Neurotransmitter Identification and Proof of Function

Neurotransmitter Identification and Proof of Function

Objectives

• Explain the criteria used to establish a chemical substance as a neurotransmitter.
• Explain the establishment of noradrenaline as a neurotransmitter.
• Explain why it is important for pharmacologists to identify neurotransmission systems.

Neurotransmitters

Established Neurotransmitters

• Acetylcholine
• Noradrenaline
• Dopamine
• Glutamate
• GABA (Gamma-aminobutyric acid)
• Glycine
• Substance P
• Vasoactive Intestinal Peptide (VIP)
• Adenosine Triphosphate (ATP)
• Nitric Oxide (NO)

Putative Neurotransmitters

• Histamine
• Adenosine
• Taurine
• Urotensin II
• Angiotensin I
• Carbon Monoxide (CO)
• Hydrogen Sulphide (H2S)

Steps for Establishing a Neurotransmitter

1. Identification of Biological Activity of Tissue Extract
2. Identification of Active Principle
3. Identification of Physiological Roles
4. Satisfying the 7 Criteria for a Neurotransmitter
   1. Mimics the nerve response.
   2. Synthesis pathway in the nerve.
   3. Storage mechanism in the nerve.
   4. Release (often Calcium-dependent) from the nerve.
   5. Specific receptor activation.
   6. Action ceases quickly via enzymatic degradation.
   7. Action ceases quickly via neuronal or extraneuronal uptake.

Noradrenaline: Establishing as a Neurotransmitter

Identification of Biological Activity

• Oliver & Schafer (1895) studied aqueous extracts of adrenal glands.
  • Found the extract increased the rate and force of contraction of the frog isolated heart.
  • Found the extract increased blood pressure of anaesthetized dogs.
  • The biologically active extract was found only in the adrenal medulla, not in the adrenal cortex.

Identification of Active Principle

• 1898 - Abel (USA) identifies ‘epinephrine’.
• 1901 - Takamine (UK) identifies ‘adrenaline’.

Identification of Physiological Role

• 1901 - Langley: effects of adrenal extract are similar to those of sympathetic nerve activation.
• 1905 - Elliott: adrenaline mimics the effects of sympathetic nerves and may be the sympathetic transmitter (adrenaline the ‘putative’ transmitter).

Mimics the Nerve Response

• Barger & Dale (1910) - anaesthetized animals:
  • Piloerection:
    • Sympathetic nerve stimulation - strong.
    • Adrenaline - weak.
    • Noradrenaline - strong.
  • Bladder relaxation:
    • Sympathetic nerve stimulation - weak.
    • Adrenaline - strong.
    • Noradrenaline - weak.
• Noradrenaline is a closer mimic than adrenaline in a rank order of potency analysis.

Presence in the Nerve

• 1960-64 - fluorescence spectroscopy showed that sympathetic nerves release noradrenaline.
  • Identification usually requires some form of histology, for the putative transmitter itself or for one of its enzymes.
  • Noradrenaline + formaldehyde yields a fluorescent product.
  • The presence of the putative transmitter should be removed by removal of the nerve (denervation).

Synthesis in Nerve

• Characterized during the 1960s.
• The synthesis pathway is as follows:
  • Tyrosine is converted to DOPA by tyrosine hydroxylase.
  • DOPA is converted to dopamine by DOPA decarboxylase.
  • Dopamine is converted to noradrenaline (NA) by dopamine-{\beta}-hydroxylase.
• Note: in the nerve, there is no phenylethanolamine-N-methyl transferase, which, in the adrenal gland, converts noradrenaline to adrenaline.

Storage in Nerve

• Storage vesicles for NA detected by electron microscopy - ‘small granular vesicles’.
• Concentrate and protect the transmitter (from degradation by mono-amine oxidase).
• Vesicles have specialized protein structures that allow them to react quickly with the nerve membrane and release the concentrated transmitter into the synaptic gap.
• Vesicles can be isolated from nerves by ultracentrifugation, and their transmitter content can be analyzed.

Release from Nerve

• Finkleman (1930) - using the rabbit jejunum.
• Nerve stimulation leads to the release of noradrenaline.
• Modern methods involve radio-labeled transmitter and chromatography.

Activation of Specific Receptors

• Ahlquist (1948): based on agonist potency ratios, proposed the existence of distinct receptors.
  • NA > adrenaline > isoprenaline: receptor defined as alpha ($\alpha$).
  • Isoprenaline > adrenaline > NA: receptor defined as beta ($\beta$).
• Concept validated by:
  • Differing antagonist affinities.
  • Differing radioligand binding affinities.
  • Differing transduction (ion channels; second messengers).
  • Molecular biology (different genes coding for different receptors).

Mechanism for Swift Termination of Action

• During the 1960s, it was shown that noradrenaline is inactivated by uptake via transporter proteins into either the nerve or the effector cell.
  • Neuronal uptake (‘uptake-1’).
  • Extra-neuronal uptake.

Noradrenaline – An Established Transmitter

• Mimics nerve - More similarly than adrenaline.
• Presence in nerve - Fluorescence (Falck & Hillarp).
• Synthesis in nerve - From tyrosine.
• Storage in nerve - In small opaque vesicles.
• Release - Calcium-dependent.
• Specific receptors - Alpha ($\alpha$) and beta ($\beta$) adrenoceptors.
• Action ceases fast - Uptake (neuronal/extraneuronal).

Why is it Important to Identify Transmitters?

For Noradrenaline

• Hypertension:
  • Beta-blockers (propranolol).
  • Alpha-blockers (prazosin).
  • Release inhibitors (guanethidine).
  • Storage inhibitors (reserpine).
  • Synthesis inhibitors ($\alpha$-methyl-p-tyrosine – abbreviated as AMPT).
• Heart failure:
  • Beta-blockers (metoprolol).
• Depression:
  • Uptake inhibitors (imipramine).
• Facilitates understanding of disease mechanisms and allows development of selective drugs as therapeutic agents.