Natriuretic peptides are a family of hormones that regulate a number of functions, including blood pressure and cardiovascular homeostasis. The mammalian family of natriuretic peptides consists of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). Natriuretic peptides exert their physiological effects by the occupation of three membrane receptors; two are guanylyl cyclase-coupled receptors, known as NPR-A and NPR-B, while NPR-C lacks enzymatic activity (1, 2). NPR-A shows high affinity and is activated by ANP and BNP (3), NPR-B is activated by CNP (4) and, finally, the NPR-C binds all natriuretic peptides (5).

NPR-A is a membrane receptor composed of a single transmembrane domain, a variable extracellular natriuretic peptide-binding region, a conserved intracellular kinase homology domain (KHD) and a catalytic domain (6). Natriuretic peptide binding to NPR-A at a stoichiometry of 1:2 stimulates the synthesis of the intracellular second messenger, cGMP (7). We have recently suggested the existence of functional differences in NPR-A between spontaneously hypertensive rats (SHR) and age-matched normotensive WKY rats (8-11). According to this, binding of ANP1-28 with NPR-A produces cGMP at a higher rate in SHR than in age-matched WKY, suggesting that the activation of guanylyl cyclase by ANP1-28 was altered in SHR. The enhanced cGMP production in SHR occurs even before the development of hypertension, which indicates that this alteration plays a primary role in the pathogenesis of hypertension (8).

ATP has been reported to increase ANP-dependent activation of NPR-A-coupled guanylyl cyclase (12-15). ATP regulates NPR-A allosterically by binding to a motif within the KHD region of NPR-A as revealed by cDNA cloning. This motif resembles the ATP-binding glycine elbow present in a number of protein kinases (12, 16). In turn, ANP binding to NPR-A modulates the interaction of ATP with the KHD region (17). In addition, ATP-dependent regulation of NPR-A-coupled guanylyl cyclase also involves changes in its phosphorylation state, an event that is necessary for natriuretic peptide receptor activation (18; 19).

Free divalent cations are also essential for NPR-A-coupled guanylyl cyclase activity. No enzymatic activity has been detected in Mg2+-free medium (12; 20). In addition, Ca2+ has been reported to have a direct effect on guanylyl cyclase activity. Koch and Stryer (21) showed that the intracellular cGMP and free Ca2+ concentration are reciprocally controlled by negative feedback. Cyclic-AMP has been reported to regulate the natriuretic peptide system in rat thyroid cells by reducing NPR-B-coupled guanylyl cyclase activity by a Ca2+-dependent mechanism (22). In rat lung membranes Ca2+ has been reported to inhibit ATP-stimulated guanylyl cyclase activity, although little effect of Ca2+ on basal and ANP-stimulated guanylyl cyclase activity was found (23). The effect of extracellular Ca2+ on guanylyl cyclase activity has been shown to be mimicked by lanthanum, suggesting that this event is likely mediated by interaction with a specific Ca2+ binding site probably related to the ATP binding motif (23). At present, the regulation by extracellular Ca2+ of NPR-A-coupled guanylyl cyclase has not been investigated in renal glomeruli, which plays a crucial role in the development and consolidation of hypertension. In addition, the possible differences in the regulation by extracellular Ca2+ of NPR-A-coupled guanylyl cyclase between SHR and age-matched WKY have not been explored. The present work, therefore, examines the effects of extracellular Ca2+ on signal transduction associated to NPR-A-coupled guanylyl cyclase in renal glomeruli from SHR and WKY.


MATERIALS AND METHODS
Materials

[125I]-ANP1-28 and cGMP radioimmunoassay commercial kits were from Amersham, Bucks, UK. ANP1-28 and C-ANP were from Peninsula Laboratories (Merseyside, U.K.). Isobutylmethylxanthine (IBMX), bovine serum albumin (BSA), ATP and Tris were from Sigma (Poole, Dorset, UK). All other reagents were of analytical grade.

Animals

Twelve-week-old SHR and WKY rats (250-300g) were randomly selected from our inbred colonies, which were obtained from Charles River (Margate, U.K.). Animals were housed within the Animal House of the Physiology Laboratory, University of Cambridge.

Glomerular membrane preparation

Glomerular membranes were prepared as described previously (24). Briefly, kidneys were removed and placed in ice-cold Hank’s balanced salt solution (HBSS), containing (in mM): 137 NaCl, 10 HEPES, 5.4 KCl, 0.4 Mg2SO4, 0.34 Na2HPO4, 1.26 CaCl2, 4.17 Na2HCO3, 0.44 K2HPO4, 0.49 MgCl2, 0.2 % (w/v) BSA and 5.56 glucose, pH 7.2. The cortices were minced and glomeruli were isolated by differential sieving as previously described (25). Freshly isolated glomeruli were homogenized in ice-cold HBSS. The homogenate was centrifuged at 1,000 g for 10 min at 4 °C. The supernatant was then centrifuged for 60 min at 4 °C at 40,000 g. The pellet was washed, sonicated, and stored at -70 °C for less than one week before use. Preliminary experiments showed that under this storage condition, guanylyl cyclase activity remained stable.

Aliquots of glomerular membranes (500 µg) were incubated with agonists for 20 min at 20 °C, in 500 µl 50 mM Tris-HCl buffer (pH 7.6) containing 1 mM EDTA, 4 mM MgCl2, 1 mM DTT and 250 mM Sucrose. The mixture was then centrifuged at 105,000 g for 60 min at 4 °C and the pellet was resuspended in 1 ml Tris-HCl buffer. A total volume of 50 µl supernatant was used for protein content determination using the Lowry protein assay and a total volume of 950 µl supernatant was used for subsequent cGMP assay as described below.

Determination of cGMP production in glomerular membranes

Guanylyl cyclase activity in aliquots of glomerular membranes (3-5 µg of protein) was measured at 37 °C in a reaction mixture containing (in mM): 50 Tris-HCl, 4 MgCl2, 1 IBMX, 1 GTP-Mg2+, 15 creatine phosphate and 20 Units/ml creatine phosphokinase, pH 7.6, and in the presence of various concentrations of CaCl2 (0-1.26 mM). The reaction was started by addition of 1 µM ANP1-28 to the membrane suspension and the reaction was stopped with 50 mM sodium acetate (pH 5.8) followed by boiling. The samples were centrifuged at 4,000 g for 10 min and cGMP was determined by radioimmunoassay.

To perform kinetic studies, 3-5 µg glomerular membranes were incubated for 20 min at 37 °C in triplicate in the absent or present of 1 µM ANP1-28 and/or 1 mM ATP in the presence of various concentrations (0-1.5 mM) of Mg2+-GTP. Cyclic-GMP was determined by radioimmunoassay.

Ligand stability

The stability of ANP1-28 after incubation with glomerular membranes for 20 min at 20 °C was checked by collecting the incubation fluids and subjecting to RP-HPLC, in order to exclude any possibility that the different potency of ANP1-28 to stimulate cGMP production in both strains resulted from the different stability of the ligands.

Competitive inhibition of [125I]ANP1-28 binding

Aliquots (5-8 µg of protein) of glomerular membranes were incubated with 100 pM [125I]ANP1-28 (2,000 Ci/mmol) in the absence or presence of 1 mM CaCl2, with 1 mM ATP, and various concentrations (1 pM to 10 µM) of unlabelled rat ANP1-28 for 60 min at 20 °C as described previously (24). Preliminary experiments showed that specific binding of radioligand reached equilibrium at 60 min (data not shown). Incubations were stopped by centrifugation and 125I labeling was determined using a Packard Gamma Counter. In order to avoid [125I]ANP1-28 binding to NPR-C, 1 µM C-ANP was used (26).

Data analysis

The data are presented as means ± SEM. Analysis of statistical significance was performed using the Student’s t-test. The significance level was p<0.05.


RESULTS
Regulation of NPR-A guanylyl cyclase from WKY and SHR glomerular membranes

The kinetics of NPR-A-coupled guanylyl cyclase activity was investigated in glomerular membranes from normotensive (WKY) and hypertensive (SHR) rats after incubation in the absence or presence of agonists. The Lineweaver-Burk plots of effects of ANP1-28, ATP and ANP1-28/ATP on the kinetics of guanylyl cyclase in SHR and WKY are depicted in Fig. 1. Glomerular membranes from both strains were assayed using a range of substrate concentrations (0-1.5 mM GTP), with Mg2+ as the cation cofactor. Basal guanylyl cyclase activity was concentration-dependent and saturable at millimolar concentrations in both strains (data not shown). Lineweaver-Burk plot was linear (Fig. 1A) and shows a similar affinity in WKY rats (Km = 824 ± 205 µM) than in SHR (Km = 713 ± 15 µM). In contrast, NPR-A-coupled guanylyl cyclase shows a significantly greater Vmax in SHR than in WKY (Vmax = 7 ± 1 and 9 ± 0.7 pmol/mg protein/min in WKY and SHR, respectively; P<0.05; n=6).

Fig. 1. Comparison of Lineweaver-Burk plots obtained for NPR-A guanylyl cyclase from WKY and SHR glomerular membranes. Glomerular membranes from WKY (open symbols) and SHR (closed symbols) were incubated for 20 min at 37 °C in the absence (A) or presence of 1 µM ANP1-28 (B), 1 mM ATP (C) or both (D) in the presence of various concentrations (0-1.5 mM) of Mg2+-GTP. Guanylyl cyclase activity in basal conditions or after incubation with agonists was expressed as a function of GTP concentration.

Guanylyl cyclase activity as a function of GTP concentration in the presence of ANP1-28 and ATP is depicted as the Lineweaver-Burk plots in Fig. 1B and C. Incubation of NPR-A guanylyl cyclase for 20 min with 1 µM ANP1-28 or 1 mM ATP increased Vmax without modifying the affinity for the substrate in both strains, although, as for basal activity, Vmax was significantly greater in SHR than in WKY (Vmax = 8 ± 1 and 9.5 ± 2 pmol/mg protein/min in WKY and SHR, respectively; P<0.05; n=6). In the presence of both agonists, ANP1-28 and ATP, guanylyl cyclase activity markedly increased and remained saturable at millimolar concentrations of GTP (data not shown). As shown in Fig. 1D, the Lineweaver-Burk plot revealed that incubation of NPR-A-coupled guanylyl cyclase from glomerular membranes with ANP1-28 and ATP did not affect its affinity but increased Vmax more than the simple addition of the individual effects of both agonists. As shown above, Vmax was significantly greater in SHR than in WKY (Vmax = 14 ± 3 and 21 ± 4 pmol/mg protein/min in WKY and SHR, respectively; P<0.05; n=6).

Effect of extracellular Ca2+ on basal and ANP1-28-, ATP- and ANP1-28 + ATP-stimulated guanylyl cyclase activity in glomerular membranes

Consistent with the results reported above, Fig. 2 shows that in the absence of extracellular Ca2+ basal cGMP production by NPR-A-coupled guanylyl cyclase was significant higher in SHR than in age-matched WKY (Fig. 2A and B; P<0.05; n=6). Incubation of glomerular membranes for 20 min in the presence of 1 µM ANP1-28 and/or 1 mM ATP significantly enhanced cGMP production in both strains (Fig. 2; P<0.05), although this effect was significantly greater in SHR than in WKY (Fig. 2A and B; P<0.05). In the presence of physiological concentrations of extracellular Ca2+ (1.26 mM) cGMP production stimulated by 1 mM ATP or ANP1-28 plus ATP was significantly reduced in both strains (Fig. 2), whereas basal and ANP1-28-stimulated cGMP production were not significantly modified, suggesting that extracellular Ca2+, at millimolar concentrations, may directly affect the regulation of guanylyl cyclase by ATP.

Fig. 2. Effect of Ca2+ on the activation of NPR-A guanylyl cyclase from WKY and SHR glomerular membranes by ANP1-28 and ATP. Glomerular membranes from WKY (A) and SHR (B) were incubated 20 min with 1 µM ANP1-28, 1 mM ATP or both in the absence or presence of 1.26 mM CaCl2, as indicated. Cyclic-GMP production was determined as described in Material and methods. Data are presented as mean ± SEM of 6 separate determinations. *P<0.05 compared to the effect observed in the absence of Ca2+. oP<0.05 compared to the results obtained in WKY rats.

The effect of different concentrations of extracellular Ca2+ on guanylyl cyclase activity was further investigated by incubation of NPR-A-coupled guanylyl cyclase with 1 µM ANP1-28 or 1 mM ATP in the presence of a range of extracellular Ca2+ concentrations (0-200 µM). In both strains, extracellular Ca2+ increased basal and ANP1-28-stimulated guanylyl cyclase activity in a concentration-dependent manner in glomerular membranes (Fig. 3). Basal and ANP1-28-stimulated guanylyl cyclase activity were higher in SHR than in age-matched WKY in the absence or presence of extracellular Ca2+ (Fig. 3; P<0.05). In contrast, ATP-stimulated cGMP production was inhibited in the presence of extracellular Ca2+. The inhibitory effect of Ca2+ was detectable at concentrations as low as 10 µM (Fig. 3). The maximal inhibition of ATP-stimulated NPR-A-coupled guanylyl cyclase activity by extracellular Ca2+ was 25 % in WKY and 19 % in SHR.

Fig. 3. Concentration-dependent effect of Ca2+ on the activation of NPR-A guanylyl cyclase from WKY and SHR glomerular membranes by ANP1-28 and ATP. Glomerular membranes from WKY (A) and SHR (B) were incubated for 20 min in the absence (squares) or presence of 1 µM ANP1-28 (circles) or 1 mM ATP (triangles) in the absence or presence of various concentrations (0-200 µM) of CaCl2, as indicated. Cyclic-GMP production was determined as described in Material and methods. Data are presented as mean ± SEM of 6 separate determinations.

As shown in Fig. 4, in the presence of 1 µM ANP1-28, 1 mM ATP stimulated guanylyl cyclase activity in a concentration-dependent manner in the absence of extracellular Ca2+, with EC50 of 350 µM in both strains. Addition of 10, 30 and 50 µM CaCl2 to the medium reduced ATP-stimulated cGMP production in the presence of ANP1-28 by 7 %, 12 % and 15 % of the maximal enzyme activity, respectively, in WKY, and by 5 %, 13 % and 16 % of the maximal enzyme activity, respectively, in SHR (Fig. 4). However, the EC50 for ATP was almost unaffected by extracellular Ca2+ in both strains, suggesting that the effect of Ca2+ on ATP-stimulated guanylyl cyclase activity is unlikely due to the alteration of ATP binding to NPR-A-coupled guanylyl cyclase.

Fig. 4. Concentration-dependent effect of Ca2+ and ATP on the activation of NPR-A guanylyl cyclase from WKY and SHR glomerular membranes by ANP1-28. Glomerular membranes from WKY (A) and SHR (B) were incubated 20 min with 1 µM ANP1-28 and increasing concentrations (0-2 mM) of ATP, in the absence (solid circles) or presence of either 10 µM (open circles), 30 µM (solid triangles) or 50 µM (open triangles) CaCl2. Cyclic-GMP production was determined as described in Material and methods. Data are presented as mean ± SEM of 6 separate determinations.

Effect of extracellular Ca2+ on ANP1-28 binding to glomerular membranes

Since extracellular Ca2+ has no significant effect on ANP1-28-induced activation of NPR-A-coupled guanylyl cyclase, we have further examined the effect of extracellular Ca2+ on ANP1-28 binding to NPR-A. The effect of Ca2+ on ANP1-28 binding to NPR-A was examined in competitive binding experiments in normotensive WKY rats. Binding of [125I]-ANP1-28 to glomerular membranes was not modified in the presence of 1.26 mM extracellular Ca2+ (data not shown). Scatchard analysis of [125I]-ANP1-28 binding yielded linear plots. Our results indicate that Ca2+ did not significantly modify the NPR-A-ANP1-28 binding properties, including affinity (see Kd) and maximal binding capacity (Bmax; Table 1). Incubation with ATP decreased both the Kd and Bmax of NPR-A (Table 1; P<0.05), an effect that was found to be similar in the absence and presence of extracellular Ca2+ (Table 1), suggesting that Ca2+ does not modify the binding properties of NPR-A in the absence or presence of ATP.

Table 1. Effect of Ca2+ on [125I]-ANP1-28 binding to glomerular membranes from WKY

The maximum binding capacity (Bmax) and dissociation constant (Kd) were assessed from the competitive inhibition of the binding of 100 pM [125I]ANP1-28 by various concentrations (1 pM to 10 µM) of unlabelled ANP1-28 in the absence and presence of 1 mM ATP and 1.26 mM CaCl2. In order to avoid [125I]ANP1-28 binding to NPR-C 1 µM C-ANP was used. Values are means ± S.E. from 6 separate experiments. *P<0.05 compared with [125I]ANP1-28 binding in the absence of 1 mM ATP.

DISCUSSION
ANP is a cardiac hormone with potent natriuretic and vasorelaxant activities (27). ANP may affect blood pressure indirectly by modulating renal hemodynamics and excretory functions of the kidney. The plasma ANP concentration has been demonstrated to be increased in a number of experimental models of hypertension (28, 29). In addition, gene expression of NPR-A has been shown to be up-regulated in the aorta of hypertensive rats suggesting that alterations in NPR-A, as well as increase in the plasma levels of ANP, might modulate the biological actions of ANP in hypertension (30). Consistent with this, a number of studies have found that NPR-A binding characteristics are altered in SHR compared with normotensive WKY rats, resulting in an enhanced cGMP production in hypertensive rats (8-11, 31, 32).

ANP-stimulated cGMP production by occupation of NPR-A is modulated by ATP and divalent cations, such as Ca2+, which, in turn, has been shown to interfere with ATP binding to NPR-A in rat lung membranes (23). Here we show that ATP stimulates guanylyl cyclase activity and potentiates ANP1-28-evoked cGMP production in renal glomerular membranes from SHR and normotensive WKY rats. Our results indicate that NPR-A-ANP1-28 binding properties are modified by ATP. In agreement with previous studies (15), radioligand binding analysis revealed that ATP decreased the Bmax and increased the affinity of NPR-A for ANP1-28.

The relationship between Ca2+, hypertension and natriuretic peptides has been established, as demonstrated in aortic rings from SHR, where endothelium-dependent relaxations in response to the Ca2+ ionophore A23187 were significantly impaired by high salt intake (33). Hypercalcemia has been shown to be associated to hypertension and Ca2+ channel antagonists have been proposed as antihypertensive therapeutic tools (34, 35). We have found that extracellular Ca2+ did not modify basal or ANP1-28-stimulated cGMP production, but significantly inhibited ATP-enhanced guanylyl cyclase activity in the absence and presence of ANP1-28, which, to our knowledge, is the first evidence of a role for Ca2+ in the biological functions of NPR-A-coupled guanylyl cyclase in renal glomeruli.

The inhibitory effect of extracellular Ca2+ on ATP-stimulated guanylyl cyclase activity has also been reported in rat lung membranes (23). The differential regulation by extracellular Ca2+ of guanylyl cyclase activity stimulated by ATP or ANP1-28 suggests that different mechanisms are involved in the activation of guanylyl cyclase by ANP1-28 and ATP. It is unlikely that Ca2+ binds to guanylyl cyclase directly, since this protein lacks the EF-hand motif (23). Therefore, the inhibitory effect of extracellular Ca2+ on ATP-induced response is likely mediated by a Ca2+-dependent protein involved in ATP-mediated regulation of NPR-A-coupled guanylyl cyclase in glomerular membranes. Consistent with this, we have found that the effect of ATP on NPR-A binding characteristics was also independent of extracellular Ca2+, as shown in Table 1, and in agreement with previous studies in rat mesenteric artery (36), which indicates that a different mechanism, such as a Ca2+ binding protein might be involved in the inhibitory effect of Ca2+ on ATP-mediated regulation of NPR-A-coupled guanylyl cyclase activity in glomerular membranes. In support of this hypothesis guanylyl cyclase activator proteins (GCAPs) are Ca2+-binding proteins that interact with guanylyl cyclase and regulate its activity. GCAPs accelerate the synthesis of cGMP at low Ca2+ concentrations and become inhibitors of the guanylyl cyclase when GCAPs bind Ca2+ (37-39).

Studies on the effect of Ca2+ on guanylyl cyclase activity have shown that this effect might not result from simple ionic interactions since the potencies of LiCl, AlCl3 and CaCl2 do not correlated with their charges (23). The concentration of extracellular Ca2+ required for half-maximal inhibition of ATP-stimulated cGMP production was about 10-15 µM in both strains, which was relatively high compared with the intracellular Ca2+ level (cytoplasmic free Ca2+ concentration at rest is about 100 nM, (40)). Therefore intracellular Ca2+ elevation may be a regulatory factor for intracellular cGMP levels in glomerular membranes.

Activation of guanylyl cyclase by ANP1-28 and/or ATP was significantly higher in SHR than in age-matched WKY, which confirms that the guanylyl cyclase pathway is sensitized in SHR as previously reported (8-11, 31, 32). Kinetics studies on guanylyl cyclase demonstrated that ATP induced a greater Vmax in SHR than in WKY, with similar affinity for GTP, which might explain the higher cGMP production by NPR-A reported in a number of structures, including renal glomerular membranes (8, 11, 32), choroid plexus (41), olfactory bulb and hypothalamus (9) from SHR. Extracellular Ca2+ had similar effect on guanylyl cyclase activity in SHR and normotensive WKY, which suggests that NPR-A-coupled guanylyl cyclase from both strains are similarly sensitive to Ca2+ and, therefore, Ca2+ did not account for the enhanced cGMP production rate in renal glomeruli from hypertensive subjects.

In summary, this is the first study that explores the effects of extracellular Ca2+ on ATP-mediated regulation of NPR-A-coupled guanylyl cyclase in renal glomeruli. ATP is a potent regulator of NPR-A-coupled guanylyl cyclase both in SHR and WKY and potentiates the activation of guanylyl cyclase by ANP1-28 in both strains. Our results provide evidence supporting that extracellular Ca2+ exerts inhibitory effect on ATP-mediated stimulation of guanylyl cyclase activity in glomerular membranes probably by interaction with a Ca2+-binding protein. The guanylyl cyclase activity stimulated by ATP or ANP1-28 responds differently to extracellular Ca2+, suggesting that different interactions may be involved in the activation of guanylyl cyclase by both agents. Radioligand binding studies showed that Ca2+ did not affect NPR-A-ANP1-28 binding properties. The effects of extracellular Ca2+ on guanylyl cyclase activity were similar in hypertensive (SHR) and normotensive WKY rats, suggesting that a rise in intracellular or extracellular Ca2+ is not responsible for the elevated cGMP production rate observed in SHR after NPR-A occupation.

Acknowledgements: The British Heart Foundation supported this work.
Abbreviations: ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; BSA, bovine serum albumin; CNP, C-type natriuretic peptide; HBSS, Hank’s balanced salt solution; IBMX, Isobutylmethylxanthine; NPR, natriuretic peptide receptor; SHR, spontaneously hypertensive rats; WKY, Wistar-Kyoto rats.


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