Influence of intravenously administered leptin on nitric oxide production, renal hemodynamics and renal function in the rat

Regulatory Peptides 120 (2004) 59 – 67 www.elsevier.com/locate/regpep

Influence of intravenously administered leptin on nitric oxide production, renal hemodynamics and renal function in the rat Jerzy Beltowski a,*, Jerzy Jochem b, Graz˙yna Wo´jcicka a, Krystyna Z˙wirska-Korczala b a

Department of Pathophysiology, Medical University, ul. Jaczewskiego 8 20-090 Lublin, Poland b Department of Physiology, Medical University of Silesia, Zabrze, Poland

Received 13 February 2004; received in revised form 13 February 2004; accepted 19 February 2004 Available online 9 April 2004

Abstract We investigated the effect of leptin on systemic nitric oxide (NO) production, arterial pressure, renal hemodynamics and renal excretory function in the rat. Leptin (1 mg/kg) was injected intravenously and mean arterial pressure (MAP), heart rate (HR), renal blood flow (RBF) and renal cortical blood flow (RCBF), were measured for 210 min after injection. Urine was collected for seven consecutive 30-min periods and blood samples were withdrawn at 15, 45, 75, 105, 135, 165 and 195 min after leptin administration. Leptin had no effect on MAP, HR, RBF, RCBF and creatinine clearance, but increased urine output by 37.8% (0 – 30 min), 32.4% (31 – 60 min) and 27.0% (61 – 90 min), as well as urinary sodium excretion by 175.8% (0 – 30 min), 136.4% (31 – 60 min) and 124.2% (61 – 90 min). In contrast, leptin had no effect on potassium and phosphate excretion. Plasma concentration of NO metabolites, nitrites + nitrates (NOx), increased following leptin injection at 15, 45, 75 and 105 min by 27.7%, 178.1%, 156.4% and 58.7%, respectively. Leptin increased urinary NOx excretion by 241.6% (0 – 30 min), 552.6% (31 – 60 min), 430.7% (61 – 90 min) and 88.9% (91 – 120 min). This was accompanied by increase in plasma and urinary cyclic GMP. These data indicate that leptin stimulates systemic NO production but has no effect on arterial pressure and renal hemodynamics. D 2004 Elsevier B.V. All rights reserved. Keywords: Leptin; Nitric oxide; Cyclic GMP; Atrial natriuretic peptide; Obesity; Arterial hypertension

1. Introduction Leptin, a recently described peptide hormone secreted by white adipose tissue, acts on hypothalamic centers to regulate food intake and energy expenditure. Leptin is also involved in the regulation of various other physiological processes including carbohydrate and lipid metabolism, gastrointestinal and cardiovascular function, inflammation, immune response and reproduction [1]. Plasma leptin concentration is proportional to the amount of white adipose tissue and is markedly increased in obese individuals [2]. Hyperleptinemia is increasingly recognized as a key mechanism of obesity-associated hypertension [3]. Both centrally and peripherally administered leptin stimulates sympathetic nervous system (SNS) [4,5] and increases blood pressure (BP) if infused chronically [6]. In contrast to chronic, acute leptin treatment increases BP only when the hormone is * Corresponding author. Tel.: +48-81-7425837; fax: +48-81-7425828. E-mail address: [email protected] (J. Beltowski). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.02.012

administered centrally, suggesting that leptin induces counteracting depressor mechanisms outside the CNS which balance sympathoexcitation [5,7]. Nitric oxide (NO), produced by endothelial cells, is a principal mediator regulating vascular tone via a paracrine mechanism. Although leptin receptors are expressed in endothelial cells [8], its effect on NO generation is controversial. In vitro, leptin stimulates endothelial NO production [9,10] and induces NO-mediated vasorelaxation [11,12]. The results of in vivo studies are less clear. Some authors have observed the involvement of NO in leptin-induced vasodilatation [13], whereas others have not [11,14,15]. In addition, leptin may induce NO-independent vasorelaxation [11,16 –18]. Only few studies directly addressed the effect of leptin on NO generation in vivo. Fru¨hbeck [13], Fru¨hbeck and Gomez-Ambrosi [19] and Mastronardi et al. [20] observed increased plasma concentration of nitric oxide metabolites, nitrites + nitrates (NOx), following leptin administration. However, the source of NO was not identified. Apart from endothelial cells, leptin may stimulate NO synthesis in other tissues such as adipocytes

60

J. Beltowski et al. / Regulatory Peptides 120 (2004) 59–67

[20], macrophages [21] and the central nervous system [22]. In addition, plasma NOx level is affected by factors other than NO production such as dietary nitrates and renal excretory function. Therefore, simultaneous measurement of plasma and urinary NOx as well as of NO second messenger, cyclic GMP, is needed to accurately measure the generation of endothelium-derived, hemodynamically active NO [23]. We have recently reported that bolus intraperitoneal leptin administration increases plasma concentration and urinary excretion of NOx and cGMP [24]. However, intraperitoneally injected leptin could affect NO generation indirectly by acting on afferent sensory nerves [25,26]. In the present study, we investigated the effect of intravenously administered leptin on systemic NO generation in the rat. In addition, we examined renal hemodynamics and renal handling of electrolytes. We also assessed the effect of leptin on plasma and urinary atrial natriuretic peptide (ANP) which, like NO, acts through cyclic GMP and could be involved in natriuretic and/or vasodilatory effect of leptin.

maintained at 36.5 – 37.5 jC using the heating lamp throughout the experiment. After surgery, the animals were allowed to recover for 30 min. Then, urine was collected for 30-min baseline period. Subsequently, either leptin (1 mg/kg in 0.5 ml) or vehicle (0.5 ml) was injected i.v. and urine collection was continued in 30-min intervals for the next 210 min. Preliminary experiments have demonstrated that plasma leptin returns to baseline level after this time. Blood samples (0.4 ml) were withdrawn in the middle of each collection period including baseline period, i.e. 15 min before as well as 15, 45, 75, 105, 135, 165 and 195 min after leptin/vehicle injection. Blood was collected into

2. Materials and methods 2.1. Experimental protocol All experimental procedures were approved by the local ethics committee. Studies were carried out in male Wistar rats weighing 230 –250 g (5– 6 months old). The animals were housed in individual cages under controlled conditions of temperature (20 –22 jC), humidity (60 –70%), lighting (12-h light/dark cycle) and provided with food and water ad libitum. After induction of general anaesthesia with ethylurethane (1.25 g/kg intraperitoneally) and heparinisation (Heparinum, 600 IU/kg iv), rats were implanted with catheters in the right carotid artery and the right jugular vein. Another catheter was implanted into the urinary bladder to collect urine. Mean arterial pressure (MAP) and heart rate (HR) were measured using the pressure transducer RMN-201 (Temed, Poland) and the electrocardiograph Diascope 2 (Unitra Biazet, Poland), respectively. Electromagnetic probe (Type 1RB2006, Hugo Sachs Elektronik, Germany) was implanted around the right renal artery to monitor renal blood flow (RBF) using Transit Time Flowmeter Type 700 (Hugo Sachs Elektronik, Germany; Transonic System, USA) [27]. Renal cortical blood flow (RCBF) was measured in the middle part of ventral surface of the left kidney using laser flowmeter (Laser Flow type BRL-100, Bio Research Center, USA). Physiological saline was continuously infused through the venous catheter at a rate of 2 ml/ h to supplement fluid losses during the surgery and to avoid hypovolemia. Body temperature was monitored by a rectal thermometer (RMN-201, Temed, Poland) and

Fig. 1. Effect of leptin on hemodynamic variables. Leptin (1 mg/kg) or vehicle was injected intravenously into anesthetized animals. Upper panel presents MAP (left scale) and HR (right scale), and lower panel—RBF (left scale) and RCBF (right scale).

J. Beltowski et al. / Regulatory Peptides 120 (2004) 59–67

61

EDTA-containing tubes and centrifuged. Erythrocytes were washed with 1 ml of 0.9% NaCl, resuspended in 40 g/ l bovine serum albumin in 0.9% NaCl to a final volume of 0.5 ml, and returned to the animal within 15 min. Preliminary studies demonstrated that this procedure of repeated blood sampling had no effect on hematocrit, plasma protein, creatinine, Na+ and K+ levels. Plasma and urine samples were frozen and stored at 80 jC. For the measurement of cyclic GMP, 3-isobuthyl-1-methylxanthine (IBMX) was added to the samples (30 Al of 10 mmol/ l IBMX per 0.5 ml of sample) to prevent breakdown of cGMP by phosphodiesterases. 2.2. Biochemical studies

Fig. 2. Effect of leptin (1 mg/kg i.v.) on urine output (top panel) and GFR measured as creatinine clearance (bottom panel). *P < 0.05, compared to baseline pretreatment value by repeated-measures ANOVA and Newman – Keuls test.

Creatinine in plasma and urine was assayed colorimetrically using Sigma Diagnostics kit (Sigma-Aldrich, St. Louis, MO, USA). Sodium and potassium concentrations in plasma and urine were measured by flame photometry. Inorganic phosphate was assayed by the method of Hurst [28]. Nitric oxide metabolites (nitrates + nitrites, NOx) were assayed in plasma and urine by the colorimetric method of Griess after enzymatic conversion of nitrates to nitrites by

Fig. 3. Effect of leptin (1 mg/kg i.v.) on: (A) absolute (UNaV) and fractional (FENa+) excretion of sodium, (B) absolute and fractional excretion of potassium (UKV and FEK+), (C) absolute and fractional excretion of inorganic phosphate (UPiV and FEPi). ***P < 0.001, compared to pretreatment baseline value by repeated-measures ANOVA and Newman – Keuls test.

62

J. Beltowski et al. / Regulatory Peptides 120 (2004) 59–67

nitrate reductase [29,30], using Total Nitric Oxide Assay Kit (R&D Systems, Abingdon, Oxon, United Kingdom). Before assay, plasma and urine samples were deproteinized by centrifugation at 10 000  g for 10 min through 10,000 MW cut-off filters (Ultrafree 0.5, Milipore, Bedford, MA, USA). Cyclic GMP was assayed by the competitive enzyme immunoassay (EIA) using Cyclic GMP Enzyme Immunoassay Kit (Cayman Chemical, Ann Arbor, MI, USA). The antibodies used in this method show < 0.01% cross-reactivity with cAMP. Plasma and urinary ANP was assayed by competitive EIA using Rat Atriopeptin EIA kit (SPIBio, Massy, France). The intraassay/interassay coefficients of variation were 5.3%/7.0% for NOx, 4.7%/7.7% for cGMP and 6.1%/6.9% for ANP, respectively. NOx, cGMP and ANP assays have been described in details in our recent paper [24]. Plasma ANP is filtered in glomeruli and then degraded by neutral endopeptidase contained in the brush border of proximal tubule cells, therefore, urinary concentration of true ANP is very low ( < 50 pg/day) [31]. The antibodies against ANP used in our assay demonstrate 100% cross-reactivity with nephrogenous natriuretic peptide (urodilatin) which is abundant in urine. The level of urinary ANP detected by us is within the range of urodilatin excretion, which is at least three orders of magnitude

higher than excretion of ANP (see Section 3 and Ref. [32]). Thus, urinary ‘‘ANP’’ measured in this study is mainly accounted for by urodilatin. Plasma leptin was measured using Leptin Enzyme Immunoassay Kit (Cayman Chemical). The intraassay and interassay CV was 6.1% and 8.5%, respectively. 2.3. Calculation of renal parameters Glomerular filtration rate (GFR) was estimated by calculating creatinine clearance. Filtration fraction (FF) was obtained as the ratio between GFR and renal plasma flow (RBF  (1 hematocrit)). Fractional excretion of Na+, K+, inorganic phosphate and NOx was counted as the ratio between urinary excretion and the amount filtered (GFR  plasma concentration). Nephrogenous cGMP was determined as the difference between urinary excretion of this nucleotide and the amount filtered [33]. 2.4. Reagents Recombinant human leptin was purchased from Calbiochem-Novabiochem (San Diego, CA, USA). As recommen-

Fig. 4. (A) Effect of leptin on plasma concentration of nitric oxide metabolites (nitrites + nitrates, NOx), (B) urinary excretion of nitric oxide metabolites (UNOxV) in vehicle-treated and leptin-treated rats, (C) effect of leptin on fractional excretion of NO metabolites (FENOx), (D) effect of leptin on the ratio between fractional excretion of NOx and fractional excretion of sodium (FENOx/FENa+). *P < 0.05, **P < 0.01, ***P < 0.001, compared to pretreatment period by repeated measures ANOVA and Newman – Keuls test.

J. Beltowski et al. / Regulatory Peptides 120 (2004) 59–67

ded by the manufacturer, the vial containing 5 mg was dissolved in 2.5 ml of 15 mM HCl and then 1.5 ml of 7.5 mM NaOH was added to bring pH to 5.2. This solution was diluted with the 15 mM HCl/7.5 mM NaOH mixture (5:3 vol/vol) to yield the appropriate concentration, frozen, stored at 80 jC and thawed immediately before use. Until otherwise stated, all other reagents were obtained from Sigma-Aldrich. 2.5. Statistical analysis

63

baseline in vehicle-treated rats and was not significantly elevated at subsequent time points (Fig. 4A). Urinary excretion of nitric oxide metabolites increased following leptin administration by 241.6% between 0 and 30 min, by 552.6% between 31 and 60 min, by 430.7% between 61 and 90 min, and by 88.9% between 91 and 120 min. Vehicle-treated animals demonstrated mild increase in urinary NOx ( + 64.0%) only between 0 and 30 min after injection (Fig. 4B). Fractional excretion of NOx did not change in the control group, but increased significantly after leptin administration by 134.4% (0 –30 min), 114.0%

Data are reported as mean F S.E.M. from eight animals in each group. The results were subjected to Barlett’s test for homogeneity of variance. If homogeneity existed, oneway ANOVA with repeated measures was performed and, when appropriate, the individual means were compared by Newman– Keuls test. If Barlett’s test indicated heterogeneity of variance, then the values were logarithmically transformed and the test for homogeneity of variance was repeated. The results obtained in leptin-treated and control group at a given time point were compared by Student’s ttest for unrelated variables. P-values < 0.05 were considered significant.

3. Results There were no differences between groups in systemic and renal hemodynamic parameters before treatment (Fig. 1). Neither vehicle nor leptin administration had any significant effect on mean arterial pressure, heart rate, renal blood flow and renal cortical blood flow within 210 min after injection (Fig. 1). Leptin induced increase in urine output by 37.8% between 0 and 30 min, by 32.4% between 31 and 60 min, and by 27.0% between 61 and 90 min after injection, respectively (Fig. 2). In contrast, vehicle had no effect on urine output. Creatinine clearance (Fig. 2) and filtration fraction (not shown) did not change following vehicle or leptin injection. Leptin induced significant increase in natriuresis in three consecutive 30-min postinjection periods (175.8%, 136.4% and 124.2% increase above baseline, respectively). Fractional excretion of sodium (FENa+) increased following leptin injection by 160% between 0 and 30 min, by 135% between 31 and 60 min, and by 110% between 61 and 90 min (Fig. 3A). These data indicate that leptin increases natriuresis by inhibiting tubular sodium reabsorption. In contrast, leptin had no effect on both absolute or fractional excretion of potassium (Fig. 3B) and phosphate (Fig. 3C). In animals injected with leptin, plasma NOx increased at 15, 45, 75 and 105 min by 27.7%, 178.1%, 156.4% and 58.7%, respectively. In vehicle-treated group, plasma NOx increased significantly at 15 min (34.2% above baseline) and at this time point was not different from leptin-treated group. However, after this time plasma NOx returned to

Fig. 5. Effect of leptin on plasma concentration of cGMP (A), total urinary excretion of cGMP (UcGMPV), (B) and the excretion of nephrogenous cGMP (C). *P < 0.05, **P < 0.01, ***P < 0.001, compared to pretreatment period by repeated measures ANOVA and Newman – Keuls test.

64

J. Beltowski et al. / Regulatory Peptides 120 (2004) 59–67

(31 –60 min) and 95.2% (61 –90 min) (Fig. 4C). Because NO metabolites are reabsorbed together with sodium in renal tubules, we calculated FENOx/FENa+ ratio to get more insight into the relationship between Na+ and NOx reabsorption. Neither vehicle not leptin had any significant effect on this variable (Fig. 4D). Leptin-induced increase in plasma and urinary NOx was accompanied by parallel changes in NO second messenger, cyclic GMP. In leptin-treated group plasma cGMP was higher than baseline at 45, 75 and 105 min after injection by 112.2%, 214.5% and 103.8%, respectively (Fig. 5A). In vehicle-treated group, plasma cGMP tended to be higher at 45 min, but the difference did not reach the level of significance. Leptin administration induced increase in urinary excretion of cGMP between 31 and 60 min ( + 75.4%), between 61 and 90 min ( + 133.2%) and between 91 and 120 min ( + 68.6%) (Fig. 5B). Vehicle injection caused modest increase in urinary cGMP ( + 34.9%) only between 31 and 60 min. The amount of nephrogenous cGMP did not change significantly throughout the experiment in either leptin- or vehicle-treated group (Fig. 5C). Plasma concentration and urinary excretion of ANP did not change significantly following leptin or vehicle injection (Fig. 6). Vehicle administration had no effect on plasma leptin concentration. In contrast, marked increase in plasma leptin was observed in animals treated with this hormone. In this group, plasma leptin was the highest 15

Fig. 6. Effect of leptin on plasma concentration of ANP (top) and urinary excretion of urodilatin (bottom).

Fig. 7. Plasma leptin concentration in control and leptin-treated animals. *P < 0.05, **P < 0.01, ***P < 0.001, compared to pretreatment period by repeated measures ANOVA and Newman – Keuls test.

min after injection and returned to baseline at 195 min (Fig. 7).

4. Discussion In the present study, we demonstrated that bolus intravenous leptin administration increased systemic nitric oxide production in the rat, as evidenced by increase in plasma concentration and urinary excretion of NO metabolites, as well as of NO second messenger, cGMP. These data are consistent with our recent study [24], in which the same dose of leptin was injected intraperitoneally. In that study [24], plasma NOx and cGMP increased less markedly (maximally by f 60% and 80%, respectively) than in the present experiment, most likely due to lower plasma leptin level after i.p. than after i.v. injection. Nevertheless, these data suggest that either i.p. or i.v. administered leptin stimulates NO generation through the similar mechanism. Previously, Fru¨hbeck [13,19] has demonstrated that i.v. injection of the same dose of recombinant murine leptin increases plasma NOx by f 60% after 60 min and by f 90% after 90 min. Mastronardi et al. [20] observed a six-fold increase in plasma NOx following administration of the rat leptin. However, baseline NOx level was lower in that study [20] than in our animals, possibly due to different nitrate content in the diet [23], whereas maximal values of plasma NOx were similar (40 – 50 AM). These data suggest that rat and human leptin have comparable ability to stimulate NO production, whereas murine leptin is less efficient. This is a little surprising since the sequences of rat and mouse leptin share 96% homology, whereas the degree of homology between rat and human hormone is only 84%. However, the biological activity of recombinant hormones provided by different manufacturers could be affected by factors other than amino acid sequence. In addition, differences in renal clearance of NOx could affect their plasma level. Neither urinary excretion of NOx, nor plasma cGMP (a more direct indicator of NO activity) were

J. Beltowski et al. / Regulatory Peptides 120 (2004) 59–67

reported in those studies [13,19,20], making such comparisons difficult. The source of leptin-induced NO production can not be identified in the present study. Leptin receptors are expressed in endothelial cells [8] and leptin increases NO generation and induces NO-dependent vasorelaxation in vitro [9– 12]. In addition, other vasodilators which stimulate endothelial NO such as bradykinin, substance P and insulin cause comparable increase in plasma and urinary NOx [34 – 36]. However, nonendothelial sources of NO can not be excluded. Indeed, leptin may stimulate NO generation in adipocytes [20], macrophages [21], cardiac myocytes [37] and erythrocytes [38]. Interestingly, both vehicle and leptin administration caused slight increase in plasma NOx at 15 min after injection, accompanied by a tendency to higher plasma and urinary cGMP. The similar effect was observed by Mastronardi et al. [20] and may reflect stimulation of NO production due to slight volume expansion. Significant increase in plasma NOx in leptin-treated vs. control group was evident at 45 min. Leptin triggers several signaling pathways in target cells, including stimulation of JAK kinases which phosphorylate signal transducers and activators of transcription (STAT), thus affecting gene transcription and protein synthesis. In addition, leptin activates different nongenomic signaling mechanisms [39]. The time interval between leptin administration and peak plasma NOx observed in this study seems to be too short to be accounted for by changes in protein expression, but is clearly longer than observed in studies addressing the effect of bradykinin and substance P which stimulate endothelial NO synthase (NOS) by increasing intracellular Ca2 + [34]. Vecchione et al. [10] have demonstrated that leptin stimulates protein kinase B/Akt, which phosphorylates endothelial NO synthase increasing its activity even at low calcium concentration. It is likely that such covalent modification of NOS requires more time than allosteric Ca2 +-dependent activation, resulting in later increase in plasma NOx than after bradykinin or substance P [34]. Despite marked stimulation of NO and consistently with other studies [5,11,40], acutely administered leptin had no effect on systemic arterial pressure in the present experiment. Also, similarly to other reports [15,40], we observed no effect of leptin on renal blood flow. These findings may be explained in two ways. As initially suggested [5], leptin may stimulate two opposite but balanced mechanisms, i.e. vasorelaxation and sympathoexcitation. Supporting this hypothesis, Fru¨hbeck [13] observed that leptin per se had no effect on blood pressure in anaesthetized rats; increased BP in rats pretreated with NOS inhibitor, and decreased it in animals with intact NO but with blocked SNS activity. However, this concept has been challenged by more recent studies in which no effect of leptin on BP could be demonstrated even after NOS or SNS blockade [11,14,40], and leptin was unable to counteract SNS-induced vasoconstriction [15]. It would also be surprising if these two

65

effects: centrally mediated sympathoexcitation and direct endothelium-dependent vasorelaxation were so perfectly matched to cause no changes in blood pressure and renal hemodynamics throughout the 3-h experimental period in the present study. Thus, the alternative possibility should be considered, i.e. that leptin-induced NO production is not involved in the regulation of systemic and renal hemodynamics. Leptin may stimulate NO only in some parts of the vasculature such as conduit vessels [11] or, as mentioned above, in extravascular tissues. In the latter case, NO may be involved in the effects of leptin on pituitary hormones [22], lipolysis [19,20], myocardial contractility [37] and on fluidity of erythrocyte membranes [38]. Consistently with other studies [41 – 44], we have observed that acutely administered leptin increases natriuresis primarily by inhibiting tubular sodium reabsorption. Furthermore, lack of changes in either potassium or phosphate excretion suggests that leptin acts within the distal nephron, presumably in the collecting duct. Leptin receptors are expressed in the renal medulla [45,46], most likely in the medullary collecting duct [47]. Thus, it is possible that leptin inhibits Na+ transport by acting directly on tubular cells. In addition, leptin could modulate the activity of other mediators regulating natriuresis. ANP as well as urodilatin inhibit Na+ reabsorption in the inner medullary collecting duct [48]. However, we did not observe any changes in plasma and urinary ANP, suggesting that natriuretic peptides are not involved in renal effects of leptin. Nitric oxide is produced by tubular cells and inhibits Na+ reabsorption in an auto-and paracrine manner [49]. The role of NO in mediating leptin-induced natriuresis requires further study. Our experimental protocol does not allow to determine the effect of leptin on NO generation specifically within the kidney. NOx are filtered in glomeruli and then partially reabsorbed together with sodium throughout the nephron. Simultaneously, NOx originating from intrarenally produced NO are added to the tubular fluid [50 – 52]. Thus, changes of intrarenal NO production should affect fractional excretion of NOx. Although we observed marked increase in FENOx following leptin injection, this could result solely from the inhibition of tubular Na+ and NOx reabsorption because both FENa+ and FENOx were increased to the similar degree. If leptin stimulated renal NO generation, it would cause greater increase in FENOx than FENa+, i.e. would elevate FENOx/FENa+ ratio. In addition, leptin had no effect on nephrogenous cGMP, which should be observed if leptin stimulated renal NO. However, we can not exclude that leptin did stimulate NO production in some tubular segment(s) causing undetectable changes in total urinary NOx and cGMP. We are aware of several potential limitations of the present study. First, we used human leptin in the rat. We did so to make the results comparable to our previous study in which leptin was administered intraperitoneally [24]. Human leptin is active in the rat [53] and, in the case of urinary Na+ excretion, it is even more active than homolo-

66

J. Beltowski et al. / Regulatory Peptides 120 (2004) 59–67

gous hormone [42]. Second, we administered leptin at a pharmacological dose which resulted in plasma concentration far above physiological level, and even above the level observed in obesity. However, this does not necessarily exclude the physiological significance of our findings. First, to demonstrate the effect of leptin on systemic NO production in vivo on the basis of plasma and urinary NOx and cGMP, higher doses will be required than when studying isolated tissues. Second, because leptin is a highly hydrophobic peptide which binds to tissue lipids and proteins, its local concentrations, especially in adipose tissue where the hormone is produced, are probably much higher than in plasma. Third, recombinant leptin may be less active than native hormone. Another disadvantage is that we made no attempts to recognize functional interactions between NO and SNS in the regulation of hemodynamics following leptin administration. As mentioned above, the results of such studies are extremely variable, depending on experimental conditions [11,13,40]. Finally, we measured blood flow only in renal circulation and can not exclude that leptin affected local hemodynamics in other vascular beds, with no net effect on mean arterial pressure. In conclusion, we observed that bolus i.v. leptin administration stimulated systemic NO production in the rat but had no effect on arterial pressure. In addition, leptin increased urine output and urinary excretion of sodium by inhibiting its tubular reabsorption with no effect on renal hemodynamics and glomerular filtration.

Acknowledgements This study was supported by the grant PW 447/2002 from Medical University in Lublin.

References [1] Margetic S, Gazzola C, Pegg GG, Hill RA. Leptin: a review of its peripheral actions and interactions. Int J Obes Relat Metab Disord 2002;26:1407 – 33. [2] Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, et al. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med 1996;334:292 – 5. [3] Hall JE, Hildebrandt DA, Kuo J. Obesity hypertension: role of leptin and sympathetic nervous system. Am J Hypertens 2001;14:103S – 15S. [4] Dunbar JC, Hu Y, Lu H. Intracerebroventricular leptin increases lumbar and renal sympathetic nerve activity and blood pressure in normal rats. Diabetes 1997;46:2040 – 3. [5] Haynes WG, Morgan DA, Walsh SA, Mark AL, Sivitz WI. Receptormediated regional sympathetic nerve activation by leptin. J Clin Invest 1997;100:270 – 8. [6] Shek EW, Brands MW, Hall JE. Chronic leptin infusion increases arterial pressure. Hypertension 1998;31:409 – 14. [7] Casto RM, VanNess JM, Overton JM. Effects of central leptin administration on blood pressure in normotensive rats. Neurosci Lett 1998;246:29 – 32. [8] Bouloumie A, Drexler HC, Lafontan M, Busse R. Leptin, the product of Ob gene, promotes angiogenesis. Circ Res 1998;83:1059 – 66.

[9] Winters B, Mo Z, Brooks-Asplund E, Kim S, Shoukas A, Li D, et al. Reduction of obesity, as induced by leptin, reverses endothelial dysfunction in obese (Lep(ob)) mice. J Appl Physiol 2000; 89:2382 – 90. [10] Vecchione C, Maffei A, Colella S, Aretini A, Poulet R, Frati G, et al. Leptin effect on endothelial nitric oxide is mediated through Aktendothelial nitric oxide synthase phosphorylation pathway. Diabetes 2002;51:168 – 73. [11] Lembo G, Vecchione C, Fratta L, Marino G, Trimarco V, d’Amati G, et al. Leptin induces direct vasodilation through distinct endothelial mechanisms. Diabetes 2000;49:293 – 7. [12] Kimura K, Tsuda K, Baba A, Kawabe T, Boh-oka S, Ibata M, et al. Involvement of nitric oxide in endothelium-dependent arterial relaxation by leptin. Biochem Biophys Res Commun 2000;273:745 – 9. [13] Fru¨hbeck G. Pivotal role of nitric oxide in the control of blood pressure after leptin administration. Diabetes 1999;48:903 – 8. [14] Mitchell JL, Morgan DA, Correia ML, Mark AL, Sivitz WI, Haynes WG. Does leptin stimulate nitric oxide to oppose the effects of sympathetic activation? Hypertension 2001;38:1081 – 6. [15] Jalali A, Morgan DA, Sivitz WI, Correia ML, Mark AL, Haynes WG. Does leptin cause functional peripheral sympatholysis? Am J Hypertens 2001;14:615 – 8. [16] Nakagawa K, Higashi Y, Sasaki S, Oshima T, Matsuura H, Chayama K. Leptin causes vasodilation in humans. Hypertens Res 2002;25: 161 – 5. [17] Fortuno A, Rodriguez A, Gomez-Ambrosi J, Muniz P, Salvador J, Diez J, et al. Leptin inhibits angiotensin II-induced intracellular calcium increase and vasoconstriction in the rat aorta. Endocrinology 2002;143:3555 – 60. [18] Matsuda K, Teragawa H, Fukuda Y, Nakagawa K, Higashi Y, Chayama K. Leptin causes nitric-oxide independent coronary artery vasodilation in humans. Hypertens Res 2003;26:147 – 52. [19] Fru¨hbeck G, Gomez-Ambrosi J. Modulation of the leptin-induced white adipose tissue lipolysis by nitric oxide. Cell Signal 2001;13: 827 – 33. [20] Mastronardi CA, Yu WH, McCann SM. Resting and circadian release of nitric oxide is controlled by leptin in male rats. Proc Natl Acad Sci U S A 2002;99:5721 – 6. [21] Raso GM, Pacilio M, Esposito E, Coppola A, Di Carlo R, Meli R. Leptin potentiates IFN-gamma-induced expression of nitric oxide synthase and cyclo-oxygenase-2 in murine macrophage J774A.1. Br J Pharmacol 2002;137:799 – 804. [22] Yu WH, Walczewska A, Karanth S, McCann SM. Nitric oxide mediates leptin-induced luteinizing hormone-releasing hormone (LHRH) and LHRH and leptin-induced LH release from the pituitary gland. Endocrinology 1997;138:5055 – 8. [23] Baylis C, Vallance P. Measurement of nitrite and nitrate levels in plasma and urine—what does this measure tell us about the activity of the endogenous nitric oxide system? Curr Opin Nephrol Hypertens 1998;7:59 – 62. [24] Beltowski J, Wo´jcicka G, Borkowska E. Human leptin stimulates systemic nitric oxide production in the rat. Obes Res 2002;10:939 – 46. [25] Brzozowski T, Konturek PC, Konturek SJ, Pajdo R, Duda A, Pierzchalski P, et al. Leptin in gastroprotection induced by cholecystokinin or by a meal. Role of vagal and sensory nerves and nitric oxide. Eur J Pharmacol 1999;374:263 – 6. [26] Brzozowski T, Konturek PC, Pajdo R, Kwiecien S, Ptak A, Sliwowski Z, et al. Brain-gut axis in gastroprotection by leptin and cholecystokinin against ischemia – reperfusion induced gastric lesions. J Physiol Pharmacol 2001;52:583 – 602. [27] Jochem J. Central histamine-induced reversal of critical haemorrhagic hypotension in rats—haemodynamic studies. J Physiol Pharmacol 2002;53:75 – 84. [28] Hurst RO. The determination of nucleotide phosphorus with a stannous chloride – hydrazine sulphate reagent. Can J Biochem 1964;42: 287 – 92. [29] Green LC, Wagner DA, Glogowski J, Skipper L, Wishnok JS,

J. Beltowski et al. / Regulatory Peptides 120 (2004) 59–67

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39] [40]

Tannenbaum SR. Analysis of nitrate, nitrite and [15N] nitrate in biological fluids. Anal Biochem 1982;120:131 – 8. Moshage H, Kok B, Huizenga JR, Jansen PLM. Nitrite and nitrate determinations in plasma: a critical evaluation. Clin Chem 1995;41: 892 – 6. Shin SJ, Wen JD, Chen IH, Lai FJ, Hsieh MC, Hsieh TJ, et al. Increased renal ANP synthesis, but decreased or unchanged cardiac ANP synthesis in water-deprived and salt-restricted rats. Kidney Int 1998;54:1617 – 25. Frey BA, Grisk O, Bandelow N, Wussow S, Bie P, Rettig R. Sodium homeostasis in transplanted rats with a spontaneously hypertensive rat kidney. Am J Physiol Regul Integr Comp Physiol 2000;279:R1099 – 104. Pham I, Sediame S, Maistre G, Roudot-Thoraval F, Chabrier PE, Carayon A, et al. Renal and vascular effects of C-type and atrial natriuretic peptides in humans. Am J Physiol 1997;273:R1457 – 64. Nava E, Wiklund NP, Salazar FJ. Changes in nitric oxide release in vivo in response to vasoactive substances. Br J Pharmacol 1996;119: 1211 – 6. Komers R, Pelikanova T, Kazdova L. Effect of hyperinsulinaemia on renal function and nitrate/nitrite excretion in healthy subjects. Clin Exp Pharmacol Physiol 1999;26:336 – 41. Tsukahara H, Kikuchi K, Tsumura K, Kimura K, Hata I, Hiraoka M, et al. Experimentally induced acute hyperinsulinemia stimulates endogenous nitric oxide production in humans: detection using urinary NO2-/NO3-excretion. Metabolism 1997;46:406 – 9. Nickola MW, Wold LE, Colligan PB, Wang GJ, Samson WK, Ren J. Leptin attenuates cardiac contraction in rat ventricular myocytes. Role No Hypertens 2000;36:501 – 5. Tsuda K, Kimura K, Nishio I. Leptin improves membrane fluidity of erythrocytes in humans via a nitric oxide-dependent mechanism-an electron paramagnetic resonance investigation. Biochem Biophys Res Commun 2002;297:672 – 81. Sweeney G. Leptin signalling. Cell Signal 2002;4:655 – 63. Gardiner SM, Kemp PA, March JE, Bennett T. Regional haemodynamic effects of recombinant murine or human leptin in conscious rats. Br J Pharmacol 2000;130:805 – 10.

67

[41] Jackson EK, Li P. Human leptin has natriuretic activity in the rat. Am J Physiol 1997;272:F333 – 8. [42] Jackson EK, Herzer WA. A comparison of the natriuretic/diuretic effects of rat vs. human leptin in the rat. Am J Physiol 1999;277: F761 – 5. [43] Villarreal D, Reams G, Freeman RH, Taraben A. Renal effects of leptin in normotensive, hypertensive, and obese rats. Am J Physiol 1998;275:R2056 – 60. [44] Villarreal D, Reams G, Freeman RH. Effects of renal denervation on the sodium excretory actions of leptin in hypertensive rats. Kidney Int 2000;58:989 – 94. [45] Serradeil-Le Gal C, Raufaste D, Brossard G, Pouzet B, Marty E, Maffrand JP, et al. Characterization and localization of leptin receptors in the rat kidney. FEBS Lett 1997;404:185 – 91. [46] Hoggard N, Mercer JG, Rayner DV, Moar K, Trayhurn P, Williams LM. Localization of leptin receptor mRNA splice variants in murine peripheral tissues by RT-PCR and in situ hybridization. Biochem Biophys Res Commun 1997;232:383 – 7. [47] Martinez-Anso E, Lostao MP, Martinez JA. Immunohistochemical localization of leptin in rat kidney. Kidney Int 1999;55:1129 – 30. [48] Brenner BM, Ballermann BJ, Gunning ME, Zeidel ML. Diverse biological actions of atrial natriuretic peptide. Physiol Rev 1990;70: 665 – 99. [49] Ortiz PA, Garvin JL. Role of nitric oxide in the regulation of nephron transport. Am J Physiol Renal Physiol 2002;282:F777 – 84. [50] Suto T, Losonczy G, Qiu C, Hill C, Samsell L, Ruby J, et al. Acute changes in urinary excretion of nitrite + nitrate do not necessarily predict renal vascular NO production. Kidney Int 1995;48:1272 – 7. [51] Godfrey M, Majid DS. Renal handling of circulating nitrates in anesthetized dogs. Am J Physiol 1998;275:F68 – 73. [52] Rahma M, Kimura S, Yoneyama H, Kosaka H, Nishiyama A, Fukui T, et al. Effects of furosemide on the tubular reabsorption of nitrates in anesthetized dogs. Eur J Pharmacol 2001;428:113 – 9. [53] Eckel LA, Langhans W, Kahler A, Campfield LA, Smith FJ, Geary N. Chronic administration of OB protein decreases food intake by selectively reducing meal size in female rats. Am J Physiol 1998;275: R186 – 93.