Introduction
The macula densa (MD) cells play an important role in the function of the Juxta Glomerular Apparatus (JGA). It senses the fluid load to the distal tubule and adjusts the glomerular filtration rate through the tubuloglomerular feedback control mechanism (TGF) and can also control renin release. The signal detected by the MD cells is possibly transmitted via mesangial cells to the glomerulus and the glomerular arterioles. Important modulation of JGA functions can occur but it is not known where this takes place. It is possible that this modulation occur in the MD cells themself and a detailed analysis of the function of these cells is important. We have investigated the existence of purinergic receptors on the macula densa cells.
Methods
Glomeruli were dissected from New Zealand white rabbits and put in a low NaCl (35 mM) buffer solution. The glomeruli were mounted with two manipulators on a thermostated (37° C) microscope chamber focusing the MD cells in profile. The Nikon microscopes were either attached to a Noran Odyssey confocal system or to Applied Imaging intensified video camera system. At confocal microscopy, T-series laser scanning was used in fura-red loaded preparations while using video microscopy Fura-2 was used to monitor intracellular MD cell calcium concentration. The agonists used were ATP, UTP, 2MeS-ATP, ADP and adenosine. In the microperfusion preparation, individual cTALs with attached glomeruli were dissected. The cTAL was cannulated and perfused with the low NaCl buffer solution. The preparation was bathed in the normal buffer solution (which is the same as the low NaCl buffer solution except the concentration of NaCl is 135 mM) continuously. The macula densa cell were loaded from the luminal side, and challenged with only ATP (0.1M) either from luminal or from bath solution using the same time procedure.In the calcium free solution, CaCl2 was replaced by 5 mM EGTA.
Results
The results showed that there was no response to adenosine, UTP and ATP caused greatest [Ca2+]i in macula densa cells, ADP and 2Mes-ATP only had weak effect on [Ca2+]i. Compared to the normal calcium experiment, [Ca2+] i response to ATP in calcium free solution was litter lower, but no significant difference. In the microperfusion experiment, [Ca2+] i response to ATP only got from the side of bath solution. There was no [Ca2+] i increase when ATP was challenged from the luminal side.
Conclusions
Our result indicate that it is possible to study MD cell calcium concentrations using confocal microscopy. We found that purinoreceptors in rabbit macula densa cells are P2Y2 type and not located on the luminal side, which can be an important modulator in the function of TGF and for renin release.
References