by pretreating the animals with OSSO. Co-administration of diazepam (1 mg/kg, sc), a benzodiazepine (BZD), with OSSO (1 ml/kg, ip) enhanced the effect of OSSO on RS-induced changes in both humoral and cell-mediated immune responses. Further, flumazenil (5 mg/kg, ip), a central BZD receptor antagonist inhibited the immunomodulatory action of OSSO on RS-induced immune responsiveness. Thus, OSSO appears to modulate both humoral and cell-mediated immune responsiveness and these immunomodulatory effects may be mediated by GABAergic pathways.

Receptor mechanisms involved in the anticonvulsant effect of melatonin in maximal electroshock seizures.

The present study investigates the mechanisms involved in the anticonvulsant effect of melatonin in maximum electroshock (MES) seizures. Melatonin (25-100 mg/kg) dose-dependently decreased the duration of tonic hindlimb extension (THLE). The anticonvulsant effect of melatonin was blocked by bicuculline, a GABA(A) receptor antagonist, and luzindole, an ML(1) receptor antagonist, while prazosin, an ML(2) receptor antagonist, enhanced the anticonvulsant actions of melatonin in this seizure model. Administration of serotonergic agents, mianserin and ondansetron, along with melatonin, increased the antiseizurogenic activity of melatonin, while buspirone had no effect. Pretreating the animals with diazepam, carbamazepine or lamotrigine enhanced the anticonvulsant effect of melatonin. Melatonin thus appears to be an effective anticonvulsant, and melatonin ML(1) receptors, GABAergic and serotonergic mechanisms may play an important role in mediating the anticonvulsant activity of melatonin in electroshock seizures.

Possible mechanism of anticonvulsant effect of ketamine in mice.

The study was designed to investigate the effect of ketamine on convulsive behaviour using maximal electroshock (MES) test. An attempt was also made to study the possible receptor mechanisms involved. MES seizures were induced in mice via transauricular electrodes (60 mA, 0.2sec). Seizure severity was assessed by the duration of tonic hindlimb extensor phase and mortality due to convulsions. Intraperitoneal administration of ketamine produced a dose-dependent (5-50 mg/kg) protection against hindlimb extensor phase. The anticonvulsant effect of ketamine was antagonized neither by naloxone (low as well as high doses) nor sulpiride, but was attenuated by haloperidol, a dopamine (D2)/sigma receptor antagonist. Co-administration of gamma-aminobutyric acid (GABA)-ergic drugs (GABA, muscimol, diazepam and baclofen) and N-methyl-D-aspartate (NMDA) receptor antagonist, dizocilpine (MK801) with ketamine facilitated the anticonvulsant action of the latter drug. In contrast, flumazenil, a benzodiazepine (BZD)-GABAA receptor antagonist, reversed the facilitatory effect of diazepam on the anti-MES effect of ketamine. Similarly, delta-aminovaleric acid (DAVA), antagonized the facilitatory effect of baclofen on anti-MES action of ketamine. These BZD-GABAergic antagonists, flumazenil or DAVA per se also attenuated the anti-MES effect of ketamine given alone. The results suggest that besides its known antagonistic effect on NMDA channel, other neurotransmitter systems i.e. sigma, GABAA-BZD-chloride channel complex and GABAB receptors may also be involved in the anti-MES action of ketamine.

Effects of Azadirachta indica A Juss on some biochemical, immunological and visceral parameters in normal and stressed rats.

Effects of A. indica (AI) were evaluated on some biochemical, immunological and visceral parameters in normal and stress rats. AI (100 mg/kg) lowered blood glucose, triglyceride and SGOT levels in normal rats, and attenuated stress-induced elevations of cholesterol and urea levels. In rats immunized with SRBC, AI enhanced the humoral antibody response to the antigen. Further, AI facilitated the footpad thickness response to SRBC in sensitized mice and also enhanced leucocyte migration in immunized rats. In stressed rats, AI significantly attenuated the stress-induced (a) suppression of humoral immune response and (b) gastric ulcerogenesis. These results are discussed in light of the possible mechanisms involved in the effects of AI in normal and stressful situations.

Potentiation of analgesia and reversal of tolerance to morphine by calcium channel blockers.

Effect of four calcium channel blockers (CCBs) belonging to different chemical classes, alone and in combination with morphine was investigated on two models of pain sensitivity, i.e. formalin and tail flick tests in mice. All the studied CCBs, i.e. diltiazem, flunarizine, nimodipine and verapamil inhibited formalin-induced pain responses; however, with verapamil, though there was a trend towards a reduction of paw-licking response to formalin, it was not found to be statistically significant. In contrast, none of the CCBs affected the tail flick latency at any of the doses studied. Morphine, a mu-receptor agonist exerted a significant analgesic effect in formalin as well in tail flick tests. Pretreatment with all CCBs significantly enhanced the analgesic effect of morphine in both tests of nociception. Further, concomitant administration of one of the CCBs, diltiazem with morphine prevented the development of tolerance to the latter. However, combination of diltiazem with morphine, like morphine alone was found to be ineffective in morphine tolerant animals. Results, thus, show that CCBs produced an analgesic effect of their own in formalin-induced tonic pain and potentiated the analgesic activity of morphine. They also modulated opioid-induced tolerance.

Effects of stress on immune responsiveness, gastric ulcerogenesis and plasma corticosterone in rats: modulation by diazepam and naltrexone.

Effects of restraint stress (24 hr at room temperature) were evaluated on some immunological, visceral and endocrinal responses in rats. In animals sensitized with sheep RBC (SRBC), restraint stress (a) prevented the booster-induced rise in anti-SRBC antibody titre, (b) induced gastric mucosal erosions, and (c) elevated plasma corticosterone, when compared to non-stressed controls. Diazepam (1 or 10 mg/kg) consistently attenuated the effects of stress on all three parameters studied. The opioid antagonist, naltrexone (1 or 5 mg/kg) tended to aggravate these stress-induced effects. These concurrent biological changes during stress and their modulation by drugs are discussed in light of a possible correlation between endocrinal, immunological and visceral changes during such aversive stimuli.

Antinociceptive activity of a neurosteroid tetrahydrodeoxycorticosterone (5alpha-pregnan-3alpha-21-diol-20-one) and its possible mechanism(s) of action.

The present study investigates the effects of a neurosteroid tetrahydrodeoxycorticosterone (5alpha-pregnan-3alpha-21-diol-20-one) in two experimental models of pain sensitivity in mice. Tetrahydrodeoxycorticosterone (2.5, 5 mg/kg, i.p.) dose dependently decreased the licking response in formalin test and increased the tail flick latency (TFL) in tail flick test. Bicuculline (2 mg/kg, i.p.), a GABA(A) receptor antagonist blocked the antinociceptive effect of tetrahydrodeoxycorticosterone in TFL test but failed to modulate licking response in formalin test. Naloxone (1 mg/kg, i.p.), an opioid antagonist effectively attenuated the analgesic effect of tetrahydrodeoxycorticosterone in both the models. Tetrahydrodeoxycorticosterone pretreatment potentiated the antinociceptive response of morphine, an opioid compound and nimodipine, a calcium channel blocker in formalin as well as TFL test. Thus, tetrahydrodeoxycorticosterone exerts an analgesic effect, which may be mediated by modulating GABA-ergic and/or opioid-ergic mechanisms and voltage-gated calcium channels.

Tramadol, a centrally acting opioid: anticonvulsant effect against maximal electroshock seizure in mice.

The present study was designed to investigate the pro- or anticonvulsant effect of tramadol using maximal electroshock (MES) test. An attempt was also made to determine the possible opioid receptor mechanism involved. MES seizures were induced through transauricular electrodes (60 mA, 0.2s) and the seizure severity was assessed by the duration of tonic hindlimb extensor phase. Intraperitoneal (i.p.) administration of tramadol resulted in a dose-dependent anticonvulsant action; the ED50 for the effect was 33 mg/kg. The anti-MES effect of tramadol was antagonized by the low doses (0.05 and 0.1 mg/kg, s.c.) of MR 2266, a selective kappa receptor antagonist and also by the high doses (1.0 and 5.0 mg/kg, i.p.) but not the low doses (0.1 and 0.25 mg/kg) of naloxone. The results suggest that the anti-MES effect of tramadol is mediated by kappa receptors, since MR 2266 and naloxone (in high doses) are known to block these receptors.

Development of differential tolerance to the sedative and anti-stress effects of benzodiazepines.

Differential degree of tolerance has been reported to develop for anticonvulsant, sedative and skeletal muscle relaxant effects of benzodiazepines (BZDs). Acute treatment with BZDs reportedly reduces the formation of gastric stress ulcers and attenuates stress-induced immunosuppression. The present study investigates whether tolerance develops to these antistress effects of BZDs by using diazepam and chlordiazepoxide as representative drugs. A single dose of diazepam (5 mg/kg, i.p.) or chlordiazepoxide (20 mg/kg, i.p.) produced a significant reduction in locomotor activity, a measure of sedative effect and antagonized the effect of restraint stress (RS) on gastric mucosal lesions and anti-sheep red blood cell (SRBC) antibody titre. With chronic treatment (X 7 d), there was a marked tolerance to the sedative effect of both the studied BZD drugs, while much less tolerance developed to their ulcer protective action. However, no tolerance was observed to the attenuating effect of diazepam and chlordiazepoxide on RS-induced immunosuppression. Thus, the results of the present study indicate that different mechanisms may be involved in the development of tolerance to the sedative, antiulcer and immunomodulatory effects of BZDs.

Interaction of pentazocine with calcium channel blocking drugs during chemical and thermal pain in mice.

The present study was designed to investigate the antinociceptive interaction of a clinically used opioid, pentazocine which produces its analgesic effect mainly through kappa receptors, with some calcium channel blockers (CCBs, viz. Diltiazem, flunarizine, nimodipine and verapamil--each representing one chemical class) in formalin and tail flick tests in mice. All the CCBs, except verapamil, significantly inhibited the formalin-induced pain response in a dose-dependent manner. However, none of these drugs affected tail flick latency at any of the studied doses. Pentazocine showed a significant antinociceptive response in both pain models, although a high dose was required to increase the tail flick latency. Pretreatment with all CCBs, individually enhanced the analgesic effect of pentazocine in both formalin and tail flick tests. In the latter test of nociception, a per se ineffective dose of pentazocine, showed a significant analgesic response in presence of CCB dose which itself was not effective in the test. Chronic concomitant administration of diltiazem with pentazocine did not prevent the development of tolerance to the opioid compound. However, diltiazem when given in combination with pentazocine to pentazocine-tolerant animals, it effectively reversed the tolerance. Results of the study thus suggest that concomitant treatment with CCBs, irrespective of their chemical nature, not only potentiate the antinociceptive effect of pentazocine in opioid naive animals in both tonic and acute nociceptive tests but also reverse the pentazocine tolerance.

Action of acetylcholine on the atropinised frog heart during the winter months.

During the winter months, high doses of acetylcholine produced positive inotropic action without any chronotropic action on the perfused atropinised frog heart, in 5 out of 24 preparations. In the remaining preparations acetylcholine failed to produce any action and positive inotropic effect of acetylcholine on these preparations was noticed if they were perfused with the medium containing excess of calcium. However, the rate remained unchanged. The positive inotropic action was blocked by the local anaesthetic amethocaine and thus may be due to increased penetration of calcium into the cardiac cell.

Action of acetylcholine on the atropinised heart of the frog during summer months.

During summer months high doses of acetylcholine produced positive inotropic and chronotropic actions in 19 out of 22 preparations of atropinised perfused heart of the frog. Hexamethonium failed to block both the positive inotropic and chronotropic actions of Ach. Interestingly another ganglion blocking agent mecamylamine as well as beta adrenergic blocking agent propranolol blocked the positive inotropic action but not the positive chronotropic action.

Attenuation of the effect of progesterone and 4'-chlordiazepam on stress-induced immune responses by bicuculline.

The present study investigates the effect of progesterone, a pregnane precursor of neurosteroids, and 4'-chlordiazepam (4'-CD), a specific ligand for mitochondrial diazepam binding inhibitor receptor (MDR) involved in neurosteroidogenesis, on restraint stress (RS)-induced modulation of humoral and cell-mediated immune responses. RS produced a significant reduction in anti-sheep red blood cells (SRBC) antibody titre, a measure of humoral immune response, and % leucocyte migration inhibition (LMI) and foot-pad thickness test, measures of cell-mediated immune responses. These effects of RS on immune responses were effectively blocked by pretreating the animals with progesterone (10 mg/kg, sc) or 4'-CD (0.5 mg/kg, sc) administered just before subjecting the animal to RS. The effect of both progesterone and 4'-CD on RS-induced immune modulation was significantly attenuated by bicuculline (2 mg/kg, ip) but not by flumazenil (10 mg/kg, ip). Unlike its effect on RS-induced immune responsiveness, progesterone (5, 10 mg/kg, sc) when administered to non-stressed animals produced a significant suppression of both humoral and cell-mediated immune responses which was not reversed by bicuculline. However, 4'-CD failed to modulate immune response in naive non-stressed animals. These results suggest that progesterone and 4'-CD affect stress-induced immune responses by modulating GABA-ergic mechanism. However, GABA-A receptor system does not appear to be involved in progesterone-induced immunosuppression in nonstressed animals.

Possible role of histamine receptors in the central regulation of immune responses.

The present study was designed to delineate the role of H1- and H2-histamine receptors in the neuro-immune regulation in rats. The effects of H1- and H2-receptor antagonists on humoral and cell-mediated immune (HI and CMI) responses were investigated after intraperitoneal (i.p.) and intra-cerebroventricular (i.c.v.) administration. HI response was assayed by anti-sheep red blood cell (SRBC) antibody titre in presence and absence of 2-mercaptoethanol (2-ME). The CMI responses were evaluated by delayed type hypersensitivity (DTH) reaction (in vivo), i.e., measurement of footpad thickness, and lymphokine activity such as leucocyte migration inhibition (LMI) test (in vitro). On i.p. administration, both H1- (pheniramine and astemizole) and H2-receptor antagonists (ranitidine and cimetidine) were observed to produce significant enhancement of anti-SRBC antibody response. However, only H2- and not H1-receptor blockers were observed to stimulate CMI response significantly. When administered by icv route, only H2-receptor antagonists caused a statistically significant increase in both HI and CMI responses, while the H1-receptor blockers failed to modify the same. Thus, H2-receptors appear to play a major role in the histaminergic mechanisms involved in immunomodulation both at the level of immunocompetent cells active in the peripheral tissues as well as through the central nervous system structures involved in the central regulation of neuro-immune interaction.

Differential effects of benzodiazepines on immune responses in non-stressed and stressed animals.

Benzodiazepines (BZDs) used extensively as antianxiety agents are known for their low toxicity. However, a long lasting depression of mitogen stimulated secretion of macrophage-derived cytokines has been shown in offsprings of rats that were exposed to diazepam during pregnancy. The Present study investigates the effects of long term administration of diazepam and alprazolam on humoral and cell-mediated immune responses in adult male Wistar rats and Swiss albino mice. Administration of diazepam (5 mg/kg/day x 7-14 d) and alprazolam (1 mg/kg/day x 7-14 d) produced a significant reduction of anti-SRBC antibody titre, a measure of humoral immune response, and foot pad thickness and % leucocyte migration inhibition (% LMI), measures of cell-mediated immune responses. Administration of diazepam (5 mg/kg, i.p.) or alprazolam (1 mg/kg, i.p.) before subjecting the animals to restraint stress (RS) reversed the immunosuppressive effects of RS. Both per se immunosuppressive effects and attenuation of RS-induced immunosuppression of BZDs was antagonized by flumazenil (10 mg/kg, i.p.), a central BZD receptor antagonist. Thus, BZDs appear to modulate the immune system in non-stressed and stressed adult animals in a differential manner and these effects are mediated via central benzodiazepine receptors.

A review on recent development of common cold therapeutic agents.

(1) The common cold is a frequently occurring illness caused by rhinoviruses. Inspite of its ubiquitous occurrence the disease has defied all efforts of finding a cure. The current approaches to the treatment of common cold can be divided into two important categories: the antiviral and antiinflammatory; both of these leave a lot to be desired. Most of the rhinovirus serotypes use a single cellular receptor, i.e. the intercellular Adhesion Molecule-1 (ICAM-1) for attachment to the cells. This has lead to the development of blockers of this receptor in an effort to find a cure for the common cold. (2) Recently tremacarmra, a synthetic ICAM-1 glycoprotein has been investigated in human volunteers as an antiadhesion molecule towards an approach to common cold therapy. Two dosage forms of the compound-phosphate buffered saline spray and carboxymethyl cellulose-mannitol powder spray were administered intra-nasally in two modes--pre-inoculation (7 h prior) and post-inoculation (24 h after) time periods of rhinovirus type 39 challenge to different groups of human volunteers. Both the treatment modes produced a significant decrease in the symptoms score of clinical illness and concentration of interleukin-8 in the nasal lavage. Saline spray was found to be devoid of any side effects, whereas powder spray produced some nasal irritation initially. The encouraging results of clinical trial with tremacamra show that a cure for common cold is not far off. However, it remains to be seen what would be the impact of such synthetic protein administration on the immune response of the body, should such compounds be used repeatedly. Further, since all colds are not due to rhinovirus it would be wise to restrict the use of tremacamra during autumn and spring when rhinoviruses are known to be the causative organisms of common cold.