Effect of the Carbamate ( Physostigmine ) on the Activity of Nitric Oxide Synthase in Different Parts of Rat Brain ( Invivo Studies )

Physostigmine (eserine) is the methyl carbamic ester of phenolic trimethyl ammonium compound. It is a powerful inhibitor of acetylcholinesterase enzyme activity and used in some therapeutic preparation. In this work, experiments were carried out in-vivo to study the intraperitoneal (i.p.) infusion of eserine on the activity of nitric oxide synthase (NOS) enzyme, obtained from whole and five different parts of rat brain, namely: basal ganglia, frontal cortex, medulla oblongata, pons, and cerebellum. In this work ,two experiments were carried out. Experiment A: to study the dose dependence of i.p. infusion of eserine on NOS activity. Experiment B: to study the time dependence post-infusion of constant dose of eserine (the dose which caused 50% inhibition of the enzyme activity: I50). The results showed that the inhibition of the enzyme occurred in each part studied, and the inhibition increased with increasing the infused dose of eserine, and the time post i.p. infusion, i.e., the inhibition is dose and time dependent. The highest inhibition occurred in the pons and medulla oblongata extracts; these parts are responsible for the reflex centers of cough and vital centers.


INTRODUCTION
Carbamate pesticides poisoning tends to be less severe than organophosphorus pesticides (OP) because they bind reversibly to the active site of the enzyme, in contrast to OP pesticide that binds irreversibly. (1)Carbamate pesticides inhibit rat brain nitric oxide synthase (NOS) activity through inhibiting the regulatory protein calmodulin (CaM ) (NOS stimulator) activity without affecting the basal enzyme activity; leading to brain toxicity . (2)tric oxide (NO) is a regulatory biological substance and an important intracellular messenger that acts as a specific mediator of various neuropathological disorders. (3)NO plays an Bull High Inst Public Health Vol.37 No. 2 [2007]   important role as a neurotransmitter in the nervous system, as vasodilator in the cardiovascular system and as a cytotoxin in the host defence mechanism of macrophages. (4)Nitric Oxide Synthase research. (5)Brain NOS is a constitutive enzyme and the function of which is to produce NO on demand for diverse neurophysiological activities. (6)ysostigmine (eserine) is a tertiary nitrogen atom derivative which blocks conduction and enzyme activity . (7)e aim of this work is to study the in-  25 rats were used in this experiment.
with saline as experiment A, while the experimental groups (5 rats each) were injected with a constant dose 3.0 mg/kg body weight (I50) of eserine.The rats were decapitated at various time interval (3, 6, 9, and 12 min).
In the above experiments A as well as B the whole as well as the different parts of the brain were isolated, washed with ice cold saline and weighed each separately.
The parts were homogenized in ice cold phosphate buffer Na2HPO4: NaH2PO4, for NOS. (8,9)e to the transient and volatile nature of NO which makes it unstable, NO were oxidized to the stable compounds nitrite  The inhibition of NOS in the different parts increased with increasing the infused dose of eserine.Rats were killed after 9 min post injection, due to the signs of tremors and creeping with their hind limbs at 3.0 mg/kg BW.The highest inhibition occurred in the pons and medulla oblongata extracts (Fig. 1), and the lowest in the cerebellum, basal ganglia and frontal cortex extracts.

b) Dependence on the different time post infusion of constant dose of eserine (experiment B)
Table 2 shows that the activity of NOS post injection of constant dose of eserine 3.0 mg/KgBw of rats.The highest inhibition occurred in the medulla oblongata and the pons extracts, (Fig. 2), while the lowest inhibition occurred in basal ganglia and frontal cortex extracts.

DISCUSSION
The experiments showed considerable difference in NOS activity in the various parts of rat brain.The highest activity was detected in the cerebellum, lower in basal ganglia, and the lowest in the frontal cortex.(12)(13) In the

(
NOS) was first identified and described in 1989.The demonstration of the formation of NO by an enzyme (NOS) in vascular endothelial cells opened up what can be considered a new area of biological p. with eserine in doses of 1.0, 2.0, 3.0, and 4.0 mg/kg body weight.The rats were decapitated 9 minutes post-injection.At this time, signs of tremors, shivering, tachypnea, and craving for air appeared on the rats and most of them died 15 min.post infusion of 4.0 mg eserine/Kg BW.Experiment B: (Dependence on the time course post injection).

pH 7 .
4, 0.1 mole/L and used for the enzyme assay.Assay of Nitric oxide synthase (NOS) enzyme activity: Brain Nitric oxide synthase (NOS) is a constitutive enzyme and the function of which is to produce nitric oxide (NO).Nitric oxide (NO) is also a neurotransmitter formed in addition to citrulline from L-arginine through mediation of the enzyme nitric oxide synthase (NOS).Bull High Inst Public Health Vol.37 No.2 [2007] L-Arginine NOS ⎯⎯⎯ → L-citrulline + NO NOS was determined by estimating nitrite.Nitrite was being the most stable metabolite of oxide.It is used as an index

(NO 2
) and nitrate (NO 3 ).The most convenient detection method for NO was measured in term of NO 2 which can be detected by photometric method using Griess reagent(8) at 546 nm.Griess reagent consists of one part of naphthylethelenediamine dihydrochloride (NED) in distilled water and one part of 1% sulfanilamide in 5% concentrated phosphoric acid (H3PO4).The two parts being mixed together within 12 h of use and kept chilled.Each part may be stored separately refrigerated up to 2 months.The mixture of the two parts is incubated with a NO2-continaing sample (ratio 1:1) to form purple azo dye and its absorbance is measured at a wave length of 546 nm.(8,9)To determine NO2 in brain tissue, 150 μl brain homogenate containing 20 mg tissue was mixed with 1.5 ml of Griess reagent and the mixture incubated for 5 min at room temperature, the absorbance was measured at 546 nm.Nitrite were calculated from NO2 standard curve.RESULTS a) Dependence on the different doses of eserine (experiment A) present work, the i.p injection of different doses of the carbamate eserine resulted in the inhibition of NOS activity.Likewise, previous studies showed that the organochlorine and organophosphorus compounds owing to their lipophilic nature, bind to the hydrophobic region of CaM and thus impair CaM dependent NOS activities like Ca 2+ ATPase phosphodiestrase.(13)Also, Roa et al.,2 found that the Carbamate sevin inhibits rat brain nitric oxide synthase (NOS) activity through inhibiting the regulatory protein calmodulin (CaM) (NOS stimulator) activity without affecting the basal enzyme activity; leading to brain toxicity.The present work showed that the inhibition of NOS activity decreased with increasing the concentrations of eserine, i.e., dose dependence.The NOS obtained from medulla oblongata as well as pons showed the highest inhibition while the lowest inhibition was obtained in the cerebellum, basal ganglia and frontal Bull High Inst Public Health Vol.37 No.2 [2007] cortex.This indicated that the eserine affects the NOS of pons and medulla oblongata more than other parts.In this respect eserine resembles sevin in its inhibition of rat brain NOS .(14)Regarding the effect of time post injection of constant dose of eserine, the results indicated that the inhibition reached about 50% after 12 min post injection of 3.0 mg/KgBw eserine.This inhibition increased with increasing the time post injection, i.e., it is also time dependent.It has been found that the results obtained from experiment B coincises with those obtained from experiment A. Complete inhibition of NOS wasn't obtained in any part of the brain through the 12-minute course post infusion in spite of the signs of tremors and suffocation of the rats.Finally; it is note-worthy to mention that the difference in the rate of NOS inhibition by eserine in different parts of rat brain could be due to: a.The inhibition NOS in the various regions of the brain is dependent on the concentration or the form of the enzyme, i.e., isoenzyme phenomenon.b.The rate of supply of the inhibitor differs because blood flow differs between areas of the brain.c.The high effect of eserine on NOS activity may be due to the solubility of this compound in the lipid layer in the brain as eserine is a tertiary nitrogen compound and affects the parts containing white matter more than the parts containing gray matter, as previously proved by Osman et al .(15)•The parts which were the most affected with eserine were the pons

Figure ( 2 )
Figure (2): The effect of constant dose of eserine (3.0mg/KgBW) on whole and five different parts of rat brain NOS at various time post injection