Which of the two proposed mechanisms for AA production in these cells do the observations support? Explain why by discussing what information each  experiment (a,b,c,d,or e) provides, and which experimental observations (a,b,c,d,or e) allow you to distinguish between the pathways used for IP3 and AA generation.

In a cell line derived from normal rat thyroid, stimulation of the alpha1-adrenergic receptor increases both IP3 formation and release of arachidonic acid (AA). IP3 elevates cytosolic Ca, which mediates thyroxine efflux, whereas AA serves as a source of prostaglandin E2, which stimulates DNA synthesis. It is not clear how AA release is connected to the adrenergic receptor. AA could arise by cleavage from the DAG that accompanies IP3 production. Alternatively, AA could arise through an independent effect of the receptor on PLA2, which can directly release AA from intact phosphoglycerides.

Consider the following experimental observations:

1. a) Addition of noradrenaline to cell cultures stimulates production of both IP3 and AA.
2. b) If the alpha1-adrenergic receptors are made unresponsive to noradrenaline by treatment with phorbol esters (which act through PKC to cause phosphorylation, and inactivation, of the receptor), addition of noradrenaline causes no increase in IP3 or AA.
3. c) When cells are made permeable to GTPγS (a nonhydrolyzable analogue of GTP), production of both IP3 and AA is increased.
4. d) If cells are treated with neomycin (which blocks the action of PLC), subsequent treatment with either noradrenaline alone or with GTPγS stimulates AA production but causes no increase in IP3.
5. e) If cells are treated with pertussis toxin, subsequent treatment with

either noradrenaline alone or with GTPγS alone stimulates production of IP3 but causes no increase in AA.

Which of the two proposed mechanisms for AA production in these cells do the observations support? Explain why by discussing what information each  experiment (a,b,c,d,or e) provides, and which experimental observations (a,b,c,d,or e) allow you to distinguish between the pathways used for IP3 and AA generation.