DƯỢC LÍ Goodman & Gilman's The Pharmacological Basis of Therapeutics 12th, 2010

and is responsible for providing choline for ACh
synthesis. Once ACh is released from cholinergic
neurons following the arrival of action potentials, ACh
is hydrolyzed by acetylcholine esterase (AChE) to
acetate and choline. Choline is recycled after reuptake
into the nerve terminal of cholinergic cells and reused
for ACh synthesis. Under many circumstances this
reuptake and availability of choline appear to serve as
the rate limiting step in acetylcholine synthesis.
The gene for the high-affinity choline transporter (CHT1) has
been cloned from a variety of species including human and it is
homologous to the Na + -dependent glucose transport family. The
location of CHT1 is in intracellular vesicular structures rather than
the nerve terminal plasma membrane and co-localizes with synaptic
vesicle markers such as vesicle-associated membrane protein 2
(VAMP2) and vesicular ACh transporter (VAChT). Although the
details are incomplete there is evidence that in response to the cascade
of events that culminate in transmitter release, there is also
increased trafficking of CHT1 to the plasma membrane, where it
functions to take up choline after hydrolysis of acetylcholine. Since
choline transport is rate limiting for acetylcholine synthesis,
increased availability of choline via its transport by CHT1 would
favor an increase in ACh stores to maintain high levels of transmitter
release during neuronal stimulation. This also suggests that the
availability of CHT1 at the cell surface is dynamically regulated in
a manner very similar to the regulation of the exocytosis of synaptic
vesicles. The precise mechanisms involved in maintaining the distribution
of CHT1 predominantly in intracellular vesicles rather than at
the terminal surface like other neurotransmitter transporters are
unclear (Ferguson and Blakely, 2004; Chaudhry et al. 2008).
Storage of Acetylcholine. Following the synthesis of
acetylcholine, which takes place in the cytoplasm of the
nerve terminal, ACh is transported into synaptic
vesicles by VAChT using a proton electrochemical gradient
to move ACh to the inside of the organelle.
VAChT is thought to be a protein comprising 12 transmembrane
domains with hydrophilic N- and
C-termini in the cytoplasm. By sequence homology,
VAChT appears to be a member of a family of transport
proteins that includes two vesicular monoamine
transporters. Transport of protons out of the vesicle is
coupled to uptake of ACh into the vesicle and against a
concentration gradient via the acetylcholine antiporter.
There appear to be two types of vesicles in cholinergic terminals:
electron-lucent vesicles (40-50 nm in diameter) and densecored
vesicles (80-150 nm). The core of the vesicles contains both
ACh and ATP, at an estimated ratio of 10:1, which are dissolved in
the fluid phase with metal ions (Ca 2+ and Mg 2+ ) and a proteoglycan
called vesiculin. Vesiculin is negatively charged and is thought to
sequester the Ca 2+ or ACh. It is bound within the vesicle, with the
protein moiety anchoring it to the vesicular membrane. In some
cholinergic terminals there are peptides, such as VIP, that act as cotransmitters
at some junctions. The peptides usually are located in
the dense-cored vesicles. Vesicular membranes are rich in lipids, primarily
cholesterol and phospholipids, as well as protein. The proteins
include ATPase, which is ouabain-sensitive and thought to be
involved in proton pumping and in vesicular inward transport of
Ca 2+ . Other proteins include protein kinases (involved in phosphorylation
mechanisms of Ca 2+ uptake), calmodulin, atractylosidebinding
protein (which acts as an ATP carrier), and synapsin (which
is thought to be involved with exocytosis).
The vesicular transporter allows for the uptake of ACh into
the vesicle, has considerable concentrating power, is saturable, and
is ATPase-dependent. The process is inhibited by vesamicol
(Figure 8–4). Inhibition by vesamicol is noncompetitive and
reversible and does not affect the vesicular ATPase. The gene for
choline acetyltransferase and the vesicular transporter are found at
the same locus, with the transporter gene positioned in the first intron
of the transferase gene. Hence a common promoter regulates the
expression of both genes (Eiden, 1998).
Estimates of the ACh content of synaptic vesicles range from
1000 to over 50,000 molecules per vesicle, and it has been calculated
that a single motor nerve terminal contains 300,000 or more
vesicles. In addition, an uncertain but possibly significant amount
of ACh is present in the extravesicular cytoplasm. Recording the
electrical events associated with the opening of single channels at
the motor end plate during continuous application of ACh has permitted
estimation of the potential change induced by a single molecule
of ACh (3 × 10 –7 V); from such calculations it is evident that
even the lower estimate of the ACh content per vesicle (1000 molecules)
is sufficient to account for the magnitude of the mepps.
Release of Acetylcholine. Release of acetylcholine and
co-transmitters (e.g., ATP and VIP or in some neurons,
NO) occurs on depolarization of the nerve terminals
and takes place by exocytosis. Depolarization of the terminals
allows the entry of Ca 2+ through voltage-gated
Ca 2+ channels. Elevated Ca 2+ concentration promotes
fusion of the vesicular membrane with the plasma
membrane, allowing exocytosis to occur.
The molecular mechanisms involved in the release and regulation
of release are not completely understood (Südhof, 2004). ACh,
like other neurotransmitters, is stored in vesicles located at special
release sites, close to presynaptic membranes and ready for release
following the appropriate stimulus. The vesicles initially dock and
are primed for release. A multiprotein complex appears to form and
attach the vesicle to the plasma membrane close to other signaling
elements. The complex involves proteins from the vesicular membrane
and the presynaptic neuronal membrane, as well as other components
that help link them together. Various synaptic proteins,
including the plasma membrane protein syntaxin and synaptosomal
protein 25 kDa (SNAP-25), and the vesicular membrane protein,
synaptobrevin, form a complex. This complex interacts in an ATPdependent
manner with soluble N-ethylmalemide-sensitive fusion
protein and soluble SNAPs. The ability of synaptobrevin, sytaxin,
and SNAP-25 to bind SNAPs has led to their designation as SNAP
regulators (SNARES). It has been hypothesized that most, if not all,
intracellular fusion events are mediated by SNARE interactions.
Important evidence supporting the involvement of SNARE proteins
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CHAPTER 8
NEUROTRANSMISSION: THE AUTONOMIC AND SOMATIC MOTOR NERVOUS SYSTEMS