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Nitric Oxide Production by Primary Liver Cells Isolated from Amino Acid Diet–Fed Rats
[45]
diet-induced NO production in rat primary liver cells
535
[45] Nitric Oxide Production by Primary Liver Cells Isolated from Amino Acid Diet–Fed Rats By YASHIGE KOTAKE, HIDEKI KISHIDA, DAI NAKAE , and ROBERT A. FLOYD Abstract
Primary mixed liver cells were isolated from rats that had been fed an amino acid (AA) diet in which natural protein was replaced with a defined mixture of pure AAs. Nitric oxide (NO) production from these cells in vitro was monitored using a nitric oxide (NO)–selective fluorescent probe, diaminofluorescein, followed by flow cytometric analysis. High levels of NO fluorescence were seen in approximately half of liver cells isolated from rats fed an AA diet for 1–7 days, whereas there was baseline fluorescence in cells obtained from regular diet–fed rats. The apparent size of NO‐producing cells was smaller than those not producing NO. The production of NO was inhibited when rats were treated with either inducible NO synthase (iNOS)– or endothelial NOS–specific inhibitor, and an inhibitor for iNOS induction during AA diet feeding. L‐Arginine or L‐glutamine (material for L‐arginine biosynthesis) enriched diet showed the same NO augmentation as in AA diet. It is speculated that a high content of free L‐arginine in AA diet may have caused enhanced NO production.
Introduction
Amino acid (AA) diet is a diet that contains a mixture of L‐amino acids in a defined ratio, as a substitute for natural protein (Rogers et al., 1965). Although long‐term consumption of AA diet in rats caused no major deleterious effects, when combined with a choline deficient diet, it significantly enhanced hepatocellular carcinoma formation (Nakae, 1999). Because AA diet is rich in free L‐arginine, a substrate for nitric oxide (NO) synthase, NO production in the liver may be modulated by AA diet consumption. The objective of this study was to determine NO production in isolated primary liver cells in rats that had been fed AA diet for various durations. NO formation from isolated liver cells was determined by labeling NO with NO‐specific fluorescence‐labeling agent, followed by flow cytometric analysis.
METHODS IN ENZYMOLOGY, VOL. 396 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)96045-X
536
cell biology and physiology
[45]
Feeding Animals with Specific Diets Rats were treated strictly following the animal‐use protocol approved by the institutional laboratory animal care and use committee in the Oklahoma Medical Research Foundation. Male Wistar rats (8–10 weeks old) were obtained from Charles River Laboratory (Indianapolis, IN). L‐Amino acid diet, synthetic defined, was purchased from two vendors, ICN Biomedicals, Inc. (Irvine, CA), and Dyets, Inc. (Bethlehem, PA). The composition of L‐amino acid in the two diets is similar; for example, each contains 1.3% L‐arginine hydrochloride and 2.9% L‐glutamic acid (starting material for L‐arginine biosynthesis). The contents of L‐arginine and L‐glutamine in these AA diets are the same as choline‐deficient L‐amino acid–defined (CDAA) diet, which was used by Nakae (1999) to clarify issues regarding diet in a choline deficiency hepatocarcinogenesis model. Basal diet was a Purina 5005 rodent chow (Ralston Purina, St. Louis, MO). Small batches of L‐arginine or L‐glutamate–enriched diet were prepared in our laboratory by mixing the powdered basal diet with 10% by weight of the amino acid obtained from Sigma Chemical Co. (St. Louis, MO). Rats were fasted overnight (15 h), and then the specific diet was fed for 1 (24 h), 3, 7, 14, and 30 days. Basal diet control animals were also fasted overnight before continuing the feeding. Nitric oxide synthase (NOS) inhibitor, either N‐nitro‐L‐arginine or aminoguanidine (Sigma, 150 mg/kg), was intraperitoneally administered twice, 12 h and 1 h before cell isolation. The inhibitor of inducible NOS (iNOS) induction ‐phenyl‐tert‐butylnitrone (PBN) (Sigma) was administered in the diet by adding at the rate of 0.3% w/w to the powdered AA diet. Primary Cell Isolation Primary liver cells were isolated following the method previously reported (Alpini et al., 1994; Jeejeebhoy et al., 1975). After rats were fed the diet for a specified period, they were anesthetized with isoflurane (Abbott Laboratories, Chicago, IL) using 95% oxygen–5% carbon dioxide as a carrier gas for a vaporizer (Surgevet/Anesco, Waukesha, WI). Under anesthesia, the abdomen was opened with a middle incision and the portal vein cannulated with polyethylene tubing (PE 205, Intramedic Becton‐Dickenson). To remove blood from the liver, calcium‐free Hanks’ HEPES buffer was perfused into the cannulae with a peristaltic pump (Gilson Optima 2000, PerkinElmer, Boston, MA) at the flow rate of 30 ml/min for 10 min. Perfusate was allowed to drain from the opened inferior artery. The temperature of incoming perfusate was carefully maintained at 37 1 at the liver inlet by controlling the perfusate‐reservoir temperature. The perfusate was switched to the same buffer containing 0.05% collagenase B
[45]
diet-induced NO production in rat primary liver cells
537
(Sigma) and allowed to perfuse with the same flow rate for 30 min. Well‐ digested liver became soft and flat and was readily dispersible in buffer with a plastic spatula. Cells were gently suspended in the buffer, and connecting tissues and cell debris were removed with a 220 nylon mesh filter. Cells were washed in buffer three times, and viability was determined using the Trypan blue exclusion method. A majority of liver cells isolated with the collagenase perfusion method is known to be hepatocytes (>90%) (Alpini et al., 1994). The average size of hepatocyte (25 m) is about 2.5 times larger than other liver cells, such as Kupffer cells and endothelial cells (Alpini et al., 1994). Forward‐ and side‐scattering dot‐histograms obtained in flow cytometry indicated homogeneous size distribution (Fig. 1D), suggesting that the cell preparation was mainly hepatocytes. Flow Cytometry For flow cytometric analysis of NO production, isolated cells were suspended in fresh William’s carbonate buffer (Sigma), and the NO‐labeling agent diaminofluorescein acetate (DAFA, Sigma) (Itoh et al., 2000; Nagano et al., 2002) was added to 20 M and incubated for 20 min at 37 , and then kept on ice before subjecting them to analysis. Histograms were obtained using a FACScan flow cytometer (BD Biosciences, San Jose, CA), and fluorescence intensities were analyzed with BD CellQuest for greater than 10,000 gated cells. Flow cytometry histograms indicated that liver cells that had been isolated from rats fed an AA diet for 1 day (or longer) increased NO production as compared to cells obtained from basal diet (regular rodent chow)–fed rats (Fig. 1). A summary of these experiments is as follows: 1. Approximately 50% of liver primary cells obtained from rats that had been fed AA diet 1 day or more produced DAFA‐stainable NO (Fig. 1A). The average fluorescence intensity per cell in AA diet–fed rats was approximately four times higher than those on basal diet. NO production level has little dependence on the duration of AA diet feeding period up to 30 days (Fig. 3). The scattering histograms (Fig. 1B) indicate that the size of NO‐producing cells was distinctively smaller than those not producing NO (Fig. 1B). Functional heterogeneity of hepatocytes from different lobular zone has been demonstrated (Alpini et al., 1994). Periportal hepatocytes have smaller size, higher oxygen tension, and higher amino acid catabolism than pericentral hepatocytes. It is possible that NO‐producing cells are distributed mainly in the periportal region. However, it has been demonstrated that the size of hepatocytes is readily influenced by administered drugs, such as pentobarbital (Willson et al., 1984), so it is also possible that NO production could have caused the size change.
538
cell biology and physiology
[45]
FIG. 1. Rats were fed with a defined L‐amino acid (AA) diet or a basal diet for 3 days, and primary liver cells were isolated using in situ collagenase perfusion. Intact cells were then incubated with the nitric oxide (NO)–labeling compound diaminofluorescein acetate (DAFA) and subjected to flow cytometric analysis. (A) Flow cytometry histogram of liver cells isolated from a rat fed AA diet for 3 days, displaying NO fluorescence (log scale) versus counts (approximately equal to cell numbers). Lightly shaded peak is from cell population that produces more NO than that dark‐shaded peak. (B) Dot plot displays forward scattering (FSC) height versus side scattering (SSC) height. Each dot corresponds to individual cells. Dots with light gray are from those NO‐producing cells [i.e., lightly shaded peak in histogram (A)]. (C) Histogram obtained from liver cells isolated from basal diet–fed rats. (D) Dot plot for FSC versus SSC for liver cells isolated from basal diet–fed rats.
2. L‐Arginine– or L‐glutamine–enriched diet (10 weight %) caused the appearance of NO‐producing cell population (Fig. 2), but such NO‐ producing cells disappeared when the diet was switched from the enriched diet or an AA diet back to the basal diet for 1 day (Figs. 2 and 3).
[45]
diet-induced NO production in rat primary liver cells
539
FIG. 2. Flow cytometry histograms obtained from liver cells isolated from rats fed with various diets and inhibitors. (A) Fed amino acid (AA) diet for 3 days; (B) fed basal diet; (C) fed AA diet for 3 days, and on the third day of feeding nitric oxide synthase (NOS) inhibitor N‐nitroarginine (NNA) was administered i.p. 6 h and 30 min before cell isolation; (D) fed AA diet for 3 days, and on the third day of feeding NOS inhibitor, aminoguanidine (AG) was administered i.p. 6 h and 30 min before cell isolation; (E) fed AA diet supplemented with 0.3% a‐phenyl‐tert‐butylnitrone (PBN) for 3 days; (F) fed basal diet supplemented with 10% L‐arginine for 3 days; (G) fed basal diet supplemented with 10% Lglutamine for 3 days; (H) fed AA diet for 3 days then switched to basal diet feeding for 1 day.
3. The administration of NOS inhibitors, N‐nitro‐L‐arginine (NNA) or aminoguanidine (AG) during AA diet feeding abolished NO production in these cells, indicating that NO was produced through L‐arginine–dependent NOS pathways. NNA is considered an eNOS‐specific inhibitor (specificity:
540
cell biology and physiology
[45]
FIG. 3. Average fluorescence intensity per cell for liver cells isolated from rats on various diet regimens (three to five rats for each treatment). Rats were fed basal diet (Basal), or amino acid (AA) diet for 1 day (1d), 3 days (3d), 1 week (1wk), 2 weeks (2wk), and 4 weeks (4wk). Rats were fed AA diet for 3 days, and on the third day of feeding, nitric oxide synthase (NOS) inhibitor N‐nitroarginine (NNA) or aminoguanidine (AG) was administered i.p. 6 h and 30 min before hepatocyte isolation, or fed AA diet supplemented with 0.3% a‐ phenyl‐tert‐butylnitrone (PBN) for 3 days. Rats were fed 10% L‐arginine (L‐Arg)– or L‐ glutamine (L‐Glu)–supplemented basal diet for 3 days. Liver cells were isolated from rats fed AA diet for 3 days then switched to basal diet feeding for 1 day (AA!B).
eNOS/iNOS ¼ 30/1) (Salerno et al., 1997), and AG an iNOS‐specific inhibitor (specificity: eNOS/iNOS ¼ 1/30) (Southan et al., 1996). In the present experiments, both NNA and AG inhibited NO formation to a similar extent (Figs. 2 and 3), suggesting that both eNOS and iNOS are involved in the NO production. Co‐feeding of an inhibitor of iNOS induction PBN (0.3% w/w, 150 mg/kg/rat/day) (Kotake et al., 1998) also suppressed NO formation (Figs. 2 and 3). We speculate that initial NO production through eNOS‐mediated iNOS induction is the major source of NO in primary liver cells.
Acknowledgments We thank Dr. Lester A. Reinke and Mr. Danny Moore, University of Oklahoma Health Sciences Center, for the assistance in primary cell isolation. Support of this work was provided by National Institutes of Health grant CA82506.
[46]
NO-dependent dilation in humans
541
References Alpini, G., Phillips, J. O., Vroman, B., and LaRusso, N. F. (1994). Recent advances in the isolation of liver cells. Hepatology 20, 494–514. Itoh, Y., Ma, F. H., Hoshi, H., Oka, M., Noda, K., Ukai, Y., Kojima, H., Nagano, T., and Toda, N. (2000). Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry. Anal. Biochem. 287, 203–209. Jeejeebhoy, K. N., Ho, J., Greenberg, G. R., Philips, M. J., Bruce‐Robertson, A., and Sodtke, U. (1975). Albumin, fibrinogen and transferrin synthesis in isolated rat hepatocyte suspensions. A model for the study of plasma protein synthesis. Biochem. J. 146, 141–155. Kotake, Y., Sang, H., Miyajima, T., and Wallis, G. L. (1998). Inhibition of NF‐kappaB, iNOS mRNA, COX2 mRNA, and COX catalytic activity by phenyl‐N‐tert‐butylnitrone (PBN). Biochim. Biophys. Acta 1448, 77–84. Nagano, T., and Yoshimura, T. (2002). Bioimaging of nitric oxide. Chem. Rev. 102, 1235–1244. Nakae, D. (1999). Endogenous liver carcinogenesis in the rat. Pathol. Int. 49, 1028–1042. Rogers, Q. R., and Harper, A. E. (1965). Amino acid diets and maximal growth in the rat. J. Nutr. 87, 267–273. Salerno, J. C., Martasek, P., Williams, R. F., and Masters, B. S. (1997). Substrate and substrate analog binding to endothelial nitric oxide synthase: Electron paramagnetic resonance as an isoform‐specific probe of the binding mode of substrate analogs. Biochemistry 36, 11821–11828. Southan, G. J., and Szabo, C. (1996). Selective pharmacological inhibition of distinct nitric oxide synthase isoforms. Biochem. Pharmacol. 51, 383–394. Willson, R. A., Wormsley, S. B., and Muller‐Eberhard, U. (1984). A comparison of hepatocyte size distribution in untreated and phenobarbital‐treated rats as assessed by flow cytometry. Dig. Dis. Sci. 29, 753–757.
[46] Update on Nitric Oxide–Dependent Vasodilation in Human Subjects By CRAIG J. MCMACKIN and JOSEPH A. VITA Abstract
There currently is great interest in translating findings about the importance of nitric oxide (NO) in vascular biology to the clinical arena. The bioactivity of endothelium‐derived NO can readily be assessed in human subjects as vasodilation of conduit arteries or increased flow, which reflects vasodilation of resistance vessels. This chapter provides an update on the available noninvasive methodology to assess endothelium‐dependent vasodilation in human subjects.
METHODS IN ENZYMOLOGY, VOL. 396 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)96046-1
diet-induced NO production in rat primary liver cells
535
[45] Nitric Oxide Production by Primary Liver Cells Isolated from Amino Acid Diet–Fed Rats By YASHIGE KOTAKE, HIDEKI KISHIDA, DAI NAKAE , and ROBERT A. FLOYD Abstract
Primary mixed liver cells were isolated from rats that had been fed an amino acid (AA) diet in which natural protein was replaced with a defined mixture of pure AAs. Nitric oxide (NO) production from these cells in vitro was monitored using a nitric oxide (NO)–selective fluorescent probe, diaminofluorescein, followed by flow cytometric analysis. High levels of NO fluorescence were seen in approximately half of liver cells isolated from rats fed an AA diet for 1–7 days, whereas there was baseline fluorescence in cells obtained from regular diet–fed rats. The apparent size of NO‐producing cells was smaller than those not producing NO. The production of NO was inhibited when rats were treated with either inducible NO synthase (iNOS)– or endothelial NOS–specific inhibitor, and an inhibitor for iNOS induction during AA diet feeding. L‐Arginine or L‐glutamine (material for L‐arginine biosynthesis) enriched diet showed the same NO augmentation as in AA diet. It is speculated that a high content of free L‐arginine in AA diet may have caused enhanced NO production.
Introduction
Amino acid (AA) diet is a diet that contains a mixture of L‐amino acids in a defined ratio, as a substitute for natural protein (Rogers et al., 1965). Although long‐term consumption of AA diet in rats caused no major deleterious effects, when combined with a choline deficient diet, it significantly enhanced hepatocellular carcinoma formation (Nakae, 1999). Because AA diet is rich in free L‐arginine, a substrate for nitric oxide (NO) synthase, NO production in the liver may be modulated by AA diet consumption. The objective of this study was to determine NO production in isolated primary liver cells in rats that had been fed AA diet for various durations. NO formation from isolated liver cells was determined by labeling NO with NO‐specific fluorescence‐labeling agent, followed by flow cytometric analysis.
METHODS IN ENZYMOLOGY, VOL. 396 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)96045-X
536
cell biology and physiology
[45]
Feeding Animals with Specific Diets Rats were treated strictly following the animal‐use protocol approved by the institutional laboratory animal care and use committee in the Oklahoma Medical Research Foundation. Male Wistar rats (8–10 weeks old) were obtained from Charles River Laboratory (Indianapolis, IN). L‐Amino acid diet, synthetic defined, was purchased from two vendors, ICN Biomedicals, Inc. (Irvine, CA), and Dyets, Inc. (Bethlehem, PA). The composition of L‐amino acid in the two diets is similar; for example, each contains 1.3% L‐arginine hydrochloride and 2.9% L‐glutamic acid (starting material for L‐arginine biosynthesis). The contents of L‐arginine and L‐glutamine in these AA diets are the same as choline‐deficient L‐amino acid–defined (CDAA) diet, which was used by Nakae (1999) to clarify issues regarding diet in a choline deficiency hepatocarcinogenesis model. Basal diet was a Purina 5005 rodent chow (Ralston Purina, St. Louis, MO). Small batches of L‐arginine or L‐glutamate–enriched diet were prepared in our laboratory by mixing the powdered basal diet with 10% by weight of the amino acid obtained from Sigma Chemical Co. (St. Louis, MO). Rats were fasted overnight (15 h), and then the specific diet was fed for 1 (24 h), 3, 7, 14, and 30 days. Basal diet control animals were also fasted overnight before continuing the feeding. Nitric oxide synthase (NOS) inhibitor, either N‐nitro‐L‐arginine or aminoguanidine (Sigma, 150 mg/kg), was intraperitoneally administered twice, 12 h and 1 h before cell isolation. The inhibitor of inducible NOS (iNOS) induction ‐phenyl‐tert‐butylnitrone (PBN) (Sigma) was administered in the diet by adding at the rate of 0.3% w/w to the powdered AA diet. Primary Cell Isolation Primary liver cells were isolated following the method previously reported (Alpini et al., 1994; Jeejeebhoy et al., 1975). After rats were fed the diet for a specified period, they were anesthetized with isoflurane (Abbott Laboratories, Chicago, IL) using 95% oxygen–5% carbon dioxide as a carrier gas for a vaporizer (Surgevet/Anesco, Waukesha, WI). Under anesthesia, the abdomen was opened with a middle incision and the portal vein cannulated with polyethylene tubing (PE 205, Intramedic Becton‐Dickenson). To remove blood from the liver, calcium‐free Hanks’ HEPES buffer was perfused into the cannulae with a peristaltic pump (Gilson Optima 2000, PerkinElmer, Boston, MA) at the flow rate of 30 ml/min for 10 min. Perfusate was allowed to drain from the opened inferior artery. The temperature of incoming perfusate was carefully maintained at 37 1 at the liver inlet by controlling the perfusate‐reservoir temperature. The perfusate was switched to the same buffer containing 0.05% collagenase B
[45]
diet-induced NO production in rat primary liver cells
537
(Sigma) and allowed to perfuse with the same flow rate for 30 min. Well‐ digested liver became soft and flat and was readily dispersible in buffer with a plastic spatula. Cells were gently suspended in the buffer, and connecting tissues and cell debris were removed with a 220 nylon mesh filter. Cells were washed in buffer three times, and viability was determined using the Trypan blue exclusion method. A majority of liver cells isolated with the collagenase perfusion method is known to be hepatocytes (>90%) (Alpini et al., 1994). The average size of hepatocyte (25 m) is about 2.5 times larger than other liver cells, such as Kupffer cells and endothelial cells (Alpini et al., 1994). Forward‐ and side‐scattering dot‐histograms obtained in flow cytometry indicated homogeneous size distribution (Fig. 1D), suggesting that the cell preparation was mainly hepatocytes. Flow Cytometry For flow cytometric analysis of NO production, isolated cells were suspended in fresh William’s carbonate buffer (Sigma), and the NO‐labeling agent diaminofluorescein acetate (DAFA, Sigma) (Itoh et al., 2000; Nagano et al., 2002) was added to 20 M and incubated for 20 min at 37 , and then kept on ice before subjecting them to analysis. Histograms were obtained using a FACScan flow cytometer (BD Biosciences, San Jose, CA), and fluorescence intensities were analyzed with BD CellQuest for greater than 10,000 gated cells. Flow cytometry histograms indicated that liver cells that had been isolated from rats fed an AA diet for 1 day (or longer) increased NO production as compared to cells obtained from basal diet (regular rodent chow)–fed rats (Fig. 1). A summary of these experiments is as follows: 1. Approximately 50% of liver primary cells obtained from rats that had been fed AA diet 1 day or more produced DAFA‐stainable NO (Fig. 1A). The average fluorescence intensity per cell in AA diet–fed rats was approximately four times higher than those on basal diet. NO production level has little dependence on the duration of AA diet feeding period up to 30 days (Fig. 3). The scattering histograms (Fig. 1B) indicate that the size of NO‐producing cells was distinctively smaller than those not producing NO (Fig. 1B). Functional heterogeneity of hepatocytes from different lobular zone has been demonstrated (Alpini et al., 1994). Periportal hepatocytes have smaller size, higher oxygen tension, and higher amino acid catabolism than pericentral hepatocytes. It is possible that NO‐producing cells are distributed mainly in the periportal region. However, it has been demonstrated that the size of hepatocytes is readily influenced by administered drugs, such as pentobarbital (Willson et al., 1984), so it is also possible that NO production could have caused the size change.
538
cell biology and physiology
[45]
FIG. 1. Rats were fed with a defined L‐amino acid (AA) diet or a basal diet for 3 days, and primary liver cells were isolated using in situ collagenase perfusion. Intact cells were then incubated with the nitric oxide (NO)–labeling compound diaminofluorescein acetate (DAFA) and subjected to flow cytometric analysis. (A) Flow cytometry histogram of liver cells isolated from a rat fed AA diet for 3 days, displaying NO fluorescence (log scale) versus counts (approximately equal to cell numbers). Lightly shaded peak is from cell population that produces more NO than that dark‐shaded peak. (B) Dot plot displays forward scattering (FSC) height versus side scattering (SSC) height. Each dot corresponds to individual cells. Dots with light gray are from those NO‐producing cells [i.e., lightly shaded peak in histogram (A)]. (C) Histogram obtained from liver cells isolated from basal diet–fed rats. (D) Dot plot for FSC versus SSC for liver cells isolated from basal diet–fed rats.
2. L‐Arginine– or L‐glutamine–enriched diet (10 weight %) caused the appearance of NO‐producing cell population (Fig. 2), but such NO‐ producing cells disappeared when the diet was switched from the enriched diet or an AA diet back to the basal diet for 1 day (Figs. 2 and 3).
[45]
diet-induced NO production in rat primary liver cells
539
FIG. 2. Flow cytometry histograms obtained from liver cells isolated from rats fed with various diets and inhibitors. (A) Fed amino acid (AA) diet for 3 days; (B) fed basal diet; (C) fed AA diet for 3 days, and on the third day of feeding nitric oxide synthase (NOS) inhibitor N‐nitroarginine (NNA) was administered i.p. 6 h and 30 min before cell isolation; (D) fed AA diet for 3 days, and on the third day of feeding NOS inhibitor, aminoguanidine (AG) was administered i.p. 6 h and 30 min before cell isolation; (E) fed AA diet supplemented with 0.3% a‐phenyl‐tert‐butylnitrone (PBN) for 3 days; (F) fed basal diet supplemented with 10% L‐arginine for 3 days; (G) fed basal diet supplemented with 10% Lglutamine for 3 days; (H) fed AA diet for 3 days then switched to basal diet feeding for 1 day.
3. The administration of NOS inhibitors, N‐nitro‐L‐arginine (NNA) or aminoguanidine (AG) during AA diet feeding abolished NO production in these cells, indicating that NO was produced through L‐arginine–dependent NOS pathways. NNA is considered an eNOS‐specific inhibitor (specificity:
540
cell biology and physiology
[45]
FIG. 3. Average fluorescence intensity per cell for liver cells isolated from rats on various diet regimens (three to five rats for each treatment). Rats were fed basal diet (Basal), or amino acid (AA) diet for 1 day (1d), 3 days (3d), 1 week (1wk), 2 weeks (2wk), and 4 weeks (4wk). Rats were fed AA diet for 3 days, and on the third day of feeding, nitric oxide synthase (NOS) inhibitor N‐nitroarginine (NNA) or aminoguanidine (AG) was administered i.p. 6 h and 30 min before hepatocyte isolation, or fed AA diet supplemented with 0.3% a‐ phenyl‐tert‐butylnitrone (PBN) for 3 days. Rats were fed 10% L‐arginine (L‐Arg)– or L‐ glutamine (L‐Glu)–supplemented basal diet for 3 days. Liver cells were isolated from rats fed AA diet for 3 days then switched to basal diet feeding for 1 day (AA!B).
eNOS/iNOS ¼ 30/1) (Salerno et al., 1997), and AG an iNOS‐specific inhibitor (specificity: eNOS/iNOS ¼ 1/30) (Southan et al., 1996). In the present experiments, both NNA and AG inhibited NO formation to a similar extent (Figs. 2 and 3), suggesting that both eNOS and iNOS are involved in the NO production. Co‐feeding of an inhibitor of iNOS induction PBN (0.3% w/w, 150 mg/kg/rat/day) (Kotake et al., 1998) also suppressed NO formation (Figs. 2 and 3). We speculate that initial NO production through eNOS‐mediated iNOS induction is the major source of NO in primary liver cells.
Acknowledgments We thank Dr. Lester A. Reinke and Mr. Danny Moore, University of Oklahoma Health Sciences Center, for the assistance in primary cell isolation. Support of this work was provided by National Institutes of Health grant CA82506.
[46]
NO-dependent dilation in humans
541
References Alpini, G., Phillips, J. O., Vroman, B., and LaRusso, N. F. (1994). Recent advances in the isolation of liver cells. Hepatology 20, 494–514. Itoh, Y., Ma, F. H., Hoshi, H., Oka, M., Noda, K., Ukai, Y., Kojima, H., Nagano, T., and Toda, N. (2000). Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry. Anal. Biochem. 287, 203–209. Jeejeebhoy, K. N., Ho, J., Greenberg, G. R., Philips, M. J., Bruce‐Robertson, A., and Sodtke, U. (1975). Albumin, fibrinogen and transferrin synthesis in isolated rat hepatocyte suspensions. A model for the study of plasma protein synthesis. Biochem. J. 146, 141–155. Kotake, Y., Sang, H., Miyajima, T., and Wallis, G. L. (1998). Inhibition of NF‐kappaB, iNOS mRNA, COX2 mRNA, and COX catalytic activity by phenyl‐N‐tert‐butylnitrone (PBN). Biochim. Biophys. Acta 1448, 77–84. Nagano, T., and Yoshimura, T. (2002). Bioimaging of nitric oxide. Chem. Rev. 102, 1235–1244. Nakae, D. (1999). Endogenous liver carcinogenesis in the rat. Pathol. Int. 49, 1028–1042. Rogers, Q. R., and Harper, A. E. (1965). Amino acid diets and maximal growth in the rat. J. Nutr. 87, 267–273. Salerno, J. C., Martasek, P., Williams, R. F., and Masters, B. S. (1997). Substrate and substrate analog binding to endothelial nitric oxide synthase: Electron paramagnetic resonance as an isoform‐specific probe of the binding mode of substrate analogs. Biochemistry 36, 11821–11828. Southan, G. J., and Szabo, C. (1996). Selective pharmacological inhibition of distinct nitric oxide synthase isoforms. Biochem. Pharmacol. 51, 383–394. Willson, R. A., Wormsley, S. B., and Muller‐Eberhard, U. (1984). A comparison of hepatocyte size distribution in untreated and phenobarbital‐treated rats as assessed by flow cytometry. Dig. Dis. Sci. 29, 753–757.
[46] Update on Nitric Oxide–Dependent Vasodilation in Human Subjects By CRAIG J. MCMACKIN and JOSEPH A. VITA Abstract
There currently is great interest in translating findings about the importance of nitric oxide (NO) in vascular biology to the clinical arena. The bioactivity of endothelium‐derived NO can readily be assessed in human subjects as vasodilation of conduit arteries or increased flow, which reflects vasodilation of resistance vessels. This chapter provides an update on the available noninvasive methodology to assess endothelium‐dependent vasodilation in human subjects.
METHODS IN ENZYMOLOGY, VOL. 396 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)96046-1