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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

The Pancreatohepatorenal cAMP-Adenosine Mechanism

Departments of Medicine (E.K.J., Z.M., L.C.Z., S.P.T., R.K.D.) and Pharmacology (E.K.J.), Center for Clinical Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and Clinic for Endocrinology, University Hospital Zurich, Zurich, Switzerland (R.K.D.)

Received December 24, 2006; accepted February 16, 2007.

	Abstract

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Stimulation of adenylyl cyclase causes cellular efflux of cAMP, and cAMP (unlike adenosine) is stable in blood. Therefore, it is conceivable that cAMP could function as a circulating adenosine prohormone by local target-organ conversion of distally released cAMP to adenosine via the sequential actions of ectophosphodiesterase and ecto-5'-nucleotidase (cAMP AMP adenosine; called the cAMP-adenosine pathway). A possible specific representation of this general concept is the pancreatohepatorenal cAMP-adenosine mechanism. The pancreas secretes glucagon into the portal circulation, and glucagon is a stimulant of hepatic adenylyl cyclase. Therefore, we hypothesize that the pancreas, via glucagon, stimulates hepatic cAMP production, which provides circulating cAMP for conversion to adenosine in the kidney via the cAMP-adenosine pathway. In normal rats, intravenous cAMP increased urinary and renal interstitial (assessed by renal microdialysis) cAMP and adenosine. Intraportal infusions of glucagon increased plasma cAMP 10-fold, it did not affect plasma adenosine, and it increased urinary and renal interstitial cAMP and adenosine. Local renal interstitial blockade (by adding inhibitors directly to the microdialysis perfusate) of ectophosphodiesterase (using 3-isobutyl-1-methylxanthine or 1,3-dipropyl-8-p-sulfophenylxanthine) or ecto-5'-nucleotidase (using ,-methyleneadenosine-5'-diphosphate) prevented the cAMP-induced and glucagon-induced increases in renal interstitial adenosine, but not cAMP. In ZSF1 rats with the metabolic syndrome, an oral glucose load increased plasma glucagon and urinary cAMP and adenosine excretion. We conclude that circulating cAMP is a substrate for local conversion to adenosine via the cAMP-adenosine pathway. A specific manifestation of this is the pancreatohepatorenal cAMP-adenosine mechanism (pancreas portal glucagon liver circulating cAMP kidney local cAMP-adenosine pathway).

A potential specific example of this general concept is the pancreatohepatorenal cAMP-adenosine mechanism: the pancreas releases glucagon into the portal circulation, which delivers high concentrations of glucagon to hepatocytes, which increases hepatic adenylyl cyclase activity, which causes cAMP efflux into the systemic circulation, which delivers cAMP to the kidneys where cAMP is converted locally to adenosine via the extracellular cAMP-adenosine pathway (pancreas portal glucagon hepatocytes circulating cAMP kidney local conversion of cAMP to adenosine). Our rationale for the general hypothesis that cAMP is a prohormone that is converted to adenosine by organs remote from the site of cAMP secretion and for the specific pancreatohepatorenal cAMP-adenosine mechanism is based on several considerations. When adenylyl cyclase is activated, cAMP effluxes from cells. Cellular egress of cAMP evolved as a survival mechanism in the slime mold Dictyostelium discoideum, which secretes cAMP into the environment to initiate chemotaxis leading to starvation-induced aggregation of single-celled amoebae into a migrating slug (Albert et al., 1989). Efflux of cAMP occurs in a large array of tissues, including cervical ganglia (Cramer and Lindl, 1974), fibroblasts (Kelly and Butcher, 1974), heart (O'Brien and Strange, 1975), adipose tissue (Zumstein et al., 1974), and adipocytes (Finnegan and Carey, 1998). Most important for the pancreatohepatorenal cAMP-adenosine mechanism is the fact that cAMP egresses from hepatocytes (Kuster et al., 1973). The liver is a large organ in the body; therefore, it would be a particularly plentiful source of circulating cAMP whenever hepatic adenylyl cyclase is stimulated by pancreatic hormones that are released directly into the portal circulation. For example, glucagon is secreted by the pancreas into the portal circulation where it reaches the hepatocytes at elevated concentrations and stimulates adenylyl cyclase. Indeed, Bankir et al. (1997, 2002) postulate that liver-derived cAMP released by glucagon is delivered to the renal tubules by filtration and acts on apical cell surface cAMP receptors to alter proximal tubular function. Although our hypothesis is not mutually exclusive with the concept that cAMP acts directly on cell surface cAMP receptors on renal epithelial cells, searches of mammalian databases reveal no cAMP receptors in mammals with homology to the cAMP receptors found in D. discoideum (Bankir et al., 2002). However, adenosine receptors are abundantly expressed in the kidney, and they alter renal function (Vallon et al., 2006).

Another part of the rationale for the aforementioned hypothesis is based on our previous studies demonstrating the existence of ectophosphodiesterase, which metabolizes extracellular cAMP to AMP in a number of cell types, including aortic vascular smooth muscle cells (Dubey et al., 1996, 1998) and cardiac fibroblasts (Dubey et al., 2000, 2001). Most important for the rationale of the pancreatohepatorenal cAMP-adenosine mechanism is the fact that preglomerular vascular smooth muscle cells (Jackson et al., 1997), preglomerular microvessels (Jackson and Mi, 2000), mesangial cells (Dubey et al., 1997), proximal tubular epithelial cells and proximal tubules (Jackson et al., 2006), and collecting duct epithelial cells and collecting ducts (Jackson et al., 2003) also express ectophosphodiesterase activity. Acting "downstream" of ectophosphodiesterase is ecto-5'-nucleotidase, a ubiquitous enzyme fastened to the extracellular aspect of cell membranes by a lipid-sugar linkage (Zimmerman, 1992). Ecto-5'-nucleotidase rapidly metabolizes AMP to adenosine in many organs, including the kidney (Huang et al., 2006; Satriano et al., 2006; Vekaria et al., 2006).

The general concept that emerges from the aforementioned discussion is that cAMP may function as a prohormone, and a specific example of this concept is that cAMP secreted by the liver in response to glucagon is delivered to the kidneys where the cAMP is metabolized to AMP by ectophosphodiesterase and hence to adenosine by ecto-5'-nucleotidase. The main goal of this study was to test the hypothesis that the pancreatohepatorenal cAMP-adenosine mechanism exists. This hypothesis was tested using nine protocols.

	Materials and Methods

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Animals and Chemicals. Protocols 1 to 8 used adult (approximately 18-week-old) male rats obtained from either Taconic Farms (Germantown, NY) or Charles River Laboratories, Inc. (Wilmington, MA). Protocol 9 was performed in adult (approximately 20-week-old) male "lean" ZSF1 rats obtained from Genetic Models Inc. (Indianapolis, IN). The Institutional Animal Care and Use Committee approved all procedures. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

Protocol 1: Effects of Intravenous Infusions of cAMP on the Urinary Excretion of Adenosine. Rats (n = 16) were anesthetized with Inactin (90–100 mg/kg i.p.) and placed on a Deltaphase isothermal pad (Braintree Scientific, Braintree, MA). Body temperature was monitored with a rectal temperature probe (Physitemp Instruments, Clifton, NJ) and maintained at 37 ± 0.5°C by adjusting a heat lamp positioned above the rat. A short section of polyethylene (PE)-240 tubing was placed in the trachea to facilitate respiration. A PE-50 cannula was inserted into the right jugular vein, and an infusion of 0.9% saline was initiated at 50 µl/min. A PE-50 catheter was placed in the left carotid artery for measurement of mean arterial blood pressure (MABP) via a digital blood pressure analyzer (model BPA; Micro-Med Inc., Louisville, KY). A PE-10 catheter was inserted into the left ureter for urine collection, and a flow probe (model 1RB; Transonic Systems Inc., Ithaca, NY) was placed on the left renal artery and connected to a transit time flowmeter (model T206; Transonic Systems) for determination of renal blood flow (RBF). After at least a 1-h rest period, parameters were recorded in all animals during three 20-min urine collection periods during which the animals received intravenously 0.9% saline, 30 µg/kg/min cAMP, or 100 µg/kg/min cAMP. Urinary adenosine was measured by high-pressure liquid chromatography (HPLC) with fluorescence detection as described previously (Jackson et al., 1996).

Protocol 2: Effects of Intrarenal Artery Infusions of cAMP on the Urinary Excretion of Adenosine. Rats (n = 17) were prepared similarly as described for protocol 1 with the exception that, rather than a jugular infusion, a 32-gauge needle was also inserted into the left renal artery, and it received an infusion (50 µl/min) of 0.9% saline. In addition, the protocol was similar to protocol 1 except that cAMP was administered directly into the renal artery, rather than intravenously. Urinary adenosine was measured by HPLC with fluorescence detection as described previously (Jackson et al., 1996).

Protocol 3: Effects of Intravenous Infusions of cAMP on the Urinary Excretion of Adenosine and cAMP in Rats Pretreated with Probenecid to Block Renal Tubular cAMP Transport. Rats (n = 12) were prepared similarly as described for protocol 1. In addition, the protocol was similar to protocol 1 except that half of the rats received probenecid (10 mg/kg bolus followed by 0.1 mg/kg/min infusion) via intravenous administration beginning 30 min before a single intravenous infusion of cAMP at 10 µg/kg/min. Both urinary cAMP and adenosine was measured by HPLC with fluorescence detection as described previously (Jackson et al., 1996).

Protocol 4: Effects of Intravenous Infusions of cAMP on Renal Microdialysate Levels of cAMP and Adenosine in the Absence and Presence of Inhibitors of cAMP-Adenosine Pathway. Rats (n = 18) were anesthetized, body temperature was monitored and maintained, respiration was protected, and MABP was measured as described in protocol 1. A PE-10 cannula was inserted into the femoral vein and advanced into the vena cava until the tip of the catheter was approximately at the level of the hepatic vein, and then an infusion of 0.9% saline was initiated at 50 µl/min. A microdialysis probe (CMA/20 microdialysis probe, 4 mm; BAS Bioanalytical Systems, West Lafayette, IN) was placed in the renal cortex of both the left and right kidneys. Microdialysis probes had a membrane outer diameter of 0.5 mm and a 20,000-Da membrane cut-off. Using infusion pumps (model BSP 99; Braintree Scientific), the microdialysis probes were perfused at 2 µl/min with 0.9% saline. Preparations were allowed to rest for at least 2 h while perfusion of the probe was continued to allow adenosine levels in the interstitium to recovery from the trauma of probe insertion. Probe positioning was confirmed by visualization of the probe tip in the kidney at the end of the experiment. After the rest period, microdialysate was collected for 30 min. Next, cAMP (30 µg/kg/min) was infused into the vena cava at the level of the hepatic vein, and microdialysate was collected for another 30 min. In some kidneys, the probe perfusate, from the outset of the protocol, also contained 2.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.25 mM ,-methyleneadenosine-5'-diphosphate (AMPCP), or 2.5 mM 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX). Both cAMP and adenosine in the microdialysate were measured using a Thermo Finnigan high-pressure liquid chromatographic system coupled to a Thermo Finnigan LCQ Duo ion trap mass spectrometer equipped with an electrospray ionization source (Thermo Electron Corporation, Waltham, MA) as described recently (Jackson et al., 2006).

Protocol 5: Effects of Intravenous Infusions of cAMP on Renal Microdialysate Levels of cAMP Assessed Immediately after Probe Insertion. Insertion of a microdialysis probe into tissue stimulates adenosine production due to local tissue trauma. Therefore, it is necessary to allow the preparation to rest for at least 2 h after probe insertion to allow background adenosine production to return to normal. However, with time, the efficiency of the microdialysis process is compromised as cells and macromolecules, such as fibrin, gradually occlude the microdialysis membrane. Therefore, higher rate infusions of cAMP are necessary to achieve detectable levels of cAMP and adenosine in the microdialysate. The purpose of protocol 5 was to focus on cAMP and to determine the dose-response relationship between intravenous infusions of cAMP and cAMP in the microdialysate with sample collection occurring soon after probe insertion, and, therefore, with optimal probe efficiency. The protocol was similar to protocol 4, except that the long rest period after probe insertion was eliminated, and the rate of infusion of cAMP was reduced to 3 and 10 µg/kg/min, rather than 30 µg/kg/min. This protocol involved 20 rats (40 kidneys).

Protocol 6: Effects of Intraportal Infusions of Glucagon on the Urinary Excretion of cAMP and Adenosine. Rats (n = 10) were prepared similarly as described for protocol 1 with the exception that, rather than a jugular infusion, a 32-gauge needle was inserted into the portal vein and received an infusion (50 µl/min) of 0.9% saline. After at least a 1-h rest period, parameters were recorded in all animals during six 20-min urine collection periods during which the animals received increasing doses of glucagon (0, 0.05, 0.15, 0.5, 1.5, and 5 µg/kg/min) infused into the portal vein. Before infusing glucagon and after the highest dose of glucagon, a 1-ml blood sample (from carotid artery) was rapidly drawn into a syringe containing 1 ml of ice-cold stopping solution [containing 6 µg of erythro-9-(2-hydroxy-3-nonyl)adenine, 3 µg of EDTA, and 0.1 mg of dipyridamole]. Urinary and plasma adenosine and cAMP were measured by HPLC with fluorescence detection as described previously (Jackson et al., 1996).

Protocol 7: Effects of Intraportal and Intravenous Infusions of Glucagon on Renal Microdialysate Levels of cAMP and Adenosine. Rats (n = 18) were anesthetized, body temperature was monitored and maintained, respiration was protected, and MABP and RBF were measured as described in protocol 1. A PE-50 cannula was inserted into the jugular vein for infusion of 0.9% saline at 25 µl/min, and a 32-gauge needle was inserted into both the portal vein and the vena cava (at the level of the hepatic vein), and then an infusion of 0.9% saline was initiated at 25 µl/min in each. A microdialysis probe (same type as described in protocol 4) was placed in the renal cortex of the left kidney, and the probe was perfused as described in protocol 4. As in protocol 4, the preparations were allowed to rest for at least 2 h while perfusion of the probe was continued to allow adenosine levels in the interstitium to recovery from the trauma of probe insertion. After the rest period, microdialysate was collected for five 30-min experimental periods. In six animals, only saline was administered into the portal vein and vena cava throughout the five experimental periods. In the other 12 animals, saline was administration into the portal vein and vena cava during the first period; however, during subsequent periods, glucagon at 1.5 µg/kg/min was infused either into the vena cava at the level of the hepatic vein (six animals) or into the portal vein (six animals). Both cAMP and adenosine in the microdialysate were measured as described above.

Protocol 8: Effects of Intraportal Infusions of Glucagon on Renal Microdialysate Levels of cAMP, AMP, Adenosine, and Inosine in the Absence and Presence of Inhibitors of cAMP-Adenosine Pathway. Rats (n = 36) were anesthetized, body temperature was monitored and maintained, respiration was protected, and MABP was measured as described in protocol 1. A 32-gauge needle was inserted into the portal vein, and an infusion of 0.9% saline was initiated at 50 µl/min. A microdialysis probe (same type as described in protocol 4) was placed in the renal cortex of one or both kidneys, and it was perfused with 0.9% saline at 2 µl/min. In some kidneys, the probe perfusate, from the outset of the protocol, also contained 2.5 mM IBMX, 0.25 mM AMPCP, or 2.5 mM DPSPX. The preparations were allowed to rest for at least 2 h while perfusion of the probe was continued to allow adenosine levels in the interstitium to recovery from the trauma of probe insertion. After the rest period, microdialysate was collected for two 30-min experimental periods. Saline was administration into the portal vein during the first period, and during the second period glucagon at 1.5 µg/kg/min was infused into the portal vein. Purines, including cAMP, AMP, adenosine, and inosine, were measured in the microdialysate using a Thermo Finnigan high-pressure liquid chromatographic system coupled to a Thermo Finnigan LCQ Duo ion trap mass spectrometer equipped with an electrospray ionization source as described recently (Jackson et al., 2006).

Protocol 9: Effects of an Oral Glucose Load on Urinary Excretion of cAMP and Adenosine and on Plasma Levels of Glucagon in ZSF1 Rats. ZSF1 rats (n = 14) were prepared similarly as described for protocol 1. In addition, the protocol was similar to protocol 1, except that the stimulus was oral administration of saline (4 ml/kg; n = 7) or glucose (2 g/kg/4 ml; n = 7), rather than an intravenous infusion of cAMP. Furthermore, blood samples were taken during each of the three periods for analysis of plasma glucose (Precision Q-I-D Blood Glucose Test Strips kit; Medisense, Inc., Bedford, MA) and plasma levels of glucagon (Glucagon RIA kit; Linco Research, Inc., St. Charles, MO). Urinary adenosine and cAMP were measured by HPLC with fluorescence detection as described previously (Jackson et al., 1996).

Statistics. Data were analyzed by repeated measures one-factor and two-factor analysis of variance followed by a Fisher's least significant difference test or by a paired Student's t test as appropriate. The criterion of significance was P < 0.05. All values in text and figures are means ± S.E.M.

	Results

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Protocol 1: Effects of Intravenous Infusions of cAMP on the Urinary Excretion of Adenosine. If circulating cAMP is converted to adenosine in the kidneys, then intravenous infusions of cAMP should increase the renal production of adenosine. To test this prediction, cAMP was infused intravenously while urinary adenosine, as an index of kidney adenosine production, was measured. As shown in Fig. 1 (top), intravenous infusions of cAMP at 100 µg/kg/min but not 30 µg/kg/min significantly increased urinary adenosine excretion approximately 3-fold without altering MABP or RBF (Table 1). Although rats used in protocols 1 to 8 were genetically normotensive, MABPs were somewhat elevated because the animals were older (approximately 18 weeks), they were invasively instrumented, and they were volume expanded due to the high rate of saline infusion. Absolute RBFs also were high due to the large size of the animals used in these protocols (approximately 600 g).

View larger version (32K): [in this window] [in a new window]   Fig. 1. Bar graphs illustrate the effects of intravenous (IV; top) and intrarenal artery (IRA; bottom) infusions of cAMP (30 and 100 µg/kg/min) on the urinary excretion rate of adenosine. a, indicates significantly different from basal. Values represent means ± S.E.M.

Protocol 2: Effects of Intrarenal Artery Infusions of cAMP on the Urinary Excretion of Adenosine. In protocol 1, cAMP was infused intravenously to mimic the release of cAMP by the liver. It was possible, however, that the increased urinary adenosine excretion was due to systemic (nonrenal) conversion of cAMP to adenosine followed by delivery of circulating adenosine to the kidney. If, however, intravenous cAMP elevated renal biosynthesis of adenosine by delivering cAMP to the kidney, then direct infusions of cAMP into the renal artery should also increase the urinary excretion of adenosine and at lower doses compared with intravenous infusions. As shown in Fig. 1 (bottom), intrarenal artery infusions of cAMP at both 30 and 100 µg/kg/min significantly increased urinary adenosine excretion, with the lower dose increasing urinary adenosine excretion approximately 50% and without altering MABP or RBF (Table 1). RBFs in this protocol were higher than those in the other protocols, because to prepare the renal artery for insertion of the needle, the artery was thoroughly isolated and cleaned. This procedure denervated the kidney, interrupted renal sympathetic tone, and thereby increased RBF.

Protocol 3: Effects of Intravenous Infusions of cAMP on the Urinary Excretion of Adenosine and cAMP in Rats Pretreated with Probenecid to Block Renal Tubular cAMP Transport. The increased urinary excretion of adenosine in response to cAMP infusions is most probably due to conversion of cAMP to adenosine in the lumen of the renal tubules. For this to occur, cAMP must first enter the tubular lumen either by glomerular filtration or by secretion via the organic acid secretory mechanism in the proximal tubule. If tubular secretion is involved, blocking the secretion of cAMP into the tubules with an inhibitor of cAMP transport should attenuate the urinary excretion of both cAMP and adenosine. Figure 2 illustrates the effects of intravenously administered cAMP on urinary cAMP and adenosine excretion in the absence and presence of probenecid (10 mg/kg bolus followed by an intravenous infusion of 0.1 mg/kg/min), an inhibitor of epithelial cAMP transport in the kidney (Gogel et al., 1983). Consistent with the findings of protocol 1, an intravenous infusion of cAMP at 10 µg/kg/min did not elevate urinary adenosine excretion in the absence of probenecid. In contrast, this same dose of cAMP approximately doubled urinary adenosine excretion in animals pretreated with probenecid. cAMP at 10 µg/kg/min did not elevate urinary cAMP excretion in the absence of probenecid; yet, it increased urinary cAMP excretion by approximately 8-fold in rats pretreated with probenecid. There was a statistically significant interaction between cAMP and probenecid with regard to both cAMP and adenosine urinary excretion. These data indicate that presentation of cAMP to the tubular lumen is mainly by filtration, rather than by secretion, and that inhibition of cAMP transport increases, rather than decreases, cAMP in the tubular lumen. cAMP infusions did not affect MABP or RBF either in the absence or presence of probenecid (Table 1).

View larger version (38K): [in this window] [in a new window]   Fig. 2. Bar graphs illustrate the effects of intravenous (IV) infusions of 30 µg/kg/min cAMP on the urinary excretion of cAMP (top) and adenosine (bottom) in the absence (left) and presence (right) of probenecid (10 mg/kg + 0.1 mg/kg/min). a, indicates significantly different from basal. Values represent means ± S.E.M. The P values are for interaction term in two-factor analysis of variance.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Bar graphs illustrate effects of intravenous infusions of 30 µg/kg/min cAMP on levels of cAMP in the renal microdialysate in the absence (top, left) and presence of IBMX (2.5 mM; top, right), DPSPX (2.5 mM; bottom, left) or AMPCP (0.25 mM; bottom, right) added to the probe perfusate. a, indicates significantly different from basal. b, values represent means ± S.E.M.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Bar graphs illustrate effects of intravenous infusions of 30 µg/kg/min cAMP on levels of adenosine in the renal microdialysate in the absence (top, left) and presence of IBMX (2.5 mM; top, right), DPSPX (2.5 mM; bottom, left), or AMPCP (0.25 mM; bottom, right) added to the probe perfusate. a, indicates significantly different from basal. Values represent means ± S.E.M.

Protocol 5: Effects of Intravenous Infusions of cAMP on Renal Microdialysate Levels of cAMP Assessed Immediately after Probe Insertion. The renal microdialysis experiments in protocol 4 were performed several hours after probe insertion to allow adenosine levels to return to baseline following tissue injury. During this time interval, probe efficiency decreased as cells and macromolecules occluded the dialysis membrane, thus underestimating the increases in purines in the interstitial space. To examine with more fidelity the relationship between circulating cAMP and renal interstitial cAMP, we measured microdialysate levels of cAMP immediately after probe insertion and during low infusion rates of cAMP. As shown in Fig. 5, an intravenous infusion of only 3 µg/kg/min cAMP into the vena cava at the level of the hepatic vein increased renal microdialysate levels by 4-fold, and an intravenous infusion of 10 µg/kg/min increased renal microdialysate levels 13-fold. These data demonstrate that very low infusion rates of cAMP into the systemic circulation can cause large increases of cAMP in the renal interstitial compartment. MABP was not affected by the cAMP infusion (Table 1).

View larger version (28K): [in this window] [in a new window]   Fig. 5. Bar graph illustrate effect of intravenous infusions of 3 and 10 µg/kg/min cAMP on levels of cAMP in the renal microdialysate assessed immediately after probe insertion. a, indicates significantly different from 0 µg/kg/min dose (basal). Values represent means ± S.E.M.

Protocol 6: Effects of Intraportal Infusions of Glucagon on the Urinary Excretion of cAMP and Adenosine. Our hypothesis predicts that intraportal glucagon should stimulate renal urinary excretion of both cAMP and adenosine. As shown in Fig. 6, intraportal infusions of glucagon caused a dose-related increase in urinary cAMP and urinary adenosine, with the highest dose of glucagon increasing urinary cAMP 9-fold and urinary adenosine 3-fold. Intraportal infusions of 5 µg/kg/min glucagon increased plasma levels of cAMP approximately 10-fold, yet they had no effect on plasma levels of adenosine (Fig. 7). This indicates that the increase in urinary adenosine by intraportal glucagon was not secondary to increases in plasma levels of adenosine, but rather it was most probably due to increases in circulating cAMP. Intraportal glucagon infusions did not affect RBF, but they did cause a modest dose-related decrease in MABP (Table 2).

View larger version (20K): [in this window] [in a new window]   Fig. 6. Bar graphs illustrate effects of intraportal infusions of 0.05 to 5 µg/kg/min glucagon on urinary excretion of cAMP (top) and adenosine (bottom). a, indicates significantly different from 0 µg/kg/min dose (basal). Values represent means ± S.E.M.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Bar graphs demonstrate effects of intraportal infusions of 5 µg/kg/min glucagon on plasma levels of cAMP (top) and adenosine (bottom). a, indicates significantly different from 0 µg/kg/min dose (basal). Values represent means ± S.E.M.

Protocol 7: Effects of Intraportal versus Intravenous Infusions of Glucagon on Renal Microdialysate Levels of cAMP and Adenosine. In addition to increasing urinary cAMP and adenosine, our hypothesis predicts that intraportal, but not intravenous, glucagon should also increase renal interstitial levels of cAMP and adenosine. In rats receiving only saline administration during five 20-min experimental periods, levels of cAMP and adenosine in renal cortical microdialysate remained stable (Fig. 8). Intraportal, but not intravenous, infusions of glucagon were associated with significant increases in both cAMP and adenosine levels in the renal cortical microdialysate, with changes in cAMP occurring first followed by increases in adenosine (Fig. 8). Intraportal and intravenous glucagon infusions did not affect RBF, but they did cause a modest and sustained decrease in MABP (Table 2).

View larger version (20K): [in this window] [in a new window]   Fig. 8. Line graphs illustrate effects of 1.5 µg/kg/min glucagon infusions either into the vena cava at level of hepatic vein or into the portal vein on renal microdialysate levels of cAMP (top) and adenosine (bottom). Time/vehicle control rats received only saline infusions for all five experimental periods. a, indicates significantly different from the first experimental period before glucagon was infused. Values represent means ± S.E.M.

Protocol 8: Effects of Intraportal Infusions of Glucagon on Renal Microdialysate Levels of cAMP, AMP, Adenosine, and Inosine in the Absence and Presence of Inhibitors of cAMP-Adenosine Pathway. The role of the cAMP-adenosine pathway in the formation of renal interstitial adenosine during intraportal infusions of glucagon was tested by delivering inhibitors of the pathway directly into the renal cortex via the inflow of a microdialysis probe. Intraportal infusions of glucagon were associated with a significant increase in renal microdialysate levels of cAMP (Fig. 9), AMP (Fig. 10), adenosine (Fig. 11), and inosine (metabolite of adenosine; Fig. 12). Neither IBMX, DPSPX, nor AMPCP added to the probe perfusate blocked the ability of glucagon to increase microdialysate levels of cAMP (Fig. 9). With regard to AMP (Fig. 10), both IBMX and DPSPX blocked glucagon-induced increases in microdialysate levels of AMP, whereas AMPCP enhanced glucagon-induced increases in microdialysate levels of AMP. IBMX, DPSPX, and AMPCP blocked glucagon-induced increases in microdialysate levels of both adenosine (Fig. 11) and inosine (Fig. 12). Intraportal glucagon infusions caused a modest decrease in MABP (Table 2).

View larger version (28K): [in this window] [in a new window]   Fig. 9. Bar graphs illustrate effects of intraportal infusions of 1.5 µg/kg/min glucagon on levels of cAMP in the renal microdialysate in the absence (top, left) and presence of IBMX (2.5 mM; top, right), DPSPX (2.5 mM; bottom, left), or AMPCP (0.25 mM; bottom, right) added to the probe perfusate. a, indicates significantly different from basal. Values represent means ± S.E.M.

View larger version (26K): [in this window] [in a new window]   Fig. 10. Bar graphs illustrate effects of intraportal infusions of 1.5 µg/kg/min glucagon on levels of AMP in the renal microdialysate in the absence (top, left) and presence of IBMX (2.5 mM; top, right), DPSPX (2.5 mM; bottom, left) or AMPCP (0.25 mM; bottom, right) added to the probe perfusate. a, indicates significantly different from basal. Values represent means ± S.E.M.

View larger version (31K): [in this window] [in a new window]   Fig. 11. Bar graphs illustrate effects of intraportal infusions of glucagon (1.5 µg/kg/min) on levels of adenosine in the renal microdialysate in the absence (top, left) and presence of IBMX (2.5 mM; top, right), DPSPX (2.5 mM; bottom, left) or AMPCP (0.25 mM; bottom, right) added to the probe perfusate. a, indicates significantly different from basal. Values represent means ± S.E.M.

View larger version (26K): [in this window] [in a new window]   Fig. 12. Bar graphs illustrate effects of intraportal infusions of 1.5 µg/kg/min glucagon on levels of inosine in the renal microdialysate in the absence (top, left) and presence of IBMX (2.5 mM; top, right), DPSPX (2.5 mM; bottom, left), or AMPCP (0.25 mM; bottom, right) added to the probe perfusate. a, indicates significantly different from basal. Values represent means ± S.E.M.

Protocol 9: Effects of an Oral Glucose Load on Urinary Excretion of cAMP and Adenosine and on Plasma Levels of Glucagon in ZSF1 Rats. In animals and humans with the metabolic syndrome, oral glucose stimulates, rather than inhibits, glucagon release from the pancreas (Laube et al., 1974; Iannello et al., 1998; Velliquette et al., 2002). To test the role of endogenous glucagon on renal adenosine production, glucose was administered to ZSF1 rats with the metabolic syndrome (Tofovic and Jackson, 2003). In ZSF1 rats, oral administration of 2 ml/kg saline did not influence the urinary excretion rate of cAMP or adenosine, and it did not change plasma levels of glucagon (Fig. 13). In contrast, oral administration of 2 g/kg/4 ml glucose increased both urinary cAMP and adenosine, and it also increased plasma levels of glucagon (Fig. 13). Oral administration of glucose did not affect MABP or RBF (Table 2). Baseline MABP, however, was elevated in these ZSF1 rats, because these animals are genetically related to spontaneously hypertensive rats and express hypertensive genes (Tofovic and Jackson, 2003). Basal plasma glucose levels were high: 167 ± 15 and 178 ± 7 mg/dl during the basal period for the animals to receive saline and glucose, respectively. Oral glucose increased plasma glucose levels to 186 ± 9 and 202 ± 13 mg/dl during the second and third 20-min experimental periods, respectively, after administration of glucose. In contrast, in the group that received oral saline, plasma glucose levels were 159 ± 14 and 171 ± 15 mg/dl during the second and third 20-min experimental periods, respectively, after administration of saline.

View larger version (23K): [in this window] [in a new window]   Fig. 13. Line graphs illustrate effects of oral administration of 4 ml/kg saline (left) or 2 g/kg/4 ml glucose (right) on the urinary excretion rate of cAMP (top), urinary excretion rate of adenosine (middle), or plasma levels of glucagon (bottom) in rats with the metabolic syndrome (ZSF1 rats). a, indicates significantly different from basal. Values represent means ± S.E.M.

	Discussion

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Consistent with the pancreatohepatorenal cAMP-adenosine mechanism, intravenous and intrarenal artery cAMP increases the urinary excretion of adenosine. Lower doses of cAMP administered directly into the renal artery, compared with doses administered intravenously, cause a significant increase in urinary adenosine excretion. This would be expected if cAMP is being converted to adenosine primarily in the kidney, rather than in other organ systems.

Increases in urinary adenosine following systemic administration of cAMP are probably due to conversion of cAMP to adenosine in tubular lumens, because apical membranes of proximal tubules (Jackson et al., 2006) and collecting ducts (Jackson et al., 2003) are known to convert cAMP to adenosine via the cAMP-adenosine pathway. cAMP in the renal arterial blood could be delivered into the tubular lumen either by filtration or secretion by proximal tubular epithelial cells. Administration of probenecid, an inhibitor of cAMP transport in the kidney (Gogel et al., 1983), does not attenuate the relationship between cAMP dose and the urinary excretion rate of either cAMP or adenosine. This suggests that glomerular filtration, rather than tubular secretion, is the primary mechanism by which cAMP reaches the tubular lumen for conversion to adenosine. Probenecid increases, rather than decreases, the appearance of cAMP and adenosine in the urine following systemic administration of cAMP, suggesting that when organic acid transport systems are blocked, either higher plasma levels of cAMP are achieved with a given infusion rate of cAMP or more of the filtered cAMP is retained in the tubular lumen.

Blood-borne cAMP probably reaches the renal interstitial space and is there converted to adenosine. The endothelium of the afferent arterial is fenestrated, which allows for flow of fluid from afferent arterioles into the renal interstitium (Rosivall et al., 2006). In addition, there is probably diffusion of cAMP out of peritubular capillaries into the renal interstitium. Once in the renal interstitium, cAMP could be converted to adenosine by vascular, mesangial, or proximal tubular cells. Indeed, studies demonstrate metabolism of cAMP to AMP and adenosine by preglomerular vascular smooth muscle cells (Jackson et al., 1997), preglomerular microvessels (Jackson and Mi, 2000), mesangial cells (Dubey et al., 1997), and proximal tubules (Jackson et al., 2006). In support of this concept, the present study demonstrates that intravenous infusions of cAMP increase renal interstitial levels of cAMP and adenosine (as assessed by renal microdialysis). Moreover, local administration (via the microdialysis probe) of inhibitors of phosphodiesterase (IBMX) or ectophosphodiesterase (DPSPX) enhance renal interstitial levels of cAMP during intravenous infusions of cAMP, suggesting local interstitial metabolism of cAMP by ectophosphodiesterase. In addition, local administration of inhibitors of phosphodiesterase, ectophosphodiesterase, or ecto-5'-nucleotidase (AMPCP) block the increase in renal interstitial adenosine induced by intravenous cAMP, suggesting that the increase levels of adenosine are mediated by conversion of cAMP to AMP and hence to adenosine.

The present study underestimates the relationship between systemic delivery of cAMP and renal interstitial levels of cAMP and adenosine. Insertion of a microdialysis probe into kidneys perturbs the cells at the local insertion site, thus generating adenosine. It is necessary to allow several hours for adenosine levels to recover before conducting an experiment that focuses on adenosine measurements; however, during this time the microdialysis probe becomes less efficient as cells and macromolecules clog the dialysis membrane. Indeed, the present study demonstrates that 1/10 as much cAMP infused intravenously just after probe insertion causes a similar increase in renal microdialysate levels of cAMP compared with 10 times higher intravenous infusions of cAMP given several hours after probe insertion. Although renal microdialysis is a valuable technique to assess intrarenal levels of purines, the results will underestimate the magnitude of changes that the pancreatohepatorenal cAMP-adenosine mechanism can generate in the renal interstitium.

The pancreatohepatorenal cAMP-adenosine mechanism proposes that secretion of glucagon into the portal circulation provides a stimulus for hepatic cAMP production. The venous drainage of the pancreas enters the portal circulation, an anatomical arrangement that maximizes hepatic concentrations of pancreatic hormones. Glucagon is a pancreatic hormone secreted into the portal circulation in response to dietary factors, metabolic status, and activation of the autonomic nervous system (Sperling, 1979). When released into the portal vein, glucagon reaches the hepatic sinusoids before any metabolic clearance of glucagon can occur. Thus, the effective concentration of pancreatic glucagon in the hepatic sinusoids is maximized. However, because both the liver (Polonsky et al., 1983) and the lungs (Geddes et al., 1979) contribute importantly to the metabolic clearance of glucagon, a significant percentage of glucagon released into the portal vein is removed by the liver and lungs before reaching the systemic circulation. Therefore, glucagon is substantially cleared and diluted by the time it reaches target tissues outside the liver. Finally, the high clearance rate of glucagon (Houslay, 1986) ensures that accumulation of glucagon in the systemic circulation is minimal. For these reasons, pancreatic release of glucagon would have a much more profound effect on hepatocytes compared with other target tissues. It is noteworthy that glucagon is a powerful stimulant of hepatic adenylyl cyclase (Broadus et al., 1970; Houslay, 1986), an observation that takes on particular significance in view of the possible role of intracellular cAMP as a substrate for the extracellular production of adenosine.

In the present study, intraportal infusions of glucagon caused dosed-related increases in the urinary excretion of cAMP and adenosine. It is noteworthy that glucagon increased plasma cAMP levels 10-fold, but it did not increase plasma levels of adenosine. This suggests that the ability of glucagon to increase the urinary excretion of adenosine is not due to augmentation of adenosine delivery to the kidney but rather to increased delivery of an adenosine precursor to the kidney. In addition to increasing urinary excretion of cAMP and adenosine, our studies demonstrate that intraportal, but not intravenous, infusions of glucagon augment renal interstitial levels of cAMP and adenosine. These data indicate that release of endogenous cAMP not only increases urinary cAMP and adenosine excretion but also raises renal interstitial cAMP and adenosine. The fact that intraportal, but not intravenous, infusions of glucagon increase renal interstitial cAMP and adenosine indicates the importance of secretion of glucagon directly into the portal circulation where concentrated glucagon can optimally drive hepatic cAMP production. Our experiments also rule out a direct effect of glucagon on renal cAMP and adenosine production, indicating the essential role of the liver in the mechanism.

Intraportal glucagon also increases renal interstitial levels AMP. The observed increases in interstitial levels of AMP are predictable from the cAMP-adenosine pathway hypothesis. Predictable also is the fact that blockade of phosphodiesterase and ectophosphodiesterase inhibits glucagon-induced increases in AMP and adenosine, but it leaves unaffected glucagon-mediated changes in renal interstitial levels of cAMP. Also as expected from the hypothesis, inhibition of ecto-5'-nucleotidase blocks the glucagon-induced increase in renal interstitial adenosine, but it tends to augment glucagon-mediated increases in AMP. Changes in inosine tracked changes in adenosine, as would be anticipated because inosine is a "downstream" metabolite of adenosine.

An important issue is whether release of endogenous glucagon can increase the renal production of adenosine. Generally, glucagon secretion by the pancreas is inhibited by hyperglycemia and stimulated by hypoglycemia. However, in animals and humans with the metabolic syndrome, glucagon secretion is augmented, rather than decreased, by an oral glucose load (Laube et al., 1974; Iannello et al., 1998; Velliquette et al., 2002). In the present study, we demonstrate that oral administration of glucose to rats with the metabolic syndrome increases glucagon levels, and this increase is accompanied by a significant increase in urinary excretion of both cAMP and adenosine. These results are consistent with a physiologically functioning pancreatohepatorenal cAMP-adenosine mechanism.

Glucagon is a hormone released in response to severe physiological stresses such as hypoglycemia and intense exercise (Sperling, 1979). Under such stressful conditions, increases in renal interstitial levels of adenosine by glucagon would be adaptive, because adenosine reduces glomerular filtration rate and increases reabsorption of glucose and sodium (Vallon et al., 2006). The reduction in glomerular filtration rate would reduce renal energy demand and preserve energy for other tissues in need of energy, and the increase in reabsorption of glucose and sodium would provide more glucose for tissues and cause volume expansion to defend the cardiovascular system.

The metabolic syndrome, characterized by obesity, hypertension, insulin resistance, and hyperlipidemia, contributes to heart, vascular, and kidney disease. Glucose loading normally decreases circulating glucagon (Taborsky et al., 1998). However, in obese Koletsky rats, an animal model of the metabolic syndrome, an oral glucose load increases circulating glucagon severalfold (Velliquette et al., 2002). Moreover, in obese hyperglycemic mice, a glucose challenge causes a paradoxical increase, rather than the expected decrease, in glucagon release (Laube et al., 1974). Although normal human subjects show a fall in glucagon levels in response to oral glucose, in human obese, type 2 diabetic subjects, an oral glucose increases plasma glucagon levels (Iannello et al., 1998). A₁ adenosine receptors in the renal cortex mediate vasoconstriction of the preglomerular microcirculation and augment sodium reabsorption in proximal tubules (Vallon et al., 2006). Therefore, if carbohydrate intake stimulates adenosine production in the renal cortex of patients with the metabolic syndrome via the pancreatohepatorenal cAMP-adenosine mechanism, this could contribute to the etiology of hypertension. However, this inference remains speculative at present.

	Footnotes

 
This study was supported by National Institutes of Health Grants HL69846 and DK68575.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.119164.

ABBREVIATIONS: PE, polyethylene; MABP, mean arterial blood pressure; RBF, renal blood flow; HPLC, high-pressure liquid chromatography; IBMX, 3-isobutyl-1-methylxanthine; AMPCP, ,-methyleneadenosine-5'-diphosphate; DPSPX, 1,3-dipropyl-8-p-sulfophenylxanthine.

Address correspondence to: Dr. Edwin K. Jackson, Departments of Medicine and Pharmacology, Center for Clinical Pharmacology, University of Pittsburgh School of Medicine, 100 Technology Dr., Suite 450, Pittsburgh, PA 15219-3130. E-mail: edj{at}pitt.edu

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