Abstract. The luminal NaCl concentration ([NaCl]) at the macula densa (MD) controls both tubuloglomerular feedback (TGF) and renin release. Nitric oxide (NO) inhibits TGF sensitivity to a great extent. The NO concentration in the MD cells is not known. This study measured this concentration in MD cells with confocal microscopy in the isolated perfused thick ascending limb using a NO-sensitive fluorophore 4,5-diaminofluorescein (DAF-2). Calcein was used to measure cell volume changes. The loop perfusion fluid was a modified Ringer solution containing 10, 35, or 135 mM NaCl with a constant total osmolarity (290 mOsm), and the bath was perfused with the 135 mM NaCl solution. The results show that MD cell volume and NO concentration measured with DAF-2 DA increased considerably with increasing luminal[NaCl] and with calcium-free solutions in the lumen and bath. L-arginine (5 mM) increased NO concentration in the MD cells by 30%. 7-nitroindazole could totally inhibit the NO production caused by L-arginine and by increased luminal [NaCl]. In conclusion, the MD cell volume changes caused by the changes of luminal [NaCl] were quantitatively measured, and it was found that increasing the luminal [NaCl] resulted in an increase in cell volume. It was also found that NO formation in MD cells could be measured with DAF-2 and that NO production was increased through neuronal NO synthase activation with an increased luminal [NaCl]. An increased NO production will inhibit the vasoconstriction induced by the TGF and at the same time will reduce TGF sensitivity.The juxtaglomerular apparatus (JGA) is a complex assembly of specialized structures related to each other anatomically, forming the vascular pole of the glomerulus. The JGA is comprised of the macula densa (MD), the extraglomerular mesangium, and the afferent and efferent arterioles. The MD is a plaque of 20 to 30 specialized epithelial cells belonging to the end portion of the thick ascending limb. The luminal NaCl concentration ([NaCl]) at the MD has two established effects: (1) regulation of glomerular arteriolar resistance through tubuloglomerular feedback (TGF); and (2) control of renin release (1,2). The first step in these signal transmissions involves NaCl transport by the MD, which is relatively well understood. NaCl uptake occurs primarily through the Na ϩ -K ϩ -2Cl Ϫ cotransporter, which has been demonstrated both on functional and transcriptional levels (3,4). The next step is not yet clear. The possible mediators and modulators of the information transmitted between the MD and its target cells include the ion concentration, ATP, angiotensin II, adenosine, arachidonic acid metabolites, and nitric oxide (NO) (5-7). Among these, NO appears to play an important role in the vascular response to changes in luminal [NaCl]. Many studies have been done on the effects of NO on TGF regulation (8 -14), but there has been no report on instantaneous NO concentration changes caused by changes in the luminal [NaCl]. In the present study, an NO-sensitive probe was used for di...

Abstract-A fluorescent nitric oxide (NO) indicator, 4,5-diaminofluorescein diacetate, and the calcium indicator, indo-1, with 488 nm and 364 nm UV confocal laser scanning microscopy were used to detect NO and calcium concentration in rabbit macula densa (MD) cells challenged by angiotensin II (Ang II The findings of a constitutive isoform of nitric oxide (NO) synthase, namely neuronal NO synthase (nNOS), 5,6 and the angiotensin AT 1 receptor 7 in MD cells have raised speculations about their possible relationship. It is well documented that angiotensin II (Ang II) can potently sensitize the tubuloglomerular feedback mechanism, while it is equally well known that NO desensitizes this mechanism. 8 -10 Studies have shown that Ang II can stimulate the nNOS pathway measured by the grade of immunostaining for nNOS in the MD of the rat kidney 11 and total renal nNOS mRNA levels. 12 However, there have been no reports hitherto of changes in the cytosolic calcium concentration ([Ca 2ϩ ] i ) and in NO production in MD cells stimulated by Ang II. In the present study, the calcium indicator indo-1 and the NO indicator, 4,5-diaminofluorescein diacetate (DAF-2 DA) were used to detect [Ca 2ϩ ] i and NO production, respectively, in MD cells challenged by Ang II, using a confocal laser scanning microscopy. Methods Experimental Preparation and MeasurementsGlomeruli with attached cortical thick ascending limbs and containing the MD plaque were isolated and microperfused using a method similar to that described. 13 The cortical thick ascending limbs was cannulated and perfused with the 35 mmol/L NaCl buffer solution. The preparation was bathed continuously in a 135 mmol/L NaCl buffer solution (containing in mM: 135 NaCl, 1.3 CaCl 2 , 1 MgSO 4 , 1.6 K 2 HPO 4 , 5 glucose, and 20 HEPES, with pH adjusted to 7.4 and an osmolality of 290 mOsm).The preparation was loaded with 10 M cell-permeable fluorescent Ca 2ϩ indicator indo-1 AM, 0.5% dimethyl sulfoxide plus 0.1% pluronic acid from the lumen for 30 to 40 minutes. Indo-1-loaded MD-glomerular preparations were studied in the confocal system (Noran) with UV laser. A Nikon ϫ60/1.2 water-immersion objective lens was used to visualize MD cells. The confocal slit was set at a width of 15 nm. Indo-1 was excited at 364 nm with a UV laser. Emission was measured at 405 nm and 485 nm and transmitted to photomultiplier tubes (Figure 1). The measurements of calcium were made as described by other investigators. 14,15 A cell-permeable fluorescent NO indicator, DAF-2 DA, was used to detect NO production in MD cells. The cells were loaded with 10 M DAF-2 DA (in 0.5% dimethyl sulfoxide) from the lumen for 40

Abstract. Purinergic receptors are important in the regulation of renal hemodynamics; therefore, this study sought to determine if such receptors influence macula densa cell function. Isolated glomeruli containing macula densa cells, with and without the cortical thick ascending limb, were loaded with the Ca 2ϩ sensitive indicators, Fura Red (confocal microscopy) or fura 2 (conventional video image analysis). Studies were performed on an inverted microscope in a chamber with a flowthrough perfusion system. Changes in cytosolic calcium concentration ([Ca 2ϩ ] i ) from exposed macula densa plaques were assessed upon addition of adenosine, ATP, UTP, ADP, or 2-methylthio-ATP (2-MeS-ATP) for 2 min added to the bathing solution. The tubuloglomerular feedback (TGF) mechanism is a very important regulator of renal hemodynamics. This mechanism operates as a negative feedback loop, sensing changes in distal nephron fluid flow rate by detecting flow-dependent alterations in luminal sodium chloride concentration ([NaCl]) at the macula densa. Signals are then sent by the macula densa cells, which contract the afferent arteriole, thereby adjusting the level of GFR and renal blood flow (1-3). Furthermore, macula densa cells can also influence release of renin from the granular cells in the afferent arteriole (4). The primary mechanism by which the macula densa cells detect changes in [NaCl] is through an apically located Na:2Cl:K cotransport mechanism (5,6). It has also been found that the sensitivity of this TGF mechanism can be reset by a large number of different factors. For instance, renal interstitial pressure and angiotensin II are potent regulators of TGF sensitivity (7,8). Nitric oxide, prostaglandin, thromboxanes, and many other local factors and hormones can also influence the sensitivity of the TGF mechanism (9). The site(s) at which all these factors exert their influence is not well understood. One possibility is that modulation of TGF can occur at the level of the macula densa cells, and it is therefore of particular interest to investigate the receptors expressed by macula densa cells and what effects occur in these cells with receptor activation. For instance, it has recently been reported that macula densa cells possess AT 1 receptors for angiotensin II and that activation of these receptors increases sodium-proton exchange of the apical NHE2 isoform (10 -12).Purinergic receptors have been identified as playing a large role within the juxtaglomerular apparatus (13). These receptors are activated by extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) and are important signaling molecules that mediate various biologic effects in the kidney. They may serve as paracrine regulators of renal microvascular resistance (14,15) and may modulate mesangial cell contraction, alter epithelial ion transport, and influence the TGF mechanism (16 -20) via cell surface receptors for purines.There are two main families of purine receptors: adenosine or P1 receptors and P2 receptors, the latter recogni...

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