ADVERTISEMENTS:
After reading this article you will learn about:- 1. Identification Criteria for a Neuro Transmitter 2. Basic Principles of Transmission 3. Molecular Basis of Transmitter Action.
Identification Criteria for a Neuro Transmitter:
A neuronal chemical should qualify certain criteria before it can be declared as a transmitter.
These include:
ADVERTISEMENTS:
1. It should be present in the presynaptic terminal; it should be synthesized by the neuron in perikaryon (precursors of neuropeptides) or nerve terminal (others), and stored inside synaptic vesicles. Therefore, its precursors and enzymes of its biosynthesis should be present in the presynaptic neuron.
2. It should be released by the nerve terminal upon stimulation (or arrival of action potential) into the synapse or junction in sufficient amounts.
3. It should be able to elicit response when applied to post- synaptic sites exogenously in an identical manner to that when acting following its endogenous release from its presynaptic source. This implies that endogenously applied chemical should faithfully mimic the actions of endogenously released chemical; and also the factors (or chemicals) that interfere with the actions mediated by the endogenous chemical should also have similar effects on exogenously applied chemical.
4. It should be removed from the site of action by specific or non-specific mechanisms so that its action is terminated within reasonable limits of time. It also implies that the chemical should not remain at the site of action for a longer period than that required for its action.
ADVERTISEMENTS:
The criteria are based on neurotransmission process that has been established mostly with respect to PNS. It is very difficult to apply these criteria at central synapses (inaccessibility to experimental manipulation); and many substances that are almost certainly transmitters in the vertebrate CNS have still not satisfied all the criteria.
It is more proper to qualify a transmitter as putative or most probable if it fulfils most of the criteria (direct or indirect evidences) but does not fail to qualify any one; if it qualifies all, it is an established transmitter; and if it fails to qualify any one of the listed criteria, it is not a transmitter.
There is another major problem with these criteria. They pertain to ‘Dale doctrine’ that one neuron releases only one transmitter at its nerve terminals. The doctorine has been reformulated as ‘one neuron releases the same transmitters, not necessarily only one, from its nerve terminals, as the evidence for co-localization, co-synthesis, co- storage, co-release and even co-actions of more than one transmitter substances from the same neurons have become established facts in CNS as well as in PNS:
It is now believed that in many instances the synaptic transmission may be mediated by the release of more than one transmitter. Therefore, the criteria need to be reframed to accommodate the newer eventuality related to co-release and co- action.
Basic Principles of Transmission:
The process of transmission involves a sequential cycle of steps, that are repeated continuously to enable the neurons to make optimal utilization of the transmitters for required actions.
These steps include:
(i) Biosynthesis,
(ii) Storage,
ADVERTISEMENTS:
(iii) Release,
(iv) Action (s),
(v) Inactivation, and
(vi) Regulation of intra-neuronal transmitter levels.
(i) Biosynthesis:
ADVERTISEMENTS:
Most known transmitters are obtained or derived from amino acids with or without modification except acetyl-chloline which is derived from immediate precursor choline. The synthesis of transmitter takes place at nerve terminal from precursors (neuropeptide precursors are synthesized at perikaryon and transported to the site by axoplasmic flow).
Some neurons utilize amino acids directly (glutamate, aspartate, glycine) or modify them to form amines (e.g. gamma- amino butyric acid, dopamine, norepinephrine, serotonin, histamine) or polymerize them into polypeptides (at perikaryon) and then process at the nerve terminals (neuropeptides).
(ii) Storage:
The transmitter molecules are stored inside nerve terminal within miniscule membraneous bags called synaptic vesicles. Each vesicle contains several thousands molecules of the transmitter, and constitute one quantum (one packet of action). The vesicles serve as storage depot, a mechanism to prevent loss of transmitter from leakage and/or enzymatic breakdown if left free, and as a unit to evoke graded response.
The vesicles are normally repelled by inner membrane surface of the presynaptic nerve terminal as both possess net negative charge on their opposing surfaces owing to dominant anionic environment (proteins and phosphates) of axoplasm.
(iii) Release:
ADVERTISEMENTS:
The opening of voltage-gated Ca++ ion channels (by arriving action potential or depolarization wave i.e. axonal conduction) leads to influx of Ca++ ions into the terminal. The ions (being cationic) bring vesicles closer to presynaptic site; the two membranes use; openings appear at fusion points; vesicles extrude transmitter into the synapse (or junction).
The process of release is called exocytosis. The amount of transmitter to be released depends on the frequency of opening of the Ca++ channel gates , as dictated by appropriate nerve activity. The release is accordingly graded or quantal.
(iv) Action:
The transmitter interacts with its specific receptors (s) to elicit effector mechanisms that are characteristic of the receptor type or subtype. The net result is alteration of postsynaptic neuronal activity with or without changes in its metabolic activity. The two usual actions are either excitation or inhibition of neuronal function.
(v) Inactivation:
It refers to termination of action of the transmitter.
ADVERTISEMENTS:
It involves one or more of the following processes:
(i) Transmitter is actively pumped back into nerve terminal (called uptake1) or taken by glia cells (uptake2),
(ii) Transmitter diffuses into available space and tissues so reducing effective concentration at the site and/or,
(iii) Transmitter is enzymatically inactivated at synaptic site, inside nerve terminal or in surrounding tissues.
(vi) Regulation:
The neuron maintains intra-neuronal content of the transmitter relatively constant, and independent of nerve activity or inactivity. This is accomplished by varying – (i) rate of precursor synthesis or precursor uptake or transmitter reuptake, and (ii) activity of regulatory enzymes for its biosynthesis, processing and/or breakdown.
To ensure these functions, the neuron responds to the signals – (i) obtained from postsynaptic receptor activation or blockade, and/or, (ii) generated within or at presynaptic sites (Rs, R3, R4). The DNA plays crucial role in this regards.
Molecular Basis of Transmitter Action:
The effects of a particular transmitter on its receptor site are not necessarily specific for the transmitter but are exclusively determined by the nature of the receptor site. The receptor sites are of two main types: ionotropic and metabotropic.
ADVERTISEMENTS:
Differential features of the receptors are given in table 1.A transmitter may activate only ionotropic receptors (e.g. glycine, glutamate/aspartate) or only metabotropic receptors (e.g. dopamine, norepinephrine, histamine, neuropeptides) or both types (e.g. acetylcholine, 5-HT, GABA, purines).
Ionotropic Receptors:
Ionotropic receptor is a multi-subunit protein that surrounds an ion channel. The channel is called ligand gated ion channel as it is opened by the interaction of a specific chemical with the receptor or channel protein.
The channel differs from voltage- gated ion channel as it can allow one or more than one ionic species to flow simultaneously through the same channel (whereas in voltage-gated ion channel the ionic flow is sequential and through independent channels, one for each ionic species).
Potential features evoked are characterized by:
(i) Fast onset,
ADVERTISEMENTS:
(ii) High amplitude up to several millivolts and
(iii) Brief duration-milliseconds to seconds.
Potential changes are independent of second messenger system, and are evoked by the transmitter directly. Ionotropic receptors do not alter metabolic activity in the target neuron/cell. The physiologic effects are of two types; excitation or inhibition.
Excitation:
The neuron is excited when opening of ion-channel leads to increase in the conductances of Na+ (influx) and K+ (efflux), and at certain places that of Ca++ (influx). The ionic fluxes generate fast EPSP (excitatory postsynaptic potential). Transmitters that evoke such effects include glutamate/aspartate (at most sites), acetylcholine (nicotinic sites), 5-HT (at site 3) and purines (at P2x site).
(a) Gates regulated by protein kinases (second messenger-dependent) and/or by G protein fragments directly
(b) Regulated by protein kinases (second messenger-specific)
(c) Fast excitatory response due to opening of the channel for Na+, K+ and at some sites for Ca++, fast inhibitory due to opening of channel for Cl– or at some sites for K+.
(d) Responses with respect to metabolic activity and neuronal excitability.
Inhibition:
The neuron is inhibited when opening of ion channel leads to an increase in the conductance of Cl– ions (influx). This leads to generation of fast IPSP (inhibitory postsynaptic potential). Transmitters that evoke such a response include glycine, and GAB A (at site A).
Metabotropic Receptors:
Metabotropic receptor is a single unit hydrophobic protein consisting of a long polypeptide chain arranged into seven helical segments; external membrane site(s) bind to a specific transmitter, and internal (cytoplasmic) site is coupled to a specific G protein (i.e. protein with high binding affinity for GTP).
These receptors are also called G protein coupled receptors. The physiologic effects include metabolic alterations that may be accompanied by changes in ionic conductances (pertaining mainly to cationic species).
Potential features, wherever pertinent, are characterized by:
(i) Slow onset,
(ii) Low amplitude-one to several millivolts, and
(iii) Long duration up to minutes.
Biochemical alterations include activation or inhibition of adenylate cyclase, or activation of phospholipase C.
The effects are governed by:
(i) Levels of second messengers, &
(ii) G protein fragment activities. The activity spectrum varies with nature of G protein that is specific for the receptor site.
G Protein Based Effectors Mechanisms:
There are at-least four types of G proteins; Gs, Gi, Go and Gq. Each type has several subtypes. Each G protein is composed of three distinct polypeptide units; alpha, beta and gamma. The nature of G protein is determined by the nature of its alpha unit. The alpha unit also determines which type of enzyme will be affected. Beta-gamma fragment of G protein may also alter metabolic and/or ionic conductances.
The operational mechanism of G protein may be summed up as:
(i) GDP is bound to alpha unit of a G protein at rest, and G protein is inactive,
(ii) Transmitter interacts with the receptor,
(iii) Interaction causes dissociation of GDP from G protein, and association of GTP to alpha unit,
(iv) G protein gets activated, &
(v) Fragments into two; GTP- alpha and beta-gamma that mediate cellular responses independently or in co-operation with each other,
(vi) Once GTP-alpha has accomplished its function(s) it expresses its intrinsic GTPase activity that causes removal of phosphate from its bound GTP to form GDP-alpha,
(vii) GDP-alpha attaches to beta-gamma fragment to regenerate original G protein (i.e. GDP-alpha-beta-gamma complex). The cycle is completed within a few seconds.
The effector mechanisms may be conveniently categorized as under:
1. Gs Mediated Actions (Adenylate Cyclase Activation):
Alpha-s stimulates adenylate cyclase that raises cAMP and increases Ca++ currents by opening Ca++ channels. cAMP and Ca++ ions act as second messengers. They activate specific protein kinases (enzymes that phosphorylate proteins including enzymes). Beta-gamma fragment is known to activate certain isoenzymes of adenylate cyclase in presence of alpha-s; thus it serves to amplify receptor signal.
Phosphorylation of proteins through activated protein kinases alters one or more of the functions depending upon that kinases are present in the target cell:
(i) Phosphorylation of the membrane proteins involved in ion permeability changes,
(ii) Phosphorylation of cytoplasmic proteins involved in control of cellular metabolism,
(iii) Phosphorylation of intracellular transport proteins to facilitate transport of intracellular chemicals or components, and
(iv) Phosphorylation of nuclear proteins regulating gene-expression. The transmitters activating Gs protein effector mechanisms include; norepinephrine (beta sites), dopamine (D1 sites); histamine (H2 site), vasopressin (V2 site) and 5-HT (sites 4-7). The physiologic effects are metabolic activation with slow neuronal depolarization; latter by increased Ca++ conductance or at certain sites by closing K+ (see later).
2. Gi Mediated Actions (Adenylate Cyclase Inhibition and Alterations in Ionic Conductances):
ADVERTISEMENTS:
Alpha-i inhibits adenylate cyclase thus decreases cAMP production. Beta-gamma fragment also inhibits certain isoenzyme forms of adenylate cyclase. These alterations decrease cellular processes dependent on cAMP actions. Alpha-i closes Ca++ channels to decrease Ca++ currents. Beta-gamma fragment increases K+ currents by opening K+ channels.
The consequent physiologic effects are inhibitory to membrane excitability and to metabolic functions. The transmitters evoking such responses include; dopamine (D2 site), norepinephrine (alpha-2 site), acetylcholine (M2 and M4 sites), 5HT (site-1), GABA (site-B), endogenous opioid peptides (mu, kappa and delta sites), somatostatin and pancreatic polypeptide.
3. Gq Mediated Actions (Phospholipase C-beta Activation):
Alpha-q activates membrane bound phospholipase C-beta that hydrolyses membrane-bound phosphatidyl inositol-4, 5-diphosphate generating, thereby, two second messengers; inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 mobilizes Ca++ from internal storage sites thus raises cytosolic Ca++ pool. Ca++ and DAG activate their specific protein kinases.
Ca++ may also participate, independent of protein kinase activation, to alter cellular metabolism. The consequent physiologic effects are stimulatory to cellular metabolism. The transmitter that elicit such responses include norepinephrine (alpha- 1 site), acetylcholine (M1 and M3 sites), vasopressin (Vi site), histamine (H1 site), 5-HT (site-2), oxytocin, tachykinins, and purines (P2Y site).
4. Closure of Potassium Channels that are Normally Open:
At certain sites G protein-coupled receptor activation leads to second messenger mediated closure of potassium channels. This consequently decreases K+ currents (efflux) and tends to cause slow depolarization of the neuronal membrane. The neuronal threshold for excitatory inputs is reduced. Effect of concurrently acting excitatory input is potentiated.
The receptor sites and coupled G protein variety include:
5 – HT2A (5-HT), alpha-1 (norepinephrine) and M1 (acetylcholine) – all Gd coupled; 5-HT4 (5-HT) – Gs coupled. The endogenous ligands are indicated within parenthesis.
Signal Amplification:
The G protein is considered a molecular switch. It is turned on by transmitter-receptor interaction. It bifurcates the receptor-signal vide its fragment activities. It turns itself off automatically after doing its job to resume original inactive form.
An individual neuron (or effector cell) may respond in one of the three ways:
(i) The cell may express multiple G proteins, each responding to different transmitter, thus regulating several different effectors (parallel transduction),
(ii) Several receptors in a cell may activate a single G protein (convergent transduction), or
(iii) One receptor may regulate more than one type of G protein in the same cell (divergent transduction).
