Abstract:
DF2, a heptapeptide, is a member of the family of FMRFamide-like peptides and
has been shown to increase the amount of transmitter released at neuromuscular junctions
of the crayfish, Procambarus clarkit Recent evidence has shown that protein kinase C
(PKC), calcium/calmodulin-dependent protein kinase II (CaMKII) and the cAMPdependent
protein kinase (PKA) play a role in the neuromodulatory pathway of DF2. The
involvement of these kinases led to the prediction that a G-protein-coupled receptor
(GPCR) is activated by DF2 due to the role that each kinase plays in traditional GPCR
pathways seen in other organisms and in other cells. G-proteins can also act on an
enzyme that generates cyclic guanosine monophosphate (cGMP) which mediates its
effects through a cGMP-dependent protein kinase (PKG). This thesis addresses the
question of whether or not DF2's effects on synaptic transmission in crayfish are
mediated by the cyclic nucleotides cAMP and cGMP.
The effects of DF2 on synaptic transmission were examined using deep abdominal
extensor muscles of the crayfish Procambarus clarkii. An identified motor neuron was
stimulated, and excitatory post-synaptic potentials (EPSPs) were recorded in abdominal
extensor muscle LI . A number of activators and inhibitors were used to determine
whether or not cAMP, PKA, cGMP and PKG mediate the effect of this peptide.
Chemicals that are known to activate PKA (Sp-cAMPS) and/or PKG (8-pCPTcGMP)
mimic and potentiate DF2's effect by increasing EPSP amplitude. Inhibitors of
either PKA (Rp-cAMPS) or PKG (Rp-8-pCPT-cGMPS) block a portion of the increase in
EPSP amplitude induced by the peptide. When both kinase inhibitors are applied
simultaneously, the entire effect of DF2 on EPSPs is blocked. The PKG inhibitor blocks the effects of a PKG activator but does not alter the effect of a PKA activator on EPSP
amplitude. Thus, the PKG inhibitor appears to be relatively specific for PKG. A trend in
the data suggests that the PKA inhibitor blocks a portion of the response elicited by the
PKG activator. Thus, the PKA inhibitor may be less specific for PKA.
Phosphodiesterase inhibitors, which are known to inhibit the breakdown of cAMP
(IBMX) and/or cGMP (mdBAMQ), potentiate the effect of the peptide. These results
support the hypothesis that cAMP and cGMP, acting through their respective protein
kinase enzymes, mediate the ability of DFi to increase transmitter output.
