It is now generally accepted that adenosine is capable of regulating a wide range of physiological functions. Nowhere is the diversity of this action better illustrated than in the kidney. When adenosine binds to plasma membrane receptors on a variety of cell types in the kidney, it stimulates functional responses that span the entire spectrum of renal physiology, including alterations in hemodynamics, hormone and neurotransmitter release, and tubular reabsorption. These responses to adenosine appear to represent a means by which the organ and its constituent cell types can regulate their metabolic demand such that it is maintained at an appropriate level for the prevailing metabolic supply. Extracellular adenosine, produced from the hydrolysis of adenosine 5'-monophosphate and stimulated by increased substrate availability and enzyme induction, acts on at least two types of cell surface receptors to stimulate or inhibit the production of cyclic adenosine-3',5'-monophosphate and also acts in some renal cells to stimulate the production of inositol phosphates and elevation of cytosolic calcium concentration. To understand when and why this complicated system becomes activated, how it interacts with other known extracellular effector systems, and ultimately how to manipulate the system to therapeutic advantage by selective agonists or antagonists, requires a detailed knowledge of renal adenosine receptors and their signaling mechanisms. The following discussion attempts to highlight our knowledge in this area, to present a modified hypothesis for adenosine as a feedback regulator of renal function, and to identify some important questions regarding the specific cellular mechanisms of adenosine in renal cell types. T he kidney, like many organs, is endowed with intrinsic mechanisms that allow it to self-regulate many of its functions. Two well-known examples are the relatively constant glomerular filtration rate (GFR) and renal blood flow over a wide range of arterial pressure (i.e., autoregulation) and the ability to release renin in response to changes in renal perfusion pressure independent of neural or humoral signals. It was the search to understand the renal mechanism responsible for this intrinsic regulation that prompted investigators to turn their interest toward adenosine. Previous reviews 1 -5 have dealt specifically with a proposed role for adenosine in the control of GFR and renin release. Although this may have prompted much of the original interest in intrarenal adenosine, recent investigations have revealed adenosine to have a much broader regulatory role. The present discussion focuses From the Departments of Physiology and Biochemistry, Michigan State University, East Lansing, Mich.

Adenosine is produced by renal tissue and has potent effects on renal blood flow and its distribution, glomerular filtration rate (GFR), and the secretion of renin. Intrarenal infusion of adenosine decreases GFR primarily by decreasing glomerular hydrostatic pressure through its effects in increasing afferent arteriolar resistance and possibly decreasing efferent arteriolar resistance. The fall in GFR due to adenosine is accompanied by little change or an increase in total organ blood flow. Regional renal blood flow during adenosine infusion is redistributed, with a greater percentage of total flow going to the juxtamedullary cortex. Intrarenal adenosine produces marked decreases in water and sodium excretion that are proportionally greater than its effect on GFR, suggesting a possible direct tubular action. Intrarenal adenosine also produces a rapid and pronounced inhibition of renin release that appears to be independent of its hemodynamic or tubular effects. A metabolic hypothesis for the control of glomerular filtration rate and renin release with adenosine acting as a mediator is considered, and criteria for establishing an intrarenal role for adenosine in the regulation of renal function are discussed.

No abstract

No abstract

RAMPs (1-3) are single transmembrane accessory proteins crucial for plasma membrane expression, which also determine receptor phenotype of various G-protein-coupled receptors. For example, adrenomedullin receptors are comprised of RAMP2 or RAMP3 (AM1R and AM2R, respectively) and calcitonin receptor-like receptor (CRLR), while a CRLR heterodimer with RAMP1 yields a calcitonin gene-related peptide receptor. The major aim of this study was to determine the role of RAMPs in receptor trafficking. We hypothesized that a PDZ type I domain present in the C terminus of RAMP3, but not in RAMP1 or RAMP2, leads to protein-protein interactions that determine receptor trafficking. Employing adenylate cyclase assays, radioligand binding, and immunofluorescence microscopy, we observed that in HEK293 cells the CRLR-RAMP complex undergoes agonist-stimulated desensitization and internalization and fails to resensitize (i.e. degradation of the receptor complex). Co-expression of N-ethylmaleimide-sensitive factor (NSF) with the CRLR-RAMP3 complex, but not CRLR-RAMP1 or CRLR-RAMP2 complex, altered receptor trafficking to a recycling pathway. Mutational analysis of RAMP3, by deletion and point mutations, indicated that the PDZ motif of RAMP3 interacts with NSF to cause the change in trafficking. The role of RAMP3 and NSF in AM2R recycling was confirmed in rat mesangial cells, where RNA interference with RAMP3 and pharmacological inhibition of NSF both resulted in a lack of receptor resensitization/recycling after agonist-stimulated desensitization. These findings provide the first functional difference between the AM1R and AM2R at the level of post-endocytic receptor trafficking. These results indicate a novel function for RAMP3 in the post-endocytic sorting of the AM-R and suggest a broader regulatory role for RAMPs in receptor trafficking.

Receptor activity-modifying proteins (RAMPs 1-3) are single transmembrane accessory proteins critical to various G-protein coupled receptors for plasma membrane expression and receptor phenotype. A functional receptor for the vasodilatory ligand, adrenomedullin (AM), is comprised of RAMP2 or RAMP3 and calcitonin receptor-like receptor (CRLR). It is now known that RAMP3 protein-protein interactions regulate the recycling of the AM2 receptor. The major aim of this study was to identify other interaction partners of RAMP3 and determine their role in CRLR-RAMP3 trafficking. Trafficking of G-protein-coupled receptors has been shown to be regulated by the Na ؉ /H ؉ exchanger regulatory factor-1 (NHERF-1), an adaptor protein containing two tandem PSD-95/Discs-large/ZO-1 homology (PDZ) domains. In HEK 293T cells expressing the AM2 receptor, the complex undergoes agonist-induced desensitization and internalization. However, in the presence of NHERF-1, although the AM receptor (CRLR/RAMP3) undergoes desensitization, the internalization of the receptor complex is blocked. Overlay assays and mutational analysis indicated that RAMP3 and NHERF-1 interact via a PDZ type I domain on NHERF-1. The internalization of the CRLR-RAMP complex was not affected by NHERF-1 when CRLR was co-expressed with RAMP1 or RAMP2. Mutation of the ezrin/radixin/moesin (ERM) domain on NHERF-1 indicated that NHERF-1 inhibits CRLR/RAMP3 complex internalization by tethering the complex to the actin cytoskeleton. When examined in a primary culture of human proximal tubule cells endogenously expressing the CRLR-RAMP3 complex and NHERF-1, the CRLR-RAMP complex desensitizes but is unable to internalize upon agonist stimulation. Knock-down of either RAMP3 or NHERF-1 by RNA interference technology enabled agonist-induced internalization of the CRLR-RAMP complex. These results, using both endogenous and overexpressed cellular models, indicate a novel function for NHERF-1 and RAMP3 in the internalization of the AM receptor and suggest additional regulatory mechanisms for receptor trafficking.The recent discovery of receptor activity-modifying proteins (RAMPs) 1 has broadened the field of G-protein-coupled receptor (GPCR) regulation. RAMPs were discovered as required accessory proteins to an orphan GPCR, now termed the calcitonin receptor-like receptor (CRLR) (1). The three RAMP isoforms (1-3) are products of three distinct genes and yield unique single transmembrane accessory proteins. RAMPs are required for the plasma membrane expression and determination of receptor phenotype for CRLR (1, 2). RAMPs have recently been found to associate with additional members of the Class II family of GPCRs (3). Co-expression of RAMP1 with CRLR yields a functional calcitonin gene-related peptide (CGRP) receptor, whereas co-expression of RAMP2 or RAMP3 with CRLR produces a receptor responsive to adrenomedullin (AM) (AM1-R and AM2-R, respectively) (4, 5). AM and CGRP are multifunctional peptides with many overlapping functions, ranging from potent vasodilation to proliferatio...

~en adenosine binds to plasma-membrane receptors on a variety of cell types in the kidney, it stimulates functional responses that span the entire spectrum of renal cellular physiology, including alterations in hemodynamics, hormone and neurotransmi tter release, and tubular reabsorption (Table 1). This array of diverse responses, appears to represent a means by which the kidney and its constituent cell types can regulate the metabolic demand such that it is maintained at an appropriate level for the prevailing metabolic supply (Figure 1). With the increased recognition of this wide array of renal cellular actions, and the continuing development of relatively specific adenosine receptor agonist and antagonist ligands, investigators have undertaken the task of assigning the different renal actions of adenosine to the known adenosine receptor types, by comparison of relative agon1st and antagonist potencies. It is apparent from the inspection of a list of the renal actions of adenosine, that not on1y does adenosine control a variety of functions but it appears to have a "dual-control" over many aspects of renal function mediated by separate receptors. This approach, while providing useful information on the action and the possible receptor subtypes leaves some questions as to the coupling to secend messenger systems, and does not provide molecular information of the subcellular events that may be involved.With the exception of their ability to respond to adenosine and adenosine analogs, nothing as yet has been described that distinguishes adenosine receptors from the wide variety of receptors that modify adenylate cyclase activity and are therefore likely members of a large class of hormone receptors that, like the visual pigment rhodopsin, are coupled to their intracellular effector systems by guanine nucleotide binding proteins. In some systems, however, it has been impossible to correlate physiological responses to adenosine with changes in levels of cAMP, and therefore, i t has been proposed that adenosine may be coupled to other signal transduction systems as well. In the kidney, several of the actions of adenosine associated with activation of the Al receptor (i. e. vasoconstriction, renin release inhibition, and inhibition of neurotransmitter release) are effects that have been proposed to be mediated by changes in cytosolic calcium (Churcbill and Churchill, 1988). Wehave recently reported in primary cultures of rabbit cortical collecting tubule cells (Arend et al., 1988) andin an established cell derived from RCCT cells (Arend et al., 1989) that in addition to the classical Al and A2 receptors coup1ed to the the inhibition and stimulation of adenylate cyclase (Arend et al., 1987), adenosine stimulates the turnover of inositol phosphates and 220

No abstract