Ring-deleted Analogs of Atria1 Natriuretic Factor Inhibit Adenylate Cyclase/cAMP System POSSIBLE COUPLING OF CLEARANCE ATRIAL NATRIURETIC FACTOR RECEPTORS TO ADENYLATE CYCLASE/cAMP SIGNAL TRANSDUCTION SYSTEM*

We have recently shown that atria1 natriuretic factor (ANF) inhibits adenylate cyclase activity in rat platelets where only one population of ANF receptors (ANF-R2) is present, indicating that ANF-R2 receptors may be coupled to the adenylate cyclase/cAMP system. In the present studies, we have used ring-deleted peptides which have been reported to interact with ANF-R2 receptors also called without affecting the guanylate cydase/cGMP system, to examine if these peptides can also inhibit the adenylate cyclase/cAMP

Atria1 natriuretic factor (ANF)' has been reported to regulate a variety of physiological processes (1) affecting cardiovascular homeostasis.
ANF inhibits adenylate cyclase and CAMP levels (2-9) and stimulates guanylate cyclase/cGMP accumulation (10,11) in several tissues suggesting that these two second messenger systems may be responsible in mediating the physiological responses of ANF. The inhibition of adenylate cyclase by ANF has been reported to be dependent on the presence of guanine nucleotides (7,8) indicating that the inhibition is a receptor-coupled phenomenon.
Recently, the involvement of Gi regulatory protein in the coupling of ANF receptors to the adenylate cyclase system has also been demonstrated (12,13). Cross-linking studies have identified at least two classes of ANF receptors with an apparent molecular weight of 130,000 and 66,000 designated as ANF-Rl and ANF-R2 (14)(15)(16). ANF-Rl receptors are coupled to the guanylate cyclase system (14), whereas the coupling of ANF-R2 receptors to other signal transduction systems is not clearly demonstrated.
Recently, Maack and his colleagues (17) by using a ringdeleted analog of ANF, C-ANF4-23 (des[Gln", Serlg, Gin", Let?', Gly22]ANF4m,:,-NH2), have reported that this analog binds with high affinity to ANF-R2 receptors without affecting the cGMP levels as well as any of the renal vascular, hemodynamic, and excretory effects of ANF (17). However, the ring-deleted analog of ANF was able to lower the blood pressure and increase sodium excretion in intact anesthetized rats. From these studies, these investigators postulated that the majority of the renal and vascular receptors are biologically silent receptors and the primary function of this receptor is sequestration and metabolic clearance of ANF and have therefore designated them as clearance receptors (C-ANF receptors  (20). In the present studies, we have used C-ANF,.,:, and other ring-deleted analogs of ANF which interact with only ANF-R2 or so-called "C-ANF" receptors to investigate if these analogs were also able to inhibit the adenylate cyclase/cAMP system. and was homogenized using a Teflon-glass homogenizer in a buffer containing 10 mM Tris-HCl and 1 mM EDTA (pH 7.5). The homogenate was centrifuged at 20,000 X g for 10 min. The supernatant was discarded, and the pellet was homogenized in the above buffer with a glass-Teflon homogenizer. This preparation was used for adenylate cyclase determination. Preparation of Brain Striatal Membranes-The brain striatal membranes were prepared as described previously (22). Sprague-Dawley rats weighing about 250-300 g were killed by decapitation. Brains were removed, and the striata were dissected out and placed in icecold buffer containing 10 mM imidazole and 1 mM EDTA, pH 7.5. Striata (two striata per 3 ml) were homogenized by hand with a glass-Teflon homogenizer. The homogenate was centrifuged at 1,000 x g for 10 min. The pellet was suspended in 1 mM NaHCOII (pH 7.5) and centrifuged at 16,000 x g for 10 min. The supernatant fraction was discarded, and the pellet was finally suspended in a buffer containing 10 mM imidazole and 1 mM EDTA (pH 7.5) and used for adenylate cyclase determination.

RESULTS
Effect of C-ANF4-23 on Adenylate Cyclase Actioity-To investigate if the C-ANF receptors are coupled to the adenylate cyclase system, the effect of ring-deleted analog C-ANF,.,, on adenylate cyclase activity from several tissues was studied and was compared with the effects observed with ANF99-126. Fig. 1 shows that C-ANF,.,, like ANF9gmlZ6 inhibited the adenylate cyclase activity in a concentration-dependent manner in anterior pituitary homogenates, aorta-washed particles, brain striatal and adrenal cortical membranes. Both the peptides were equally effective in inhibiting the enzyme activity. The maximal inhibitions elicited by both these peptides were between 50 and 60% in anterior pituitary, aorta, and brain striatum, whereas about 30% inhibition was observed in adrenal cortical membranes.
Both peptides were almost equipotent (apparent K, between 0.1 and 1 nM) in eliciting the inhibitory effects on the adenylate cyclase activity. C-ANF,-23 was also able to inhibit adenylate cyclase activity by about 40% in Leydig tumor cells (data not shown).
Effect of C-ANF4.23 on CAMP and cGMP Levels- Fig.  2 shows the effect of C-ANF,.,, and ANF99-126 on CAMP and cGMP levels in cultured vascular smooth muscle cells from rat aorta. Both peptides decreased CAMP levels in a concentration-dependent manner with an apparent Ki of about 1 nM. The maximal inhibition observed was about 60-70%. On the other hand, C-ANF,_,, as reported earlier (17) was ineffective in stimulating cGMP levels, whereas cGMP levels were dose dependently increased by ANF99-1Z6. These results indicate that C-ANF,_,:, does not interact with ANF-Rl receptors associated with the guanylate cyclase/cGMP system but interacts with ANF-RP or C-ANF receptors and results in an FIG. 1 Dependence of C-ANFbmz3 Inhibition of Adenylate Cyclase on Guanine Nucleotides-The inhibitory effects of ANF on adenylate cyclase have been reported to be dependent on the presence of guanine nucleotides (5,6). If C-ANFM, interacts with the same receptor populations associated with adenylate cyclase, the inhibitory effect of C-ANFbmZ3 should also require the presence of guanine nucleotides. Fig. 3 illustrates the effect of C-ANF4-23 on adenylate cyclase from brain striatum in the absence and presence of various concentrations of GTP@.
C-ANF,-2, did not exert any effect on adenylate cyclase activity in the absence of GTP?S; however, in the presence of various concentrations of GTPyS, C-ANF4-23 inhibited the enzyme activity in a concentration-dependent manner. The maximal inhibition (-60%) was observed at 30 pM GTP-& These results indicate that C-ANF receptors are coupled to adenylate cyclase through guanine nucleotide regulatory protein.
Effect of PT on C-ANF4-23-mediated Inhibition of Adenylute Cyclase-The coupling of ANF receptors to adenylate cyclase through inhibitory guanine nucleotide regulatory protein (Gi) has been demonstrated recently (12,13). To examine if C-ANF receptors are also coupled to adenylate cyclase through Gi, the effect of PT treatment on C-ANF-mediated inhibition was studied. As shown in Fig. 4, C-ANFdmZ3 inhibited the enzyme activity in a concentration-dependent manner, and this inhibition was attenuated by PT treatment indicating the involvement of Gi regulatory protein in the coupling of C-ANF receptors to adenylate cyclase.
Interaction of C-ANFdmz3 and ANF9+,26 on Adenylate Cyclase Actioity-Since both ANFggm126 and C-ANF4-23 were equipotent in inhibiting the adenylate cyclase activity, it was of interest to investigate if both peptides interact with the same receptor population to elicit inhibition or interact with two distinctly different receptor populations. Fig. 5 shows that C-ANF4-23 and ANF99-126 inhibit adenylate cyclase activity by 28 and 32%, respectively, and when the effect of an optimal concentration of both these peptides was studied together on adenylate cyclase, the percent inhibition remained the same. These data indicate that both these peptides interact with the same population of ANF receptors. Effect of C-ANF4-23 on Agonist-stimulated Adenylate Cyclase and CAMP Leuels- Table  I shows that NECA, dopamine, and forskolin stimulated adenylate cyclase activity to various degrees in brain striatum and C-ANF,_,, as shown before inhibited basal activity as well as the stimulatory effects of all these agonists on enzyme activity. For example, NECA, stimulated the enzyme activity by about 300% which was inhibited to about 130% (-55% inhibition) in the presence of 0.1 pM C-ANFdm23. Similarly, the stimulation of adenylate cyclase by forskolin (-500%) was inhibited to about 200% in the presence of 0.1 pM C-ANF4-23. Furthermore, C-ANFamS3 was also able to decrease the basal as well as dopamine-or forskolinstimulated CAMP levels in cultured vascular smooth muscle cells from rat aorta. The similar inhibitory effects of ANFlol-ltlG or ANF99.126 on hormone-responsive adenylate cyclase activities and CAMP levels have also been reported previously in several other tissues (2)(3)(4)(5)(6)(7)(8)(9) reported that a linear truncated analog of ANF: des-Cy#'", C~S'*~-ANF (104-126) specifically labeled a 65,000-dalton protein referred to ANF-R2 receptors and did not activate guanylate cyclase in rabbit lung membranes. To investigate if the linear analog of ANF can also interact with the adenylate cyclase/cAMP system, the effects of various truncated linear analogs of ANF on CAMP levels and adenylate cyclase activity in cultured aorta cells were studied and compared to the effects elicited by ANFggelzs and C-ANF4-23. As shown in Table  II, all the truncated linear fragments of ANF were able to inhibit/decrease adenylate cyclase activity and CAMP levels, respectively, to various degrees. The inhibitions were quite comparable with those exerted by ANF99-126 or C-ANFdmZ3. These results indicate that truncated linear analogs of ANF like C-ANFddZ3 may also interact, with ANF-R2 receptors.
Effect of C-ANF,-,, on CAMP Levels and Progesterone Secretion in Leydig Tumor Cells-ANF has been reported to inhibit CAMP levels and progesterone secretion in Leydig tumor cells (30). In order to examine if the observed decrease in progesterone secretion by ANF was due to its interaction with ANF-R2 receptors, we studied the effect of C-ANF4-2D on progesterone secretion using the Leydig tumor cell line (MA-10 cells). As shown in Table III, C-ANF4-23 and ANF9+ 126 did not show any significant effect on basal progesterone secretion up to 10e9 M; however, luteinizing hormone-stimulated progesterone secretion was significantly inhibited (-25-35%) by both C-ANFamZ3 and ANFggmlZ6. In addition, C-ANF,. 23 as well as ANF99-126 were also able to decrease CAMP levels (-40%) in these cells stimulated by luteinizing hormone (data not shown). DISCUSSION The data presented in the current studies demonstrate that ANF-R2 or C-ANF receptors are coupled to the adenylate cyclase/cAMP signal transduction system. ANF elicits its physiological responses by interacting with its receptors. Two subclasses of ANF receptors, ANF-Rl and ANF-R2 have been reported to be present in most of the tissues (14)(15)(16). The ANF-Rl receptor has a molecular mass of 130 kDa and co-purifies with particulate guanylate cyclase (14). The interaction of ANF with ANF-Rl receptors results    tissues. The functions of this receptor are not known, and it has been reported that this receptor is a non-guanylate cyclase-coupled receptor. Since ANF inhibits adenylate cyclase activity in a variety of tissues (2)(3)(4)(5)(6)(7)(8)(9) in a GTP-dependent manner, it is clear that the ANF-mediated inhibition of adenylate cyclase is a receptor-coupled phenomenon, and it is highly likely that one of the ANF receptors is coupled to this signal transduction system. We have recently shown that platelets which are devoid of particulate guanylate cyclase activity have ANF-R2 receptors and since ANF inhibits adenylate cyclase activity in platelets in a GTP-dependent manner, we suggested that ANF-RX receptors are coupled to the adenylate cyclase/cAMP system (20).
Recently, Maack and colleagues (17) by using the ringdeleted analog of ANF (C-ANF4& have suggested that ANF-R2 receptors are biologically inactive because of the fact that the interaction of C-ANFbet3 with ANF-R2 receptors did not result in an enhancement of cGMP levels. However, in the present studies we have shown that C-ANF4-23 like ANFW-X~ also inhibits adenylate cyclase activity in anterior pituitary, aorta, brain striatum, and adrenal cortex in a concentrationdependent manner. The maximal inhibition and the potency of C-ANF4-23 to inhibit enzyme activity was in the same range as was observed with ANFg9-iS6. In addition, C-ANFbm23 was also able to decrease CAMP levels in cultured vascular