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The Facilitatory Post Tetanic Potentiation (Fptp) as an Expression of Presynaptic Nicotinic Stimulatory Autoreceptors at the Mammalian Neuro-muscular Junction


Gawade Shivaji P.  
Dept.of Pharmacology, Sahyadri College of Pharmacy, Methwade, Tal.Sangola, Dist.Solapur-413307, M.S., India.

ABSTRACT 

The sea snake Enhydrina schistosa is major neurotoxin, enhydrotoxin-and produced irreversible neuromuscular blockade of single twitch responses to indirect electrical stimulation. The tetanic stimulation elicited sustained tetanus during the neuromuscular blockade.  The sustained tetanus was transformed to an exaggerated twitch response, following treatment of enhydrotoxin-a plus monovalent sea snake antivenin and washout when the mammalian neuromuscular preparation completely restored to normal twitch responses to single indirect electrical stimulation. The exaggerated twitch response as a facilitatory post tetanic potentiation (fPTP) is discovered as an expression of the presynaptic nicotinic stimulatory autoreceptor at the mammalian neuromuscular junction. The observation of fPTP as a presynaptic nicotinic stimulatory autoreceptor was analogous to the experiments of tetanic fade induced by d-tubocurarine on mammalian neuromuscular junction subjected to high frequency stimulation.

Key Words: Exaggerated twitch response, facilitatory post tetanic potentiation, presynaptic nicotinic stimulatory auto receptor.

INTRODUCTION

The neurotoxin with presynaptic and postsynaptic site of action enhydrotoxin-a, a principal strongly basic neurotoxin was isolated from the venom of common sea snake Enhydrina schistosa from western Indian Coast 1 . The presynaptic site of action of enhydrotoxin-a was evident from the transformation of sustained tetanus to an exaggerated twitch response and restoration of sustained tetanus by pre-treatment with anti-acetylcholinesterase, physostigmine at the mammalian neuromuscular junction following exposure to enhydrotoxin-a and horse monovalent E.schistosa antiveni 2. The exaggerated twitch response is the response, higher than the conventional post tetanic potentiation (PTP) that occurred when neuromuscular preparation was exposed to enhydrotoxin-a and  monovalent E. schistosa antivenin and restored normal twitch responses to a single indirect electrical stimulation (IES).

The PTP is the potentiation of normal twitch response to single indirect electrical stimulation immediately following exposure of the synapse to high frequency (10-100 Hz) stimulation for 2-5 seconds. It is in the form of short lived synaptic plasticity that results into an increase in the frequency of miniature excitatory postsynaptic potentials (mEPSPs) or miniature postsynaptic currents (mEPSCs) without affecting amplitude of spontaneous postsynaptic potentials 3. It was shown that at low concentration of enhydrotoxin-a (4.8 X 10 -7 g/ml), tetanic stimulation (20 Hz, for 5 sec) elicited well sustained tetanus with the presence of PTP except in the terminal phase of n-m blockade. Whereas d-tubocurarine produced well defined wednesky inhibition with the absence of PTP 4, 5. It was reported that   α –bungarotoxin showed a well sustained tetanus with the absence of PTP whereas β-bungarotoxin exhibited typical wednesky inhibition with the presence of PTP 6.

In the present review, the observation of exaggerated twitch response as a facilitatory PTP is reiterated as an expression of presynaptic nicotinic stimulatory autoreceptor in the context of, presynaptic nicotinic receptor, presynaptic nicotinic stimulatory/ facilitatory autoreceptor in the functioning of cholinergic neurotransmission.

MATERIALS AND METHODS

Experimental method of isolated rat phrenic nerve diaphragm preparation described by Bulbring was followed 7. The twitch responses to single indirect electrical stimulation at 0.2Hz and 0.5 msec. duration were recorded at supramaximal voltage. The nerve muscle preparation was used to evaluate the effect of enhydrotoxin-a (EsNTx-a), monovalent sea snake antivenin (ASSVS), 8 EsNTx-a + ASSVS combination and eserine pre-treated EsNTx-a + ASSVS combination on a single and repetitive indirect electrical stimulation and PTP.

Besides, various conventional and modern pharmacological, neurophysiologic and neurobiochemical methods reported in the literature explaining the role of nicotinic acetylcholine receptors in pre and post synaptic cholinergic neurotransmission have been reviewed.

RESULTS AND DISCUSSION

The present review article encompasses state of art about (I) postsynaptic nicotinic acetylcholine receptor, (II) presynaptic acetylcholine receptor, (III) presynaptic nicotinic autoreceptor and (IV) perspectives on fPTP as the presynaptic nicotinic stimulatory autoreceptor at the mammalian neuromuscular junction.

(I) Postsynaptic nicotinic acetylcholine receptor 

The snake venom neurotoxin α-bungarotoxin isolated from Bungarus multicinctus,, because of stronger affinity, blocks irreversibly the nicotinic ACh R at the motor end plate. The α-bungarotoxin have been successfully employed to isolate and purify nACh- R from Torpedo electric organ Electrophorus electricus 9. and its primary structure was determined. Radio isotope labelled neurotoxin was used for the identification of nicotinic cholinergic receptor in vitro comprising five subunits resembling the petals of lily flower10, 11 . The nicotinic muscle AChRs are divided into two types based on binding properties to α-bungarotoxin. Homopentamer of α7, α8 or α9 subunits or heteropentamer α 2βε (γ) δ. The second type of AChR not bound to α-bungarotoxin include, α26 and β24 subunits. Acetylcholine binds at the αγ and αδ sites, prominently in the α1 subunits thereby induce conformational changes resulting into activation and opening of receptor operated ion channel with consequent depolarisation and excitation due to increased cellular permeability for Na+ and Ca2+ 12 .The neuronal nicotinic AChR expressed in the peripheral ganglia, adrenal medulla and in the brain are homopentamers of five α78 and α9 subunits and heteropentamers of  α78 α910 subunits 13.  It has been reported that the binding of acetylcholine to open neurotransmitter gated ion channel causes a localised disturbances in the extracellular domain and initiates small rotations in the protein subunits which triggers  conformational changes in α helices lining the membrane spanning pore 14. 

  1. Presynaptic nicotinic acetylcholine receptor

By using monoclonal antibodies, α bungarotoxin insensitive presynaptic receptors containing α3 protomers at the motor nerve terminals selective for Ca2+ in the open state, were investigated 15. Nicotine and nicotinic agonists such as epibatidine, anatoxin-a, cytidine, isoarecolone induced release of 3H ACh from superfused rat hippocampal synaptosome was abolished by nicotinic antagonist dihydro β-erythroidine, mechamylamine and pempidine. However this ACh release was not inhibited by α7 selective antagonists, methylaconitine. 3H ACh release was calcium and concentration dependent however; K+ induced ACh release was not calcium or concentration dependent 16. Patch clamp studies were performed on rat hippocampus and human cerebral cortex slice preparation using nonselective agonist ACh and α7 nAChR –selective agonist, choline. The activation of different nACh R subtypes present at the preterminal and terminal areas triggers TTx sensitive release of GABA. The modulation of GABA release is either due to inhibition or disinhibition of pyramidal neuron. It was observed that desensitization of α7 nAChR was faster and longer lasting than that of choline. It was implicated that presynaptic α-7 nAChR may be involved in the cognitive function in Alzheimer’s disease 17.

III. Presynaptic nicotinic acetylcholine autoreceptor  

Auto receptors are release regulating receptors localized externally on presynaptic axon terminal.18 Receptors controlling the release following depolarization are suggested to be presynaptic auto receptors 19.  Presynaptic nicotinic autoreceptors probably representing α4β2 nAChR localised on cholinergic nerve endings, mediate enhancement of ACh release. The effects of nicotinic receptor activation on the basal release of 3H ACh from human neocortex synaptosomes were studied. It was shown that nicotine or ACh + atropine increased the spontaneous release of tritium in a concentration dependent manner [EC50 value (-) nicotine (1.16 + 0.33 µM) and that of ACh + atropine (2.0 + 0.04 µM)] 20. Nicotine and nicotine with scopolamine, piracetam, mechamylamine, ondansetron and sulpiride treated male wistar rats were evaluated on various psychopharmacological animal models. The maximum increase in step down latency in scopolamine group during initiation of learning, piracetam group during learning process and mechamylamine group during retrieval  was an indication of differential involvement of cholinergic activation mediated via presynaptic mechanism during initiation of learning ,learning process and memory retention 21 .

IVa]:  Stimulatory / Facilitatory presynaptic nicotinic autoreceptor

The existence of facilitatory presynaptic nicotinic autoreceptors was shown from maximum inhibition of tritiated efflux using radiolabelled technique. The effect of snake venom neurotoxins, β-bungarotoxin, κ-bungarotoxin, α-cobra neurotoxin and erabutoxin-b and that of plant neurotoxin d-tubocurarine (d-tc) were evaluated on evoked release of newly synthesized 3H ACh following incubation of rat left hemi diaphragm with 3H choline.The d-tc reduced the 3H ACh by about 50% of basal tritiated efflux indicative of blockade of presynaptic nicotinic autoreceptors at the motor endplates 22. Oxotremorine, a muscarinic receptor agonist inhibited release of 3HACh whereas atropine, a muscarinic antagonist stimulated release of 3HACh evoked at 0.3 and 0.1 Hz from rat cerebrocortex slice prelabeled with [3H] choline. Nicotine enhanced [3H] ACh whereas nicotinic antagonist, mechamylmine inhibited [3H] ACh release at 0.1 Hz without affecting [3H] ACh at 3 Hz. Nicotinic autoreceptors responded to endogenous ACh and in conditions when ACh release was affected by exogenously applied nicotine agonist and antagonist 23. Evoked release of newly synthesized 3HACh following incubation of rat left hemi diaphragm with 3H choline was investigated in the absence of anticholinesterase. The effect of snake venom neurotoxins β -bungarotoxin, k-bungarotoxin, α-cobra-neurotoxin and erabutoxin-b along with d-tubocurarine on evoked release of 3HACh was evaluated. It was observed that d-tubocurarine reduced the 3HACh release by about 50% whereas erabutoxin-b enhanced basal tritiated efflux. It was inferred that d-tubocurarine by blocking presynaptic nicotinic autoreceptors at the end plate, motor nerve caused reduction in the tritiated efflux of [3H] Ach 24 .

Chronic exposure of nicotine to the rat enhanced nicotinic 3H methyl carbamylcholine (3HMCC) binding sites and acetylcholine release from cholinergic nerve terminal in the frontal cortex, parietal cortex, striatum and hippocampus of rat brain. Muscarinic binding sites M1 3H pirenzepine and M2 3H ACh   binding however, remain unaffected indicating selectivity of presynaptic nicotinic autoreceptor function at the central cholinergic presynapse in rat brain. The treatment of nicotine in rat increased N3H methylcarbonyl choline (N3HMCC) binding sites by up regulation in the frontal cortex. in the rat brain 25. In atropinised preparation and in the preparation of high K+, muscarinic inhibition of ACh response was reversed into a nicotinic potentiation. The desensitization produced by acetylcholine in high K+ was reversed to normal by washings. In high K+ medium, lowering of Ca2+ or chelation of Ca2+ by BAPTA–AM [(1, 2-bis (O-aminophenoxy) ethane-N, N, N’, N’-tetra acetic acid) (Acetoxy methyl)], synaptosomal preparation produce nicotine response of acetylcholine. However, BAPTA treatment could not avoid desensitization by acetylcholine indicating the role of Ca2+ between muscarinic and nicotinic autoreceptor function 26.  

In the presence of d-tubocurarine (0.2 μM) , at 2 mM Ca 2+ concentration , quantal content was decreased by 30 % when motor nerve terminal was stimulated at high frequencies (50-150 Hz), whereas quantal content was  enhanced  by 20 % when the preparation was stimulated at low frequencies ( 0.5 -1 Hz). The calcium independent decrease in the quantal content in curarised motor nerve terminal subjected to high frequency stimulation was suggested to be inhibitory action on +ve feed back presynaptic nicotinic muscle autoreceptors 27.  It has been suggested that nicotinic agonist induced synaptic facilitation contributes of both presynaptic nAChR and voltage sensitive Ca 2+ channel for the entry of calcium into the synaptic nerve terminal in central neurons of the rat 28. The effect of neuronal acetylcholine receptor antagonists, α bungarotoxin, d-tubocurarine, mechamylamine and hexamethonium on twitch response to indirect stimulation, 3H ACh release and response to high frequency stimulation (50 Hz, 5 sec.) on isolated phrenic nerve diaphragm were investigated. The nicotinic receptor antagonists reduced single twitch response and also caused tetanic fade. The α bungarotoxin however, at the concentration 0.3 µM, blocked single twitch by interaction with post junctional nicotinic ACh receptor. The nACh receptor antagonists reduced single twitch response and also caused tetanic fade however, did not affect [3H] ACh release. It was inferred that, the reduction in the single twitch was by interaction with postjunctional nicotinic ACh receptor consisting of α1 subunit whereas occurrence of tetanic fade following high frequency stimulation, may be by blocking presynaptic nicotinic facilitatory autoreceptor consisting α3β2 subunits 29. The skeletal muscle relaxants panchuronium, cisatracurium, d-tubocurarine and ganglionic blocking agent hexamethonium decreased muscle tension of train to four fade [(TOF) fade]. The TOF fade parameter was used to assess intensity of neuromuscular blockade in curarised patient.  The TOF fade was attributed to the blockade of facilitatory nicotinic receptor on motor nerve terminals 30.

IVb]. The f- PTP as a presynaptic stimulatory nicotinic autoreceptor  

The f-PTP as a presynaptic stimulatory nicotinic autoreceptor was first time revealed when author was a post doctoral fellow with Prof.Anthony T.Tu at Department  of  Biochemistry ,Colorado State University, Fortcollins, Colorado (1997-98), while comparing mechanism of neuromuscular blockade produced enhydrotoxin-a and d-tubocurarine at two concentrations on mammalian neuromuscular junction from his Ph.D.(Faculty of technology) in pharmacology thesis submitted to University of Bombay (1980) 31. The existence of presynaptic stimulatory autoreceptor was reported by Prof.W.C.Bowman and co-workers using d-tubocurarine on mammalian neuromuscular junction subjected to tetanic stimulation . The elapid short chain neurotoxin ,cobrotoxin (62-4) inhibited enhanced release of neurotransmitter, ACh by +ve feed back during tetanic stimulation at the neuromuscular junction indicating tetanic run-down or tetanic fade which was evident when neuromuscular junction was restored following washout to a  normal twitch response to a single indirect electrical stimulation 32,33.   

Stimulation of presynaptic receptors by accumulated acetylcholine due to inhibition of AChE, gave rise to the antidromic impulses in the motor nerve as well as orthodromic excitation of transmitter release. It has been implicated that cholinergic nerve terminal autoreceptors play physiological role in mobilizing acetylcholine from the reserve to the readily releasable pool of acetylcholine, in order that the vesicular release may keep pace with the demands of high frequency stimulation of motor nerve impulse, characteristic to transmission to skeletal muscle at the mammalian neuromuscular junction.33 Tubocurarine blocked these stimulatory type of presynaptic nicotinic autoreceptor as evidenced from ‘tetanic fade’ that occurred during partial neuromuscular blockade 34 . 

Using principal sea snake neurotoxin enhydrotoxin-a and monovalent E.schistosa horse antivenin (ASSVS) treatment on rat phrenic nerve diaphragm subjected to high frequency stimulation,  it has been possible to unravel the existence of presynaptic stimulatory nicotinic autoreceptor as a facilitating post tetanic potentiation (f-PTP) when sustained tetanic response was transformed into an exaggerated twitch response following washout when neuromuscular transmission was restored to a normal single twitch response 35. Antibodies directed to enhydrotoxin-a present in monovalent E.schistosa antivenin, while displacing the toxin from postsynaptic nicotinic cholinoceptive sites, able to restore the twitch response to a single electrical stimulation. However the sustained response to high frequency stimulation was lost and transformed into an initial heightened twitch, as the prolonged exposure of antibodies at the postsynaptic nicotinic receptors, altered its conformational state, required to elicit the normal sustained tetanic form. In the context of nonavailability of the postsynaptic cholinoceptive sites, the accumulated acetylcholine elicited the exaggerated twitch response as f-PTP by interaction with presynaptic nicotinic autoreceptor expressed to elicit facilitatory twitch response, termed as presynaptic nicotinic stimulatory type of autoreceptor. AntiAChE, physostigmine (eserine) pre-treatment restored the sustained form of tetanus in toxin-antivenin treated neuromuscular preparation when hydrolysis of acetylcholine was prevented and sufficient acetylcholine was available at the post synaptic cholinoceptive site to restore the altered conformation of the post synaptic nicotinic receptors for the normal sustained form of tetanus .Using model studies of   enhydrotoxin-a and ASSVS, it was possible to unravel the existence of facilitatory presynaptic nicotinic stimulatory autoreceptors as f-PTP when sustained tetanus was disappeared at the rat skeletal neuromuscular junction subjected to high frequency stimulation.  

CONCLUSION

The existence of f-PTP would provide an insite as a diagnostic tool to detect defective presynaptic acetylcholine release mechanism in experimental animals with neuromuscular diseases and in depth studies would contribute in the basic mechanism of diseases affecting neuromuscular system.

Acknowledgements

The author wish to acknowledge the valuable guidance and constant encouragement of Ph.D. guide Dr.B.B.Gaitonde Director Haffkine Institute, Bombay, during the period 1977-1980.

Conflicts of Interests

The author declares that there is no conflict of interest to disclose.  

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