INVITED REVIEW
Role of the extracellular cAMP-adenosine pathway in renal physiology

Edwin K. Jackson1,2 and Raghvendra K. Dubey2,3

Center for Clinical Pharmacology, Departments of 1 Pharmacology and 2 Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; and 3 Department of Obstetrics and Gynecology, Clinic for Endocrinology, University Hospital Zurich, 8091 Zurich, Switzerland


    ABSTRACT
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Adenosine exerts physiologically significant receptor-mediated effects on renal function. For example, adenosine participates in the regulation of preglomerular and postglomerular vascular resistances, glomerular filtration rate, renin release, epithelial transport, intrarenal inflammation, and growth of mesangial and vascular smooth muscle cells. It is important, therefore, to understand the mechanisms that generate extracellular adenosine within the kidney. In addition to three "classic" pathways of adenosine biosynthesis, contemporary studies are revealing a novel mechanism for renal adenosine production termed the "extracellular cAMP-adenosine pathway." The extracellular cAMP-adenosine pathway is defined as the egress of cAMP from cells during activation of adenylyl cyclase, followed by the extracellular conversion of cAMP to adenosine by the serial actions of ecto-phosphodiesterase and ecto-5'-nucleotidase. This mechanism of extracellular adenosine production may provide hormonal control of adenosine levels in the cell-surface biophase in which adenosine receptors reside. Tight coupling of the site of adenosine production to the site of adenosine receptors would permit a low-capacity mechanism of adenosine biosynthesis to have a large impact on adenosine receptor activation. The purposes of this review are to summarize the physiological roles of adenosine in the kidney; to describe the classic pathways of renal adenosine biosynthesis; to review the evidence for the existence of the extracellular cAMP-adenosine pathway; and to describe possible physiological roles of the extracellular cAMP-adenosine pathway, with particular emphasis on the kidney.

cAMP egress; ecto-5'-nucleotidase; phosphodiesterase; adenosine receptors; kidney; vascular smooth muscle


    PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL ROLES OF ADENOSINE IN THE KIDNEY
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REGULATION OF EXCRETORY ORGANS by adenosine receptors is a cellular strategy adopted early in vertebrate evolution. For example, primordial adenosine receptors strongly influence the rate of NaCl transport and elimination in the shark rectal gland (46), an excretory organ that evolved over 400 million years ago. It comes as no surprise, therefore, that adenosine receptors modulate many aspects of renal function in mammals (Fig. 1).


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Fig. 1.   Some of the known roles for adenosine receptors in mammalian kidneys. TGF, transforming growth factor; GFR, glomerular filtration rate; NE, norepinephrine.

Adenosine receptors are typical heptahelical G protein-coupled receptors, and four subtypes exist, i.e., A1, A2A, A2B, and A3 (for review, see Ref. 102). Details regarding the structure, genes, signal transduction mechanisms, and pharmacology of the human adenosine receptors are summarized in Table 1. A1 and A2A receptors are known to participate in renal physiology, and it is likely that A2B receptors do as well. Presently, next to nothing is known about A3 receptors in renal function; however, Western blots detect A3 receptors in preglomerular microvessels (Jackson EK, Zhu C, and Tofovic SP, unpublished observations), and future studies may yet uncover a role for A3 receptors in renal physiology.

                              
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Table 1.   Summary of adenosine receptors

Delivery of A1-receptor agonists directly into the renal interstitium reduces blood flow to both superficial and deep nephrons (1). In this regard, A1 receptor-mediated vasoconstriction in the outer cortex is most likely mediated by contraction of preglomerular, rather than postglomerular, microvessels (56, 64, 94, 95). However, in juxtaglomerular nephrons, A1 receptors cause vasoconstriction of not only preglomerular microvessels (98) but also efferent arterioles (98) and outer medullary descending vasa recta (126). In most experimental paradigms, angiotensin II strongly potentiates A1 receptor-mediated preglomerular vasoconstriction (93, 135, 139). The ability of A1 receptors to sensitize and/or directly contract preglomerular microvascular smooth muscle cells explains the mediator role of adenosine in tubuloglomerular feedback (for review, see Ref. 61); accounts for the ability of adenosine to reduce glomerular filtration rate (for review, see Ref. 60); and explains the ability of adenosine to potentiate postjunctional vasoconstrictor responses to renal sympathetic neurotransmission (54, 55).

Juxtaglomerular A1 receptors may function to restrain renin release responses, a theory called the "adenosine-brake hypothesis" (for review, see Ref. 59). In this regard, A1 receptors are negatively coupled to adenylyl cyclase via inhibitory G proteins (81, 141), and stimulation of renin release from juxtaglomerular cells by many stimuli involves activation of adenylyl cyclase (for review, see Ref. 78). Thus stimulation of renal A1 receptors inhibits renin release, and blockade of renal A1 receptors stimulates renin release (for review, see Ref. 61).

A1 receptors mediate enhancement of transport in proximal tubular epithelial cells. Activation of A1 receptors in cultured epithelial cells that express a proximal tubular phenotype increases Na+-glucose symport and Na+-phosphate symport (18). Moreover, in microperfused proximal convoluted tubules, stimulation of A1 receptors increases basolateral Na+-3HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> symport (132), and selective blockade of A1 receptors inhibits Na+-dependent phosphate transport in renal proximal tubular cells by increasing cAMP (14, 15). In intact kidneys, selective blockade of A1 receptors invariably induces a brisk natriuretic response (72, 75) that is uncoupled from tubuloglomerular feedback (140).

A1 receptors may also be involved in renal pathophysiology. Selective blockade of A1 receptors reduces the adverse renal effects of a number of nephrotoxins, including cisplatin (71, 96, 130), gentamicin (142), cephaloridine (97), glycerol (68, 106, 125), and radioconstrast media (4, 43). Also, selective A1-receptor antagonists may find utility as eukaluretic natriuretics in sodium-retaining states such as heart failure (52).

Unlike selective A1-receptor agonists, selective A2A-receptor agonists increase renal blood flow (84, 85). In this regard, A2A receptor activation in the renal microcirculation leads to vasodilation in the medulla, but not in the cortex (1). A2A receptor-mediated increases in medullary blood flow are most likely due to vasodilation of afferent and efferent arterioles of juxtaglomerular nephrons (98), as well as vasodilation of outer medullary descending vasa recta (126). Activation of A2A receptors in microvessels of juxtaglomerular nephrons (98) and in the medullary microcirculation (1) enhances medullary blood flow (146), thus altering peritubular forces that modulate sodium reabsorption. The overall result is increased excretion of NaCl (146). Consequently, in contrast to A1 receptors that directly increase NaCl reabsorption by stimulating epithelial transport, A2A receptors indirectly attenuate NaCl reabsorption by increasing renal medullary blood flow.

In vitro, A2A receptor activation strongly inhibits neutrophil-endothelial cell interactions (20), and, in vivo, A2A receptor activation attenuates neutrophil-endothelial interactions after ischemia-reperfusion injury (99). In kidneys subjected to ischemia-reperfusion injury, selective activation of A2A receptors markedly decreases the renal infiltration of neutrophils and attenuates renal dysfunction (101).

Our studies in freshly isolated preglomerular microvessels reveal that A2B receptors are expressed in this tissue (Jackson EK, Zhu C, and Tofovic SP, unpublished observations). Moreover, our studies in cultured mesangial cells (33) and vascular smooth muscle cells (29, 31, 38-40) indicate that activation of A2B receptors inhibits cellular proliferation and extracellular matrix production. Additional studies, however, are required to determine the full range of actions mediated by A2B receptors on renal structure and function.


    BIOCHEMICAL MECHANISMS OF RENAL ADENOSINE PRODUCTION: THE CLASSIC PATHWAYS
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Multiple pathways exist to produce appropriate extracellular levels of adenosine. The intracellular ATP pathway entails sequential dephosphorylation of intracellular ATP to adenosine (ATP right-arrow ADP right-arrow AMP right-arrow adenosine). Therefore, the intracellular flux through this pathway is increased when energy demand exceeds energy supply. In cells that utilize ATP at a rapid pace and/or have limited ability to accelerate the rate of ATP production, enhanced demand for ATP and/or decreased production of ATP will increase the intracellular production rate of adenosine (123). Because many cell types contain bidirectional nucleoside transporters that facilitate diffusion of adenosine across cell membranes (17), activation of the intracellular ATP pathway in cell types susceptible to energy imbalances would result in increased exposure of nearby cells to adenosine due to diffusion of intracellular adenosine into the interstitial space.

There are two noteworthy examples of this mechanism of adenosine formation in the kidney. Increased NaCl delivery to the thick ascending limb of Henle increases epithelial basolateral Na+-K+-ATPase activity in the renal medulla as more sodium enters epithelial cells from the luminal membrane. Because oxygen supply to the renal medulla is only marginally adequate (105), under these circumstances the rate of ATP utilization may exceed the rate of ATP production, leading to dephosphorylation of adenine nucleotides, i.e., adenosine formation. Indeed, maneuvers that increase the delivery of NaCl to the thick ascending limb stimulate the rate of adenosine biosynthesis in the kidneys. For example, exposure of isolated thick ascending limbs of Henle to high sodium concentrations increases adenosine production by this tubular site (9). Also, infusions of hypertonic radiocontrast agents (67) and a high NaCl intake (127, 147) increase intrarenal levels of adenosine. The intracellular ATP pathway of adenosine formation participates in the coupling of increased delivery of NaCl to the loop of Henle to increases in preglomerular vascular resistance (mediated by A1 receptors) and decreases in the vascular resistance of the vasa recta (mediated by A2A receptors). Increases in preglomerular vascular resistance limit single-nephron glomerular filtration and thereby decrease NaCl delivery to the distal nephron, i.e., tubuloglomerular feedback (for review, see Ref. 61). Decreases in the vascular resistance of the vasa recta inhibit NaCl reabsorption and thereby increase the urinary excretion of excess NaCl (146).

Another important example of the intracellular ATP pathway in the kidney is the phenomenon of reactive ischemia. In most vascular beds, reperfusion of a tissue after a brief interval of no or low blood flow causes an immediate, short-lived reduction in vascular resistance, so-called "reactive hyperemia." In contrast, the renal vascular bed responds to a brief period of ischemia by a short-lived interval of increased vascular resistance, i.e., reactive ischemia. Renal ischemia is associated with increased renal adenosine production via the intracellular ATP pathway (91, 104, 114), and evidence indicates that endogenous adenosine (mediated by A1 receptors) contributes importantly to reactive ischemia in the kidney (113, 122).

The extracellular ATP pathway is another source of renal adenosine (for reviews, see Refs. 58 and 124). This pathway is not dependent on an imbalance between energy supply and energy demand and is consequently a source of extracellular adenosine regardless of the metabolic status of the kidneys. Release of adenine nucleotides from renal sympathetic nerve terminals, intrarenal platelets, renal endothelial cells, renal vascular smooth muscle cells, and/or renal epithelial cells engages an extracellular ATP pathway in which ecto-enzymes (ecto-ATPases, ecto-ADPases, and ecto-5'-nucleotidase) convert adenine nucleotides to adenosine. In particular, endothelial cells and vascular smooth muscle cells can release adenine nucleotides in response to thrombin (110) and neutrophil-derived proteases (83) and can efficiently convert adenine nucleotides to adenosine (49, 50, 108). Presently, it is unknown whether the extracellular ATP pathway significantly contributes to the renal production of adenosine under physiological and/or pathophysiological conditions. However, the release of adenine nucleotides may contribute importantly to activation of renal P2 receptors (for reviews, see Refs. 58 and 124).

The transmethylation pathway is yet another biosynthetic route for adenosine. This pathway involves the hydrolysis of S-adenosyl-L-homocysteine to L-homocysteine and adenosine (86). Inasmuch as transfer of a methyl group from S-adenosyl-L-methionine to methyl acceptors results in the formation of S-adenosyl-L-homocysteine, the rate of transmethylation reactions determines the rate of adenosine formation via the transmethylation pathway. In this regard, the cellular requirement for transmethylation reactions is relatively constant. Thus, unlike the intracellular and extracellular ATP pathways, the transmethylation pathway most likely provides a "basal tone" of adenosine production that does not depend on "crisis events" such as ischemia, platelet activation, neutrophil attack, etc. In the heart, approximately one-third of the adenosine released to the extracellular space by cardiomyocytes is mediated by the transmethylation pathway (26). The importance of this pathway in the kidney is unknown.


    THE cAMP-ADENOSINE PATHWAY: CONCEPT AND BIOLOGICAL RATIONALE
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Inasmuch as the transmethylation pathway of adenosine production is more or less constitutive and the intracellular and extracellular ATP pathways for adenosine biosynthesis are triggered by crisis events, these three mechanisms of adenosine biosynthesis do not appear appropriate for physiological modulation of extracellular adenosine levels. On the other hand, the putative cAMP-adenosine pathway would provide a mechanism for adenosine production that would function during normal physiological conditions and would allow for hormonally mediated fine tuning of extracellular adenosine levels in the biophase of adenosine receptors.

The cAMP-adenosine pathway concept is illustrated in Fig. 2. It is postulated that adenosine biosynthesis by this pathway is triggered by activation of hormone receptors positively coupled to adenylyl cyclase via stimulatory G proteins. It is further hypothesized that the cAMP-adenosine pathway may have both intracellular and extracellular arms. The intracellular cAMP-pathway is envisaged to proceed with the metabolism of cAMP to AMP by cytosolic phosphodiesterase and with the conversion of AMP to adenosine via cytosolic 5'-nucleotidase. Adenosine formed from the intracellular cAMP-adenosine pathway would access the interstitial compartment via nucleoside transport across the cell membrane. However, from a quantitative perspective, under most circumstances the intracellular cAMP-adenosine pathway may be insignificant given the efficiency of the AMP kinase reaction. In this regard, at low concentrations, most intracellular AMP is rephosphorylated rather than dephosphorylated (73).


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Fig. 2.   Schematic representation of the cAMP-adenosine pathway. R, receptor coupled to stimulation of adenylyl cyclase (AC); Gs, stimulatory G protein; ADO, adenosine; ecto-PDE, ecto-phosphodiesterase; PDE, intracellular phosphodiesterase; ecto-5'-NT, ecto-5'-nucleotidase; NT, intracellular 5'-nucleotidase; AR, adenosine receptors; (?), molecular entity responsible for cAMP transport is unknown.

In contrast, in the extracellular compartment, dephosphorylation, rather than rephosphorylation, is the predominant fate of extracellular AMP. This is because ecto-5'-nucleotidase is an ubiquitous enzyme tethered to the extracellular face of the plasma membrane via a lipid-sugar linkage and efficiently converts AMP to adenosine (92, 109, 144). As first demonstrated by Davoren et al. in 1963 (22), hormonal activation of adenylyl cyclase causes egress of intracellular cAMP into the extracellular compartment. Therefore, if sufficient activity of ecto-phosphodiesterase exits, then hormonally induced cAMP stimulation would be expected to result in the highly localized extracellular metabolism of cAMP to AMP and hence to adenosine. This sequence of reactions is the extracellular cAMP-adenosine pathway.

Given the fact that cAMP egress was first described nearly 40 years ago in pigeon erythrocytes, it is surprising how little is known regarding the mechanism of this process. This lack of knowledge is even more unexpected when one considers the fact that cAMP egress evolved hundreds of millions of years ago and is a key survival mechanism in the humble slime mold Dicytostelium discoideum. In this regard, D. discoideum secretes cAMP into the environment, and the extracellular cAMP binds to cell-surface cAMP receptors on neighboring cells to initiate chemotaxis, leading to starvation-induced aggregation of single-celled amoebas into a migrating slug (2). Egress of cAMP has been observed not only in pigeon erythrocytes and D. discoideum but also in the rat superior cervical ganglia (19), rat glioma cells in culture (27), fibroblasts (69), perfused rat livers (79), perfused rat hearts (100), rat adipose tissue (148), and swine adipocytes (45). cAMP egress even occurs in intact human beings receiving intravenous infusions of beta -adrenoceptor agonists (6) or parathyroid hormone (65). Indeed, cAMP egress in response to activation of adenylyl cyclase is a ubiquitous phenomenon that appears to occur in most, if not all, cell types (see Ref. 8 for review).

Some properties of cAMP egress are well known. The rate of cAMP egress is related to the intracellular levels of cAMP (7, 70); occurs within minutes of activation of adenylyl cyclase (44); is mediated by an energy-dependent process (116); is temperature sensitive (13); is inhibited by the organic anion transport inhibitor probenecid (22, 118); and is partially blocked by certain prostaglandins, particularly prostaglandin A (7). The similarity of the topology of adenylyl cyclase to that of transporters suggests that adenylyl cyclase itself may be responsible for cAMP transport (74); however, to date, there are no reports that expressed adenylyl cyclase does indeed transport cAMP. Some have speculated that cAMP egress is mediated by an ATP-binding cassette protein (ABC protein) (103); however, confirmation is lacking.

Regardless of our ignorance of the molecular mechanics of cAMP egress, the fact that it exists, is robust, and is a rapid and ubiquitous process is beyond dispute. Why does such a mechanism exist in mammalian cells? Does it make teleological sense for a cell to reduce intracellular levels of cAMP by pumping it into the extracellular compartment, thus depleting the intracellular purine pool of precious resources? With scores of intracellular phosphodiesterases comprising at least nine phosphodiesterase families (129), is cAMP egress needed to control intracellular levels of cAMP? An alternative explanation is that, during evolution, cAMP egress was retained because of its usefulness as a participant in the extracellular cAMP-adenosine pathway. If so, then the extracellular cAMP-adenosine pathway must have some biological utility.

The biological utility of the extracellular cAMP-adenosine pathway is that it would give rise to local adenosine biosynthesis, and the adenosine so formed might then act in an autocrine and/or paracrine fashion to reduce, amplify, and/or enrich the local response to hormonal stimulation of adenylyl cyclase. Because adenosine would be synthesized in the unstirred water layer by spatially linked enzymatic reactions taking place on the cell surface (cAMP transport onto the cell surface, metabolism of cAMP to AMP by ecto-phosphodiesterase, and conversion of AMP to adenosine by membrane-bound ecto-5'-nucleotidase), quantitatively small increases in cAMP production could give rise to significant concentrations of adenosine in the biophase of cell-surface adenosine receptors.

Three of the four components of the putative extracellular cAMP pathway have been well characterized: adenylyl cyclase and ecto-5'-nucleotidase have been described at the molecular level, and cAMP egress has been characterized functionally. However, the possibility of biologically important ecto-phosphodiesterase has received much less attention. Nonetheless, during the past decade numerous ecto-enyzmes that metabolize purine nucleotides have been discovered and cloned, including ecto-nucleoside 5'-triphosphate diphosphohydrolases (E-NTPDases 1, 2, 3, 4, 5, and 6); ecto-nucleotide pyrophosphatases (E-NPP 1, 2, and 3); alkaline phosphatases; and NAD-glycohydrolases (see Ref. 145 for a comprehensive review). By analogy, the existence of ecto-phosphodiesterases would be expected, and, as described below, data are accumulating to support the existence of ecto-phosphodiesterases that, combined with adenylyl cyclases, cAMP transporters, and ecto-5'-nucleotidases, produce a functioning cAMP-adenosine pathway.


    EVIDENCE SUPPORTING THE EXISTENCE OF THE EXTRACELLULAR cAMP-ADENOSINE PATHWAY IN THE KIDNEY
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The extracellular cAMP-adenosine pathway as an explicitly articulated hypothesis was first proposed by Jackson in 1991 (59) as a "transmembrane negative feedback loop" mechanism in which adenosine formed from cAMP exiting from the juxtaglomerular cell was proposed to limit the renin release response to factors that stimulated renin release by activating adenylyl cyclase. However, at that time there was no direct evidence supporting the existence of an extracellular cAMP-adenosine pathway in the kidney. In 1992, Friedlander et al. (47) investigated the mechanism by which cAMP infusions caused phosphaturia. In this regard, they observed that opossum kidney cells, a model system for epithelial cells in the proximal tubule, metabolized exogenous cAMP to AMP and adenosine and that this conversion was blocked by inhibitors of phosphodiesterase and ecto-5'-nucleotidase. However, these investigators proposed that adenosine derived from cAMP produced its biological effects, in this case inhibition of phosphate transport, by being taken up by epithelial cells and acting internally rather than by acting on cell-surface receptors. This interpretation was proposed because dipyridamole, an inhibitor of adenosine uptake, blocked the ability of cAMP to inhibit sodium-dependent phosphate uptake in cell culture and phosphaturia induced by exogenous cAMP infusions in parathyroidectomized rats. Beginning in the mid-1990s, a number of publications appeared that provided direct evidence for the extracellular cAMP-adenosine pathway in the kidney (Table 2).

                              
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Table 2.   Summary of evidence supporting the existence of the extracellular cAMP-adenosine pathway in the kidney

Studies in the isolated, perfused rat renal vascular bed are consistent with the existence of the extracellular cAMP-adenosine pathway in the kidney (88). For example, cAMP added to the perfusate markedly increases the renal secretion rates of AMP, adenosine, and inosine (a metabolite of adenosine formed by deamination of adenosine by adenosine deaminase). Moreover, the conversion of exogenous cAMP to AMP, adenosine, and inosine is blocked by IBMX, a "broad-spectrum" inhibitor of phosphodiesterases that penetrates cell membranes (10), and by 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX). DPSPX, like IBMX, is a xanthine, but unlike IBMX it is restricted to the extracellular compartment due to a negative charge at physiological pH (134). At low concentrations, DPSPX is a nonselective adenosine-receptor antagonist (21), and at high concentrations DPSPX blocks ecto-phosphodiesterase (88, 143). In the perfused kidney, alpha ,beta -methyleneadenosine-5'-diphosphate (AMPCP), an inhibitor of ecto-5'-nucleotidase but not cytosolic 5'-nucleotidase (144), inhibits the conversion of cAMP to adenosine and inosine but does not inhibit the metabolism of cAMP to AMP (88).

The aforementioned data strongly support the existence of the cAMP-adenosine pathway in the kidney. Moreover, several lines of reasoning suggest that the metabolism of exogenous cAMP to adenosine occurs mostly in the extracellular compartment. First, cAMP is hydrophilic, and exogenous cAMP would not be expected to penetrate cell membranes. Second, AMPCP inhibits ecto-5'-nucleotidase, not cytosolic 5'-nucleotidase (144). Therefore, the inhibition of cAMP conversion to adenosine and inosine by AMPCP suggests an extracellular site of metabolism. Third, DPSPX is restricted to the extracellular space (134) and yet inhibits the conversion of cAMP to AMP, adenosine, and inosine. Again, this is a result most consistent with an extracellular site of cAMP conversion to AMP and adenosine. Although not ruling out the intracellular cAMP-adenosine pathway, these data support the existence of an extracellular cAMP-adenosine pathway in the kidneys.

Studies with exogenous cAMP underscore the plausibility of the extracellular cAMP-adenosine pathway but do not address whether this biochemical pathway actually functions during hormonal activation of adenylyl cyclase. Importantly, another study (89) demonstrates that isoproterenol, a beta -adrenoceptor agonist that stimulates adenylyl cyclase, increases adenosine and inosine secretion from the isolated, perfused rat kidney. This effect is inhibited by propranolol, a beta -adrenoceptor antagonist, indicating the involvement of beta -adrenoceptors in the stimulatory effects of isoproterenol on adenosine biosynthesis. Moreover, inhibition of phosphodiesterase with IBMX and inhibition of ecto-5'-nucleotidase with AMPCP also block isoproterenol-induced adenosine production in the isolated, perfused rat kidney. Taken together, these studies provide solid support for the extracellular cAMP-adenosine pathway in the kidney, at least in vitro.

In what renal tissues does the extracellular cAMP-adenosine pathway exist? In perfused kidneys, blockade of cAMP transport into the renal tubules with probenecid augments, rather than inhibits, the recovery of adenosine in the venous effluent after addition of cAMP to the perfusate (63). This suggests that exogenous cAMP is converted to adenosine in part in the vascular compartment but does not rule out an extracellular cAMP-adenosine pathway in the tubules. The conclusion that the renal vasculature supports an extracellular cAMP-adenosine pathway is confirmed by experiments in freshly isolated, preglomerular microvessels (62). In this regard, incubation of preglomerular microvessels with cAMP increases extracellular adenosine levels ~60-fold, and this effect is markedly attenuated by blockade of phosphodiesterase with IBMX or by blockade of ecto-phosphodiesterase with DPSPX. Moreover, incubation of preglomerular microvessels with isoproterenol plus IBMX increases the amount of extracellular cAMP ~30-fold, suggesting robust cAMP egress after activation of adenylyl cyclase in this tissue. This conclusion is corroborated by the linear relationship between intracellular and extracellular cAMP levels in preglomerular microvessels. As in the perfused kidney, in preglomerular microvessels isoproterenol increases the amount of extracellular adenosine, and this effect is blocked by propranolol, IBMX, and DPSPX. These results are highly consistent with the hypothesis that preglomerular microvessels transport endogenous cAMP to the extracellular compartment and metabolize extracellular cAMP to adenosine. Thus the preglomerular microvessels are at least one site in the renal vasculature that expresses the extracellular cAMP-adenosine pathway.

What cell types in the preglomerular microvessels express the cAMP-adenosine pathway? In cultured rat preglomerular vascular smooth muscle cells, addition of cAMP increases extracellular adenosine levels ~40-fold, and this effect is abolished by IBMX (63). The extracellular cAMP-adenosine pathway also exists in glomerular mesangial cells (GMCs). In this renal cell type, exogenous cAMP increases extracellular levels of adenosine by ~25-fold, and this response is inhibited by blockade of phosphodiesterase with IBMX, blockade of ecto-phosphodiesterase with DPSPX, and inhibition of ecto-5'-nucleotidase with AMPCP (32). Thus the presently available evidence indicates the existence of the extracellular cAMP-adenosine pathway in preglomerular vascular smooth muscle cells and in GMCs; however, it is important to stress that the extracellular cAMP-adenosine pathway may well exist in many other renal cell types, such as microvascular and glomerular endothelial cells, tubular epithelial cells, and interstitial fibroblasts. Additional studies are required to address the full range of renal cell types that express the extracellular cAMP-adenosine pathway.

Does the extracellular cAMP-adenosine pathway exist in vivo? IBMX is an effective inhibitor of the extracellular cAMP-adenosine pathway; however, when administered systemically at high doses, IBMX causes profound cardiovascular effects that render such experiments problematic. However, IBMX can be delivered locally into the renal cortical interstitial space via a microdialysis probe, thus achieving effective high local concentrations of IBMX without inducing confounding hemodynamic changes. Such experiments reveal that IBMX reduces by ~50% the recovery of both adenosine and inosine from the interstitial space of the renal cortex (87). These data support the hypothesis that cAMP is metabolized to adenosine and contributes substantially to the levels of adenosine in the renal cortical interstitium in vivo. Experiments in rats also demonstrate that intrarenal and intravenous infusions of cAMP and intrarenal infusions of isoproterenol increase urinary excretion rate of both cAMP and adenosine (Jackson EK and Mi Z, unpublished observations).


    EVIDENCE SUPPORTING THE EXISTENCE OF THE EXTRACELLULAR cAMP-ADENOSINE PATHWAY AT EXTRARENAL SITES
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Early studies by Gorin and Brenner (51), Smoake et al. (128), Rosenberg and Dichter (117), and Kather (66) provided hints that an extracellular cAMP-adenosine pathway may exist at extrarenal sites; however, the hypothesis was not well articulated in those early days, and evidence was scant. Beginning in the mid-1990s, however, a number of studies began to appear with a better-formulated hypothesis. A large database now supports the existence of the cAMP-adenosine hypothesis in extrarenal tissues (Table 3). Because this information has direct bearing by analogy on the existence of the extracellular cAMP-adenosine pathway in the kidney, it is reviewed below.

                              
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Table 3.   Summary of evidence supporting the existence of the extracellular cAMP-adenosine pathway at extrarenal sites

Evidence suggests that the extracellular cAMP-adenosine pathway exists at multiple extrarenal sites. In cultured rat aortic vascular smooth muscle cells (42) and cardiac fibroblasts (35), cAMP is converted to AMP, adenosine, and inosine in a time-dependent and concentration-dependent fashion. In this regard, steady-state levels of adenosine are achieved in the medium ~5 min after exogenous cAMP is added, and significant increases in extracellular adenosine levels occur with concentrations of cAMP as low as 1 µM in the bulk medium. In both cell types, the conversion of cAMP to AMP, adenosine, and inosine is blocked by IBMX and DPSPX, and the conversion of cAMP to adenosine and inosine, but not to AMP, is blocked by AMPCP. In cardiac fibroblasts, stimulation of adenylyl cyclase with either norepinephrine, isoproterenol, or forskolin increases extracellular levels of cAMP and adenosine (36), and this response is blocked by 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase. Importantly, in vascular smooth muscle cells, the first-order rate constant for the egress of cAMP is markedly increased by occupancy of A2 receptors (44). This implies that the vascular extracellular cAMP-adenosine pathway may be controlled in a positive-feedback fashion. That is, increases in extracellular adenosine by the cAMP-adenosine pathway may cause further activation of the pathway by both stimulating adenylyl cyclase and enhancing the efficiency of cAMP transport. Taken together, the aforementioned studies provide strong evidence that the cAMP-adenosine pathway exists in vascular smooth muscle cells from conduit arteries and in cardiac fibroblasts.

There is also evidence for the extracellular cAMP-adenosine pathway in the brain. In rat cerebral cortex in dissociated cell culture, isoproterenol increases extracellular levels of cAMP, and, in this experimental system, radiolabeled cAMP is converted to AMP and adenosine (117). In astrocyte-rich, but not neuron-enriched, cerebral cortex in culture, isoproterenol increases extracellular levels of cAMP and adenosine (118). Vasoactive intestinal peptide (119), norepinephrine (120), epinephrine (120), and forskolin (121) also increase extracellular cAMP and adenosine in cerebral cortical cultures. In rat cerebral cortex in dissociated cell culture, the increase in adenosine induced by activation of adenylyl cyclase is blocked by inhibition of cAMP transport with probenecid (118, 119), inhibition of phosphodiesterase with IBMX plus RO 20-1724 (118, 119) or RO 20-1724 alone (121), and inhibition of ecto-5'-nucleotidase with GMP (119, 121). In addition to the cerebral cortex, the extracellular cAMP pathway also appears to exists in the hippocampus (12). In superfused hippocampal slices, both forskolin and exogenous cAMP increase extracellular adenosine levels and induce adenosine-mediated electrophysiological effects (12). Taken together, it appears that the extracellular cAMP-adenosine pathway exists in the brain due to egress of cAMP from astrocytes. It also appears that the ecto-phosphodiesterase in the cerebral cortex is different from the renal enzyme because IBMX per se blocks the cAMP-adenosine pathway in the kidney, whereas in cerebral cortical cultures this is not the case.

The extracellular cAMP pathway not only is present in the brain parenchyma but is also expressed in the cerebral vasculature. In rats prepared with an implanted closed cranial window, suffusion of pial vessels with cAMP increases the formation of extracellular adenosine, and this response is blocked by IBMX, DPSPX, and AMPCP (57). Thus the extracellular cAMP pathway exits in microvessels outside the kidney.

In human adipocytes, catecholamines stimulate cAMP production and concomitantly increase extracellular levels of the adenosine metabolites inosine and hypoxanthine (66). Plasma membranes obtained from swine adipocytes metabolize cAMP to AMP and adenosine (143). Moreover, in swine adipocytes the plasma membrane-bound ecto-phosphodiesterase is pharmacologically distinct from the microsomal membrane-bound phosphodiesterase. In this regard, the plasma membrane ecto-phosphodiesterase is not inhibited by selective blockers of types 1, 2, 3B, 4, or 5 phosphodiesterase but is inhibited by DPSPX. In contrast, the microsomal enzyme is inhibited by blockers of type 3B phosphodiesterase (143). Thus the adipocyte appears to be yet another cell type that harbors a cAMP-adenosine pathway.

Hepatocytes may also express the extracellular cAMP-adenosine pathway. Freshly isolated hepatocytes (51, 128) and hepatocytes in culture (128) metabolize exogenous cAMP to AMP and nucleosides. This activity is blocked by aminophylline and trypsin (128), suggesting the involvement of an ecto-phosphodiesterase.

In summary, presently available data indicate that the cAMP-adenosine pathway may exist not only in renal tissues/cells but also in numerous extrarenal tissues and/or cells including aortic vascular smooth muscle cells, cardiac fibroblasts, cerebral cortical cells, hippocampal tissue, cerebral microvessels, adipocytes, and hepatocytes. Future studies will no doubt identify this pathway in many other important tissues and/or cells both within and outside the kidney.


    PHYSIOLOGICAL ROLES OF THE EXTRACELLULAR cAMP-ADENOSINE PATHWAY
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The extracellular cAMP-adenosine pathway may contribute importantly to the regulation of renin release. In juxtaglomerular cells, renin release is stimulated in response to increases in intracellular cAMP (48). Increases in intracellular cAMP result in the egress of cAMP and therefore may engage the extracellular cAMP-adenosine pathway. Inasmuch as adenosine inhibits renin release via A1 receptors (for review, see Ref. 61), the extracellular cAMP-adenosine pathway may function to provide negative-feedback control of renin release.

Multiple lines of evidence support the hypothesis that the extracellular cAMP-adenosine pathway functions to regulate renin release. First, the extracellular cAMP-adenosine pathway is expressed in preglomerular microvessels (62) and vascular smooth muscle cells cultured from preglomerular microvessels (63). Thus the pathway functions in cells from which juxtaglomerular cells are derived (53) and is likely therefore to exist in juxtaglomerular cells. Second, intrarenal infusions of cAMP (131), AMP (131), and adenosine (23, 25) inhibit renin release. Third, inhibition of A1 adenosine receptors with nonselective antagonists such as theophylline, caffeine, and DPSPX or with A1-selective antagonists such as 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and (R)-1-[(E)-3-(2-phenylpyrazolo[1,5a]- pyridin-3-yl)acryloyl]-2-piperidine ethanol (FK-453) augments renin release. For example, theophylline increases the release of renin in dogs (80, 115), in isolated rabbit afferent arterioles (16), and in isolated perfused rabbit (138) and rat (111) kidneys. Caffeine augments the renin release response caused by renal artery hypotension in dogs (24); by furosemide (107), hydralazine (134), and salt depletion (136) in rats; and by diazoxide in humans (11). DPSPX potentiates renin release in rats in response to sodium restriction (76), hydralazine (77, 134), and renal artery clipping (77). DPCPX increases renin release induced by isoproterenol in rats (112) and stimulates basal renin release in superfused juxtaglomerular cells (3), and FK-453 stimulates renin release in rats (112) and humans (5, 137).

The extracellular cAMP-adenosine pathway may function to regulate GMC proliferation and extracellular matrix production. As reviewed above, evidence suggests that GMCs express the extracellular cAMP-adenosine pathway (32). Moreover, exogenous cAMP inhibits proliferation of GMCs, and this effect is prevented by A2-receptor blockade with either DPSPX (nonselective adenosine-receptor antagonist at low concentrations) or (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF-17837; selective A2-receptor blocker) (32). Adenosine-receptor agonists with A2B-receptor activity, including adenosine, 2-chloroadenosine, 5'-N-methylcarboxamidoadenosine, and 5'-N-ethylcarboxamidoadenosine, inhibit GMC proliferation and collagen synthesis (33). In contrast, selective A1-receptor agonists such as N6-cyclopentyladenosine and selective A2A-receptor agonists such as 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) do not. Moreover, blockade of adenosine deaminase with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) or inhibition of adenosine kinase with iodotubercidin increases endogenous adenosine levels in GMCs and inhibits DNA and collagen synthesis and cell proliferation (33). The inhibitory effects of 2-chloroadenosine, EHNA, and iodotubercidin on GMC biology are reversed by DPSPX, but not by DPCPX. Taken together, these data indicate that the extracellular cAMP-adenosine pathway is expressed in GMCs and functions to restrain GMC proliferation and collagen synthesis via A2B receptors.

The extracellular cAMP-adenosine pathway may also function to regulate proliferation and extracellular matrix production by renal preglomerular vascular smooth muscle cells and extrarenal vascular smooth muscle cells. As reviewed above, evidence suggests that the extracellular cAMP-adenosine pathway is present in both of these cell types. In cultured vascular smooth muscle cells, exogenous and endogenous adenosine and adenosine analogs with A2B receptor-stimulating activity inhibit vascular smooth muscle cell proliferation as well as collagen and protein synthesis (29, 31, 38-40). In this regard, studies with a spectrum of adenosine-receptor agonists and antagonists and with antisense oligodeoxynucleotides against the A2B receptor strongly support the conclusion that the growth-inhibitory effects of adenosine on vascular smooth muscle cells are mediated by A2B receptors (29, 31, 38-40). In cultured aortic vascular smooth muscle cells, exogenous cAMP inhibits DNA synthesis, and EHNA and dipyridamole (a blocker of adenosine transport) enhance this effect (42). Both KF-17837 and DPSPX attenuate the inhibitory effects of cAMP on DNA synthesis, whereas these adenosine-receptor antagonists do not reduce the inhibitory effects of 8- bromo-cAMP (not metabolized to adenosine) on DNA synthesis (42). These results indicate that cAMP-derived adenosine can inhibit vascular smooth muscle cell growth. Hence, the extracellular cAMP-adenosine pathway may contribute importantly to the regulation of vascular biology, both within and outside the kidney.

Unlike most other vascular beds, in the kidney adenosine potentiates renovascular responses to sympathetic nerve stimulation (54, 55), most likely via activation of postjunctional A1 receptors. In the rat kidney, stimulation of renal sympathetic nerves releases adenosine and adenosine metabolites, and this response is inhibited by beta -adrenoceptor blockade (90). It is conceivable, therefore, that noradrenergic neurotransmission in the renal vasculature is augmented by the cAMP-adenosine pathway. In this regard, release of norepinephrine from sympathetic nerve terminals may activate postjunctional beta -adrenoceptors, cause the release of cAMP, and engage the cAMP-adenosine pathway to increase renovascular adenosine levels. Adenosine could then function to augment renal vascular responses to sympathetic nerve stimulation by potentiating the postjunctional response of vascular smooth muscle cells to norepinephrine.

As noted above, tubuloglomerular feedback is thought to be mediated by adenosine formed from the intracellular ATP pathway, i.e., from cytosolic 5'-nucleotidase. A benchmark study by Thomson et al. (133) demonstrates conclusively that inhibition of ecto-5'-nucleotidase with AMPCP effectively reduces the efficiency, range, and slope of tubuloglomerular feedback. Thus it is conceivable that the cAMP-adenosine pathway in some manner contributes to tubuloglomerular feedback, and this possibility needs to be investigated.

It is likely, although unproven, that the extracellular cAMP-adenosine pathway is importantly involved in the regulation of transport by renal epithelial cells, particular in proximal tubules. Activation of adenylyl cyclase increases cAMP levels in proximal tubular epithelial cells, and cAMP is well known to inhibit epithelial transport by decreasing the activity of the Na+/H+ antiporter in the luminal membrane (132) and the Na+-3HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> symporter in the basolateral membrane (132). Because proximal epithelial cells are rich in ecto-5'-nucleotidase (82), egress of cAMP into the tubular lumen during activation of adenylyl cyclase would result in considerable adenosine formation, provided that proximal epithelial cells contain ecto-phosphodiesterase. In this regard, studies by Friedlander et al. (47) demonstrate that exogenous cAMP is rapidly converted to extracellular adenosine in cultured opossum kidney cells (a cell model system with a proximal epithelial phenotype) by a mechanism that is blocked by inhibition of either ecto-5'-nucleotidase or phosphodiesterase. Thus it appears that proximal epithelial cells do express ecto-phosphodiesterase and rapidly convert extracellular cAMP to adenosine. As recently reviewed (61), activation of A1 receptors mediates enhancement of sodium transport in proximal tubules. For example, stimulation of A1 receptors in cultured epithelial cells that express a proximal tubular phenotype increases Na+-glucose symport and Na+-phosphate symport (18), and, in microperfused proximal convoluted tubules, stimulation of A1 receptors increases basolateral Na+-3HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> symport (132). This action of adenosine is most likely mediated by inhibition of adenylyl cyclase because the natriuretic-diuretic effects of A1-receptor antagonists are blocked by inhibition of inhibitory G proteins with pertussis toxin (72). Taken together, the present data suggest a negative-feedback mechanism in which activation of adenylyl cyclase inhibits proximal epithelial transport and that this effect is moderated by the following events: egress of cAMP, conversion of cAMP to adenosine, stimulation of A1 receptors, and inhibition of adenylyl cyclase. Thus the extracellular cAMP-adenosine pathway may provide a transmembrane negative-feedback loop that limits the natriuretic-diuretic response to stimulators of adenylyl cyclase. Interruption of this negative-feedback mechanism may explain in part the well-described natriuretic-diuretic response to A1-receptor antagonists (for review, see Ref. 61). Additional studies are required to confirm and extend this hypothesis.

In vascular smooth muscle cells, the cAMP-adenosine pathway may also contribute to the regulation of nitric oxide (NO) production (28). Treatment of cultured aortic vascular smooth muscle cells with adenosine, 2-chloroadenosine, and agents that elevate endogenous adenosine (EHNA and iodotubercidin) increase nitrite-nitrate (stable metabolites of NO) levels in the medium and enhance the conversion of arginine to citrulline by cytosolic extracts obtained from smooth muscle cells pretreated with these agents. In contrast, CGS-21680 and N6-cyclopentyladenosine, selective A2A- and A1-receptor agonists, respectively, do not increase NO production. The stimulatory effects of 2-chloroadenosine and EHNA plus iodotubercidin are inhibited by KF-17837 and DPSPX but not by DPCPX. Also, neither inhibition of adenylyl cyclase nor blockade of protein kinase A affects the ability of adenosine to increase NO synthesis. Incubation of smooth muscle cells with exogenous cAMP at concentrations that increase adenosine levels in the medium also increase nitrite-nitrate levels and citrulline formation, and the effects of cAMP on NO synthesis are blocked by DPSPX and KF-17837 but not by DPCPX. These findings support the concept that extracellular cAMP induces NO synthesis via conversion to adenosine and activation of A2B adenosine receptors.

Evidence suggests that the extracellular cAMP-adenosine pathway may also participate in estradiol-induced inhibition of vascular smooth muscle cell growth (37). In this regard, the inhibition by estradiol of vascular smooth muscle cell proliferation and protein and collagen synthesis is attenuated by DPSPX, KF-17837, and 2',5'-dideoxyadenosine (adenylyl cyclase inhibitor) but not by DPCPX. Also, the inhibitory effects of estradiol are enhanced by stimulation of adenylyl cyclase with forskolin and by inhibition of adenosine metabolism with EHNA plus iodotubercidin. Estradiol increases extracellular levels of cAMP and adenosine, and these effects are blocked by 2',5'-dideoxyadenosine. These results are consistent with the concept that the extracellular cAMP-adenosine pathway may contribute importantly to the effects of estradiol in vascular biology.

The extracellular cAMP-adenosine pathway may also participate, at least in some vascular beds, in the regulation of vascular tone. The best evidence in this regard is in rat pial arteries (57). Suffusion of rat pial arteries with cAMP increases the levels of extracellular adenosine on the cortical surface, an effect that is attenuated by IBMX, DPSPX, and AMPCP. Also, suffusion of rat pial arteries with cAMP and adenosine causes a concentration-dependent vasodilation. In contrast, cAMP analogs that are not metabolized to adenosine have little effect on vascular resistance of pial arteries. Moreover, the decreases in pial vascular resistance induced by cAMP, adenosine, and hypotension are significantly attenuated by 3,7-dimethyl-1-propargylxanthine (A2-receptor antagonist), IBMX, DPSPX, and AMPCP but not by 8-cyclopentyltheophylline (A1-receptor antagonist). Thus the cAMP-adenosine pathway may contribute importantly to the phenomenon of hypotension-induced cerebral autoregulation via activation of A2 receptors.

In addition to regulating vascular smooth muscle cell proliferation and extracellular matrix production, the extracellular cAMP-adenosine pathway may also modulate cardiac fibroblast proliferation and collagen production. In cultured cardiac fibroblasts, exogenous and endogenous adenosine and adenosine analogs with A2B receptor-stimulating activity inhibit cardiac fibroblast proliferation as well as collagen and protein synthesis (30, 34, 41). In this regard, studies with a spectrum of adenosine-receptor agonists and antagonists and with antisense oligodeoxynucleotides against the A2B receptor strongly support the conclusion that the growth-inhibitory effects of adenosine on cardiac fibroblasts are mediated by A2B receptors (30, 34, 41). Addition of exogenous cAMP to cardiac fibroblasts increases extracellular levels of adenosine and inosine, and these effects are attenuated by IBMX, DPSPX, and AMPCP (35). Also, in cardiac fibroblasts, exogenous cAMP inhibits DNA synthesis, cell proliferation, and protein synthesis, and antagonism of A2 receptors with KF-17837, but not A1 receptors with DPCPX, blocks the growth-inhibitory effects of exogenous cAMP but not those of 8-bromo-cAMP (35). Moreover, the growth-inhibitory effects of exogenous cAMP are enhanced by inhibition of adenosine deaminase with EHNA plus adenosine kinase with iodotubercidin (35). Stimulation of adenylyl cyclase with forskolin or isoproterenol increases cAMP biosynthesis and extracellular levels of adenosine in cardiac fibroblasts, and these effects are inhibited by blockade of adenylyl cyclase with 2',5'-dideoxyadenosine (36). Also, forskolin and isoproterenol inhibit DNA synthesis, and these effects are augmented by inhibition of adenosine deaminase with EHNA plus adenosine kinase with iodotubercidin (36). Furthermore, inhibition of adenylyl cyclase with 2',5'-dideoxyadenosine or antagonism of A2 receptors with KF-17837 prevents the effects of forskolin and isoproterenol on DNA synthesis. Forskolin inhibits protein synthesis and cell proliferation, and these effects are blocked by inhibition of adenylyl cyclase with 2',5'-dideoxyadenosine or antagonism of A2 receptors with KF-17837 (36). Taken together, these data indicate that the extracellular cAMP-adenosine pathway is expressed in cardiac fibroblasts and functions to restrain cardiac fibroblast proliferation and collagen synthesis via A2B receptors.


    UNANSWERED QUESTIONS AND FUTURE DIRECTIONS
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As discussed above, there is strong evidence for a physiological role of the extracellular cAMP-adenosine pathway in juxtaglomerular cells, GMCs, vascular smooth muscle cells, cerebral microvessels, and cardiac fibroblasts. There is also strong evidence for the existence of the extracellular cAMP-adenosine pathway in the cerebral cortex, hippocampus, adipocytes, and liver; however, whether the pathway has physiological significance in those tissues and/or cells is presently unknown and should be addressed. It is likely, although unproven, that the extracellular cAMP-adenosine pathway participates importantly in the regulation of epithelial transport, particularly in the proximal tubule. Additional research is needed to elucidate the functional significance of the extracellular cAMP-adenosine pathway in many other tissues and/or cells, both outside and within the boundaries of the kidneys.

The extracellular cAMP-adenosine pathway functions because of four molecular entities, i.e., adenylyl cyclase, cAMP transporter(s), ecto-phosphodiesterase(s), and ecto-5'-nucleotidase. Only adenylyl cyclases and ecto-5'-nucleotidase have been characterized at the molecular level. An important task for future research is to clone and characterize, at the molecular level, the cAMP transporters and ecto-phosphodiesterases that participate in the extracellular cAMP-adenosine pathway.

In conclusion, the extracellular cAMP-adenosine pathway appears to contribute to the production of extracellular adenosine and to modulate a wide range of renal and extrarenal systems. In this regard, much has been learned, yet much remains to be discovered.


    ACKNOWLEDGEMENTS

This work was supported by the National Heart, Lung, and Blood Institute Grant HL-55314.


    FOOTNOTES

Address for reprint requests and other correspondence: E. K. Jackson, Center for Clinical Pharmacology, University of Pittsburgh School of Medicine, 623 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (E-mail: edj+{at}pitt.edu).


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