Role of macula densa neuronal nitric oxide synthase in renal diseases

Macula densa cells have an important role in the regulation of glomerular blood flow and glomerular filtration by its regulation of afferent arteriolar vascular tone. Nitric oxide derived from neuronal nitric oxide synthase (nNOS) in macula densa can dilate afferent arterioles. Macula densa nNOS is important for renin secretion, and its expression is regulated by dietary salt, renal angiotensin II, intracellular pH, and other factors. In salt-sensitive hypertension, nNOS is suppressed, whereas in SHR or in the early phase of diabetes, nNOS is increased in macula densa along with NADPH oxidase, which limits NO bioavailability. Renal damage induced by hypertension, diabetes, and hyperlipidemia could be prevented by enhancement of nNOS in macula densa with ACEI, dipyridamole, α₁-receptor blocker, a low-salt diet, or sodium bicarbonate. Sodium bicarbonate is a safe and clinically available enhancer of nNOS in macula densa that increases glomerular blood flow and prevents the reduction of GFR in radiocontrast nephropathy and chronic renal failure. In conclusion, the enhancement of nNOS in the macula densa can be a promising strategy to prevent reduction of renal function.     

ATPase activity in macula densa cells of the rabbit kidney

Na-K- and Mg-activated ATPase activities were determined in maculae densae and glomeruli dissected from both superficial and juxtamedullary nephrons of normal rabbits, using an ultramicro method including a cycling reaction. Activities were expressed as P_i generated per macula densa or per glomerulus and normalized for tissue volume. Results indicate that the mean volume of superficial and juxtamedullary macula densa samples was not statistically different, while glomeruli from deep nephrons had sample volumes that were 29% larger than those from superficial nephrons (P<0.001). Correcting for volume both superficial and juxtamedullary macula densa samples had an Na-K-ATPase activity of 0.37±0.21 fmol · h^–1 · (m³)^–1. Mg-ATPase activity in both pools was also similar [0.41±0.07 and 0.52±0.1 fmol · h^–1 · (m³)^–1]. Na-K-ATPase activity in macula densa cells is estimated to be about 1/40th the activity of surrounding cortical thick ascending limb cells. Total glomerular ATPase per unit volume was significantly higher in glomeruli from superficial than from deep nephrons [0.41±0.04 vs. 0.28±0.04 fmol · h^–1 · (m³)^–1 P<0.05]. There was no statistically significant activity of Na-K-ATPase in either superficial or deep glomeruli. These results suggest that in contrast to previous reports, the macula densa contains Na-K-ATPase, but at a low level relative to surrounding tubular cells. Further, in normal rabbits, this activity is invariant in superficial and juxtamedullary samples.     

Low [NaCl]-induced neuronal nitric oxide synthase (nNOS) expression and NO generation are regulated by intracellular pH in a mouse macula densa cell line (NE-MD)

Changes in the luminal NaCl concentration ([NaCl]) at the macula densa (MD) modulate the tubuloglomerular feedback (TGF) responses via an affect on the release of nitric oxide (NO). This study was performed in a newly established mouse macula densa cell line (NE-MD) to investigate the effects of lowering [NaCl] on the neuronal NO synthase (nNOS) protein expression and l-arginine (Arg)-induced NO release. Expression of nNOS protein and release of NO were evaluated by Western blot analysis and an NO-sensitive electrode, respectively. Intracellular pH (pH_i) was monitored by the BCECF assay. Although there was weak staining of the nNOS protein expression, l-Arg-induced NO generation was negligible in normal (140 mM NaCl) solution. Both were significantly (P < 0.05) increased either in the presence of furosemide (12 μM), an inhibitor of the Na⁺–K⁺–2Cl⁻ cotransporter, or in a low (23 mM) Cl⁻ solution. Furosemide- and low Cl⁻-induced NO generation was completely inhibited by 50 μM 7-nitroindasole (7-NI), a nNOS inhibitor. Moreover, these increases were significantly (P < 0.05) inhibited by the addition of 100 μM amiloride, an inhibitor of the Na⁺/H⁺ exchanger, or by its analogue 5-(N)-ethyl-N-isopropyl amiloride (EIPA), and also at a lower pH of 7.1. Furthermore, nNOS expression and NO release were not stimulated in as low as 19 mM Na⁺ solution. In conclusion, low [Cl⁻], but not low [Na⁺] in the lumen at the MD, increased nNOS protein expression and NO generation. Changes in the luminal [NaCl] may modulate the TGF system via an effect on the NO generation from the MD.     

Expression of Na+/Cl–/betaine and Na+/myo-inositol transporters, aldose reductase and sorbitol dehydrogenase in macula densa cells of the kidney

 It has been suggested that macula densa cells may be exposed to hyperosmotic stress. Since chronic exposure to hypertonic stress causes the amount of intracellular organic osmolytes to increase, the expression of transporters and enzymes that participate in the intracellular accumulation of organic osmolytes was examined using non-radioactive in situ hybridization in the macula densa region of control rats and furosemide-treated animals. Both the sodium- and chloride-dependent betaine transporter (BGT) and sodium-dependent myo-inositol transporter (SMIT) were expressed preferentially in macula densa cells and for both mRNAs the signal intensity was visibly reduced by furosemide. The enzymes aldose reductase (which mediates the conversion of glucose to sorbitol) and sorbitol dehydrogenase (which converts sorbitol into fructose) were expressed not only in macula densa cells but also in the surrounding tubular cells, and the expression was insensitive to furosemide. Thus it remains unclear whether the expression of BGT and SMIT is related to a putative hypertonic juxtaglomerular region. Received: 27 April 1998 / Accepted: 8 July 1998     

Nitric oxide producing neurones in the rat medulla oblongata that project to nucleus tractus solitarii

The production of nitric oxide in neurones of the rat medulla oblongata that project to the nucleus tractus solitarii (NTS) was examined by simultaneous immunohistochemical detection of nitric oxide synthase (NOS) and of cholera toxin B-subunit (CTb), which was injected into the caudal zone of the NTS. Neurones immunoreactive for CTb and neurones immunoreactive for NOS were widely co-distributed and found in almost all the anatomical divisions of the medulla. Dual-labelled cells, containing both CTb and NOS immunoreactivities were more numerous ipsilaterally to the injection sites. They were concentrated principally in the more rostral zone of the NTS, raphé nuclei, dorsal, intermediate and lateral reticular areas, spinal trigeminal and paratrigeminal nuclei and the external cuneate and medial vestibular nuclei. Isolated dual-labelled neurones were also scattered throughout most of the divisions of the reticular formation. These observations indicate that many areas of the medulla that are known to relay somatosensory and viscerosensory inputs contain NOS immunoreactive neurones that project to the NTS, and may, therefore, contribute to the dense NOS-immunoreactive innervation of the NTS. The release of nitric oxide from the axon terminals of these neurones may modulate autonomic responses generated by NTS neurones in relation to peripheral sensory stimuli, and thus ultimately regulate sympathetic and/or parasympathetic outflow.     

Nitric oxide inhibitors attenuate ischemic degeneration in the CA1 region of rat hippocampal slices

We examined the involvement of nitric oxide (NO) in ischemic brain damage using hippocampal slices prepared from 30 day old albino rats and exposed to 20 min of oxygen/glucose deprivation (ischemia) followed by 90 min postincubation in oxygen- and glucose-containing media. Damage in the CA1 region was rated on a 0 (intact) to 4 (severe neuronal damage) scale by a rater blind to the experimental condition. Control slices exposed to ischemia were rated as 2.8 ± 0.4 (N = 12). -N^G-Monomethylarginine (100 μM) and -N^G-nitroarginine (100 μm), non-selective NO synthase (NOS) inhibitors, diminished ischemic damage (0.6 ± 0.3, N = 8, and 1.0 ± 0.5, N = 4, respectively). An inhibitor of brain NOS, 7-nitroindazole (30 μM), was also effective against ischemic degeneration (0.7 ± 0.3, N = 5). These results suggest that activation of NOS is involved in ischemic degeneration in the CA1 region.     

Nitric oxide modulates acetylcholine‐induced vasodilatation in the hepatic arterial vasculature of the dual‐perfused rat liver

The role of nitric oxide in the modulation of hepatic arterial vascular reactivity was investigated in an isolated dual‐perfused rat liver preparation. Twelve male Wistar rats (200–250 g) were anaesthetized with sodium pentobarbitone (60 mg kg^–1 i.p.). The livers were then excised and perfused in vitro through hepatic arterial and portal venous cannulae at constant flow rates. Concentration‐dependent dose–response curves to acetylcholine (10^–8–10^–5 M ), sodium nitroprusside (10^–6–5 × 10^–4 M ), and adenosine triphosphate (ATP) (10^–8–10^–5 M ) in the hepatic artery were constructed after the tone was raised by addition of methoxamine (3 μM L^–1). Acetylcholine‐induced vasodilatation in the hepatic artery was significantly attenuated with inhibition of nitric oxide synthase by using N^G‐nitro‐L ‐arginine methyl ester (30 μM ), E_max=51.7 ± 2.8 vs. 32.5 ± 3.1 mmHg, before vs. after N^G‐nitro‐L ‐arginine methyl ester, respectively. ATP‐induced hepatic arterial vasoconstriction which was significantly enhanced with L ‐NAME, E_max=94.0 ± 9.3 vs. 127.0 ± 8.0 mmHg, before vs. after N^G‐nitro‐L ‐arginine methyl ester, respectively. Sodium nitroprusside‐induced hepatic arterial vasodilatation remained unchanged with N^G‐nitro‐L ‐arginine methyl ester, E_max=57.0 ± 3.4 vs. 57.0 ± 4.1, before vs. after N^G‐nitro‐L ‐arginine methyl ester, respectively. The data from the present study suggest that acetylcholine‐induced vasodilatation in the intrahepatic arterial vasculature of the rat liver is at least, in part, mediated by the release of nitric oxide. In addition, ATP‐induced hepatic arterial vasoconstriction is also modulated by the release of nitric oxide (*P < 0.05, Student’s paired t‐test).     

Contribution of endothelin to the coronary vasoconstriction in the isolated rat heart induced by nitric oxide synthase inhibition

The possible involvement of endothelin-1 (ET-1) and angiotensin II in the coronary vasoconstriction induced by nitric oxide synthase (NOS) inhibition was investigated in isolated Langendorff-perfused rat hearts. Fifteen minutes of perfusion with the NOS inhibitor N ^G-nitro-L -arginine (L -NNA, 0.1 mM ) reduced coronary flow by 31%. Pre-treatment with the non-selective ET_A/ET_B receptor antagonist bosentan (1 and 10 μM ) attenuated this reduction in coronary flow to 16% (P < 0.05) and 8% (P < 0.01), respectively. The selective ET_A receptor antagonist BQ-123 (1 μM ) induced a similar inhibitory action, whereas the selective ET_B receptor antagonist BQ-788 and the angiotensin II type 1 receptor antagonist candesartan did not affect the vasoconstrictor response to L -NNA. In addition, bosentan administered after 15 min of L -NNA perfusion reversed the L -NNA-induced reduction in coronary flow in a dose-dependent manner. The high concentration of bosentan (10 μM ) increased the basal coronary flow by 17%, while the lower concentration of bosentan, BQ-123, BQ-788 and candesartan did not affect basal coronary flow. Bosentan (10 μM ) increased the level of ET-like immunoreactivity (-LI) in the coronary effluent twofold. L -NNA did not affect ET-LI level. These results indicate that ET-1 contributes to the coronary vasoconstrictor effect of L -NNA in the isolated rat heart, and that this action of ET-1 is mediated through ET_A receptor activation. Angiotensin II does not seem to contribute to L -NNA-induced vasoconstriction under the present condition.     

Possible release of nitric oxide from cholinergic axons in the thalamus by stimulation of the rat laterodorsal tegmental nucleus as measured with voltammetry

By means of the differential direct current voltammetry technique with carbon fiber electrodes in urethane-anesthetized rats, we monitored nitric oxide (NO) concentrations in the thalamus in the basal condition and following electrical stimulation of the laterodorsal tegmental nucleus (LDT), whose neurons have the strongest activity of NADPH-diaphorase, or NO synthase, together with acetylcholine. NO levels, measured as the height of the peak at +970–1000 mV in the voltammetry (NO was soon oxidized in vivo to be detected at the voltage of this peak, so that NO levels in this report are, in the strict sense, levels of the oxidized metabolites reflecting very possibly those of NO in physiological conditions; see Section 2, Methods), increased just after repetitive stimulation of the LDT. Stimulation of the surrounding areas or the cerebellum produced virtually no change in NO levels. An intravenous injection of -nitroarginine methyl ester reduced the basal level of NO, but stimulation of the LDT still increased NO levels, which may be due to very strong activity of NO synthase in the LDT neurons. These results are consistent with the notion that NO can be released from axons of the LDT neurons by their excitation.     

Nitric oxide mediated modulation of free radical generation response in the rat polymorphonuclear leukocytes: A flowcytometric study

Nitric oxide (NO) synthesis and free radical generation from polymorphonuclear leukocytes (PMNs) play an important role in several pathological conditions. It is therefore important to understand the regulatory mechanisms of free radical generation from PMNs. Flowcytometry can be used to assess generation of reactive oxygen and nitrogen species from PMNs by using fluorescent probes. In the present study regulation of NO synthesis in the control and lipopolysaccharide (LPS) treated rat PMNs has been investigated. Free radical generation was assessed by flow cytometry using a dye, 27-dichlorodihydrofluorescein diacetate (DCFDA), dihydrorhodamine-123 (DHR) and 4,5-diaminofluorescein diacetate (DAF). Superoxide dismutase (SOD), and catalase significantly attenuated the arachidonic acid (AA, 1 × 10^–6 M) induced free radical generation, while 4-aminobenzoicacid hydrazide (ABH), myeloperoxidase (MPO) inhibitor had no significant effect. Intracellular and extracellular calcium levels also modulated FR generation. AA induced free radical generation from PMNs was also enhanced significantly after LPS treatment. NO synthase (NOS) inhibitors, aminoguanidine (AG) and 7-nitroindazole (NI) inhibited arachidonic acid induced free radical generation from LPS treated PMNs, while in control PMNs NOS inhibition had no effect. Augmentation of free radical generation from rat PMNs following LPS treatment seems to be regulated by NO.