Transcript Pharmacology of stretch activated

Examining the pharmacology of stretch activated ion channels on mechanosensory proprioceptor
responses in crayfish, crab and Drosophila
Simpson, L.C. 1, Malloy C.1, DMahmood, D. 1, Dabbain, N. 1, Van Doorn, J.1, Uradu, H.S.1, Spence, A.E. 1, Martha, S.R.2, Potter, S.J.1, Mattingly,
M.X. 1, Kington, P.D. 1, King, M. 1, Ho, A. 1, Hickey, T.N. 1, Goleva, S.B. 1, Chukwudolue, I.M. 1, Alvarez, B.A. 1, Cooper, R.L. 1
Bio 446 & Bio 650; 1Dept. of Biology, 2College of Nursing, Univ. of Kentucky.
Introduction
Proprioceptors are a group of specialized receptors
which detect position and movement (kinesthetic). They
monitor joint position, direction, speed, and muscle-length.
Arthropods, like vertebrates have articulated appendages. It
is, therefore, not surprising that the proprioceptors described
for vertebrates have their counterparts in arthropod limbs and
joints.
The physiology of mechanosensory transduction is
diverse in that there are many types of receptors which
transduce mechanical forces into an electrical neural
impulses across taxa. Receptors that are sensitive to
mechanosensation are used to monitor both external and
internal forces and are essential in transferring information
that allow for appropriate behavioral responses to stimuli and
body positioning. Invertebrates serve as models in
understanding the physiology of mechanoreception due to the
diversity of receptor types and the relative ease with which
one can utilize these models in experimentation. The family of
stretch activated ion channels is broad and is currently being
characterized by gene/protein sequences and
pharmacological profiles. Common types of
mechanoreceptors are those associated with chordotonal
organs (COs), which monitor joint movements within the limbs
of arthropods. The MRO in the crayfish abdomen is a welldescribed model system but only preliminary studies have
been conducted for examining pharmacology of these stretch
activated receptors. In addition, the pharmacology of the COs
in the limbs of crabs has not previously been investigated.
The crab CO preparation offers unique properties as
the sensory endings are embedded in an elastic strand with
cell bodies and endings that are relatively exposed.
The crayfish MRO is more complex as the sensory
endings are embedded within muscle fibers. When the
muscle fibers are stretched the displacement stretches the
sensory ending and opens stretch activated ion channels.
There are two types of sensory neurons, each associated to
their own distinct muscle fiber. One MRO is referred to as the
rapidly adapting receptor and the other the slow adapting
neuron (see Rydqvist et al., 2007 for a review).
We examined the effect of amiloride and ruthenium
red in an attempt to profile the pharmacology of these
receptors. For comparison, we also examine cuticular
mechanosensors in larval Drosophila known to be sensitive to
amiloride. Our goal is to enhance understanding of the
physiology of COs which can serve as models for
mechanosensation.
The laboratory exercises we used in this
neurophysiology class demonstrates the use of these two
model preparations to address authentic scientific based
questions in regards to the topic of examining
pharmacological agents known in other models to block
stretch activated ion channels.
Methods
The dissection procedures for recording neural activity
from the crab PD organ and the crayfish MRO are previously
described in detail with text and video format (Majeed et al.,
2013; Leksrisawat et al., 2010).
The respective proprioceptive nerves are exposed and
recordings are made with extracellular suction electrodes. The
signals are amplified and recorded on a computer. All data
were recorded by a computer via PowerLab/4s A/D converter
(ADInstruments).
The salines used are the normal salines described
previously (Majeed et al., 2013; Leksrisawat et al., 2010).
Amiloride was used at a concentration of 1mM where as
ruthenium red was used at 30 mM.
Initial nerve recordings were made with moving the PD
segment in crabs or the joint associated for the MRO in
crayfish with normal saline, then exchanged to one containing
a compound of interest followed by exchange back to normal
saline to regain the initial responsiveness.
Summary
Crayfish - MRO
Crab - PD organ
LEFT: Overview of general dissection to isolate
abdomen. A, B, and C are the series of steps in
dissecting the crayfish.
Joint proprioceptive organ in a walking leg
BELOW: The schematic of an abdominal segment illustrates the
muscle groups (A) and a stained preparation with methylene blue (B)
helps to delineate the muscle groups in an intact preparation. The
outlined area in A is shown in B with an enlarged view. In B, the DEL1
and 2 muscle groups are not cut away as shown in the lower half of
the schematic as shown in A. Nerve is pulled into recording electrode.
1. The stretch activated channels in these model
proprioceptors are not sensitive to the common
pharmacological agents for some types of stretch activated
channels.
2. Amiloride (1 mM) and ruthenium red (30 uM) did not block
the channels even after 1 hour of incubation for the crab
preparation. The crayfish MRO was only examined for 15
minutes
RUTHENIUM RED 30 mM
SALINE
½ sec
½ sec
AMILORIDE 1 mM
3. Since some TRP channels (i.e.TRP4), mammalian
Deg/ENaC channels and the stretch activated channels
comprised of the Piezo protein are mechanosensitive which
blocked by ruthenium red or amiloride, we assume these
crustaceans stretch activated channels with proprioception
do not fall into these classes.
½ sec
SALINE
1 sec
1 sec
= bend
1 sec
4. In mammals, it is noted that ENaCs & ASICs (acid sensing
mechanosensory channels) are inhibited by amiloride. The
mammalian muscle spindle proprioceptors are amiloride
sensitive, but have yet to be fully classified
pharmacologically or by protein structure (Bewick and
Banks, 2015) .
AMILORIDE
1 mM
(after 5 min)
2 sec
2 sec
2 sec
5. As for mammals, the mechanosensory receptors in these
crustacean proprioceptor preparations have not been fully
identified to the coding genes or protein composition.
SALINE
wash
4 sec
4 sec
6. Screening various compounds known to work on other
mechanosensory receptors helps in further identifying these
particular receptors.
4 sec
7. Future studies will be to increase or sample size of the MRO
preparations and to try intracellular recordings for recording
receptor potentials for assessing more subtle changes in the
effects of various pharmacological agents.
RUTHENIUM
RED 30 mM
(after 10 min)
5 sec
5 sec
5 sec
Another set of 5 preparations were amiloride was tried before ruthenium red
Response
condition
amiloride
Normal
Saline
ruthenium red
Normal
saline
Activity
Prep1
Prep 2
No Change
No Change
Prep 3
Prep 4
Prep 5
No Change
No Change
No Change
Response
condition
amiloride
Normal
Saline
Activity
Prep1
No Change
No Change
Prep 2
No Change
No Change
ruthenium red
No Change
Normal
saline
References
No Change
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