Macula densa cells are located within the thick ascending limb;they detect changes in luminal fluid composition and transmit signals to the afferent arteriole, a process called tubuloglomerular feedback. Considerable effort has been directed towards elucidation of transport characteristics of these cells, in identifying the nature of mediator(s) that are released by macula densa cells and the signals that are sent to the underlying mesangial/afferent arteriolar complex. We have found that increases in luminal fluid sodium chloride concentration ([NaCI]L) activate a maxi anion channel leading to the movement of ATP across the basolateral membrane and direct, or with conversion to adenosine, afferent arteriole vasoconstriction and decreased GFR. This renewal is focused on an in depth analysis of this ATP signaling pathway. Studies will use the isolated perfused thick ascending limb-glomerular preparation and 2-photon microscopy, patch clamp, and molecular biology. Aim one is to elucidate the intracellular events that lead to the release of ATP across the basolateral membrane. We will test the hypothesis that macula densa cells exhibit unique properties of cytosolic calcium concentration [Ca2+], regulation and that [Ca2+]j plays a critical role in macula densa signaling. Studies will also define roles for macula densa cell volume and tubular flow in [Ca2+]j regulation and macula densa signaling. Aim two will test the hypothesis that increased macula densa [Ca2+]j results in mitochondrial Ca2+ uptake and enhanced ATP generation. Other work will define the intracellular events that activate the maxi anion channel and finally we will work towards defining the molecular identity of this ATP conductive pathway. Aim three will explore the role of ATP signaling to the mesangial/afferent arteriolar complex. Overall these studies should provide important new information regarding the role of ATP in macula densa signaling, activation of the tubuloglomerular feedback mechanism, and control of glomerular hemodynamics.