1. We have shown earlier that pharmacologic inhibition or genetic deletion of A1 adenosine receptors (A1AR) causes marked reductions in tubuloglomerular feedback (TGF) responses, but the location of the A1AR involved in TGF is not known. To investigate this issue we have generated mice with overexpression of A1AR in smooth muscle cells by pronuclear injection of the mouse A1AR cDNA linked to a 5.38 kb fragment of the rat smooth muscle alpha actin containing 2.6 kb of promoter and 2.78 kb of first intron (gift of G.K. Owens, University of Virginia). Two of the 8 founder mice that showed the highest A1AR expression in kidney and heart were selected to establish independent lines of transgenic mice (A1AR-tg). Renal A1AR mRNA determined by real time RT-PCR was 353 42 % (n=8) and 575 43 % (n=5) of Wt in line 4 and 7 of A1AR-tg, respectively. A1AR mRNA was also elevated in heart, but not in brain. Ambient urine osmolarity was similar in both A1AR-tg lines (A1AR-tg4: 1678214, n=10, and A1AR-tg7: 1668276 mosmol/l, n=10), not significantly different from wild type (1294163 mosmol/l, n=13). TGF responses of glomerular pressure to a flow increase from 0-30 nl/min were increased from 8.4 0.9 mm Hg in wild type (n=21) to 14.1 0.7 mm Hg in A1AR-tg4 (n=20;p<.0001), and to 12.3 1.4 mm Hg in A1AR-tg7 (n=10;p<0.02). TGF responses were significantly greater in A1AR-tg than wild type especially in the intermediate flow ranges of 7.5-10 nl/min and 10-15 nl/min. Basal plasma renin concentration (PRC, ng angI/ml/hr), and by inference renin secretion, was slightly increased in A1AR-tg compared to Wt (A1AR-tg4: 1918 193;A1AR-tg7: 1983 666;Wt: 1251 142;p=.01 and .24 vs. Wt). Suppression of PRC after acute salt loading (5% body weight saline i.v.) was similar in A1AR-tg4 (-36%), A1AR-tg7 (-33%), and Wt (-35%). Thus, macula densa control of renin secretion appeared largely unaltered, possibly reflecting a low activity of the smooth muscle actin promoter in granular JG cells and consequently low levels of transgene expression. We conclude that vascular overexpression of A1AR results in markedly enhanced TGF responsiveness indicating that A1AR expression in vascular smooth muscle cells is critical for TGF signaling. 2. Previous evidence has indicated that adenosine 1 receptors (A1AR) are required for TGF responsiveness and that their ligand adenosine is at least in part derived from the extracellular metabolism of nucleotides. We have tested whether transmembrane movement of adenosine mediated by the equilibrative nucleoside transporter 1 (ENT1) may also play a modifying role by assessing JGA function in ENT1-/- mice on a C57BL/6 background (breeder pairs of ENT1-/- mice were kindly provided by Doo-Sup Choi, Dept. Mol. Pharmacol. Psych, Mayo Clinic, Rochester, MN). Plasma concentrations of adenosine and inosine were markedly higher in ENT1-/- (117978 and 22548 pmol/ml) than WT mice (17924 and 47.59 pmol/ml). Telemetric measurements of 24-hour mean arterial blood pressure (mm Hg) and heart rates (bpm) were not significantly different between ENT1-/- and WT mice (11414 and 55739 vs. 114.38.1 and 59446) despite the fact that spontaneous activity levels (6.55.7 vs. 9.38 movement cpm) as well as voluntary wheel running activity was lower in ENT1-/- animals. GFR of conscious female ENT1-/- mice (347 59 microl/min;n=8) was not different than that of wild type mice (345 26 microl/min;n=11). Responses of stop flow pressure (Psf) to maximum flow stimulation were significantly reduced in ENT1-/- compared to WT mice (1.80.4 mm Hg, n=25 vs. 5.81.1 mm Hg, n=17;p=.0003). Levels of renal mRNA expression of all 4 subtypes of adenosine receptors were not significantly different between WT and ENT1-/- mice. One may conclude that TGF responsiveness is significantly attenuated in the absence of ENT1, and that this effect may be related to A1AR saturation resulting from the marked increase of extracellular adenosine levels. The reduced spontaneous and voluntary locomotor activity is likely to result from the central inhibitory effect of adenosine. 3. Gap junctions are widely used structures to provide a pathway for the functional coupling of individual cells, and such coupling may be important in the communication mechanisms that link the different cells of the juxtaglomerular apparatus. In fact, the gap junctional protein connexin 40 (Cx40) is highly expressed in the extraglomerular mesangium connecting the tubular epithelium at the macula densa and the afferent arteriole. Participation of Cx40 in juxtaglomerular communication is suggested by the finding that the expression of renin and the localization of renin-expressing cells are highly abnormal in connexin 40-deficient mice. We have therefore used wild type and connexin 40-/- mice to determine the role of connexin 40 in tubuloglomerular feedback (TGF), a regulatory system that may involve gap junctions for the propagation of tubule-vascular signals. TGF responsiveness was assessed by micropuncture as the change of glomerular capillary pressure (Psf) caused by a saturating increase in loop of Henle perfusion rate. Psf fell from 40.0 1.7 to 34.5 1.7 mm Hg in C57Bl/6 wild type mice, and from 31.3 1.0 to 26.9 0.9 mm Hg in Cx40-/- mice. Psf changes of 5.5 0.5 mm Hg in wild type and of 4.4 0.5 in Cx40-/- mice were not significantly different (p=0.125). Psf values were significantly lower in Cx40-deficient than wild type mice at all flow rates. Arterial blood pressure in the animals prepared for micropuncture were not different between wild type and Cx40-/- mice. GFR of both conscious and anesthetized mice was lower in Cx40-/- than wild types and this difference increased with aging. These observations showing that TGF responsiveness is well maintained in connexin 40-deficient mice indicate that coupling through this dominant mesangial protein is not required for tubulo-vascular communication. Renal vascular resistance appears to be elevated in Cx40-/- mice perhaps as a consequence of the elevated plasma renin levels.
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