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pharmacology
1/14/98

Davies: Introduction to the ANS

Dr. 
Davies remarks that there was something different, something missing, in 
the first week of class, and now he realizes it was that nobody was 
reading the DP during lecture because it wasn't being printed yet. :) You 
can now all return to your regular crossword puzzle activity.

This will 
be the last lecture that isn't about drugs. We're returning briefly to 
neuroscience, and he's sorry about this. This is material we've had over 
and over again, and Dr. D would really prefer to have the hour off, and 
maybe we can put in the evaluations that we didn't really want this intro 
to the autonomic nervous system....but for now, we'll discuss it. This is 
the first in a series of lectures on excitable tissues.

The ANS is not 
easy to present, there are lots of complexities and it isn't always 
logical. There's more known about it than we'll want to know or than is 
important to know. 

We'll start with some generalizations, then some 
anatomy, some physiology, and a bit about junctional transmission.


Generalizations:

1. the name autonomic NS comes from "auto" self because 
it's regulated by processes that aren't normally under conscious control.

2. it innervates tissues with a high degree of intrinsic activity and 
responsiveness - tissue that would be active even without the ANS, like 
the heart
3. innervates organs that are subject to constant modulatory 
drive via the ANS - system is tonically active.
4. plays a major role in 
homeostasis - controls the steady state of internal environment, 
coordinates physiological processes and visceral activity.

Anatomy:

1. 
major differences b/w visceral and somatic neural activity are on the 
efferent side.
2. original concept - ANS was all efferent outflow - 
therefore, the term is often used only in reference to the efferent 
neurons supplying the peripheral effector organs
3. however, you should 
remember that a major means of evoking ANS activity or changes is by 
sensory nerves and integrative centers - reflex activation. we talked 
about carotid sinus, carotid body, chemoreceptors - this implies sensory 
nerves coming from organs, synapsing in the relay areas/ integrative 
centers in the hypothalamus and medulla. The differences on sensory side 
b/w visceral and somatic ANS are very small, if any, so we'll concentrate 
on efferent/motor side.

ANS: peripheral efferents

1. originate in CNS, 
either the lateral horn of spinal cord or cranial nerve motor nuclei
2. 
efferent projections consist of two neurons in series. somatic 
projections (SNS), you recall, have only one single motor neuron with 
soma in ventral horn and axon going to muscle. in ANS, there are 
preganglionic fibers with cell bodies in CNS and myelinated axons running 
to ganglia, and postganglionic fibers, with cell bodies in peripheral 
ganglia, and nonmyelinated axons going to end organs - smooth muscle, 
secretory cells, etc. 

your job is to remember if something is 
preganglionic or postganglionic. Drugs can act at the ganglionic synapse, 
or on the postganglionic terminal at the effector organ. Or they can act 
in both places. Different drugs will affect different places. This will 
be covered later in detail.

Structurally and functionally, ANS is 
divided into sympathetic and parasympathetic ANS. See diagram in handout. 
We'll do sympathetic (SANS) first.

sympathetic preganglionic fibers:
1. 
cell bodies in lateral horn of the thoracolumbar spinal cord - T1-L5,6 in 
dog.
2. efferent axons are myelinated, leaving the cord in the ventral 
root, forming the white rami entering the ganglion.
3. they terminate in 
the sympathetic ganglia. entrance of fiber into ganglion is called the 
white ramus. see diagram .

sympathetic ganglia: three types

1. 
vertebral (also called paravertebral, chain, sympathetic trunk). These 
lie in a chain on either side of vertebral column, and are paired. The 
preganglionic fibers may pass up or down the chain before synapseing. 
long postganglionic fibers emerge from the trunk - see the diagram in 
handout. note that fibers do not have to synapse at the same level from 
which they emerged from the cord. It can travel up or down - lots of 
divergence of these fibers in the SANS. 

2A. prevertebral (collateral). 
these are in the abdominal and pelvic regions (celiac, mesenteric). they 
have long preganglionic fibers, that have to go from cord out into the 
abdominal or pelvic region, emerging from  the trunk (chain) and 
synapsing in a ganglion. then they have long postganglionic fibers too, 
going out to the gut, etc.

2B. adrenal medulla is a modified 
prevertebral ganglion innervated by typical preganglionic fibers. 

3. 
terminal ganglia - these are limited in number. Mainly in pelvic organs - 
bladder, rectum, and genitalia. They are close to end organ so have long 
preganglionic fibers and short postganglionic fibers.

sympathetic 
postganglionic fibers:

1. cell bodies are in the ganglia
2. axons are 
nonmyelinated - form grey rami
3. axons travel in grey rami to spinal 
nerves - fibers go to end organs in somatic tissues - blood vessels, 
sweat glands, piloerector muscles, things like that. There are also 
special sympathetic nerves of postganglionic fibers - the cardiac, the 
mesenteric, the carotid, the hypogastric nerves - these go to the head 
and the viscera. There is a lot of sympathetic innervation to the heart 
of this type. 

Parasympathetic system:

1. preganglionic cell bodies 
located in brainstem and sacral cord (s1 and 2 in dog). 
2. ganglia are 
close to, or in the wall of, the innervated organ. preganglionic fibers 
are very long
3. postganglionic fibers are very short. synapse often in 
the wall of organ.

Physiology of the ANS:

1. SANS system, compared to 
PANS, is rather diffuse. many organs are brought into play at the same 
time - activates many muscles and sweat glands together. greater 
activation as a unit. this is due to divergence - a single preganglionic 
fiber synapses with many postganglionic fibers, as discussed, as it runs 
up/down the chain; and a single postganglionic fiber will synapse with 
many effector cells. Also we see convergence - a single postganglionic 
fiber gets input from many preganglionic fibers. so the system acts as a 
unit. This is unlike the PANS.

2. most fibers (PANS and SANS) don't have 
true junctions, that we learned about in terms of the neuromuscular 
junctions, where there is point to point connectivity from presynaptic to 
postsynaptic. most of the axons have bead-like varicosities which contain 
NT. They come close to the effector cell, they pass by the effector cell, 
they come within microns of it, but do not "touch" it like the somatic 
ones do. They secrete NT into fluid a few microns from effector cell, and 
this is called "volume transmission" - secretion into a volume, not into 
a synaptic cleft. A few microns is a long way for diffusion to occur, so 
this isn't as rapid as SNS either. effector cells are not rapidly 
activated, they are slowly activated. This isn't good for say, playing 
the guitar where you need rapid activation of muscles. slow activation 
over time is what occurs. the varicosities on the slide look like little 
beads on a chain of axon or something. 

3. many end organs have a dual 
innervation - SANS and PANS. effects may be and often are opposite but do 
not have to be. action of each isn't always on same cell eg in iris, SANS 
and PANS innervate different cells, but actions are still opposite. level 
of function depends on balance b/w SANS and PANS input. So HR is in 
balance b/w SANS and PANS events.

4. activity is normally tonic (low 
frequency) - always operating - cells always firing, neurons always 
firing at low frequency. so you can control activity of end organ with a 
single system to increase or decrease the activity of the end organ.

5. 
many organs have intrinsic controls (heart, gut) that are modified by 
ANS. ANS modifies the intrinsic activity

6. response to ANS stimulation 
is determined by the characteristics of the receptor cell. in the gut, 
stimulation causes smooth muscle to relax, and sphincters to contract. 
nerves or NT are not "excitatory" or "inhibitory" per se - can't say ACH 
is excitatory per se, or SANS is excitatory - excitation or inhibition is 
determined by the consequences of receptor activation in the particular 
postjunctional cell. 

a few more things to introduce re: pharmacology of 
the nervous system in general....

Junctional Transmission - what goes on 
at a junction - neuromuscular, or interneuronal, or whatever. We know 
that the idea of chemical transmission was first established through 
research on ANS. You know that certain symbols are necessary for one's 
image...if you want to be brave, you need a medal, if you want to have a 
brain, you need a diploma, if you want to be a vet you need a diploma 
-not knowledge. just a diploma. you need these symbols. to be a 
pharmacologist you have to mention the name "Otto Lloyd." That is the guy 
who did the heart experiment, stimulating the vagus nerve to a heart 
causing it to slow down, and then bathing another heart with the fluid 
from the first heart, causing it to slow down too. So something in the 
stimulation of vagus nerve caused a chemical to be released which had an 
effect on the second heart. so something was released by nerve 
stimulation. 

ANS Fiber types, classed by substance released:

1. 
cholinergic fibers - release ACH - all preganglionic fibers in ANS 
release ACH. all postganglionic PANS fibers, some postganglionic SANS (to 
sweat glands and blood vessels, not in all species), and somatic motor 
fibers to muscles relase ACH.

2. adrenergic fibers - release 
noradrenaline - whole postganglionic SANS system (except the few 
mentioned above going to sweat glands and blood vessels).

3. other 
fibers- purines (adenosine, ATP)), serotonin, gut peptides.

these 
substances act at junctions.

general sequence of junctional 
transmission:

1. first an action potential invades nerve terminal, 
causes depolarization
2. depolarization causes calcium influx
3. calcium 
causes NT release from vesicles
4. transmitter molecules diffuse across 
cleft (or intercellular space)
5. NT binds to receptor sites
6. NT 
binding causes a conformational change in the receptor protein
7. 
conformational change leads to change in membrane permeability, either 
directly coupled to ion channel or indirectly coupled via 2nd messenger 
system
8. transmitter is then destroyed or recycled via reuptake 
mechanism

so we can say there are many things that go on in junctional 
transmission. lots of pharmacology of excitable tissue has to do with 
different aspects of these steps, and designating points of attack where 
drugs block or augment junctional transmission. you could anesthetize the 
nerve to prevent the action potential from invading the terminal - 
prejunctional effects, junctional effects, or postjunctional effects can 
occur that will affect the system being drugged. 
If you affect a 
cholinergic synapse, you can do it at about 6 points in the system at 
least. For a noradrenergic synapse, there are 8 to 10 points. Fluharty 
(or Yee) will go over this later. Dopaminergics, serotenergics, same 
thing. GABAergics too. You get the point.

Varieties of Modulation of 
nervous system that drugs can be used for:

1. different transmitters
2. 
different receptors (14 for serotonin alone)
3. different 2nd messengers

4. different channels
5. opening vs closing a channel
6. pre and post 
synaptic sites

so if you think about this, which you should not do b/c 
it will upset you, but if you do, you will see that pharmacology can't be 
simple. what our job will be, the task we're taking, is to make it 
simple. We need to learn the basics of what's going on.

----break----


Kirsten was sleeping during the break and now she has a big red 
waffle-weave mark on her face. hehehehehehe.

Anyway.

10-11
1/14/98

pharm

Kotlikoff: Parasympathetic Nervous System and Cholinergic 
Neurotransmission

This is the first of many lectures on cholinergic 
pharmacology. We'll be doing processes of cholinergic neurotransmission 
today, going through and discussing them in detail, keeping in mind how 
we can intervene pharmacologically, what predicted outcomes of particular 
interventions would be and if they would be specific, etc. We'll discuss 
cholinergic receptors - nicotinic and muscarinic and their subtypes. 
We'll discuss processes of receptor-effector coupling, and specific 
parasympathomimetic and parasympathetic drugs. In the third hour we'll 
get to anticholinesterase drugs - like organophosphates - drugs used that 
block the removal of ACH from the synapse, and neuromuscular blocking 
agents that are used in anesthesia and so forth. 

Overview of lecture 
goals today:

1. review cholinergic neurotransmission
2. examin key 
aspects of ACH synthesis, release, and breakdown
3. cholinergic 
receptors, location and function of nicotinic and muscarinic types
4. 
effect of receptor coupling


We won't go into to much detail of the 
history - we already discussed Otto Lowie, before him was Dale who 
observed something brilliant. They knew about muscarine products in 
mushrooms that mimickd the effects of vagal nerve stimulation when 
ingested - those were called parasympathomimetic drugs. Later, they 
determined the structure of ACH, and Dale, based on that, said that the 
system works by ACH being released, and then some esterase broke it into 
choline and acetate, terminating its action. So before it was worked out, 
he hypothesized this, and was totally right. 

Fix in your mind as firmly 
as possible the sites of cholinergic neurotransmission. ** as you think 
about using cholinergic drugs, think location location location ** if you 
know where it ACH is released, you can logically predict effects of 
agonists and antagonists without memorizing them. First - as Dr Davies 
remarked, there are two important sites of cholinergic transmission in 
PANS - 1st is the ganglionic site of synapse b/w preganglionic and 
postganglionic parasympathietic fibers - that is the preganglionic 
releases ACH, stimulates the postganglionic, which synapses largely on 
smooth muscle and glands. 2nd, that postganglionic fiber releases ACH at 
the synapse with the effector, from the postganglionic postsynaptic 
membrane.
Now, the ganglionic synapse of the SANS is also cholinergic - 
the preganglionic fiber releases ACH to the postsynaptic/postganglionic 
receptor. So - all autonomic ganglionic sites are sites of cholinergic 
neurotransmission. The ganglionic postsynaptic membrane sites use 
nicotinic receptors. The PANS postganglionic fibers release ACH that 
stimulates muscarinic receptors on the postsynaptic membranes. The 
receptors are named after their agonists. In the sympathetic system, the 
postganglionic fiber releases norepinephrine (NE) not ACH, to stimulate 
an adrenergic receptor. The somatic neuromuscular junctions are also 
cholinergic synapses, using nicotinic receptors in the postsynaptic 
membrane of the motor end plate. The nicotinic receptor on skeletal 
muscle, Nm, is structurally different from the nicotinic receptor on the 
postganglionic PANS fiber, the Nn - they have different affinities and 
stuff and can be selectively blocked. Do not forget - there are also 
cholinergic transmission sites through the brain, serving diverse 
functions. drugs, esp the organophosphates, which act at cholinergic 
transmission sites and can access the CNS, can have big effects on brain 
function. so this is a diffuse system and the hallmark of pharmacologic 
interaction will be selectivity - we want to selectively interfere at one 
point or another, not block the whole system.

bottom of p 1 of handout 
has the 5 sites of cholinergic transmission listed - make sure to 
remember them*** put them in memory***

ACH biosynthesis:


serine-->ethanolamine--enzyme--->choline (taken up into nerve terminals 
with high affinity - rate limiting step) --choline acetyl transferase, 
acetyl coA-> acetylcholine

So, one way to intervene is to block 
synthesis of ACH using something like hemicholinium, which blocks 
transport of choline. Would that be an effective pharmacologic agent? no. 
It lacks specificity. If you block all ACH synthesis, you fall down and 
die. This isn't an effective means of modulating cholinergic transmission 
practically. 

important - note that ACH is made of choline + acetate, 
and can be cleaved back into those parts, deactivating it. Also realize 
that ACH is charged, which is important - this prevents ACH or mimics of 
ACH to pass through BBB, but some uncharged mimics of ACH do pass through 
BBB to affect CNS. 

So - choline is the important precursor for uptake 
into nerve terminal. Then ACH is made, packaged into granules for release 
into synaptic space. It's there ready and waiting to go when needed. 


There are no blockers of the acetylcholine transferase enzyme. Wouldn't 
be specific enough, anyway.

Parasympathetic neurotransmission:

sites of 
cholinergic neurotransmission - 5.
1. preganglionic fibers to all ANS 
ganglia
2. parasympathetic postganglionic fibers - major site of blocking 
action of ACH on smooth muscles, glands
3. sympathetic postganglionic 
fibers to sweat glands and skeletal muscle vessels - this is an 
exception. these are modified symp postgang. fibers. horses have them, 
not all spp do.
4. extremely important place - neuromuscular junction
5. 
CNS

more specific stuff about PANS neurotransmission:

you have the 
process first of all of (see diagram p 2) axonal conductance, where Na+ 
and K+ channels mediate depolarization down nerve terminal, this is 
general - calcium release also somewhat general - you start losing Na+ 
channels before terminal; at axon ending you have specific Ca++ channels 
(there are drugs to block these too), but when depolarization comes down, 
you move from sodium AP to calcium AP, intracellular Ca++ rises abruptly, 
activates cellular processes, causing vesicles to fuse with membrane - 
dock and fuse- and release ACH into the synapse. at the synapse, you can 
think about it as a forest of ACHesterase, which rapidly breaks down ACH. 
there are ACH molecules extending into the synapse which is already full 
of the ACHesterase which wants to break it down. About 10% of the 
released ACH at neuromuscular junction makes it through to the motor end 
plate and stimulates the receptor. So, normally, ACH release is dampened 
a lot by the ACHesterase. you can block the ACHesterase with drugs to 
heighten the effect of ACH. after breakdown, choline receirculates into 
the nerve terminal for reuse.

post-synaptic receptors - 

muscarinic 
cholinergic receptor - g-protein coupled, 7 transmembrane domain protein


nicotinic Mm receptor or Mn receptor - these are ligand gated ion 
channels.

questions? next, we're going to specifically discuss structure 
and coupling of muscarinic and nicotinic receptors and drugs that work on 
these receptors. 

handout p 4 - see diagram - talk about where the 
receptors are - 

at the parasympathetic postganglionic neuroeffector 
junction are muscarinic receptors - and there are three subtypes of it, 
M1, M2, and M3. This is a bit simplified; molecularly there are 6 or 7 
subtypes, but drugs only really discriminate between 1 - 3. 

at the 
preganglionic autonomic cholinergic site, postganglionic fiber has 
nicotinic Nn receptors. There are also a few muscarinic receptors here 
but they aren't really important or understood. Also there is a 
preganglionic muscarinic receptor at this site (and the site above), that 
controls NT release. 

at the somatic motor site there are Nm nicotinic 
receptors.

in CNS are nicotinic and muscarinic receptors mediating 
diverse functions.

agonist/cholinomimetics and 
antagonist/anticholinergics are in the table in the handout. We do not 
have very selective agents at the Nn site, and some of the agents used to 
block receptors in neuromuscular junction work here, which we don't want. 
And if we did have drugs that worked here with selectivity, we couldn't 
really use them anyway, because this site activates PANS and SANS or 
inhibits PANS and SANS, which generally oppose each other. wait. he 
restates - the receptor at the autonomic ganglion is similar to that in 
CNS. the neuromuscular junction one is distinct, but drugs that work 
there cross react with the other two types.

Ok. A nicotinic receptor is 
a ligand gated ion channel. For Nm, we know it is a pentamer made of 5 
subunits - 2 alpha ones that bind ACH and are identical, and 3 other 
subunits. Both alpha sites must bind ACH to open the channel. The point 
that is very important is that you can block the action of the channel by 
blocking the ACH binding so it can't open, or by putting too much ACH 
there so that it is always bound, the channel opens, and then it 
inactivates. Remember channel inactivation from neuro - if we have a 
muscarinic gprotein coupled receptor flooded with ACH that's ok - it 
keeps making 2nd messengers. for an ion channel, you effectively block it 
by adding too much agonist. So if you block ACHesterase, all the channels 
open initially, and then they inactivate or they so depolarize the 
endplate that effectively skeletal muscle no longer contracts. so the 
implications are if you keep it open, the cell will depolarize - won't be 
able to maintain ionic gradient and be able to control calcium levels as 
is required for an effector cell. The channel will inactivate. This isn't 
true for the G protein coupled receptors or non-ligand gated ion 
channels. 

Potency of nicotinic receptors - nicotine is a more potent 
agonist than ACH - eg, false NT, this alkaloid from tobacco, opens the 
channels with higher affinity than does ACH. Ganglionic and NMJ nicotinic 
receptors have different agonist/antagonist sensitivity. so, NT binding 
opens a nonselective cation channel, big pore. some calcium goes in but 
not much, mostly monovalent specific, lots of sodium goes in and a bit of 
K+ comes out, based on the concentration gradients and equilibrium 
potentials (you're already at -70 mV, near K+ equilibrium potential), and 
you depolarize the membrane, making an EPSP which summates, etc.  That's 
generally how the nicotinic receptor works. we'll get back to how to 
block it and what that causes.

muscarinic receptors - we have in general 
two classes of ion channels - ligand gated = ionotropic channels. the 
receptor itself is a channel. we just discussed that. Other receptors, 7 
transmembrane domain spanning ones, are coupled to an ion channel - 
metabotropic. This is how muscarinic receptor works, via a G protein and 
second messenger system. It's activated by receptor binding indirectly. 


mechanisms of receptor-effector coupling for muscarinic receptors - you 
have G protein coupled receptor. for M2 receptors, there are two major 
processes of receptor-effector coupling. one is that the G protein is 
called GI, which is called GI because it inhibits adenyl cyclase. The 
effect of activating an M2 receptor is to inhibit adenyl cyclase and 
decrease cAMP production. This lowers the concentration of cAMP in the 
smooth muscle cell. in smooth muscle, cAMP is a relaxing, inhibitory 
agent. we decrease activity of cAMP dependent protein kinase, and this 
activates the smooth muscle cell. a second important mechanism is that 
one or more of the G protein subunits, after dissociation into alpha and 
beta gamma, will activate an ion channel. In the heart, the way the vagus 
slows the heart, is that ACH is released by vagal fibers, activates M2 
receptors, the betagamma subunit dissociates, binds a K+ channel, opens 
that metabotropic channel, hyperpolarizes the SA node, and slows the 
heart. this is a major mechanism by which vagal stimulation decreases HR. 
realize there is differential signalling based on effector cell - if the 
receptor is on smooth muscle, it reduces cAMP and activates the smooth 
muscle cell and perhaps some depolarizing metabotropic ion channels. but 
in cardiac smooth muscle, the same receptor results in hyperpolarization 
and inactivation, slowing the heart. also, in the heart, cAMP is 
excitatory, so decreasing cAMP in the heart is inhibitory. so the same 
receptor has different effects on the different effector targets - in the 
heart, rate and strength of contraction end up getting decreased. in the 
smooth muscle, you have disinhibition of the pre-existing inhibitory 
system (cAMP) and stimulation of activity. So, muscarinic input into 
smooth muscle is mainly excitatory and causes contraction (except where 
the innervation is indirect, for blood vessels, where it goes to 
endothelium and releases NO to relax the vessel), and in cardiac tissue, 
muscarinic activity decreases strength and rate of contraction of the 
heart.

summary: these are the ** important points **

his email address 
- mik@vet.upenn.edu

first point: location, location, location. remember 
the sites of cholinergic neurotransmission. that helps to sort out 
actions of various drugs. 

second point: ACH synthesis - choline uptake 
is rate limiting - gets acetate added by acetyl transferase and is 
preformed and available for release. not a site for pharmacological 
regulation

three: for neurotransmission, the ACH is relesed into 
synaptic cleft that is full of ACHesterase. it's critical to understand 
that 90% of the ACH released is immediately broken down. There's a huge 
safety margin. if you interact with it you have major effects.

four: 
postsynaptic receptors that bind ACH - nicotinic Nm, Nn: Nn in ganglionic 
postsynaptic membrane and all over CNS. Both are ligand gated ion 
channels, similar in structure but Nm is pentameric, Nn not always 
pentameric.

five: Muscarinic receptors - M1, M2, M3. M2 is coupled to 
GI. it inhibits cAMP. inhibits heart, stimulates smooth muscle. GI 
inhibits cAMP and opens metabotropic ion channels eg K+ channel in heart. 
it is by virtue of this coupling that vagal stimulation slows the heart.


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