Modulation of neurotransmitter release by G protein-coupled receptors (GPCRs) is a feature of the nervous system and may be essential for events such as synaptic plasticity. There is evidence that some of the modulation by G_q-coupled receptors is via protein kinase C (PKC) and changes in the exocytotic machinery (Teschemacher et al., 2000). As part of a study into this we examined signalling, including PKC activation, by different GPCRs in chromaffin cells.

Confocal imaging of fluo-3 loaded cells (all experiments at 37°) showed expression of receptors for histamine and angiotensin II (AII) coupled to Ca²⁺ signalling. Histamine (100µM throughout) evoked a robust (2.7±0.4 (160 cells) fold of basal (fob)) peak response followed by a lower (1.5±0.3 (160 cells) fob) sustained plateau (data are mean+sem (n cells 3 experiments)). In contrast, AII (100nM throughout) evoked a peak (1.8±0.2 (120 cells) fob) but no sustained [Ca²⁺] elevation. Confocal imaging of a transiently expressed eGFP-tagged PH domain of PLC1 (eGFP-PHPLC1) was used to monitor real-time activation of phospholipase C (PLC) as this binds to membrane PtdIns(4,5)P₂ but upon PLC activation translocates to the cytosol (Nash et al., 2001). Histamine caused a rapid (25s) peak and sustained lower plateau phase of increased cytosolic fluorescence (1.3±0.3 and 1.1±0.5 (18 cells) fob respectively). However, AII only transiently enhanced cytosolic fluorescence (1.1±0.4 (12 cells)).

Western blotting of whole cell extracts detected PKC-, -ß, - and - isoforms. We then used Western blotting of cytosol and membrane fractions followed by densitometry to assess agonist-evoked redistribution of PKC isoforms as an index of activation (Mochly-Rosen et al., 1990). Approximately 52%, 50%, 57% and 86% of PKC-, -ß, - and - respectively were cytosolically located (n=4). Histamine caused a loss of cytosolic- and increase of membrane-immunoreactivity for all isoforms over a 10 min stimulation period (~2.5, 2.8, 2.1 and 2.3 increase over unstimulated membrane levels (n=4)). In contrast, AII only recruited PKC to the membrane fraction (2.7±0.1 (n=4) fob at 10 min). We also used confocal imaging of cells transiently expressing a recombinant eGFP-tagged Cl₂ domain of PKC (eGFP- Cl₂PKC) to monitor diacylglycerol (DAG) generation in real-time. This construct is in the cytosol but is recruited to the plasma membrane by DAG (Mogami et al., 2003). Histamine caused a rapid (30s) transient, loss of cytosolic fluorescence (to 0.85±0.01 of basal, 12 cells) and a corresponding increase at the plasma membrane. AII caused a significantly (P< 0.05, Student's t-test) smaller transient decrease in fluorescence (to 0.89±0.01 of basal, 10 cells).

Thus, activation of either histamine or AII receptors in chromaffin cells causes different profiles of Ca²⁺ signalling, PLC activation and DAG formation, which suggest rapid and full desensitization of signalling by AII but partial desensitization of histamine-mediated signalling. This may underlie agonist-specific profiles of PKC activation.

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Mogami, H. et al., (2003) J. Biol. Chem. 278, 9896-9904.
Nash, M.S. et al., (2000) Biochem. J. 356, 137-142.
Teschemacher, A.G. et al., (2000) J. Neurosci. 20,4776-4785.