Quantifying the l -arginine paradox in vivo

Microvascular Research 71 (2006) 48 – 54 www.elsevier.com/locate/ymvre

Quantifying the l-arginine paradox in vivo Nina Vukosavljevic a, Dov Jaron a, Kenneth A. Barbee a, Donald G. Buerk b,* b

a School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA 19104, USA Department of Physiology, University of Pennsylvania, A207 Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104-6086, USA

Received 8 June 2005; revised 6 September 2005; accepted 16 October 2005 Available online 28 November 2005

Abstract NO and PO2 microelectrodes were used to quantify the effects of increased availability of l-arginine in an exteriorized rat mesentery and small intestine microcirculatory preparation in n = 16 rats. During short periods of elevated l-arginine added to the superfusion bath, transient changes in perivascular NO or PO2 were measured at 171 perivascular sites near intestinal arterioles and venules, simultaneously with tissue perfusion using laser Doppler flowmetry (LDF). Excess l-arginine increased perivascular NO over twofold, by 411 T 42 nM above the baseline of 329 T 30 nM (P < 0.0001), and increased tissue perfusion by 35.5 T 7.5% (P < 0.0001). No difference between arterioles and venules was observed in the magnitude or time course of the NO responses. Both increases and decreases in perivascular PO2 were observed after excess l-arginine, with a similar increase in tissue perfusion by 42.0 T 12.3% (P < 0.0001). Our NO measurements confirm that increased bioavailability of l-arginine causes a significant increase in NO production throughout the microcirculation of this preparation, with increased tissue perfusion, and provides direct in vivo evidence for the l-arginine paradox. D 2005 Elsevier Inc. All rights reserved. Keywords: eNOS; l-Arginine; Laser Doppler flowmetry; Nitric oxide; PO2

Introduction Nitric oxide (NO) produced by all nitric oxide synthases (NOS) requires oxygen (O2) and the amino acid l-arginine, along with other essential co-factors (Palmer et al., 1988; Moncada and Higgsm, 1993). The general consensus view is that intracellular l-arginine, reported to range between 0.1 and 2 mM (Xu et al., 2004; McDonald et al., 1997; Bogle et al., 1996), is available to eNOS in endothelial cells far in excess of enzymatic requirements. The K m for l-arginine for different NO isoforms is reported to range between 1 and 32 AM (Palmer et al., 1988; Bogle et al., 1996; Rodrı´guez-Crespo et al., 1996). However, indirect evidence from several in vitro studies (Taylor and Poston, 1994; Lee et al., 2004) suggests that supplemental l-arginine causes an increase in NO production. This unexpected effect with excess substrate is known as the ‘‘l-arginine Paradox’’ (Kurz and Harrison, 1997). The K m for O2 varies with different types of NOS (Buerk, 2001), but the partial pressure (PO2) in blood near endothelial cells also generally thought to be well above the K m for eNOS (K m < 10 Torr). * Corresponding author. Fax: +1 215 573 5851. E-mail address: [email protected] (D.G. Buerk). 0026-2862/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2005.10.006

Because of the importance of NO in vasodilation, neuronal signaling, platelet aggregation, and many other physiological functions, there has been considerable research effort to better understand mechanisms responsible for regulation and activation of NOS. Dietary supplementation of l-arginine as a therapeutic measure against endothelial dysfunction appears to have positive benefits for cardiovascular disease (Lerman et al., 1998; Blum et al., 1999; Maxwell et al., 2000). However, specific mechanisms responsible for the ‘‘l-arginine paradox’’ have not been conclusively identified. In this study, we investigated whether the l-arginine paradox exists in vivo from direct NO, PO2, and tissue perfusion (blood flow) measurements. We verified that increased availability of larginine increased perivascular NO around venules and arterioles in the microcirculation, with similar increases regardless of size. Materials and methods Male Sprague – Dawley rats (n = 16) with weights ranging between 200 and 300 g were anesthetized with sodium pentobarbital (30 – 50 mg/kg initial dose), receiving supplemental anesthesia as needed during the experiment. A section of the small intestines and mesentery was exteriorized, placed in a shallow organ dish, and continuously bathed in warm, room air oxygenated phosphate buffered saline. A schematic drawing and video image of the

N. Vukosavljevic et al. / Microvascular Research 71 (2006) 48 – 54 experimental setup are shown in Fig. 1. The total fluid volume in the bath was 50 ml, and was continuously replaced at a rate of approximately 4 ml/ min using a pump. Temperature was maintained at 37-C with a circulating water bath. In order to minimize movement, a stabilizing stainless steel ring (D = 12 mm) was placed around the area under observation. A small bolus (1 ml in ¨10 s) of l-arginine (100 mM) was delivered topically in the solution covering the preparation through a small tube near the measurement site. The l-arginine solution was mixed in the same room air oxygenated phosphate buffered saline (pH 7.35) that was used to superfuse the preparation. Teflon tubing to deliver l-arginine was attached to the laser Doppler probe a few mm above the tissue. Blood flow changes in the intestinal tissue supplied by the microvascular network under investigation were recorded using laser Doppler flowmetry (LDF). A single channel instrument (Model BLF 21, Transonic Systems, Inc., Ithaca, N.Y.) with a low power (<2 mW) infrared laser diode (780 nm) was used to measure flux of red blood cells passing through capillaries in tissue under a small fiber optic probe (Type NS in 24 gauge stainless steel needle, diameter 0.58 mm). The probe was placed on the tissue close to the microelectrode, at downstream locations supplied by the networks. Perivascular NO was measured near individual microvessels using a total of five different recessed microelectrodes with sensitivities ranging from 10 – 30 pA/AM and typical time responses <1 s. Whenever possible, the same microelectrode was used for repeated experiments, until replacement was necessary, usually due to a broken tip. Between experiments, the tips were cleaned, new Nafion membranes were applied, and the microelectrodes were recalibrated. The microelectrode tip was placed as close as possible to the outer wall of the vessel, without penetrating the wall or compressing the vessel. Zero NO levels were determined before and after each trial by moving the NO microelectrode tip into the bath above the tissue, and correcting for drift if necessary. Similar methods for perivascular NO measurements near microvessels in the skinfold of conscious hamsters have been described (Tsai et al., 2005). Details regarding NO and PO2 microelectrode fabrication, calibration, and operational characteristics have been published (Buerk et al., 1996; Buerk, 2004). One to three measurements were performed at each site. NO (or PO2) and LDF signals were recorded with a computer controlled, 12 bit data acquisition system at a 10 Hz sampling rate. After adding l-arginine to the bath, NO (or PO2) and LDF responses were recorded for 4 min or longer, depending on the time it took for NO to return to the baseline value before l-arginine. The maximum change in NO (DNOmax = NOmax NObaseline) at each site was determined. Overall time courses for the NO responses were determined by averaging individual recordings up to 4 min after adding l-arginine. Since tissue perfusion units for LDF are arbitrary, each LDF response was normalized (dividing the LDF signal by the average baseline 30 s to 1 min prior to l-arginine administration and multiplying by 100%). Average changes in normalized LDF, NO, and PO2 are reported as mean T standard error (SE). Paired t test comparisons were made between baseline levels and maximum changes for each variable, with P < 0.05 considered as statistically significant. Additional in vitro and in vivo tests were performed to evaluate thermal effects with different delivery rates and injected volumes of fluid, and whether

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there were any biological effects following transient exposure to excess darginine, the inactive isomer of l-arginine.

Results NO measurements Topical administration of l-arginine in the superfusion bath caused an increase in NO concentration and tissue perfusion in this microcirculatory network. A representative response near a small arteriole is shown in Fig. 2. After adding l-arginine to the bath at t = 0, there was rapid increase in perivascular NO at the microelectrode measurement site, with an increase in tissue perfusion measured by the laser Doppler probe at a location downstream. Oscillations in the NO and LDF signals, as seen in this example, were frequently observed, but were not analyzed for their frequency content, as we have done previously for measurements in the eye (Buerk and Riva, 1998). A total of 118 perivascular NO measurements from venules and arterioles were analyzed. The results were separated by relative size into small (S), medium (M), and large (L) groups. Since the size of the vessels could not be accurately determined from video images, relative sizes were assigned according to location along the microvascular network. The largest vessels were at the arterial and venous ends of the network. The smallest vessels were defined as those farthest downstream, after several branching orders. Measurements of NO for each subgroup were compared to their respective baseline levels prior to adding larginine. Average time courses of NO responses after adding larginine at t = 0 for different relative sizes of microvessels are shown in Fig. 3A for arterioles and Fig. 3B for venules. The overall time course was similar for both venules and arterioles. As shown in Fig. 3C, there was no significant difference among different sizes of vessels for the average time that it took for NO to increase to 50% of its maximum value (t 50%). The average time (TSE) that it took for NO to reach the maximum value was 1.98 T 0.14 min for arterioles, and 2.24 T 0.20 min for venules (N.S.). Averaged results are summarized in Table 1, and illustrated for arterioles in Fig. 4A and venules in Fig. 4B. All changes were statistically significant except for the small venule group in Fig. 4B, which had the smallest number of measurements in this study. Overall means T SE for all arterioles and

Fig. 1. Schematic drawing of experimental system for perivascular NO or PO2 measurements with microelectrodes and tissue perfusion by laser Doppler flowmetry (left) and video image of exteriorized preparation (right).

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from some in vivo tests were not included in the data analyzed for Table 1. We evaluated whether saline, delivered as a bolus to the preparation, caused any changes in NO microelectrode current or LDF signal. Transient thermal cooling was observed when larger fluid volumes were delivered too quickly, which could also cause transient changes in blood flow. This could also move the microelectrode if the fluid was delivered directly over the measurement site. We therefore chose to use a small fluid volume (1 ml) delivered over a 10 s time period for the

Fig. 2. Simultaneous measurement of tissue perfusion by LDF (bottom) and perivascular NO with a recessed NO microelectrode (top) following addition of l-arginine to the superfusion bath at t = 0. The NO microelectrode was moved up into the bath at the end of the record to determine the zero NO concentration current (scaling bar indicates 2 pA current). Baseline NO and LDF are indicated by horizontal dashed lines.

venules are indicated by the dark bars in Fig. 4. These results clearly show that the large increase in NO after l-arginine was not dependent on vessel type or relative size. The average increase in LDF, normalized with respect to the baseline prior to l-arginine, was 35.5 T 7.5% (P < 0.0001) for the studies with NO microelectrodes. PO2 measurements An example for a measurement of perivascular PO2 near a small arteriole after adding l-arginine to the perfusion bath is shown in Fig. 5. At this site, l-arginine caused a relatively large decrease in PO2, despite an increase in downstream tissue perfusion. A total of 53 measurements were obtained. Overall, the perivascular PO2 changes with l-arginine were much more variable than the NO responses, and were not statistically significant. Excess l-arginine decreased perivascular PO2 for 20 of 53 (38%) measurements, with an average decrease of 32.7 T 7.0 Torr in this subset, consistent with increased use of O2 to synthesize NO. However, PO2 increased at most of the other sites (62%), consistent with an increase in blood flow. More frequent and longer decreases in PO2 were observed near venules compared to arterioles. Near arterioles, PO2 decreased in 8 of 23 (35%) cases and remained below baseline for 2.52 T 0.58 min. For venules, a decrease was observed in 12 of 24 (50%) measurements, lasting 3.78 T 0.68 min. The magnitude of the observed changes in PO2 was unrelated to vessel size. The normalized increase in tissue perfusion for the PO2 studies was slightly higher, averaging 39 T 12% above baseline (P < 0.0001), but this was not significantly different from the LDF measurements obtained in the NO studies. Additional tests Several additional tests were conducted to evaluate possible sources for errors in our measurements. Preliminary results

Fig. 3. Average time courses for changes in perivascular NO for different sizes of arterioles (A) and venules (B) after topical application of l-arginine at t = 0, with no significant differences found for average times to 50% of peak NO (C).

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Table 1 Mean T SE for baseline NO and maximum increase (DNOmax) following l-arginine, and time for 50% of the increase in NO for different sizes of blood vessels Arterioles NObaseline (nM) Small Medium Large Overall

314 310 388 338

T T T T

74 48 85 40

Venules DNOmax (nM) 423 351 270 344

T T T T

54 80 51 38

t 50% (s) 38.3 43.5 43.7 42.0

T T T T

6.3 4.6 4.8 2.9

Sites

NObaseline (nM)

15 19 18 52

402 150 343 313

T T T T

212 35 54 48

DNOmax (nM) 751 389 497 512

T T T T

327 14 105 87

t 50% (s) 34.4 51.8 46.5 43.6

Sites T T T T

6.6 7.7 5.0 3.8

5 7 23 35

Except for small venules, all NO changes were statistically significant ( P values indicated in Fig. 4) based on paired t tests. No significant differences for t 50% were found.

studies reported here, to minimize thermal and fluid flow disturbances. The saline test was repeated with O2 microelectrodes, with similar observations. We also evaluated whether the NO electrode was sensitive to l-arginine, and did not observe any changes in the oxidation current other than transient effects due to cooling, especially with larger fluid volumes. A third in vivo test was conducted to evaluate whether d-arginine influenced blood flow or NO production. A 1 ml bolus of 100 mM d-arginine did not cause significant changes in the NO current. In some cases with longer exposure times and fluid volumes, excess d-arginine caused an initial

Fig. 4. Average NO baseline (left bars) before and peak NO (right bars) after larginine for different sizes of arterioles (A) and venules (B). All vessel groups except small venules had significant increases (dashed line shows overall average baseline).

decrease in NO followed by rapid recovery, but this phenomenon was not systematically investigated. Discussion Although several in vitro studies suggest that there may be an l-arginine paradox, direct evidence that this phenomenon exists in vivo is limited. Our experiments directly confirm that excess l-arginine causes a significant increase in NO and an increase in tissue perfusion in the superfused rat mesentery and small intestine preparation. However, to fully interpret our results, dynamic changes in blood flow velocity and vessel diameter at each NO measurement site would be required. Our NO results are similar to a smaller scale in vivo study by Bohlen (1998), also conducted in a similar rat mesentery and intestine preparation, measuring changes in NO after continuous topical administration of 1 mM l-arginine. Based on the results obtained from seven large arterioles in five rats, he reported a twofold increase in NO after l-arginine administration from a baseline of 334 T 19 nM to 686 T 53 nM within 2– 3 min. We found a similar doubling of perivascular NO for the conditions of our study using a lower concentration for excess l-arginine. Prior to l-arginine administration, we measured a wide range for baseline NO levels, at many different locations. We

Fig. 5. Simultaneous measurement of tissue perfusion by LDF (bottom) and perivascular PO2 with a recessed microelectrode (top) following addition of larginine to the bath at t = 0. The PO2 microelectrode was initially positioned in the bath above the tissue at the beginning of the record to determine the calibration current in room air equilibrated solution at 37-C.

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did not attempt to fully interpret differences in the local environment, or crosstalk between vessels, due to different NO levels. This is likely to be important, especially where arteriolar – venule pairs are parallel. We cannot rule out the possibility that our measurements at some locations include some component from other nearby sources of NO. However, we found in the case of large venules, which are typically in close proximity to large arterioles, that baseline NO levels were very similar to that observed in arterioles. Bohlen (1998) measured resting NO of 353 T 28 nM around arterioles and 401 T 48 nM around venules, and found increased NO and vasodilation with glucose absorption. Our baseline NO levels (Table 1) are in a similar range but are not as high as recently reported from measurements in the skinfold of conscious hamsters, in the 600 nM range (Tsai et al., 2005). Very high tissue NO levels (>2 AM) have been measured in tumors (Kashiwagi et al., 2005), which are related to NO production by eNOS and are higher with increasing density of blood vessels. In another in vivo NO microelectrode study, Palm et al. (2005) report that intravenous injections of l-arginine cause an increase in renal tissue NO in both normal and streptozotocin (STZ)-induced diabetic rats. Our study suggests that the smaller NO response with excess l-arginine in STZ-induced diabetic rats is likely due to a reduction in l-arginine availability, which we found to be 38% lower compared to control rats. It is not clear why excess substrate would increase NO production. Since NO plays an important role in number of physiological functions, understanding the mechanism of NOS regulation and activation is of great interest. A number of different explanations for mechanisms of the l-arginine paradox have been proposed. One possibility is that NOS activity and NO production may be linked to the rate of l-arginine transport and not to intracellular concentration. There is evidence (McDonald et al., 1997; Shaul, 2003) for co-localization of eNOS and the l-arginine transporter CAT1 (Mann et al., 2003) in specialized membrane regions called caveolae. It is possible that this particular structure makes eNOS more readily accessible to extracellular l-arginine, and less accessible to intracellular l-arginine. In addition, Shaul (2003) reports that NOS activity in membrane regions rich in caveolae is much higher than in caveolae free membrane regions or cytosol. Closs et al. (2000) proposes that endothelial cells may have two intracellular l-arginine pools, one of which corresponds to the caveolar region that allows free exchange between intracellular and extracellular space, and the other that does not. If access of this second pool to NOS is impaired, intracellular l-arginine, although present in high levels, cannot be used for NO production, whereas extracellular l-arginine would be more accessible. A recent in vivo study by Zani and Bohlen (in press) reports decreased NO production and blood flow in rat intestinal arterioles, using the amino acid L-lysine to limit larginine transport into the cell. This study examined the effects of l-lysine under different physiological conditions that cause NO dependent vasodilation (hypoxia and NaCl hyperosmolarity). In each condition, l-lysine suppressed expected increases in NO and blood flow, demonstrating the importance of extracellular l-arginine transport for NO production.

Another consideration is that l-arginine is also required for other metabolic pathways (Guoyao and Morris, 1998; Morris, 2004). A major pathway is the hydrolization of l-arginine into urea and l-ornithine by the enzyme arginase. Direct competition for l-arginine between NOS and arginase may limit its availability for NO production. Chicoine et al. (2004) reported an increase in NO production and decrease in urea production after treating bovine pulmonary arterial endothelial cells with the arginase inhibitor l-valine. However, competition between arginase and NOS for l-arginine may not occur in all tissues. Miner et al. (2004) report that l-arginine uptake by other biochemical pathways cannot be detected in the human coronary circulation. Further investigations into the role of arginase in other microcirculatory networks may help elucidate whether and to what extent competition of this enzyme with NOS limits the availability of l-arginine for NO production. Several other studies offer alternative explanations for the larginine paradox. In their in vitro studies, Tsikas et al. (2000) propose that endogenous inhibitors of NOS isoforms play a role. Bo¨ ger and Ron (2005) review the evidence that asymmetric dimethylarginine (ADMA), an analogue of larginine, may directly inhibit eNOS. Elevated ADMA levels in blood plasma are thought to be a risk factor in hypercholesterolemia, diabetes mellitus, hypertension, chronic heart failure, coronary artery disease, erectile dysfunction, and other cardiovascular diseases. It is hypothesized that ADMA alters the K m for l-arginine, causing a reduction in NO production by eNOS, despite sufficient availability of substrate. Our results with excess l-arginine might be interpreted as causing a decrease in the influence of NOS inhibitors, which, in turn, increases eNOS activity and NO production. Other NOS isoforms could also contribute to the experimental observations. Our recent mathematical models for NO transport around small arterioles predict how additional sources of NO production, from other NOS isoforms located in the vascular wall or surrounding tissue, can increase NO (Buerk et al., 2003; Lamkin-Kennard et al., 2004). Kashiwagi et al. (2002) reports that there is additional NO production from nNOS in nerves around arterioles (but not venules) in the rat mesentery, based on optical measurements with NO sensitive dyes. Perhaps l-arginine had an effect on nNOS in our study, although we did not see any difference in perivascular responses around venules. Another possibility is suggested from the study by Lass et al. (2002), in which they report that l-arginine is a scavenger of superoxide anion. Excess l-arginine might increase NO by limiting the extremely rapid reaction between NO and superoxide (Buerk et al., 2003). We did not observe any significant difference in perivascular NO levels between venules and arterioles, consistent with measurements by Tsai et al. (2005) in the skinfold microcirculation of conscious gerbils. Other studies suggest that there might be differences in endothelial NO production between the arterial and venous microcirculation. Wagner et al. (2001) speculate that rat mesenteric venular endothelial cells have a higher inherent capacity for NO synthesis than endothelial cells from arterioles. This was based on their findings that venules have greater NOS activity, due to higher intracellular l-

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arginine and eNOS enzyme levels. On the other hand, the study by Kashiwagi et al. (2002) suggests that there should be higher arteriolar NO production due to nNOS in the outer wall. Although we found that venules had a slightly larger increase in NO increase with excess l-arginine compared to arterioles, we did not find any statistical significance between vessel type for either the baseline NO or the response to l-arginine. However, it is possible that some of our measurements, especially at locations with paired vessels, could be confounded by diffusion of NO from venules to arterioles (Bohlen, 1998) or vice versa. NO production from nearby capillaries in tissue might also be a factor. Another possibility is that some NO is conserved as S-nitrosohemoglobin in small arteries and arterioles and released by capillaries or venules as blood PO2 levels fall (Singel and Stamler, 2005). We also observed several cases where both NO and LDF signals appear to have increased amplitudes for spontaneous oscillations after l-arginine, or possibly an increase in the frequencies of oscillation. Slower oscillations may be due to peristaltic movement of the intestine, but faster oscillations may be due to changes in vasomotion following the enhanced rate of NO production. This would require further study to more fully characterize. Our in vivo NO measurements provide direct evidence that the l-arginine paradox is present in the microcirculation of the mesentery, and that increased availability of l-arginine causes an increase in perivascular NO levels. Acknowledgments This research was supported by HL 068164 from NIH, and BES 0301446 from NSF. References Blum, A., Porat, R., Rosenschein, U., Keren, G., Roth, A., Laniado, S., Miller, H., 1999. Clinical and inflammatory effects of dietary l-arginine in patients with intractable angina pectoris. Am. J. Cardiol. 83 (10), 1488 – 1490. Bo¨ger, R.H., Ron, E.S., 2005. l-Arginine improves vascular function by overcoming the deleterious effects of ADMA, a novel cardiovascular risk factor. Altern. Med. Rev. 10, 14 – 23. Bogle, R.G., Baydoun, A.R., Pearson, J.D., Mann, G.E., 1996. Regulation of larginine transport and nitric oxide release in superfused porcine aortic endothelial cells. J. Physiol. 490, 229 – 241. Bohlen, H.J., 1998. Mechanism of increased vessel wall nitric oxide concentrations during intestinal absorption. Am. J. Physiol. 275, H542 – H550. Buerk, D.G., 2001. Can we model nitric oxide biotransport? A survey of mathematical models for a simple diatomic molecule with surprisingly complex biological activities. Ann. Biomed. Eng. 3, 109 – 143. Buerk, D.G., 2004. Measuring tissue PO2 with microelectrodes. Methods Enzymol. 381, 665 – 690. Buerk, D.G., Riva, C.E., 1998. Vasomotion and spontaneous low frequency oscillations in blood flow and nitric oxide in cat optic nerve head. Microvasc. Res. 55, 103 – 112. Buerk, D.G., Riva, C.E., Cranstoun, S.D., 1996. Nitric oxide has a vasodilatory role in cat optic nerve head during flicker stimuli. Microvasc. Res. 52, 13 – 26. Buerk, D.G., Lamkin-Kennard, K., Jaron, D., 2003. Modeling the influence of superoxide dismutase on superoxide and nitric oxide interactions including reversible inhibition of oxygen consumption. Free Radical Biol. Med. 34, 1488 – 1503.

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