In the kidney, nitric oxide synthase (NOS) of the neuronal isoform (nNOS) is predominantly located in the macula densa cells. Unspecific chronic NOS inhibition in rats leads to elevated blood pressure (P A ), associated with increased renal vascular resistance. This study was designed to examine the effect of chronic selective inhibition of nNOS with 7-nitro indazole (7-NI) on P A , GFR, and the tubuloglomerular feedback (TGF) system. P A was repeatedly measured by a noninvasive tail-cuff technique for 4 wk in rats treated orally with 7-NI, and in control rats. After treatment, the animals were anesthetized and renal excretion rates, GFR, and TGF activity were determined. After 1 wk of 7-NI treatment P A was increased from 129 Ϯ 4 to 143 Ϯ 2 mmHg. GFR (1.85 Ϯ 0.1 vs. 1.97 Ϯ 0.2 ml/min in controls) was unchanged, but micropuncture studies revealed a more sensitive TGF than in controls. After 4 wk of 7-NI treatment P A was 152 Ϯ 4 mmHg, but no change in GFR (1.90 Ϯ 0.5 ml/min) or TGF sensitivity was detected. Acute administration of 7-NI to nontreated rats did not affect P A , but decreased GFR (1.49 Ϯ 0.1 ml/min) and increased TGF sensitivity. In conclusion, chronic nNOS inhibition leads to increased P A . Our results suggest that the elevated P A could be caused by an initially increased TGF sensitivity, leading to decreased GFR and an increased body fluid volume.

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...

We suggest that because of its low PO2, the renal outer medulla is more sensitive to hypoxia, not because of the low PO2 as such, but probably because of the competition between NO and oxygen to control respiration.

We tested whether chronic ANG II infusion into rats affects descending vasa recta (DVR) contractility, synthesis of superoxide, or synthesis of nitric oxide (NO). Rats were infused with ANG II at 250 ng.kg(-1).min(-1) for 11-13 days. DVR were loaded with dihydroethidium (DHE) to measure superoxide and 3-amino-4-aminomethyl-2',7'-difluorofluorescein (DAFFM) to measure NO. Acute constriction of DVR by ANG II (0.1, 1, and 10 nM) was diminished, and NO generation rate was raised by chronic ANG II infusion. DHE oxidation by DVR from ANG II-infused rats was similar to controls and was significantly higher when NO synthesis was prevented with N(omega)-nitro-L-arginine methyl ester (L-NAME). The superoxide dismutase mimetic Tempol (1 mM) increased NO generation compared with controls. The increased synthesis of NO by chronic ANG II-treated vessels persisted in the presence of Tempol. DVR endothelial cytoplasmic Ca(2+) response to ACh was diminished by chronic ANG II treatment, but the capacity of ACh to increase NO generation was unaltered. We conclude that DVR generation of superoxide is not affected by chronic ANG II exposure but that basal NO synthesis is increased. DVR superoxide is unlikely to be an important mediator of chronic ANG II slow pressor hypertension in rats.

Using fura 2-loaded vessels, we tested whether ouabain modulates endothelial cytoplasmic calcium concentration ([Ca(2+)](CYT)) in rat descending vasa recta (DVR). Over a broad range between 10(-10) and 10(-4) M, ouabain elicited biphasic peak and plateau [Ca(2+)](CYT) elevations. Blockade of voltage-gated Ca(2+) entry with nifedipine did not affect the response to ouabain mitigating against a role for myo-endothelial gap junctions. Reduction of extracellular Na(+) concentration ([Na(+)](o)) or Na(+)/Ca(2+) exchanger (NCX) inhibition with SEA-0400 (10(-6) M) elevated [Ca(2+)](CYT), supporting a role for NCX in the setting of basal [Ca(2+)](CYT). SEA-0400 abolished the [Ca(2+)](CYT) response to ouabain implicating NCX as a mediator. The transient peak phase of [Ca(2+)](CYT) elevation that followed either ouabain or reduction of [Na(+)](o) was abolished by 2-aminoethoxydiphenyl borate (5 x 10(-5) M). Cation channel blockade with La(3+) (10 muM) or SKF-96365 (10 muM) also attenuated the ouabain-induced [Ca(2+)](CYT) response. Ouabain pretreatment increased the [Ca(2+)](CYT) elevation elicited by bradykinin (10(-7) M). We conclude that inhibition of ouabain-sensitive Na(+)-K(+)-ATPase enhances DVR endothelial Ca(2+) store loading and modulates [Ca(2+)](CYT) signaling through mechanisms that involve NCX, Ca(2+) release, and cation channel activation.

Nitric oxide scavenging by erythrocytes has a high impact on arteriolar nitric oxide concentration and autoregulatory response. Nitric oxide measurements in endothelial cells of the afferent arteriole showed that increased perfusion pressure/shear stress increased nitric oxide release, while simultaneously endothelial cell calcium concentration decreased, possibly indicating a feedback control of this calcium by nitric oxide release.

The juxtaglomerular apparatus (JGA) has the very important functions of detecting the fluid flow rate to the distal tubule and thus controlling the glomerular filtration rate (GFR) (tubuloglomerular feedback mechanism [TGF]) and renin release from the afferent arteriole. In studies of the TGF it has been evident that the sensitivity of this mechanism can be reset. Volume expansion will reset it to a low sensitivity leading to a high GFR and urine excretion rate, while dehydration will sensitize the TGF mechanism, giving rise to a low GFR and low urine excretion rate. Furthermore, we have found that in animals that spontaneously develop hypertension there is initially a sensitization of the TGF, leading to a reduced GFR and urine excretion rate, with fluid volume retention in the body and a consequent rise in blood pressure. When the pressure is raised, the TGF characteristics are normalized. In the macula densa (MD) cells in the JGA, there is a large production of NO from neuronal NOS. This production continuously reduces TGF sensitivity and is apparently impaired in animals that spontaneously develop hypertension. When we added an nNOS inhibitor to the drinking water for several weeks while measuring blood pressure, we found an increase in blood pressure after 3-4 weeks of treatment. This effect was abolished by a high salt diet. From these investigations, it also appeared as if nNOS-derived NO inhibited renin release. Experiments have also indicated that NO may resensitize inhibited G-protein coupled purinergic receptors.

We propose that albumin interferes with arteriolar nitric oxide homeostasis, probably by scavenging nitric oxide intra-luminally. In this respect, albumin acts similarly to red blood cells in the circulation. The magnitude of the scavenging determines the effectiveness of autoregulation in the perfused preglomerular vessels. The scavenging properties of the perfusing fluid are important in setting operating levels of endothelial nitric oxide.