INTRODUCTION

TOP ABSTRACT INTRODUCTION PURINOCEPTOR CLASSIFICATION P2 RECEPTOR AGONISTS AND... P2 RECEPTOR DISTRIBUTION WITHIN... RENAL VASCULAR RESPONSE TO... ADVENTITIAL DELIVERY OF P2... P2 RECEPTOR-MEDIATED CALCIUM... DIADENOSINE POLYPHOSPHATES WHAT PHYSIOLOGICAL ROLE DO... FINAL PERSPECTIVES REFERENCES

P2 RECEPTORS ARE EMERGING as important elements by which vascular responses to physiological and pathophysiological stimuli are mediated and maintained (1, 4, 20, 26, 30, 33, 64, 76, 83, 92, 94, 111, 116, 135). Development of this field of study has been slow due to the lack of selective probes with which to manipulate receptor activation and signaling. However, in recent years, significant advances have been made, in part, through the development of more selective investigational tools, such as expression cloning and knockout models (34, 70, 108, 135). Application of these approaches has clarified our understanding of the pharmacology of P2 receptors. However, determination of the specific roles P2 receptors play in mammalian physiology or pathophysiology still remains to be established.

As interest in P2 receptor physiology has grown, so has interest in the role P2 receptors play in the physiology and pathophysiology of renal function. With this renewed interest has come a new appreciation for the roles extracellular adenine nucleotides, like ATP, can play in regulating or modulating renal function. In the last 5 years, investigators have provided compelling evidence that extracellular nucleotides, working through activation of P2 receptors, have a significant impact on renal microvascular function, mesangial cell function, and renal epithelial transport. The latter concept is the focus of a companion review in the present issue of this journal (150). P2 receptor activation has been implicated in regulating preglomerular resistance and in mediating renal microvascular autoregulatory behavior. Locally released ATP has a direct paracrine and/or autocrine effect, modulating renal epithelial transporters and tubular epithelial channels to influence tubular fluid composition. Although the specific roles for extracellular nucleotides and their receptors in the kidney have not been firmly established, it is clear that locally released ATP may play a significant role in the regulation of renal hemodynamics and tubular function. The purpose of this review is to summarize the present literature pertaining to the effect of P2 receptor activation on renal microvascular function and to detail the signal transduction mechanisms involved.

	PURINOCEPTOR CLASSIFICATION

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Purinoceptors are membrane-bound receptors that are divided into two distinct families These families are summarized in Fig. 1 (1, 4, 20, 30, 56, 57, 64, 92, 116, 118, 120, 135). P1 receptors respond to adenosine and AMP and are further divided into four major subtypes, identified as A₁, A_2A, A_2B and A₃ receptors (56, 57, 89, 118, 120, 135). A₁ receptors are thought to induce vasoconstriction by inhibiting adenylate cyclase activity, thus reducing the formation of cAMP in vascular smooth muscle. A_2A and A_2B receptors evoke vasodilation by stimulating adenylate cyclase activity and increasing the formation of cAMP in vascular smooth muscle. The more recently described A₃ receptors are thought to exert their physiological effects through activation of calcium signaling pathways and perhaps inhibition of cAMP accumulation (88, 89, 135).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   P1 and P2 receptor classification and the signaling mechanisms thought to be involved. PLC, phospholipase C; GP, G proteins; R, receptor; VOC, voltage-operated calcium channel; ROC, receptor-operated calcium channel; GI, inhibitory G proteins; GS, stimulatory G proteins; Ad Cyc, adenylate cyclase.

Functional effects of A₁ and A₂ receptors have been described in the kidney and include direct actions on preglomerular and postglomerular microvascular function (2, 5, 22, 27, 36, 61, 83, 91, 98, 99, 104, 105, 109, 124, 133, 158, 172), protection from ischemic damage (37, 38, 50), endotoxic shock (115), renin secretion (66, 99, 110, 122, 124, 143, 153), and in mediating autoregulatory adjustments in preglomerular resistance (66, 121, 132, 143, 162). The role of A₃ receptors in the kidney remains unclear (107).

P2 receptors (Fig. 1) were originally described by Burnstock (18, 19) as purinergic receptors, reflecting the fact that the original description pertained to the release of ATP as a neurotransmitter from peripheral sympathetic nerves (18, 19). Later, the term "P purinergic receptor" was revised to "P purinoceptor," and the nomenclature for identification of P1 and P2 receptors was standardized to "P2 receptors" by the International Union of Pharmacology (55, 56). Presently, P2 receptors are divided into two major families, labeled P2X and P2Y (Figs. 1 and 2), based on the facts that distinct genes code for these receptors, there are marked differences in receptor structure between the two families, and unique signal transduction pathways are invoked after receptor activation (1, 4, 20, 30, 55, 56, 64, 92, 116, 135).

View larger version (35K): [in this window] [in a new window]   Fig. 2.   P2 receptor classification and the calcium signaling mechanisms thought to be involved. This scheme is compiled from several recent reviews (1, 39, 55, 116, 135). IP3, inositol 1,4,5-triphosphate.

P2X receptors (Fig. 2) comprise multiple subtypes that can form homomeric complexes or exist as heteromeric complexes that combine with other P2X receptor subtypes. The latter results in hybrid receptors that exhibit modified pharmacological, biophysical, and electrophysiological properties compared with those of each constituent monomeric form of the receptor (1, 4, 30, 48, 64, 92, 116, 135). A single P2X receptor possesses two membrane-spanning domains linked by a large extracellular loop and with the NH₂- and COOH-terminal tails exposed to the cytoplasm. These receptors function as ligand-gated channels and can be profoundly influenced by the presence of cations (48, 135, 163). For example, high concentrations of Mg²⁺ or Ca²⁺ generally inhibit P2X receptor currents, whereas cations like Zn²⁺, Cu²⁺, and Cd²⁺ can either potentiate or inhibit cation currents in a P2X receptor-specific manner (1, 4, 30, 64, 92, 116, 135). On activation by ATP, the nonselective cation channel, which is intrinsic in the receptor structure, is opened, allowing Na⁺ and Ca²⁺ to pass from the extracellular fluid to the intracellular cytoplasm (39, 42, 135). Potassium ions can also pass through this channel (39, 42, 135). Presently, there are approximately seven P2X receptor subtypes (P2X_1-7) that have been described on the basis of pharmacological assessment of receptor activity and of receptor cloning and expression studies (48, 116, 135). Investigators have also reported the cloning of the gene for P2XM and P2X₈ receptors that are derived from skeletal muscle; however, more work needs to be completed before their places in the P2X receptor family can be determined (11, 67, 112).

A splice variant of the P2X₂ receptor isolated from the rat cerebellum has also been described (16, 135, 151). This receptor differs from other P2X₂ receptors by the absence of a 69-residue sequence from the COOH terminal of the receptor protein. The role of this receptor is presently unknown. However, because of the potential physiological alterations that splicing could represent, more attention should be focused on the in vivo study of these receptors rather than relying on data obtained from reconstituted receptor models.

As mentioned above, it has recently been determined that some P2X receptors can form heteromultimers by combining with other P2X subtypes (48, 116). On the basis of experiments conducted in heterologous expression systems, receptor pairs formed from P2X₁ and P2X₅ (P2X₁/P2X₅), P2X₂ and P2X₃ (P2X₂/P2X₃), and P2X₄ and P2X₆ (P2X₄/P2X₆) receptors have been described. P2X₇ receptors do not coimmunoprecipitate with any other known P2X receptor; however, P2X₅ receptors coimmunoprecipitate with all other known P2X receptor subtypes (116). Although the physiological role of the heteromeric P2 receptors remains to be determined, the potential for adding a new level of sophistication and selectivity to the regulatory influences of P2 receptors is quite interesting, because the behavior of each heteromeric receptor is modified according to the respective pharmacological properties of the individual subunits. For example, homomeric P2X₁ receptors are activated by ,-methylene-ATP and desensitize rapidly, and P2X₅ receptors are insensitive to ,-methylene-ATP. In contrast, the P2X₁/P2X₅ heteromer exhibits a sustained current in response to ,-methylene-ATP that does not readily desensitize (116). The unique combination of properties that can be achieved through the formation of heteromeric P2X receptors confers potentially new levels of physiological or pathophysiological control that still need to be elucidated.

P2Y receptors (Fig. 2) are composed of seven transmembrane-spanning domains and are regulated by G proteins (1, 4, 32, 97, 135). In most systems, activation of P2Y receptors is associated with activation of phospholipase C and the subsequent mobilization of calcium from inositol 1,4,5-triphosphate (IP₃)-sensitive intracellular stores. ATP has also been reported to stimulate cAMP formation in some cell types through a variety of mechanisms and intermediates (32). For example, published reports indicate that P2 receptor stimulation can induce cAMP accumulation through adenosine-mediated A₂ receptor activation after ATP hydrolysis and through the autocrine actions of P2 receptor-mediated prostaglandin E₂ formation (32). The P2Y₁₁ receptor has been suggested to couple directly to both phospholipase C and adenylate cyclase in Chinese hamster ovary cells (31, 32). The mammalian family of P2Y receptors includes the P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptor subtypes (Fig. 1) on the basis of responses to pharmacological agonists and antagonists and from receptor cloning and expression studies. Other putative P2Y receptors have been described in other species or cell types; however, verification of the receptor structures as that of unique P2Y receptors remains to be established (32, 135).

	P2 RECEPTOR AGONISTS AND ANTAGONISTS

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Rapid advances in the field of P2 receptor pharmacology and physiology have been hampered by the lack of selective pharmacological tools. Ralevic and Burnstock (135) have compiled an excellent review of the tools presently in use as P2 receptor agonists and antagonists. In their description, they highlight the primary uses of these compounds, discuss the utility of these compounds against different P2 receptors, and indicate the limitations each compound presents. None of the agonists or antagonists presently in use discriminates very definitively between P2X and P2Y receptors. This leads to considerable confusion in the pharmacological characterization of P2 receptors from one investigator to the next, and from one tissue or cell type to the next. Nevertheless, some aspects of P2 receptor pharmacology can be deciphered by using these probes. Some of the most common agonists used are ,-methylene-ATP and ,-methylene-ATP, which are stable analogs of ATP. These agents are considered essentially inactive on P2Y receptors but exhibit fairly high selectivity for P2X receptors, in particular, the P2X₁ and P2X₃ receptor subtypes. In contrast, P2Y receptor-mediated responses can best be ascertained by using ADP, ADPS, and UTP, which are weakly active or nearly inactive at P2X receptors. It is essential to remember, however, that because a significant lack of receptor subtype specificity exists with some agonists, cautious interpretation of data obtained with these probes is important.

P2 receptor antagonists should also be used with careful consideration of their limitations (135). Some of the most commonly used antagonists that are reasonably selective for P2 receptors, but do not readily discriminate between P2X and P2Y subtypes, include suramin, a symmetrical 3'-urea of 8-(benzamido)naphthalene-1,3,5-trisulfonic acid that is a derivative of suramin, pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), reactive blue 2, and reactive red. Antagonists with some specificity for P2X receptors include 8, 8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis(1,3,5-naphthalene- trisulfonic acid) and pyridoxal phosphate-6-azophenyl-2',5'-disulfonic acid (iso-PPADS). The interested reader is referred to the review by Ralevic and Burnstock (135) for a more thorough discussion of these agents.

	P2 RECEPTOR DISTRIBUTION WITHIN THE KIDNEY

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There are numerous reports describing the expression and distribution of P2 receptors within the kidney or in renal tissues (3, 24-26). Although this review will not provide a comprehensive listing of these reports, it will endeavor to provide a reasonable representation of the ubiquitous expression of P2 receptors by different segments of the nephron and renal vasculature. P2 receptors are expressed on various segments of the renal vasculature (3, 10, 25, 44, 77, 101, 102, 169), microvasculature (3, 25, 61, 80, 83, 85, 171), glomeruli (17, 24, 72, 125, 126), and cells of the glomerular mesangium (59, 62, 63, 71, 73, 96, 128, 129, 131, 139, 146, 148, 155). In addition, P2 receptors have been shown to be expressed by proximal tubular epithelial cells (3, 23, 41, 90, 156, 175, 176), distal tubule cells (3, 9, 136), and loop of Henle cells and collecting duct cells (3, 8, 24, 87, 95, 123, 154, 156). For a more thorough discussion of this subject, readers are referred to the review by Schwiebert and Kishore (150) that appears in this issue of the journal. The P2X and P2Y receptor families are present on renal tissue, and some cells may express receptors from both families. For example, the vascular smooth muscle of afferent arterioles appears to express both P2X and P2Y receptors (80, 82). Investigations continue in an effort to establish the role that P2 receptors play in regulating renal vascular and tubular function. Those investigations have resulted in the emergence of many interesting possibilities for P2 receptor involvement in the regulation of renal microvascular function, glomerular hemodynamics, renal tubular function, and even in the regulation of urinary bladder function (26). The remainder of this review will focus on the renal microvascular effects of P2 receptor activation and discuss the possible role P2 receptors may play in the physiology of renal microvascular control.

	RENAL VASCULAR RESPONSE TO ATP

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ATP concentrations in human arterial and venous plasma have been measured to be ~0.19 (0.38 µM) and 0.7 µg/ml (1.38 µM), respectively (53, 54). Much of the ATP could derive from red blood cells (35), endothelial cells (12, 13, 127), or platelets (6, 54). The interaction of circulating ATP with endothelial or vascular smooth muscle cells could play an important role in the regulation of vascular tone (35, 43, 49, 97). Exposure of renal microvascular smooth muscle to extracellular nucleotides can occur by administration of ATP into the vascular lumen. Alternatively, it can occur by direct application of ATP to the exposed vascular smooth muscle from the adventitial surface. Intrarenal infusion of ATP will deliver it directly into the vascular space and will result in its interaction with the endothelium before gaining access to the underlying vascular smooth muscle cells. Interaction of ATP with endothelial P2 receptors can result in the generation of endothelium-derived vasoactive factors that can influence the renal microvascular response. Adventitial administration allows the nucleotide to interact directly with receptors on the vascular smooth muscle cells. The order of the interaction may be an important contributor to the overall renal hemodynamic response to P2 agonists.

The impact of infused ATP or P2 agonists on renal blood flow or renal perfusion pressure is dependent on a number of factors, including the species being studied, the type of agonist infused, the ambient vascular tone, and the experimental approach being used. Infusion of ATP directly into the renal artery of the isolated perfused rat kidney evokes vasoconstriction under basal tone conditions and both vasoconstriction and vasodilation when renal vascular resistance is elevated (44, 49, 167, 168). At basal tone, infused ATP or the P2X agonist ,-methylene-ATP induces a sustained concentration-dependent vasoconstriction (28, 44, 49, 167, 168); however, ATP-mediated responses observed in kidneys perfused at high tone are less consistent. Eltze and Ullrich (44) increased renal vascular resistance with norepinephrine and reported that infusion of the P2X agonist ,-methylene-ATP consistently evoked a renal vasoconstriction (Fig. 3). Infusion of low doses of ATP induced vasodilation with a vasoconstrictor response occurring only at higher infusion doses (44). Curiously, when Vargas and co-workers (167) increased renal vascular tone with phenylephrine, low-dose ATP infusion led to a renal vasoconstriction, with vasodilation only occurring in response to infusion of high doses of ATP. The reason for these conflicting observations is unclear, but the data do suggest that the renal vascular response to infused ATP may be influenced by ambient renal vascular tone.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Renal hemodynamic response to intrarenal infusion of ATP and selected P2 agonists into the isolated perfused rat kidney under raised vascular tone conditions. Data are expressed as the %change in perfusate flow in response to increasing concentrations of agonist in the perfusate fluid. The data are selected from studies performed by Eltze and Ullrich (44).

It is interesting to note that isolated segments of human and rabbit renal arteries, preconstricted with either norepinephrine or prostaglandin F₂, relaxed on intraluminal exposure to ATP (137, 138). Subsequent studies revealed that the vasodilation occurred through P2Y receptor-mediated generation of nitric oxide and through the local generation of adenosine and activation of adenosine-sensitive P1 receptors (138). In a separate study, ATP-mediated vasoconstriction of rat afferent arterioles was found to be augmented during adenosine receptor blockade (85). The potential interaction between the P2 receptor system and the adenosine-sensitive P1 receptor system has not been carefully examined; however, the close association between the two receptor families and the prevalence of both receptor families in the cardiovascular system (52, 97, 118, 119, 135, 142) provide an interesting opportunity for a local integration of coregulatory roles between P1 and P2 receptor-dependent responses.

In the isolated perfused rabbit kidney, ATP infusion leads to a modest vasoconstriction (113), whereas in the dog kidney, infusion of ATP produces vasodilation by stimulating the synthesis and release of nitric oxide (101, 102). Infusion of P2Y receptor-selective agonists, such as 2-methylthio ATP, UTP, or adenosine 5'-O-(3-thiotriphosphate) (ATPS) at concentrations up to 10 µM, also leads to nitric oxide-dependent vasodilation in isolated, perfused rat kidneys (28, 44), but greater concentrations lead to vasoconstriction (Fig. 3) (44). In addition, intrarenal infusion of 2-methylthio ATP or ATP into the isolated perfused rat kidney stimulates renin secretion (29), but similar infusion of the P2X agonist ,-methylene-ATP did not effect renin secretion (29). These data suggest that luminally delivered P2Y agonists will interact with P2 receptors expressed by intrarenal endothelial cells, resulting in a nitric oxide-dependent relaxation of the intrarenal microvasculature and a decrease in renal vascular resistance. P2Y receptor-mediated vasodilation is converted to vasoconstriction during inhibition of nitric oxide synthesis, whereas P2X receptor-mediated vasoconstrictor responses are augmented (28, 44). These data suggest that intrarenal microvascular smooth muscle and endothelium express P2X and P2Y receptors; however, the results of these studies do not reveal the specific intrarenal microvascular segments that are responsible for agonist-mediated alterations in renal vascular resistance.

	ADVENTITIAL DELIVERY OF P2 AGONISTS

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ATP released from sympathetic nerves travels through the interstitial fluid adjacent to the varicosities and binds to postsynaptic P2 receptors located on vascular smooth muscle, where it influences vascular tone (15, 21, 40, 134, 149). ATP can also be released from smooth muscle cells (127, 157), epithelial cells (60, 68, 159), and perhaps even macula densa cells (7). ATP released from such cell types would also move through the interstitial fluid space before reaching P2 receptors on vascular smooth muscle cells or other cell types to induce appropriate physiological or pathophysiological responses. In the kidney, that response may be to provide local paracrine regulation of preglomerular renal vascular resistance via activation of P2 receptors expressed by the renal microvasculature (111).

The distribution of P2X₁ receptors along the renal vasculature was addressed by Chan and co-workers (25). Autoradiographic assessment of radiolabeled (³H-labeled) ,-methylene-ATP revealed a distribution of P2 receptors along afferent arterioles and interlobular arteries, but no binding was visible along efferent arterioles. In the same study, immunohistochemical assessment for P2X₁ receptors, using an antibody selective for P2X₁ receptors, revealed strong positive staining along all segments of the preglomerular microvasculature, whereas no visible staining was evident along efferent arterioles or glomeruli (25). These data provide strong support for the expression of P2X₁ receptors by preglomerular vascular smooth muscle cells.

Figure 4 illustrates the results of experiments conducted to determine the responsiveness of each preglomerular and postglomerular segment to ATP (85). The responsiveness of each segment was assessed at four concentrations of ATP applied in succession. These data reveal that only the arteries and arterioles comprising the preglomerular renal circulation respond to ATP administration (85). Of these segments, only the afferent arteriole exhibits a sustained vasoconstriction in response to ATP concentrations below 10 µM. Arcuate and interlobular arteries respond with a transient vasoconstriction that subsides within 2-3 min of exposure. Notably, efferent arteriolar diameter remains unchanged during ATP administration. Weihprecht et al. (171) also reported that ATP concentrations below 10 µM evoked a significant vasoconstriction of isolated rabbit afferent arterioles. These data define the functional responses of the renal microvasculature to P2 receptor activation and are in excellent agreement with the distribution of P2X₁ receptors determined by immunohistochemistry and autoradiography (25).

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Average segmental diameter responses evoked by ATP applied to the adventitial surface of arcuate and interlobular arteries (A,  and , respectively) and afferent and efferent arterioles (B,  and , respectively). After the control period (CON), increasing concentrations of ATP from 0.1, 1.0, 10, and 100 µM were applied at 5-min intervals, as indicated by the dashed lines. Each protocol ended with a 5-min recovery period (REC), when the bath solution was returned to the control conditions. Each data point represents diameter measurements taken at 12-s intervals. The data were selected from a report by Inscho et al. (85).

As described above, the absence of highly selective agonists and antagonists creates a problem with regard to identification of the types of receptors expressed in a given tissue. Conventional pharmacological approaches to this problem include determination of rank order potency profiles using a variety of P2X and P2Y receptor agonists. This approach was used to determine the receptor subtypes possibly contributing to the afferent arteriolar response to P2 receptor activation, and the data for the sustained responses are illustrated in Fig. 5 (80). Administration of the P2X-selective agonists, ,-methylene-ATP or ,-methylene-ATP, yielded rapid biphasic vasoconstrictor responses consistent with the expression of P2X receptors on afferent arterioles. A typical response to ,-methylene-ATP is illustrated in Fig. 6. The afferent arteriolar diameter response to these P2X agonists began with a rapid initial vasoconstriction that gradually declined to a smaller but sustained vasoconstriction (80). The afferent arteriolar vasoconstriction elicited by the endogenous ligand, i.e., ATP, closely paralleled the response observed after administration of the P2X agonist ,-methylene-ATP (80, 171). Desensitization is a prominent characteristic of P2X₁ and P2X₃ receptors (116, 135). Accordingly, it is interesting to note the declining effectiveness of subsequent addition of higher concentrations of ,-methylene-ATP to evoke greater vasoconstriction as would be observed in response to a single application of a high concentration. These and other data (81) suggest that the response to ,-methylene-ATP quickly desensitized. Given that P2X₁ receptors are heavily expressed along the afferent arteriole (25), it is reasonable to conclude that P2X₁ receptors participate significantly in the afferent arteriolar response to ATP and P2X agonists ,-methylene-ATP and ,-methylene-ATP.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Steady-state changes in microvascular diameter in response to P2 agonist administration. Data are expressed as the %decrease in afferent arteriolar diameter compared with the control diameter. Each point represents the mean vessel diameter averaged over the last 2 min of each treatment period. A: comparison of the steady-state afferent arteriolar responses to ATP and the P2X agonists, ,-methylene-ATP (--ATP), and ,-methylene-ATP(--ATP). B: comparison of the steady-state afferent arteriolar responses to ATP and the P2Y agonists 2-methylthio ATP, UDP, UTP, and adenosine 5'-O-(3-thiotriphosphate) (ATPS). The data were selected from a report by Inscho et al. (80).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Afferent arteriolar response to ,-methylene-ATP before and during superfusion with the dihydropyridine calcium channel blocker felodipine. Each data point represents the mean vessel diameter (in µm) measured at 12-s intervals throughout the experimental period. Arterioles were superfused with control buffer during the control and recovery periods (0-5 and 10-15 min, respectively). The arterioles were exposed to 1.0 µM ,-methylene-ATP during the periods, as indicated by the thick horizontal bars. Calcium channel blockade was imposed with 10 µM felodipine beginning at the 15-min time point and was not interrupted. The period of calcium channel blockade by using felodipine is indicated by shaded area (right) and solid bar (middle right). The data were modified from the work of Inscho et al. (84).

Administration of the P2Y agonists 2-methylthio-ATP, ADP, UDP, UTP, or ATPS in the juxtamedullary nephron preparation yielded markedly different results (Fig. 5) (78, 80). In contrast to experiments performed in the isolated perfused kidney (44), 2-methylthio ATP, ADP, and UDP induced only very slight vasoconstrictions. UTP and ATPS were less potent agonists compared with ATP or the P2X agonists but, at concentrations of 10 and 100 µM, these agents elicited very large monophasic vasoconstrictions. The afferent arteriolar diameter usually closed during exposure to 100 µM concentrations of UTP and ATPS, and afferent arteriolar blood flow ceased. Removal of the agonists from the bathing medium resulted in a rapid reversal of the vasoconstriction and restoration of afferent arteriolar blood flow (80). UDP had no significant effect on afferent diameter. The pattern of the afferent arteriolar vasoconstrictor response to these agents is in qualitative agreement with the results obtained in the isolated perfused kidney (44) with a few notable exceptions. In the concentration ranges used in the juxtamedullary nephron preparation, no significant vasodilatory response is observed even though the renal vasculature is under significant endogenous tone. This may reflect a difference in the concentrations tested as the vasodilatory responses in the isolated perfused kidney experiments were observed at agonist concentrations several orders of magnitude lower than were used in the juxtamedullary nephron experiments. In addition, the isolated perfused kidney experiments measure the whole kidney hemodynamic response as a reflection of the response of the entire renal vasculature to the infused agents, whereas the juxtamedullary nephron approach examines the responses of individual microvascular segments of a finite nephron population.

Taken together, these agonist response experiments suggest that at least two distinct P2 receptors are found on afferent arterioles of the rat kidney. One receptor belongs to the P2X family and responds strongly to the P2X agonists ,-methylene-ATP or ,-methylene-ATP. Given the existing data, it is likely that this receptor is a P2X₁ receptor subtype. A second receptor probably belongs to the P2Y receptor family and is sensitive to UTP and ATPS. Activation of either receptor will evoke a sustained, concentration-dependent vasoconstriction of afferent arterioles. In addition, assessment of the disparate response patterns observed between experiments performed in the isolated perfused kidney and in the juxtamedullary nephron technique suggests that P2 receptor activation may evoke more complex regional alterations in renal microvascular function and could result in zonal changes in vascular resistance and renal perfusion.

	P2 RECEPTOR-MEDIATED CALCIUM SIGNALING IN RENAL MICROVASCULAR SMOOTH MUSCLE

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Calcium plays a major role in agonist-mediated afferent arteriolar vasoconstrictor responses, autoregulatory responses, and tubuloglomerular feedback responses (111). P2 receptor activation has also been shown to activate varied calcium signaling mechanisms involving multiple calcium influx pathways as well as mobilization of calcium from intracellular stores (111). P2X receptor stimulation activates a nonselective cation current, which can directly increase intracellular calcium concentration (39, 135). Furthermore, the net influx of cations can depolarize the plasma membrane and activate voltage-dependent calcium channels. Alternatively, P2Y receptors have been shown to be coupled to phospholipase C and generate IP₃-dependent mobilization of calcium from intracellular stores (39, 135). Given the diversity of effects that may be elicited via calcium-dependent mechanisms, it is important to assess the calcium signaling pathways utilized by afferent arteriolar smooth muscle cells in responding to P2 receptor stimulation.

P2X receptor channels conduct calcium down its steep concentration gradient into the cell. Therefore, calcium influx from the extracellular medium is an important component of P2X receptor-mediated responses. Removal of calcium from the extracellular medium prevents the afferent arteriolar vasoconstriction normally evoked by ,-methylene-ATP (84). Replenishing the extracellular calcium concentration to the physiological range also restores the ,-methylene-ATP-mediated afferent arteriolar vasoconstriction (84). These findings demonstrate that calcium influx from the extracellular medium is an essential component in the ,-methylene-ATP-mediated afferent arteriolar vasoconstrictor response; however, they do not identify the influx pathway involved.

Many agonists activate L-type calcium channels to stimulate vasoconstriction of afferent arterioles (111). Therefore, the role of L-type calcium channels in the afferent arteriolar response to P2 receptor activation was assessed by using different P2X and P2Y agonists. As shown in Fig. 6, P2X receptor stimulation with ,-methylene-ATP produces a biphasic vasoconstrictor response under control conditions (78, 80, 84). Typical responses include a rapid initial vasoconstriction that reaches a maximum value within 20-30 s, followed by a partial recovery to a stable diameter significantly smaller than control. Removal of ,-methylene-ATP from the bathing solution results in a prompt and complete recovery to the control diameter. Blockade of L-type calcium channels with felodipine (Fig. 6) or diltiazem evokes a rapid vasorelaxation, implicating L-type calcium channel activity in maintaining ambient afferent arteriolar tone. Subsequent introduction of ,-methylene-ATP, with continued blockade of L-type calcium channels, results in attenuation of the rapid initial vasoconstriction by ~50-60% and abolition of the sustained vasoconstrictor response (84). Preliminary data reveal that stimulation of P2Y receptors with UTP evokes a more monophasic vasoconstriction that is only slightly attenuated by calcium channel blockade with diltiazem (79). Calcium channel blockade abolishes afferent arteriolar responses to low concentrations of ATP (0.1-1 µM) and significantly attenuates the responses evoked by higher concentrations of ATP (79). These data suggest that calcium influx through voltage-dependent calcium channels plays a central role in the afferent arteriolar vasoconstriction elicited by P2X receptor activation by P2X receptor-selective agonists and by low concentrations of ATP. The data also suggest that calcium channels are not the major mechanism invoked in vasoconstrictor responses evoked by P2Y receptor activation or by higher concentrations of ATP.

Subsequent studies were performed using single microvascular smooth muscle cells that were freshly isolated from rat preglomerular microvasculature. Intracellular calcium signaling in response to P2 receptor activation was examined by using the calcium-sensitive fluorescent probe fura 2. The effect of selective P2 receptor stimulation on the intracellular calcium concentration was determined under control conditions, during calcium channel blockade, and during exposure of the cells to nominally calcium-free conditions (Fig. 7). Those studies revealed that stimulation of P2Y receptors with UTP stimulated a biphasic increase in intracellular calcium concentration. The magnitude and time course of the calcium response was nearly identical compared with similarly treated cells bathed in calcium-free medium (Fig. 7) and also during calcium channel blockade with diltiazem (82). These data indicate that the renal microvascular smooth muscle cell response to UTP involves an increase in intracellular calcium that arises primarily by mobilizing calcium from intracellular stores. Preliminary data indicate that P2X receptor activation with ,-methylene-ATP stimulates an increase in intracellular calcium concentration that is highly dependent on the influx of extracellular calcium (Fig. 7). The mechanism of the calcium influx pathway remains to be determined, but preliminary studies suggest that it is Ni²⁺ sensitive and can be significantly attenuated by calcium channel blockade (173). The dependency of P2X receptor activation on the influx of calcium in these cells is consistent with the role of calcium influx noted for P2X receptor-mediated afferent arteriolar vasoconstriction (79, 84, 173).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   The effect of ,-methylene-ATP (A), ATP (B), and UTP (C) on intracellular calcium concentration in freshly isolated preglomerular microvascular smooth muscle cells. Data are presented for the calcium response under control conditions with 1.8 mM calcium in the bathing medium (thick line; Control) and under nominally calcium-free conditions (thin line; Ca2+ free). The period of agonist administration was from 100 to 300 s and is indicated by the shaded bars located along the x-axes. Maximally effective concentrations of ,-methylene-ATP (10 µM) and UTP (100 µM) were used as P2X and P2Y agonists, respectively. ATP (100 µM) was used to activate both P2X and P2Y receptors simultaneously. The data were modified from the work of Inscho et al. (82, 86, 173).

Exposure of cells to a high concentration of ATP results in an elevation of intracellular calcium concentration that depends partly on the influx of calcium through L-type calcium channels and partly on the mobilization of calcium from intracellular stores (82, 86). ATP administration elicits a biphasic increase in intracellular calcium concentration that is similar in magnitude and time course to that elicited by an equimolar concentration of UTP (Fig. 7). However, unlike the example with UTP, removal of calcium from the extracellular bathing medium reduces the magnitude of the response to ATP by ~50%. Nearly identical results are obtained when ATP is administered during calcium channel blockade (82, 86).

Conflicting results, using isolated rabbit afferent arterioles exposed to ATP or UTP, have been reported (61). ATP administration increases intracellular calcium concentration through mechanisms that appear to be entirely dependent on calcium influx. No evidence of a calcium mobilization component was observed. In addition, UTP treatment has no detectable effect on intracellular calcium concentration in similarly treated arterioles. These data suggest that there might be significant species variation in the P2 receptor population expressed by afferent arterioles of rabbit and rat kidney or that P2 receptors on these arterioles may involve alternative signal transduction pathways.

Nevertheless, these data establish that stimulation of P2 receptors on preglomerular vascular smooth muscle increases intracellular calcium concentration through multiple signal transduction pathways, possibly involving multiple P2 receptor subtypes. In rat renal microvascular smooth muscle cells, P2 receptor activation can increase calcium by stimulating calcium mobilization from intracellular stores and by stimulating calcium influx via voltage-dependent calcium influx pathways. In addition, these data lend further support to the hypothesis that afferent arteriolar smooth muscle cells express both P2X and P2Y receptor subtypes that utilize distinct calcium signaling mechanisms to influence afferent arteriolar function.

	DIADENOSINE POLYPHOSPHATES

TOP ABSTRACT INTRODUCTION PURINOCEPTOR CLASSIFICATION P2 RECEPTOR AGONISTS AND... P2 RECEPTOR DISTRIBUTION WITHIN... RENAL VASCULAR RESPONSE TO... ADVENTITIAL DELIVERY OF P2... P2 RECEPTOR-MEDIATED CALCIUM... DIADENOSINE POLYPHOSPHATES WHAT PHYSIOLOGICAL ROLE DO... FINAL PERSPECTIVES REFERENCES

Diadenosine polyphosphates are a unique group of naturally occurring nucleotide agonists that also activate P2 receptors (52, 117, 135, 142). The original descriptions of diadenosine polyphosphates in human physiology appeared in the literature in the early 1980s and described the release of diadenosine polyphosphates from human platelets (51, 100). Those early studies led investigators to suggest that diadenosine phosphates may play an important physiological role in the control of platelet aggregation. In the ensuing years, several structurally distinct and biologically active diadenosine polyphosphates have been described (142, 161, 164-166).

Diadenosine polyphosphates exist as two adenosine molecules linked by a variable number of phosphate groups. The number of phosphates is indicated by the number substituted for the X in the formula AP_XA. Therefore, diadenosine pentaphosphate is noted as AP₅A. The number of phosphates present can have a significant impact on the biological activity of each diadenosine polyphosphate species (52, 58, 161, 164-166). The vascular response can also vary markedly depending on the specific vascular tissue being studied, the ambient vascular tone of the sample tissue or vascular bed, and the status of the endothelium (52, 142). Also important is the type of P2 receptor expressed by the tissue being studied (174).

Presently, there are several reports describing the effect of these agents on renal hemodynamics and renal function (69, 93), the renal microvasculature (58, 141, 161, 164-166), mesangial cells (96, 139, 144, 160), and cortical collecting duct cells (140). Bolus intravenous injection of AP₄A, AP₅A, or AP₆A into the anesthetized rat resulted in a rapid and transient decline in renal blood flow that was coincident with a transient decline in heart rate, cardiac output, and mean arterial pressure (93). Assessment of renal function after intravenous injection of AP₆A increased urine flow and sodium excretion, whereas AP₃A or AP₄A reduced urine flow and sodium excretion (69). These changes in renal function were observed without any detectable effect on glomerular filtration rate.

Intrarenal infusion of AP₄A, AP₅A, or AP₆A into isolated perfused kidneys increases renal perfusion pressure, suggesting that these agents increase renal vascular resistance (164-166). The vasoconstriction can be observed at agonist concentrations as low as 1.0 nM (164). The renal vasoconstriction could be attenuated with the P2 receptor antagonist PPADS (164-166) and was completely blocked by combining PPADS with the P1 receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (165). This observation suggests that the vasoconstrictor response is mediated partly through activation of P2X receptors and partly through activation of adenosine-sensitive P1 receptors. Large-caliber renal resistance vessels between 200 and 250 µm respond to AP₄A, AP₅A, and AP₆A with a concentration-dependent contraction (161). The magnitude of the response was similar for each agent. The vasoconstriction evoked by AP₄A could be attenuated by removal of calcium from the extracellular medium and was blocked by the calcium channel blocker nifedipine or by the nonselective P2 receptor antagonist PPADS (161).

Subsequent experiments revealed that AP₃A and AP₅A exert differential effects on the microvasculature of the hydronephrotic kidney (58). These agents induced transient vasoconstriction of interlobular arteries and afferent arterioles but had little effect on efferent arteriolar diameter. The vasoconstrictor response to AP₃A was completely inhibited during adenosine A₁ receptor blockade and was attenuated during adenosine A₂ receptor blockade or with P2 receptor blockade, suggesting that P1 receptors play a major role in the response to this diadenosine polyphosphate. Vasoconstrictor responses elicited by AP₅A were attenuated slightly by adenosine receptor blockade but were more sensitive to P2 receptor blockade with PPADS. These data suggest that AP₅A-mediated vasoconstriction in this model is predominately mediated through P2 receptor activation (58). Therefore, it seems clear that diadenosine polyphosphates can directly alter renal microvascular diameter and resistance and that the length of the polyphosphate chain is an important consideration in determining the mechanism by which diadenosine polyphosphates influence microvascular function. The complex nature of the possible interactions between diadenosine polyphosphates and P1 and P2 receptors will certainly require considerable more study to resolve.

	WHAT PHYSIOLOGICAL ROLE DO RENAL P2 RECEPTORS FULFILL?

TOP ABSTRACT INTRODUCTION PURINOCEPTOR CLASSIFICATION P2 RECEPTOR AGONISTS AND... P2 RECEPTOR DISTRIBUTION WITHIN... RENAL VASCULAR RESPONSE TO... ADVENTITIAL DELIVERY OF P2... P2 RECEPTOR-MEDIATED CALCIUM... DIADENOSINE POLYPHOSPHATES WHAT PHYSIOLOGICAL ROLE DO... FINAL PERSPECTIVES REFERENCES

This important question is not likely to be answered in the near future. The reason for this is the wide distribution of P2 receptors throughout the kidney (3, 24, 111). P2 receptors are found on renal microvascular smooth muscle cells and endothelial cells, glomerular epithelial cells, proximal tubular cells, cultured mesangial cells, and cultured epithelial cells. Efforts are underway in several laboratories to determine the role each plays in specific tissues, but the complete picture will take time to develop.

Neural Control

Mesangial Cells

ATP and UTP have also been reported to exhibit equal potency in stimulating phosphatidylcholine hydrolysis through activation of phospholipase D (131). Pretreatment with pertussis toxin significantly attenuates IP₃ and 1,2-diacylglycerol (DAG) generation (129). Inhibition of protein kinase C enhances IP₃ and DAG formation (129), whereas enhancement of protein kinase C activity with phorbol 12-myristate 13-acetate enhances phospholipase D activity (131). A recent report indicated that P2 receptor activation by ATP or UTP inhibited the activation of adenylate cyclase activity (145). This inhibition could only be observed during measures designed to elevate adenyl cyclase activity above baseline. No effect of these agents on baseline cAMP formation could be detected (71, 145). These data suggest that regulatory G proteins and protein kinase C are important modulatory pathways in the mesangial cell response to P2 receptor activation by ATP. The role of cAMP in the response is less clear.

Exposure of cultured mesangial cells to ATP or UTP appear to have differing effects on the magnitude and time course of changes in membrane potential (71, 124). Patch-clamp experiments reveal that ATP stimulated a transient membrane depolarization (71, 124), whereas UTP stimulated a sustained depolarization (71). Similarly, during the depolarization phase, ATP induced a transient increase in whole cell conductance, whereas UTP evoked a sustained increase in whole cell conductance (71). Reduction of the extracellular chloride concentration augmented the depolarization in response to ATP and UTP, suggesting that chloride channels participate in the depolarization response (71, 124). Interestingly, the depolarization induced by ATP can be inhibited by the nonselective P2 receptor blockers suramin and reactive blue 2, but the response to UTP is unaltered. These data suggest that responses elicited by ATP and UTP may arise from different P2 receptors.

Diadenosine polyphosphates are also known to influence mesangial cell function and thus may play a role in the control of glomerular hemodynamics. Exposure of mesangial cells to diadenosine polyphosphates stimulates membrane depolarization through activation of chloride channels and nonselective cation channels (96). Consistent with membrane depolarization and activation of nonselective cation channels, AP₃A is reported to stimulate a sustained elevation in intracellular calcium concentration and mesangial cell contraction when external calcium is in the normal range but only a transient elevation in intracellular calcium concentration when external calcium is removed (139). In contrast, AP₄A, AP₅A, and AP₆A appear to increase intracellular calcium concentration exclusively by stimulating calcium influx through voltage-gated L-type calcium channels (160). Whether these conflicting data reflect agonist-specific calcium signaling pathways or are due to experimental variation is not clear. Nevertheless, elevation of intracellular calcium is a primary response of mesangial cells challenged with diadenosine polyphosphates. Most of these responses appear to be sensitive to P2 receptor blockade with suramin or PPADS but are insensitive to adenosine A1 receptor blockade, suggesting that the responses arise through selective activation of P2 receptors (160).

The physiological or pathophysiological role of P2 receptors expressed by cultured mesangial cells remains to be determined; however, a couple of conflicting possibilities have recently been proposed. P2 receptor activation has been implicated in mesangial cell proliferation (73-75, 147, 148) and also in triggering apoptosis and necrosis (146). Exposure of mesangial cells to ATP stimulated a significant elevation in cell number, thymidine incorporation, and prostaglandin E₂ production (129, 148). Similar treatment with UTP resulted in a smaller proliferative response. The mesangial cell response to ATP involves the mitogen-activated protein kinase cascade (73) and the stress-activated protein kinase cascade (74, 75). In another study, a 90-min exposure of mesangial cells to extracellular ATP caused an increase in DNA fragmentation and an increase in tumor suppressor p53 protein, which is thought to regulate apoptosis (146). Further studies implicated activation of the P2X₇ receptor in the apoptotic response to ATP and confirmed the presence of P2X₇ receptor-associated pore formation in the presence of ATP. Collectively, these studies suggest a possible role for mesangial P2 receptors in inflammatory cell proliferation and apoptosis (130, 147).

Vascular Smooth Muscle Cell Proliferation

Autoregulatory Control

Studies were conducted in vitro using the juxtamedullary nephron technique in the isolated rat kidney and examined the effect of P2 receptor inactivation on autoregulatory behavior (81). P2 receptor inactivation was accomplished by imposing P2 receptor desensitization or saturation and by pharmacological blockade. The results of those studies revealed that inactivation of P2 receptors by receptor desensitization abolished pressure-mediated autoregulatory reductions in afferent arteriolar diameter (81). An alternative approach to inactivation of the P2 receptor influence on autoregulatory responses was implemented by clamping the extracellular ATP concentration by adding high concentrations of ATP to the exogenous medium. Under these conditions, the influence of endogenously released ATP would be overwhelmed by the exogenous ATP present in the bathing medium. Under ATP-clamp conditions, pressure-mediated afferent arteriolar autoregulatory responses were completely blocked (81). Interestingly, this inhibition of autoregulatory behavior was obtained despite the fact that afferent arteriolar responsiveness to other non-P2 vasoconstrictor agonists such as KCl, angiotensin II, or norepinephrine was well preserved (81).

In another series of experiments, we examined the effect of pharmacological inactivation of P2 receptors on autoregulatory behavior (Fig. 8). Autoregulatory responsiveness was examined before and during exposure of the vasculature to the nonselective P2 receptor antagonists PPADS or suramin (81). Under control conditions, elevation of renal perfusion pressure in 30-mmHg increments from 100 to 130 and 160 mmHg resulted in appropriate pressure-mediated reductions in afferent arteriolar diameter. However, during blockade of P2 receptors with PPADS, the pressure-mediated vasoconstrictor responses were abolished and afferent arteriolar diameter remained unchanged. Suramin administration consistently increased baseline afferent arteriolar diameter. Similar to the effect of PPADS on autoregulatory behavior, P2 receptor blockade with suramin resulted in a marked attenuation of the pressure-mediated vasoconstrictor responses (81). Therefore, each experimental manipulation designed to selectively inactivate afferent arteriolar P2 receptors simultaneously blocked normal afferent arteriolar autoregulatory behavior. The confirmation that afferent arterioles retained normal responsiveness to other vasoconstrictor stimuli supports the contention that the functional integrity of the contractile apparatus remained intact during P2 receptor inactivation but that the ability of the arteriole to respond to a pressure stimulus was impaired. These fundamental observations are consistent with selective inhibition of the autoregulatory response by P2 receptor inactivation and strongly implicate P2 receptor activation as an essential step in pressure-mediated autoregula