Interactions between intracellular Ca2+, ([Ca2+]i), and cAMP provide the possibility of coordinating the activities of the two major second messengers. Although the likelihood that these pathways interact at a number of levels is widely accepted in principle, there is no concrete awareness of the value to a cell of such interactions. Stimulation by Ca2+ of adenylyl cyclase, seems to contribute to the hippocampal model of plasticity - long term potentiation, but little is known about how this interaction modulates cellular activity. An adenylyl cyclase has been cloned which can be inhibited by the intracellular range of Ca2+ concentrations; this species, type V1 (or its close relative, type V) are the most prominent forms in striatum, cardiac and anterior pituitary tissue. The utility of the Ca2+ - inhibitability of adenylyl cyclase in these sources is not clear, although it has been speculated to provide a device for generating (or reinforcing) pacemaker activity in myocardial cells. However, we are just on the threshold of exploring this phenomenon. Studies in nonexcitable cells have shown that physiological elevation of [Ca2+]i will inhibit cAMP synthesis by these enzymes. It is also clear that the nature of the [Ca2+]i-rise is critical in determining how such cyclases are regulated; in particular, Ca2+-sensitive cyclases are highly sensitive to Ca2+ -entry, rather than release from stores. The requirement for Ca2+ -entry suggests a functional colocalization between entry sites and adenylyl cyclases, which may suggest some subcellular organization of these elements. It is not known whether such cyclases are regulated by the rapid [Ca2+]i-transients that occur in excitable cells. If these cyclases could be so regulated, then their utility is greatly increased, since this could result in oscillations in cAMP. The steps proposed in the present application are to ask (mainly in the GH3 excitable cell model) i) whether the potential negative feedback that exists between [Ca2+]i-homeostasis controlled by a Ca2+-inhibitable adenylyl cyclas, is utilized ii) the impact of phosphodiesterases at reinforcing the effects of inhibition of cAMP synthesis, and iii) whether (by aequorin-tagging) adenylyl cyclases see high [Ca2+]i. In addition, the importance of the negative feedback between [Ca2+]i and cAMP will be probed by molecular biological manipulations, coupled with single cell analysis of [Ca2+]i. Finally, a program is outlined to determine whether cAMP levels change rapidly in single cells, by virtue of the Ca2+ -inhibitability of adenylyl cyclase. This proposal, then, takes the first steps at a cellular level towards a tangible understanding of how [Ca2+]i- and cAMP-signalling are intertwined to control excitable cell activity.
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