Pharmacological characterization of a receptor for calcitonin gene-related peptide on rat, L6 myocytes

1 The L6 myocyte cell line expresses high affinity receptors for calcitonin gene-related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation. 2 In intact cells at 37 degrees C, human and rat alpha- and beta-CGRP all activated adenylyl cyclase with EC50s of about 1.5 nM. A number of CGRP analogues containing up to five amino acid substitutions showed similar potencies. In membrane binding studies at 22 degrees C in 1 mM Mg2+, the above all bound to a single site with IC50s of 0.1-0.4 nM. 3 The fragment CGRP(8-37) acted as a competitive antagonist of CGRP stimulation of adenylyl cyclase with a calculated Kd of 5 nM. The Kd determined in membrane binding assays was lower (0.5 nM). 4 The N-terminal extended human alpha-CGRP analogue Tyro-CGRP activated adenylyl cyclase and inhibited [125I]-iodohistidyl-CGRP binding less potently than human alpha-CGRP (EC50 for cyclase = 12 nM, IC50 for binding = 4 nM). 5 The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology."

Depolarisation and suppression of burst firing activity in the mouse subthalamic nucleus by dopamine D1/D5 receptor activation of a cyclic-nucleotide gated non-specific cation conductance

Neuronal burst firing in the subthalamic nucleus (STN) is one of the hallmarks of dopamine depletion in Parkinson's disease. Here, we have determined the postsynaptic effects of dopamine in the STN and the functional consequences of dopamine receptor modulation on burst firing in vitro. STN cells displayed regular spiking activity at a rate of 7.9 +/- 0.5 Hz. Application of dopamine (30 mu M) induced membrane depolarisations accompanied by an increase in firing rate of mean 12.0 +/- 0.6 Hz in all 69 cells. The dopamine effect was mimicked by the dopamine D1/D5 receptor agonist SKF38393 (10 mu M, 17 cells) and the dopamine D2-like receptor agonist quinpirole (10 mu M, 35 cells), partly reduced by D1/D5 antagonist SCH23390 (2 mu M, seven cells), but unaffected by the D2 antagonists sulpiride (10 mu M, seven cells) or eticlopride (10 mu M, six cells). Using voltage ramps, dopamine induced an inward current of 69 +/- 9.4 pA at a holding potential of -60 mV (n = 17). This current was accompanied by an increase in input conductance of 1.55 +/- 0.35 nS which reversed at -30.6 +/- 2.3 mV, an effect mimicked by SKF38393 (10 AM, nine cells). Similar responses were observed when measuring instantaneous current evoked by voltage steps and in the presence of the I-h blocker, ZD7288, indicating effects independent of I-h. The increase in conductance was blocked by SCH23390 (2 mu M, n = 4), mimicked by the activator of adenylyl cyclase forskolin (10 mu M, n = 7) and blocked by H-89, an inhibitor of cyclic AMP dependent protein kinase A (10 PM, n = 6). These results indicate that the dopamine depolarisation is in part mediated by D1/D5 receptor mediated activation of a cyclic-nucleotide gated (CNG) non-specific cation conductance. This conductance contributes to the membrane depolarisation that changes STN neuronal bursting to more regular activity by significantly increasing burst duration and number of spikes per burst.

Characterisation of the plasma membrane subproteome of bloodstream form Trypanosoma brucei

Proteome analysis by conventional approaches is biased against hydrophobic membrane proteins, many of which are also of low abundance. We have isolated plasma membrane sheets from bloodstream forms of Trypanosoma brucei by subcellular fractionation, and then applied a battery of complementary protein separation and identification techniques to identify a large number of proteins in this fraction. The results of these analyses have been combined to generate a subproteome for the pellicular plasma membrane of bloodstream forms of T. brucei as well as a separate subproteome for the pellicular cytoskeleton. In parallel, we have used in silico approaches to predict the relative abundance of proteins potentially expressed by bloodstream form trypanosomes, and to identify likely polytopic membrane proteins, providing quality control for the experimentally defined plasma membrane subproteome. We show that the application of multiple high-resolution proteomic techniques to an enriched organelle fraction is a valuable approach for the characterisation of relatively intractable membrane proteomes. We present here the most complete analysis of a protozoan plasma membrane proteome to date and show the presence of a large number of integral membrane proteins, including 11 nucleoside/nucleobase transporters, 15 ion pumps and channels and a large number of adenylate cyclases hitherto listed as putative proteins.

On the mechanisms of cerebellar synaptic plasticity

Changes in the strength of signalling between neurones are thought to provide a cellular substrate for learning and memory. In the cerebellar cortex, raising the frequency and the strength of parallel fibre (PF) stimulation leads to a long-term depression (LTD) of the strength of signalling at the synapse between PFs and Purkinje cells (PCs), which spreads to distant synapses to the same cell via a nitric oxide (NO) dependent mechanism. At the same synapse, but under conditions of reduced post-synaptic calcium activity, raised frequency stimulation (RFS) of PFs triggers a long-term potentiation of synaptic transmission. The aims of the work described in this thesis were to investigate the conditions necessary for LTD and LTP at this synapse following RFS and to identify the origins and second messenger cascades involved in the induction and spread of LTP and LTD. In thin, parasagittal cerebellar slices whole cell patch clamp recordings were made from PCs and the effects of RFS of one of two, independent PF inputs to the same PC were examined under a range of experimental conditions. Under conditions designed to reduce post-synaptic calcium activity, RFS to a single PF input led to LTP and a decreases in paired pulse facilitation (PPF) in both pathways. This heterosynaptic potentiation was prevented by inhibition of protein kinase A (PKA) or by inhibition of NO synthase with either 7-nitroindazole (7-NI) or NG Nitro-L-argenine methyl ester. Inhibition of guanylate cyclase (GC) or protein kinase G (PKG) had no effect. A similar potentiation was observed upon application of the adenylyl cyclase (AC) activator forskolin or the NO donor spermine NONOate. Both of these treatments also resulted in an increase in the frequency of mEPSCs, which provides further evidence for a presynaptic origin of LTP. Forskolin induced potentiation and the increase in mEPSC frequency were blocked by 7-NI. The styryl dye FM1-43, a fluorescent reporter of endo- and exocytosis, was also used to further examine the possible pre-synaptic origins of LTP. RFS or forskolin application enhanced FM1-43 de-staining and NOS inhibitors blocked this effect. Application of NONOate also enhanced FM1-43 de-staining. When post-synaptic calcium activity was less strictly buffered, RFS to a single PF input led to a transient potentiation that was succeeded by LTD in both pathways. This LTD, which resembled previously described forms, was prevented by inhibition of the NO/cGMP/PKG cascade. Modification of the AC/cAMP/PKA cascade had no effect. In summary, the direction of synaptic plasticity at the PF-PC synapse in response to RFS depends largely on the level of post-synaptic calcium activity. LTP and LTD were non-input specific and both forms of plasticity were dependent on NOS activity. Induction of LTP was mediated by a presynaptic mechanism and depended on NO and cAMP production. LTD on the other hand was a post-synaptic process and required activity of the NO/cGMP/PKG signalling cascade.

Differential control of two forms of glutamate release by group III metabotropic glutamate receptors at rat entorhinal synapses

Neurotransmitter release at CNS synapses occurs via both action potential-dependent and independent mechanisms, and it has generally been accepted that these two forms of release are regulated in parallel. We examined the effects of activation of group III metabotropic glutamate receptors (mGluRs) on stimulus-evoked and spontaneous glutamate release onto entorhinal cortical neurones in rats, and found a differential regulation of action potential-dependent and independent forms of release. Activation of presynaptic mGluRs depressed the amplitude of stimulus-evoked excitatory postsynaptic currents, but concurrently enhanced the frequency of spontaneous excitatory currents. Moreover, these differential effects on glutamate release were mediated by pharmacologically separable mechanisms. Application of the specific activator of adenylyl cyclase, forskolin, mimicked the effect of mGluR activation on spontaneous, but not evoked release, and inhibition of adenylyl cyclase with 9-tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) blocked mGluR-mediated enhancement of spontaneous release, but not depression of evoked release. Occlusion studies with calcium channel blockers suggested that the group III mGluRs might depress evoked release through inhibition of both N and P/Q, but not R-type calcium channels. We suggest that the concurrent depression of action potential-evoked, and enhancement of action potential-independent glutamate release operate through discrete second messenger/effector systems at excitatory entorhinal terminals in rat brain. © 2007 IBRO.