Hypoxia-induced regulation of nitric oxide synthase in cardiac endothelial cells and myocytes and the role of the PI3-K/PKB pathway

The roles of endothelial nitric oxide synthase (eNOS), and its putative association with protein kinase B (PKB), and inducible nitric oxide synthase (iNOS) are not well characterized in hypoxic cardiac cells and there is a lack of studies that measure nitric oxide (NO) directly. Objective To measure NO production in cardiomyocytes and cardiac microvascular endothelial cells (CMECs) under baseline and hypoxic conditions and to evaluate the expression, regulation and activation of eNOS, iNOS and PKB. The effect of PI3-K/PKB inhibition on NO production and eNOS expression/activation was also investigated. Methods Adult rat cardiomyocytes and rat CMECs were made hypoxic by cell pelleting and low PO₂ incubation. Intracellular NO was measured by FACS analysis of DAF-2/DA fluorescence, and eNOS, iNOS and PKB were evaluated by Western blotting or flow cytometry. Upstream PKB inhibition was achieved with wortmannin. Results (1) NO levels increased in both cell types after exposure to hypoxia. (2) In hypoxic CMECs, eNOS was upregulated and activated, no iNOS expression was observed and PKB was activated. (3) In myocytes, hypoxia did not affect eNOS expression, but increased its activation. Activated PKB also increased during hypoxia. FACS analysis showed increased iNOS in hypoxic myocytes. (4) Wortmannin resulted in decreased hypoxia-induced NO production and reduced activated eNOS levels. Conclusions Cardiomyocytes and CMECs show increased NO production during hypoxia. eNOS seems to be the main NOS isoform involved as source of the increased NO generation, although there may be a role for iNOS and other non-eNOS sources of NO in the hypoxic myocytes. Hypoxia-induced PKB and eNOS activation occurred simultaneously in both cell types, and the PI3-K/PKB pathway was associated with hypoxia-induced NO production via eNOS activation.     

Increased nitric oxide production accompanies blunted hypoxic pulmonary vasoconstriction in hyperoxic rat lung

Hyperoxia may affect lung physiology in different ways. We investigated the effect of hyperoxia on the protein expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), nitric oxide (NO) production, and hypoxic pulmonary vasoconstriction (HPV) in rat lung. Twenty-four male rats were divided into hyperoxic and normoxic groups. Hyperoxic rats were placed in > 90% F1O2 for 60 h prior to experiments. After baseline in vitro analysis, the rats underwent isolated, perfused lung experiments. Two consecutive hypoxic challenges (10 min each) were administered with the administration of a non-specific NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), in between. We measured intravascular NO production, pulmonary arterial pressure, and protein expression of eNOS and iNOS by immunohistochemistry. We found that hyperoxia rats exhibited increased baseline NO production (P < 0.001) and blunted HPV response (P < 0.001) during hypoxic challenges compared to normoxia rats. We also detected a temporal association between the attenuation in HPV and increased NO production level with a negative pre-L-NAME correlation between HPV and NO (R = 0.52, P < 0.05). After L-NAME administration, a second hypoxic challenge restored the HPV response in the hyperoxic group. There were increased protein expression of eNOS (12.6 +/- 3.1-fold, n = 3) (X200) and iNOS (8.1 +/- 2.6-fold, n = 3) (X200) in the hyperoxia group. We conclude that hyperoxia increases the protein expression of eNOS and iNOS with a subsequent increased release of endogenous NO, which attenuates the HPV response.     

Naringin protects viscera from ischemia/reperfusion injury by regulating the nitric oxide level in a rat model

We investigated the effects of naringin on small intestine, liver, kidney and lung recovery after ischemia/reperfusion (I/R) injury of the gut. Rats were divided randomly into four groups of eight. Group A was the sham control; group B was ischemic for 2 h; group C was ischemic for 2 h and re-perfused for 2 h (I/R); group D was treated with 50 mg/kg naringin after ischemia, then re-perfused for 2 h. Endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) expressions were detected by immunolabeling. We also measured arginase activity, amounts of nitric oxide (NO) and total protein. iNOS was increased significantly in the small intestine, liver and kidney in group C. iNOS was decreased significantly only in small intestine and lung in group D. eNOS was increased significantly in the small intestine, liver and lung in group C. eNOS was decreased in small intestine, liver and lung in group D; however, eNOS was decreased in the kidney in group C and increased in the kidney in group D. The amount of NO was decreased significantly in all tissues in group D, but arginase activity was decreased in the small intestine and lung, increased in the kidney and remained unchanged in the liver in group D. The total protein increased in the small intestine and liver in group D, but decreased significantly in the kidney and lung in group D. Naringin had significant, salutary effects on the biochemical parameters of I/R by decreasing the NO level, equilibrating iNOS and eNOS expressions, and decreasing arginase activity.     

NO production by cNOS and iNOS reflects blood pressure changes in LPS-challenged mice

Increased nitric oxide (NO) production is the cause of hypotension and shock during sepsis. In the present experiments, we have measured the contribution of endothelial (e) and inducible (i) nitric oxide synthase (NOS) to systemic NO production in mice under baseline conditions and upon LPS treatment (100 microg/10 g ip LPS). NO synthesis was measured by the rate of conversion of l-[guanidino-15N2]arginine to l-[ureido-15N]citrulline, and the contribution of the specific NOS isoforms was evaluated by comparing NO production in eNOS-deficient [(-/-)] and iNOS(-/-) mice with that in wild-type (WT) mice. Under baseline conditions, NO production was similar in WT and iNOS(-/-) mice but lower in eNOS(-/-) mice [WT: 1.2 +/- 0.2; iNOS(-/-): 1.2 +/- 0.2; eNOS(-/-): 0.6 +/- 0.3 nmol. 10 g body wt-1. min-1]. In response to the challenge with LPS (5 h), systemic NO production increased in WT and eNOS(-/-) mice but fell in iNOS(-/-) mice [WT: 2.7 +/- 0.3; eNOS(-/-): 2.2 +/- 0.6; iNOS(-/-): 0.7 +/- 0.1 nmol. 10 g body wt-1. min-1]. After 5 h of LPS treatment, blood pressure had dropped 14 mmHg in WT but not in iNOS(-/-) mice. The present findings provide firm evidence that, upon treatment with bacterial LPS, the increase of NO production is solely dependent on iNOS, whereas that mediated by cNOS is reduced. Furthermore, the data show that the LPS-induced blood pressure response is dependent on iNOS.     

Gonadotropin-releasing hormone-agonist inhibits synthesis of nitric oxide and steroidogenesis by luteal cells in the pregnant rat

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo suppressed progesterone production and induced apoptosis in the corpus luteum (CL) of the pregnant rat. To investigate the mechanism(s) by which progesterone secretion is suppressed and apoptosis is induced in the luteal cells, we studied nitric oxide (NO) as a messenger molecule for GnRH action. Rats were treated individually on Day 8 of pregnancy with 5 microg/day of GnRH-Ag for 4, 8, and 24 h. GnRH-Ag decreased the production of progesterone and pregnenolone 8 and 24 h after the administration. Corresponding with the reduction in these steroid hormones, luteal NO concentrations decreased at 8 and 24 h. Western blotting and immunohistochemical studies of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) in the CL demonstrated that administration of GnRH-Ag was associated with a marked decrease in eNOS and iNOS compared with sham controls at 4 and 8 h, but nNOS did not change throughout the experimental period. We demonstrated, for the first time, the presence of nNOS protein in the CL of the pregnant rat. To determine if this suppressive action of GnRH-Ag is directly on the CL, luteal cells were treated with GnRH-Ag for 4, 8, 12, and 24 h in vitro. Progesterone and NO concentrations in the media decreased at 8 and 12 h after the treatment and recovered at 24 h. Western blots revealed that eNOS and iNOS decreased in luteal cells treated with GnRH-Ag compared with controls at 4 and 8 h. These results demonstrate that suppression of luteal NO synthesis by GnRH-Ag is direct and leads to a decrease in the luteal production and release of progesterone and pregnenolone and thus suggest that GnRH could induce luteolysis in pregnant rats via NO.     

CGRP stimulation of iNOS and NO release from trigeminal ganglion glial cells involves mitogen-activated protein kinase pathways

Clinical and basic science data support an integral role of calcitonin gene-related peptide (CGRP) in the pathophysiology of temporomandibular joint disorders. Recently, we have shown that CGRP can stimulate the synthesis and release of nitric oxide (NO) from trigeminal ganglion glial cells. The goal of this study was to determine the role of mitogen-activated protein kinase (MAPK) signaling pathways in CGRP regulation of iNOS expression and NO release from cultured trigeminal ganglion glial cells from Sprague–Dawley rats. CGRP treatment for 2 h significantly increased activity of the MAPK reporter genes, Elk, ATF-2, and CHOP. In addition, CGRP increased nuclear staining for the active forms of the MAPKs: extracellular signal-regulated kinase, c-Jun amino-terminal kinase, and p38. This stimulatory event was not observed in cultures pre-treated with the CGRP receptor antagonist peptide CGRP_8–37. Similarly, pre-treatment with selective MAPK inhibitors repressed increases in reporter gene activity as well as CGRP-induced increases in iNOS expression and NO release mediated by MAPKs. In addition, over-expression of MAPK kinase 1 (MEK1), MEK3, MEK6, and MEK kinase significantly increased iNOS expression and NO production in glial cells. Results from our study provide evidence that CGRP binding to its receptor can stimulate iNOS gene expression via activation of MAPK pathways in trigeminal ganglion glial cells.     

白细胞介素—10对血管一氧化氮／一氧化氮合酶系统的影响

为探讨抗炎因子－－白细胞介素－10（IL－10）对大鼠主动脉一氧化氮（NO）／一氧化氮合酶（NOS）系统的影响,应用Griess试剂、^3H－瓜氨酸生成及蛋白免疫印迹杂交等方法,测定IL－10孵育对血管NO释放、NOS活性及表达的影响。结果发现细菌脂多糖（LPS）呈浓度领带性地激活诱导型NOS（iNOS）,促进NO生成。IL－10（10^-10-10^-8g/ml）呈浓度依赖性地上调内皮型NOS（eNOS）蛋白表达及其活性,但对iNOS活性及表达无明显影响,IL－10（10^-9-10^-8g/ml）显著抑制10μg/ml LPS诱导的NO生成和iNOS激活;而高浓度IL－10（10^-7g/ml）则上调iNOS的活性,对eNOS蛋白的表达知活性无明显影响。因此IL－10对NO／NOS系统具有双重影响,一方面可抑制炎症介质诱发的作为炎性物质的iNOS的表达及激活,另一方面可上调内皮源扩血管物质NO的释放。     

Relaxation of myometrium by calcitonin gene-related peptide is independent of nitric oxide synthase activity in mouse uterus

Calcitonin gene-related peptide (CGRP) inhibits myometrial contractile activity. However, the responsiveness of the mouse myometrium to CGRP is dependent on the hormonal and gestational stage. The inhibitory effect of CGRP in the myometrium is prominent during gestation and declines at parturition. The present study was undertaken to examine if nitric oxide (NO) production by nitric oxide synthase (NOS) isoforms mediates the inhibitory action of CGRP on uterine contractions as has been suggested earlier. Transgenic mice deficient in either of the three major NOS isoforms: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were used. Isometric force measurements on myometrial strips obtained from NOS-deficient mice were carried out and the inhibitory capacity of CGRP was monitored. CGRP inhibited KCl-induced contractions of the myometrial strips obtained from eNOS(-/-), iNOS(-/-), and nNOS(-/-) mice with equal efficiency as in wild-type animals. Additionally, NOS protein expression in the mouse uterus during gestation and during the estrous cycle was examined by means of Western immunoblot analysis. No correlation between NOS expression and inhibitory activity of CGRP was evident. The results suggest that the inhibitory action of CGRP in the mouse uterus is independent of the activity of these NOS isoforms.     

Palmitate increases nitric oxide synthase activity that is involved in palmitate-induced cell death in cardiomyocytes.

The objective of this study was to test the hypothesis that nitric oxide synthase (NOS) is subjected to regulatory control by palmitate, and that nitric oxide (NO) is operative in palmitate-induced cell death. Palmitate induced a significant ( p<0.05 ) concentration-dependent increase in NOS activity measured by the conversion of [(3)H]arginine to [3H]citrulline in embryonic chick cardiomyocytes. Cellular eNOS and iNOS, determined by immunocytochemistry, were increased by palmitate. Western blotting also showed that palmitate, 500 microM for 4h, significantly increased the amount of cellular of eNOS and iNOS by 36.2+/-6.5% ( p<0.001 ) and 38.4+/-14.4% ( p<0.05 ), respectively. The NOS inhibitor L-NAME significantly ( p<0.05 ) accentuated palmitate-induced cell death These data suggest that palmitate has a bifunctional effect on cell viability--in addition to loss of cell viability, palmitate stimulates NOS activity by inducing an increase in cellular eNOS and iNOS with the resultant NO production serving to protect cardiomyocytes from palmitate-induced cell death.     

Melatonin Decreases Nitric oxide Production, Inducible Nitric oxide Synthase Expression and Lipid Peroxidation Induced by Venezuelan Encephalitis Equine Virus in Neuroblastoma Cell Cultures

Increased expression of inducible nitric oxide synthase has been shown in murine Venezuelan equine encephalitis (VEE) virus infection. In this experimental model, melatonin (MTL) treatment has shown to be beneficial. The aim of this study was to determine the effect of VEE virus on the nitric oxide (NO) production and lipid peroxidation in neuroblastoma cell cultures, and to investigate the role of MTL during cell-virus interaction. Neuroblastoma cells were co-cultured with VEE virus and treated with MTL at doses ranging from 0 to 1.8 mM, for 6, 12, 24 and 48 h. NO and lipid peroxidation were measured in culture supernatants and in the cellular content by nitrite concentration and thiobarbituric acid assay, respectively. Expression of inducible nitric oxide synthase (iNOS) was determined by indirect immunofluorescence. Increased production of NO and lipid peroxidation products were found in supernatants and cellular contents of VEE virus treated cultures. Both NO and lipid peroxidation were decreased by MTL treatment in a time dependent manner. Increased iNOS expression was observed in VEE virus infected cultures that was reduced by MTL treatment. These results could be related to the beneficial role of MTL in the VEE experimental disease and address the possible therapeutic potential of the hormone in human VEE virus infection.     

NO and NOS isoforms in the development of apoptosis in renal ischemia/reperfusion

Nitric oxide (NO) and the expression of endothelial (eNOS) and inducible (iNOS) isoforms of nitric oxide synthase (NOS) are recognized as important mediators of physiological and pathological processes of renal ischemia/reperfusion (I/R) injury, but little is known about their role in apoptosis. The ability of the eNOS/NO system to regulate the iNOS/NO system and thus promote apoptosis was assessed during experimental renal I/R. Renal caspase-3 activity and the number of TUNEL-positive cells increased with I/R, but decreased when NOS/NO systems were blocked with L-NIO (eNOS), 1400W (iNOS), and N-nitro-l-arginine methyl ester (L-NAME; a nonselective NOS inhibitor). I/R increased renal eNOS and iNOS expression as well as NO production. The NO increase was eNOS- and iNOS-dependent. Blockage of NOS/NO systems with L-NIO or L-NAME also resulted in a lower renal expression of iNOS and iNOS mRNA; in contrast, eNOS expression was not affected by iNOS-specific blockage. In conclusion, two pathways define the role of NOS/NO systems in the development of apoptosis during experimental renal I/R: a direct route, through eNOS overexpression and NO production, and an indirect route, through expression/activation of the iNOS/NO system, induced by eNOS.     

一氧化氮合酶的遗传、生理研究进展

一氧化氮具有广泛的生理功能,哺乳动物体内的NO是由NO合酶(NOS)氧化L-精氨酸而合成的,合成后的NO迅速跨膜扩散释放,NO合成失调能介导多种疾病。催化NO生物合成的NOS有三种亚型:神经元型NOS(nNOS)、内皮型NOS(eNOS)和诱导型NOS(iNOS),目前,人的三型NOS已纯化并且已分子克隆成功,对一氧化氮合酶的遗传研究确认了NOS家族的基因结构和染色体定位。     

尾加压素对新生大鼠心肌细胞一氧化氮合成的影响

应用半定量逆转录－多聚酶链反应法,观察尾加压素（urotensin Ⅱ,UⅡ）对培养的新生SD大鼠心肌细胞内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS)mRNA表达的影响,并测定UⅡ对心肌细胞内一氧化氮合酶（nitric oxide synthase,NOS)活性和一氧化氮（nitric oxide,NO)释放的影响。结果显示：UⅡ抑制培养的新生大鼠心肌细胞eNOS mRNA表达、抑制NOS的活性及NO释放;0.1μmol/L浓度的UⅡ呈时间依赖性抑制心肌细胞NOS的活性及NO生成。上述实验结果提示UⅡ的心血管作用可能与NO合成系统有关。     

Extremely low frequency magnetic field induces hyperalgesia in mice modulated by nitric oxide synthesis

We investigated an effect of extremely low frequency magnetic field (ELF-MF, 60 Hz) on hyperalgesia using hot plate test. The level of nitric oxide (NO) and the expression of nitric oxide synthase (NOS) were measured to determine if ELF-MF is engaged in NO mediated pain mechanism. Additionally, the involvement of Ca2+-dependent NO pathway in ELF-MF induced hyperalgesia was evaluated by blocking Ca2+ sources with NMDA receptor antagonist and Ca2+ channel blocker. The exposure of mice to ELF-MF lowered pain threshold and elevated NO synthesis in brain and spinal cord. An NOS inhibitor blocked these effects of ELF-MF with attenuating the reduction of pain threshold and the rise of NO level in brain and spine by the exposure of ELF-MF. The hyperalgesic effects of ELF-MF were also blocked by a Ca2+ channel blocker, nimodipine, but not by a NMDA receptor antagonist, MK-801. The expression of Ca2+ -dependent nNOS and eNOS and Ca2+ -independent iNOS were not changed by ELF-MF. These results indicated that the exposure of ELF-MF might cause Ca2+ -dependent NOS activation, which then induces hyperalgesia with the increase in NO synthesis. In conclusion, ELF-MF may produce hyperalgesia by modulating NO synthesis via Ca2+ -dependent NOS.     

Phytoestrogen α-zearalanol antagonizes homocysteine-induced imbalance of nitric oxide/endothelin-1 and apoptosis in human umbilical vein endothelial cells

Although the issue of estrogen replacement therapy on cardiovascular health is debatable, it has presumable benefits for endothelial function in postmenopausal women. However, the fear of breast cancer has intimidated women contemplating estrogen treatment and limited its long-term application. An effective alternative remedy not associated with breast carcinoma is in serious demand. This study was designed to examine the effect of phytoestrogen alpha-zearalanol (alpha-ZAL) and 17beta-estradiol (E2) on nitric oxide (NO) and endothelin (ET)-1 levels, apoptosis, and apoptotic enzymes in human umbilical vein endothelial cells (HUVEC). HUVEC cells were challenged for 24 h with homocysteine (10-3 M), an independent risk factor for a variety of vascular diseases, in the presence of alpha-ZAL or E2 (10-9 to 10-6 M). Release of NO and ET-1 were measured with enzyme immunoassay. Apoptosis was evaluated by fluorescence-activated cell sorter analysis. Expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), Bax, and Bcl-2 were determined using Western blot. NOS activity was evaluated with 3H-arginine to 3H-citrulline conversion. Our results indicated that Hcy significantly reduced NO production, NOS activity, enhanced ET-1/NO ratio and apoptosis, upregulated iNOS, Bax, and downregulated eNOS, Bcl-2 expression. These effects were significantly attenuated by alpha-ZAL and E2. ZAL displayed a similar potency compared with E2 in antagonizing Hcy-induced effects. In summary, these results suggested that alpha-ZAL may effectively preserve Hcy-induced decrease in NO, increase in ET-1/NO ratio and apoptosis, which contributes to protective effects of phytoestrogens on endothelial function.     

Sphingomyelinase causes endothelium-dependent vasorelaxation through endothelial nitric oxide production without cytosolic Ca(2+) elevation

Neutral sphingomyelinase (N-SMase) elevated nitric oxide (NO) production without affecting intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells in situ on aortic valves, and induced prominent endothelium-dependent relaxation of coronary arteries, which was blocked by N(omega)-monomethyl-L-arginine, a NO synthase (NOS) inhibitor. N-SMase induced translocation of endothelial NOS (eNOS) from plasma membrane caveolae to intracellular region, eNOS phosphorylation on serine 1179, and an increase of ceramide level in endothelial cells. Membrane-permeable ceramide (C(8)-ceramide) mimicked the responses to N-SMase. We propose the involvement of N-SMase and ceramide in Ca(2+)-independent eNOS activation and NO production in endothelial cells in situ, linking to endothelium-dependent vasorelaxation.     

Differential activation of nitric-oxide synthase isozymes by calmodulin-troponin C chimeras

The interactions of neuronal nitric-oxide synthase (nNOS) with calmodulin (CaM) and mutant forms of CaM, including CaM-troponin C chimeras, have been previously reported, but there has been no comparable investigation of CaM interactions with the other constitutively expressed NOS (cNOS), endothelial NOS (eNOS), or the inducible isoform (iNOS). The present study was designed to evaluate the role of the four CaM EF hands in the activation of eNOS and iNOS. To assess the role of CaM regions on aspects of enzymatic function, three distinct activities associated with NOS were measured: NADPH oxidation, cytochrome c reduction, and nitric oxide (*NO) generation as assessed by the oxyhemoglobin capture assay. CaM activates the cNOS enzymes by a mechanism other than stimulating electron transfer into the oxygenase domain. Interactions with the reductase moiety are dominant in cNOS activation, and EF hand 1 is critical for activation of both nNOS and eNOS. Although the activation patterns for nNOS and eNOS are clearly related, effects of the chimeras on all the reactions are not equivalent. We propose that cytochrome c reduction is a measure of the release of the FMN domain from the reductase complex. In contrast, cytochrome c reduction by iNOS is readily activated by each of the chimeras examined here and may be constitutive. Each of the chimeras were co-expressed with the human iNOS enzyme in Escherichia coli and subsequently purified. Domains 2 and 3 of CaM contain important elements required for the Ca2+/CaM independence of *NO production by the iNOS enzyme. The disparity between cytochrome c reduction and *NO production at low calcium can be attributed to poor association of heme and FMN domains when the bound CaM constructs are depleted of Ca2+. In general cNOSs are much more difficult to activate than iNOS, which can be attributed to their extra sequence elements, which are adjacent to the CaM-binding site and associated with CaM control.     

Normalization of nitric oxide flux improves physiological parameters of porcine kidneys maintained on pulsatile perfusion.

Early endothelial damage and resultant reduction in the beneficial production of nitric oxide (NO) derived from the endothelial NO synthase (eNOS) are phenomena associated with the functional degradation of transplanted kidneys. In contrast, the inflammation characteristic of kidney preservation leads to the later, detrimental expression of the inducible NO synthase (iNOS). We reasoned that provision of low-level NO (to compensate for lack of eNOS) using the chemical NO donor S-nitrosoglutathione (GSNO), along with an iNOS inhibitor (N-omega iminoethyl-L-lysine; L-NIL), might "normalize" NO levels and therefore be beneficial in maintenance of flow. Non-heartbeating donor porcine kidneys were subjected to 30-45 min warm ischemic time and stored from 3 to 30 h, simulating the time required for national sharing. The kidneys were then machine preserved with Belzer MPS (BMP) at a set systolic pressure of 40 mmHg. Eight kidneys were perfused for 5h with BMP only (Group 1 control), 8 kidneys with BMP+GSNO only (Group 2), and 8 kidneys with BMP+GSNO+L-NIL (Group 3). Lower vascular resistance (VR) is a predictor of improved end-organ function. Both Group 2 and 3 kidneys demonstrated statistically significant reduction in VR as compared to Group 1 kidneys, with Group 3 kidneys demonstrating a greater drop in VR than Group 2. Reduced oxygen saturation suggests a higher metabolic rate. Only Group 3 had lower oxygen saturation as compared to Group 1. Increased Ca2+ concentration in the perfusate is a predictor of worse end-organ function. Group 2, but not Group 3, had a higher perfusate Ca2+ concentration than Group 1. The combination of suppression of harmful amounts of NO, while supplying a constant low-level amount of NO, may improve pulsatile kidney preservation.