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Section I:
Cellular and Molecular Neurobiology

14. Neuropeptides and Nitric Oxide
Part 2 of 4

Biosynthesis and Regulation

Neuropeptides are derived from larger precursors by proteolytic processing. They are all initially synthesized within the soma (Figure 14.2).

Figure 14.2

Precursors are initially formed by translation on polyribosomes attached to the endoplasmic reticulum near the cell body. From there, they progress through the Golgi apparatus where further modifications take place, including glycosylation. They are then packaged into secretory granules that are transported to terminals by fast axonal transport. Fast is a relative term, however, and due to the relatively long distances that some neuropeptides must travel. Replenishing the pool of releasable neuropeptide might require many hours. During the transport to the nerve terminal, proteases that are packaged within the vesicle begin to cleave the precursor neuropeptide into its final mature form. This processing is essential for the activation of the neuropeptides since the precursors are biologically inactive. At least three types of processing occurs within the vesicles (Figure 14.2; click boxes for more details). First, an endopeptidase cleaves the precursor to generate two new products (First box in axon). For many precursors this cleavage occurs after basic residues (like Lys and Arg) and is accomplished by trypsin-like proteases. Next, although not for every neuropeptide, a carboxypeptidase cleaves the basic residues from the C-terminus of the new peptide (Second box in axon). Finally, a third enzyme converts the COOH (carboxy) group of a Gly residue, found at the C-terminus of many neuropeptides, to an NH₂ (amide) group to produce the mature, active form of the neuropeptide.

Multiple Mechanisms are Utilized to Produce the Diversity of Neuropeptides.

Most proteins are produced from mRNA molecules that are spliced from precursor RNAs into their final forms in the nucleus. Differential splicing is one way that a neuron uses to diversify the production of different types of neuropeptides. One well-known example is the substance P mRNA that normally also includes mRNA encoding substance K. The substance K portion of the mRNA can be differentially spliced out so that the resulting mRNA can produce only substance P (click on box over nucleus in Figure 14.2)

Figure 14.3

Neuropeptides are produced from a longer precursor protein by proteolytic processing. An excellent example is the opioid family of peptides (e.g., the processing of proopiomelanocorticotropin, POMC and Enkephalin; see Figure 14.3). As noted, the proteolytic processing takes place within the transport vesicles and most often occurs by cleaving the precursor on the N-terminal side of basic residues (arginine and lysine), although other cleavage sites have also been identified. In some instances, such as the Enkephalin precursor protein, multiple copies of the same final bioactive peptide are present. The one precursor molecule shown at the top of Figure 14.3 contains six copies of Met-enkephalin (ME) and one copy of Leu-enkephalin (LE).

Diversity can thus be generated by altering the sequence of the cleavage sites by differential splicing, by producing and/or packaging different proteases (recognizing different sequences for cleavage) into the transport vesicles, or by hiding a proteolytic site by post-translational modifications. An example of the latter is that a specific cleavage site might be hidden by the addition of a carbohydrate side chain that sterically blocks the protease from having access to that site. Another common finding is that a single precursor molecule will contain several different neuropeptides (see Figure 14.3) and therefore the types of processing that occur ultimately determines which neuropeptide is released by the neuron. The POMC precursor protein can be cleaved to form ACTH (orange) and b-lipoprotein (light blue) that each can be further cleaved to generate additional bioactive neuropeptides (Figure 14.3). For example, the b-lipoprotein (light blue) can be further cleaved into both g-lipoprotein (green stripes) and b-endorphin (dark blue). Again, depending on the processing that takes place, the same precursor protein can be modified to produce neuropeptides with dramatically different biological responses.

Release

Peptides are released by calcium-dependent exocytosis with some important differences from the release of classical neurotransmitters.

Figure 14.4

Typically, vesicles releasing neuropeptides are much larger than those that contain small molecule neurotransmitters (e.g., glutamate) and do not require a presynaptic specialization for release (see the electron micrograph in Figure 14.4). In contrast to the small vesicles that contain glutamate, the large vesicles do not appear docked at the membrane. This observation is consistent with the idea that small molecule neurotransmitters produce brief, local effects (at synaptic connections), whereas neuropeptides produce slow, long-lasting effects often encompassing a significant area surrounding the site of release. Also, recall that since neuropeptides are synthesized in the cell soma and not locally at the synapse, if their supply is exhausted from sustained release it might take several hours to replenish the releasable pools. For example, a motor neuron, with its cell body in the spinal cord and the synapse in the foot, has an axon as long as one meter. Utilizing fast axonal transport it would potentially take more than a day for a newly synthesized neuropeptide to arrive at this synapse from the soma. It should also be evident that endogenous pain-killing neuropeptides, like beta-endorphin, could be "used-up" in times of persistent stimulation leading to situations where pain can no longer be controlled by endogenous mechanisms.

A typical mature neuron will often release one small molecule neurotransmitter and one or more neuropeptides (as in the example shown in Figure 14.4). If more than one neuropeptide is released they most often come from the same single precursor molecule. An example is the co-release of both ACh and calcitonin gene-related peptide from spinal motor neurons. CGRP activates adenylate cyclase, raising cAMP levels, and potentiates the force of contraction produced by ACh activation of the nicotinic ACh receptor. In this case, the neuropeptide is modulatory as described in Figure 14.1. However, in this instance, the effect potentiates muscle contraction instead of increasing the magnitude of the EPSP. In both examples, the potentiated response is due to increased sensitivity of the system to a constant amount of released neurotransmitter.

Termination of Action

Neuropeptides are slowly removed from the extracellular space; a feature which also contributes to their relatively long lasting effects. Inactivation occurs by both diffusion and breakdown by extracellular proteases. No evidence has been found for peptide re-uptake as a means of terminating their action.

Receptors are all G-protein Linked

All known neuropeptide receptors produce their effects by altering the levels of intracellular second messengers. These receptors are seven transmembrane spanning proteins that are linked through G proteins (GPCRs) to alter the activation of other cellular enzymes. This property is consistent with neuropeptides inducing a slower response and is well suited for a modulatory role. One important distinction between small and neuropeptide molecule transmitters is that neuropeptide receptors have a high affinity for binding (nanomolar) as opposed to micro- or millimolar affinities measured for small molecule neurotransmitters (like glutamate). As neuropeptides are not released directionally into the confined volume of a synapse, their concentrations do not achieve very high levels and the receptors then must have high affinities to react to these small concentrations. This high affinity slows the dissociation of the neuropeptide from its receptor and also contributes to the persistent effects of these molecules.

Test Your Knowledge

17. Neuropeptides are present in synaptic terminals because they are:

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