Neuronal Nitric Oxide Synthase: Its Role and Regulation in Macula Densa Cells GERGELY KOVA ´ CS,* † PE ´ TER KOMLO ´ SI,* † AMANDA FUSON,* JA ´ NOS PETI-PETERDI,* † LA ´ SZLO ´ ROSIVALL, †‡ and P. DARWIN BELL* *Nephrology Research and Training Center, Division of Nephrology, Departments of Medicine and Physiology, University of Alabama at Birmingham, Birmingham, Alabama; † International Nephrology Research and Training Center, Institute of Pathophysiology, Semmelweis University, Budapest, Hungary; and ‡ Hungarian Academy of Sciences and Semmelweis University Nephrology Research Group, Budapest, Hungary Abstract. Macula densa (MD) cells detect changes in distal tubular sodium chloride concentration ([NaCl] L ), at least in part, through an apical Na:2Cl:K co-transporter. This co-trans- porter may be a site for regulation of tubuloglomerular feed- back (TGF), and recently angiotensin II (Ang II) was shown to regulate the MD Na:2Cl:K co-transporter. In addition, nitric oxide (NO) produced via neuronal NO synthase (nNOS) in MD cells attenuates MD-TGF signaling. This study investigated [NaCl] L -dependent MD-NO production, the regulation of co- transporter activity by NO, and the possible interaction of NO with Ang II. MD cell Na + concentration ([Na + ] i ) and NO production were measured using sodium-binding benzofuran isophthalate and 4-amino-5-methylamino-2’,7’-difluorescein diacetate, respectively, using fluorescence microscopy. Na: 2Cl:K co-transport activity was assessed as the initial rate of increase in [Na + ] i when [NaCl] L was elevated from 25 to 150 mM. 10 -4 M 7-nitroindazole, a specific nNOS blocker, signif- icantly increased by twofold the initial rate of rise in [Na + ] i when [NaCl] L was increased from 25 to 150 mM, indicating co-transporter stimulation. There was no evidence for an in- teraction between the stimulatory effect of Ang II and the inhibitory effect of NO on co-transport activity, and, further- more, Ang II failed to alter MD-NO production. NO produc- tion was sensitive to [NaCl] L but increased only when [NaCl] L was elevated from 60 to 150 mM. These studies indicate that MD-NO directly inhibits Na:2Cl:K co-transport and that NO and Ang II independently alter co-transporter activity. In ad- dition, generation of MD-NO seems to occur only at markedly elevated [NaCl] L , suggesting that NO may serve as a buffer against high rates of MD cell transport and excessive TGF- mediated vasoconstriction. Macula densa (MD) cells serve as a distal nephron sensor element detecting changes in tubular fluid composition and transmitting information to afferent arteriolar smooth muscle cells (tubuloglomerular feedback [TGF]) and renin-containing granular cells (1). The entry of sodium chloride into MD cells occurs primarily through a furosemide-sensitive apical Na: 2Cl:K co-transporter (NKCC2) and the Na:H exchanger NHE2 (2– 4). Alterations in luminal sodium chloride concentration ([NaCl] L )initiate a number of electrochemical processes in MD cells, including changes in intracellular ion concentrations (3,5–7), membrane potential (2,4), and cell volume (8,9). Re- cently, it has been suggested that MD cells communicate with the underlying mesangial cell/smooth muscle/granular cell jux- taglomerular complex via the basolateral release of ATP, pros- taglandin E 2 , and production of nitric oxide (NO) (10 –13). NaCl entry into MD cells seems to be a necessary step in signal generation by these cells. It is also possible that this step may be a critical regulatory point in controlling MD cell communication. For instance, with the identification of angio- tensin II (Ang II) AT 1 receptors in MD cells (14), it was found that Ang II at nanomolar concentrations stimulated Na entry and cell alkalization via Na:H exchange in MD cells (3). We have also recently reported that Ang II, at least in the same concentration range, directly stimulates Na:2Cl:K co-transport activity in MD cells (5). This stimulation of NaCl entry into MD cells by Ang II is consistent with Ang II–mediated en- hancement of TGF responses (15,16). There has been considerable interest in the role of NO in TGF and renin secretion. This was initially based on immuno- histochemical findings of a high level of neuronal NO synthase (NOS) expression in MD cells (17,18). It has been presumed that the regulation of MD NO production is under the control of MD apical transport, although this point has not been well established. However, there is a wealth of information regard- ing the role of NO in MD-TGF signaling. Local, intratubular administration of non–isoform-specific NOS blockers or 7-ni- troindazole (7-NI), which selectively blocks nNOS, was shown to enhance TGF responses (18 –22). NO has also been shown to participate in renin secretion with current evidence indicat- Received December 6, 2002. Accepted July 11, 2003. Correspondence to Dr. P. Darwin Bell, UAB Station, 865 Sparks Center, University of Alabama at Birmingham, Birmingham, AL 35294. Phone: 205- 934-3806; Fax: 205-934-1147; E-mail: pdbell@uab.edu 1046-6673/1410-2475 Journal of the American Society of Nephrology Copyright © 2003 by the American Society of Nephrology DOI: 10.1097/01.ASN.0000088737.05283.2B J Am Soc Nephrol 14: 2475–2483, 2003