Molecular and Celhdar Biochemistry 113: 83-92, 1992. © 1992Kluwer Academic Publishers. Printedin the Netherlands.

Characterization of ANF-R2 receptor-mediated inhibition of adenylate cyclase Madhu B. Anand-Srivastava Department o f Physiology, UniversitO de MontrOal, Canada

Received 4 December 1991: accepted 13 February 1992

Abstract We have characterized the ANF-R2 receptor-mediated inhibition of adenylate cyclase with respect to its modulation by several regulators. ANF (99-126) inhibits adenylate cyclase activity only in the presence of guanine nucleotides. The maximal inhibition ( - 45%) was observed in the presence of 10--30/xM GTPyS, and at higher concentrations, the inhibitory effect of ANF was completely abolished. ANF-mediated inhibition was not dependent on the presence of monovalent cations, however Na + enhanced the degree of inhibition by about 60%, whereas K + and Li + suppressed the extent of inhibition by about 50%. On the other hand, divalent cation, such as Mn 2+ decreased the degree of inhibition in a concentration dependent manner, with an apparent Ki of about 0.7 raM, and at 2mM; the inhibition was completely abolished. In addition, proteolytic digestion of the membranes with trypsin (40 ng/ml) resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase. Other membrane disrupting agents such as neuraminidase and phospholipase A~ treatments also inhibited completely, the ANF-mediated inhibition of enzyme activity. N-Ethylmaleimide (NEM), phorbol ester and Ca-~+-phospholipid dependent protein kinase (C-kinase) which have been shown to interact with inhibitory guanine nucleotide regulating protein (Gi) also resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase activity. These results indicate that in addition to the Gi, the phospholipids and glycoproteins may also play an important role in the expression of ANF-R2 receptor-mediated inhibition of adenylate cyclase. (Mol Cell Biochem 113" 83-92, 1992) Key words." ANF-R2 receptor, adenylate cyclase, Gi-protein, Phorbol ester, C-kinase Abbreviations. ANF - Atrial Natriuretic Factor, GTPyS - Guanosine 5'-0-(Thiotriphosphate), Gi - inhibitory guanine nucleotide regulatory protein, NEM - N-Ethylmaleimide, PMA - Phorbol, 12-Myristate, 13-Acetate, v+ C-kinase, Ca- , phospholipid-dependent protein kinase, PHL-Ae - Phospholipase A~

Introduction Atrial nutriuretic factor (ANF) from mammalian atria have been reported to exhibit a variety of physiological effects including diuresis, natriuresis, vasorelaxation, inhibition of aldosterone secretion from adrenal cortex and inhibition of vasopressin and renin release [1]. ANF

has been reported to stimulate guanylate cyclase/cGMP [2, 3] and to inhibit the adenylate cyclase/cAMP system in several tissues [4-11], suggesting that these two second messenger systems may be responsible in mediating the physiological responses of ANF. Using affinity

Address for offprints." M.B. Anand-Srivastava,Ddpartementde physiologic,Facultdde Medecine,Universitede Montreal,C.P. 6128,Succursale A, Montreal H3C 3J7, Quebec, Canada

84 cross-linking techniques [12] or photo-affinity labeling [1_3], two distinct ANF receptor subtypes have been identified and characterized in several tissues and cell types [12-15]. These are of high (130,000) and low Mr (66,000) receptors and have been designated as ANFR1 and ANF-R2 receptors respectively [12]. However, using molecular cloning techniques, three subtypes of ANF receptors of the ANF-family have been identified. These are type A, type B and type C receptors. The type A (ANF-A) also referred to as GC-A [16] is a particulate form of guanylate cyclase. This receptor responds to stimulation by both ANF and brain natriuretic peptide (BNP), however ANF is more potent than BNP in stimulating guanylate cyclase [16]. The second type B, (ANF-B) receptor, referred to as GC-B is also a membrane form of guanylate cyclase which is more responsive to BNP than ANF [17, 18]. The third receptor, type C is also referred to as ANF-R2 (ANF-C (C-ANF)) receptor [19], and is homologous to ANF-A and ANF-B type receptors throughout the extracellular domain of over 440 amino acids. However ANF-R2/ ANF-C receptor possesses a short carboxyl cytoplasmic domain of only 37-amino acid in contrast to the large (greater than 500 amino acids) cytoplasmic domain of ANF-A and ANF-B type receptors. A role for ANFR2/ANF-C receptor as clearance receptor has been postulated [20]. However, we and others [21, 22] have recently reported that ANF-R2 receptors are coupled to adenylate cyclase/cAMP signal transduction system through inhibitory guanine nucleotide regulatory protein (Gi). Subsequently by using C-ANF4_:3 peptide which interacts with ANF-R2/C receptors only, we have shown that ANF-R2 and C-ANF receptors are the same and are coupled to adenylate cyclase through system Gi [23] and mediate some of the physiological effects such as inhibition of progesterone secretion in Leydig tumor cells [23] and adrenergic neurotransmission in nerve growth factor-treated pheochromocytoma cells [24]. However, the regulation of this receptor has not yet been studied. We have therefore undertaken the present studies to characterize the ANF-R2 receptormediated adenylate cyclase inhibition with respect to its regulation by various modulators such as phorbol esters, (PMA), CaZ+-phospholipid dependent protein kinase (C-kinase) and N-Ethylmaleimide (NEM) which modulate the adenylate cyclase activity possibly by interacting with Gi-guanine nucleotide regulatory protein [25-27]. In addition, we have also studied the effect of some membrane disrupting agents, such as neuraminidase, phospholipase A~_,and trypsin to examine if mem-

brane integrity plays an important role in the expression of ANF-R2 receptor-mediated inhibition of adenylate cyclase.

Materials and methods Materials ATP and cyclic AMP were purchased from Sigma (St. Louis, MO, U.S.A.). Creatine kinase (EC 2.7.3.2), myokinase (EC 2.7.4.3) and guanosine 5'-[y-thio]triphosphate (GTP[S]) were purchased from BoehringerMannheim, Montreal, Quebec, Canada. Neuraminidase, trypsin, phospholipase A, and phorbol ester (PMA) were purchased from Sigma (St. Louis, MO, U.S.A.) C-kinase was purified from rat brain as described previously [26]. This enzyme preparation was stimulated between 8-10 fold by phosphatidyl serine and diolein. Rat (ANF-(99-126)-peptide was kindly provided by Bio-Mega, Laval, Quebec, Canada.

Methods Preparation of aorta washed particles (aorta membranes) Aorta washed particles were prepared as described previously [4, 9]. Aorta were dissected out from SpragueDawley rats (200-250g) and quickly frozen in liquid nitrogen. The frozen aorta were pulverized to a fine powder using a percussion mortar cooled in liquid nitrogen. The powdered aorta were stored at - 7 0 ° C until assayed. Aorta were homogenized using amotor-driven teflon glass homogenizer in a buffer containing 10 mM Tris-HC1 and 1 mM EDTA pH 7.5. The homogenates were used for PMA treatments. The homogenate was centrifuged at 16,000 x g for 10 rain. The supernatant was discarded and the pellet was finally suspended in 10mM Tris-HCl and 1 mM EDTA pH7.5 and used for adenylate cyclase determination.

Preparation of anterior pituitary homogenates Anterior pituitary homogenates were prepared as described previously [9]. Pituitaries were dissected out from Sprague-Dawley rats (200-250 g) and anterior pituitaries were separated and placed in ice-cold buffer containing 10 mM Tris-HC1 and 1 mM EDTA, pH 7.5. The anterior pituitaries were homogenized in the above buffer by hand with a glass-teflon homogenizer. The

85 homogenates thus obtained were used for adenylate cyclase determination. Neuraminidase treatment Aorta membranes (2-3mg/ml) were pre-incubated with neuraminidase at a concentration of 1 Unit/ml at 25°C for 20 rain in a medium containing 50 mM TrisHCI buffer pH 7.5,100 mM KC1 and 10 mM MgCI> The reaction was terminated by centrifugation at 16,000 x g for 10 min at 4 ° C. The supernatant was discarded and the pellet was washed twice with 10ram Tris-HC1, 1 mM EDTA buffer pH 7.5 and finally suspended in the same buffer for adenylate cyclase activity determination. Phospholipase A , treatment Aorta membranes (2-5 mg/ml) were preincubated with phospholipase A, at a concentration of 1Unit/ml at 25°C for 10 min in a medium containing 50 mM Tris/ HC1, pH 7.5, 100 mM KC1 and 2 mM CaCI2. The reaction was terminated by the addition of 5 mM EGTA. The contents were centrifuged at 16,000 × g for 10 min. The pellet was washed twice with 10mM Tris-HCl, 1 mM EDTA buffer pH 7.5 and finally suspended in the same buffer for adenylate cyclase activity determination. Trypsin treatment Aorta membranes (2mg/ml) were preincubated with trypsin at a concentration of 40 ng/ml in a medium containing 50mM Tris/HCl, pH7.5 and 20mM KCI for 5 rain at 30 ° C with constant agitation. The reaction was terminated by the addition of 3-fold excess of trypsin inhibitor followed by vigorous mixing for 10sec. The contents were centrifuged at 16,000 × g for 10 min. The membranes were washed twice with 10 mM Tris/HC1, 1 mM EDTA, pH7.5 and finally suspended in the same medium for adenylate cyclase activity determination. N E M treatment Aorta membranes ( - 2 mg/ml) were pre-incubated with NEM at a concentration of 0.1 mM at 25°C for 25 min in a medium containing 50 mM Tris/HC1 buffer pH 7.5, The reaction was terminated by the addition of 5raM dithiothreitol (DTT). The contents were centrifuged at 10,000 × g for 10rain. The supernatant was discarded and pellet was washed twice with 10 mM Tris/ HCI, 1 mM EDTA pH 7.5 and finally suspended in the same buffer for adenylate cyclase activity determination.

PMA treatment Aorta homogenates (2 mg/ml) were preincubated with PMA, at a concentration of 1/,M at 25°C for 30 min. The reaction was terminated by centrifugation at 16,000 x g for l0 rain. The pellet was washed twice with 10mM Tris/HCl, 1 mM EDTA (pH 7.5) and finally suspended in the same buffer for adenylate cyclase activity determination. Adenylate cyclase activity determination Adenylate cyclase activity was determined by measuring [cx~=P] cAMP formation from [cd2p]ATP as described previously [4, 22]. Typical assay medium contained 50 mM glycylglycine, pH 7.5, 0.5 mM MgATP, [c~3eP]ATP (1-1.5 x 10('CPM), 5 mM MgCI2 (in excess of the ATP concentration), 0.5mM cAMP, 1 mM 3isobutyl-l-methylxanthine, 0.1 mM EGTA, 10/J,M GTPyS, and ATP regenerating system consisting of 2 mM creatine phosphate, 0.ling creatine kinase per ml, and 0.1 mg myokinase per ml in a final volume of 200/xl. Incubations were initiated by the addition of the particulate fraction (30--70/xg) to the reaction mixture which had been thermally equilibrated for 2rain at 37 ° C. Reactions were conducted in triplicate for 10 rain at 37 ° C. Reactions were terminated by the addition of 0.6 ml of 120ram zinc acetate, cAMP was purified by co-precipitation of other nucleotides with ZnCO~ by the addition of 0.5 ml of 144 mM Na=CO3 and subsequent chromatography by the double column system as described by Salomon et al. [28].

Results and discussion We have previously reported that ANF-mediated inhibition of adenylate cyclase is absolutely dependent on the presence of guanine nucleotides. However, some investigators were unable to demonstrate the inhibitory effect of ANF on the enzyme activity in the presence of guanine nucleotides in anterior pituitary [29]. To investigate the reason for such an apparent discrepency, the effect of various concentrations of GTPyS on adenylate cyclase activity was studied in the absence and presence of ANF in anterior pituitary homogenates and the results are shown in Fig. 1. As reported earlier [7-9] ANF in the absence of GTPyS did not inhibit adenylate cyclase activity in rat anterior pituitary, however, the inhibition elicited by ANF was apparent in the presence of various concentrations of GTP,/S. The maximal inhibition ( - 4 5 % ) was observed in the presence of I0-

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late cyclase by ANF in rat anterior pituitary homogenates. Adenylate cyclase activity was determined at various concentrations of GTPyS in the absence control, (O) or presence (A) of 10 7M ANF as described under 'Materials and methods'. Values are means _+ S.E.M. of three separate experiments.

30/*M GTP•S, whereas at higher concentrations, the extent of inhibition was decreased and at 300/,M the ANF-mediated inhibition was completely abolished. These results indicate that ANF-R2 receptor-mediated inhibition of adenylate cyclase is absolutely dependent on the presence of guanine nucleotides. A lack of inhibitory effect of ANF on adenylate cyclase in rat anterior pituitary by Heisler et al. [29] may be due to the fact that these investigators used a very high concentration of GTP (300/,M) which completely abolished the expression of the inhibitory effect of ANF on adenylate cyclase in these membranes.

Effect of divalent cations Divalent cations play an important role in the expression of adenylate cyclase activity in three different ways as a complex with ATP as substrate (eg, MgATP), as an activator at a metal binding site [33] and at the guanine nucleotide binding site [34]. The degree of stimulation/ inhibition of adenylate cyclase by hormones has been reported to be dependent on the concentration of metal ions [35, 36]. Since Mn 2+ has been reported to uncouple the hormone receptors from the catalytic subunit of adenylate cyclase [37], it was of interest to examine if Mn 2+ could also affect the inhibitory responses of ANF on adenylate cyclase. The results indicated in Fig. 2A, demonstrate that M n 2+ decreased the ability of ANF to inhibit adenylate cyclase activity. ANF in the absence of Mn 2+ inhibited adenylate cyclase activity by about

Table 1. Effect of monovalent cations on ANF-mediated inhibition of adenylate cyclase in rat aorta.

Additions

Adenylate cyclase activity pmol c A M P (mg protein lOmin) -~

Effect of monovalent cations Monovalent cations such as Na +, K +, and Li + have been shown to be required in addition to GTP, for the inhibitory hormones to elicit full inhibition of adenylate cyclase [30]. The mechanism, by which, these monovalent cations amplify the hormonal inhibition of adenylate cyclase is not yet clear, however, it has also been shown that monovalent cations decrease [31] the receptor affinity for hormone agonists and slightly increase the receptor affinity for antagonists [31]. To investigate if ANF-mediated inhibition of adenylate cyclase is also dependent on the presence of monovalent cations, we examined the effect of Na +, K + and Li + on ANFmediated inhibition of enzyme activity in rat aorta. The

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Adenylate cyclase activity was determined as described under 'Materials and methods'. Values are mean +- S. E.M. of three separate experim en ts.

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2. A. Effect of Mn ~+ on A N F - m e d i a t e d inhibition of adenylate cyclase in rat aorta membranes. Adenylate cyclase activity was determined in the absence or presence of various concentrations of Mn 2÷ as described under 'Materials and methods'. MgATP was substituted by MnATP. Values are m e a n s _+ S.E.M. of three separate experiment. The basal adenylate cyclase activities in the absence and presence of A N F were 26 + 4 and 14 _+ 5 pmol c A M P (rag protein. 10 rain) 1 respectively. B. Effect of trypsin treatment on A N F - R 2 receptor-mediated inhibition of adenylate cyclase in rat aorta membranes. Aorta m e m b r a n e s were treated without (control) or with trypsin (treated) as described under 'Materials and methods'. Adenylate cyclase activity was determined in the absence or presence of various concentrations of A N F in control untreated ( I ) and trypsin treated ( A ) aorta m e m b r a n e s as described under ~Materials and methods'. Values are m e a n + S.E.M. of three separate experiments. The basal enzyme activities in control and trypsin-treated aorta m e m b r a n e s were 100 + 5 and 80 _+ 8 pmol of c A M P , (rag protein, i0 min) ~ respectively.

45%, however, in the presence of Mn 2+ the extent of ANF-mediated inhibition was decreased in a concentration dependent manner. At 2 raM, the ANF-mediated inhibition was completely abolished. These data suggest that ANF-R2 receptors like other hormone receptors can also be uncoupled by high concentrations of Mn 2+. On the other hand, the ANF-mediated inhibition of adenylate cyclase was also dependent on Mg 2+ concentration, but the extent of inhibition was never abolished in the presence of high concentrations of Mg 2+ and remained unaltered (data not shown).

Effect of trypsin Trypsin has been shown to activate the basal adenylate cyclase activity as well as the sensitivity of several hormones to stimulate the adenylate cyclase in various tissues [38-40]. However, the inhibitions elicited by hormones on adenylate cyclase have been reported to be attenuated by trypsin treatment [41] which may be due to its interaction with Gi-regulatory protein. Recently trypsin has also been shown to affect ANF binding as well as to inhibit the ANF-mediated stimulation of guanylate cyclase, which involves ANF-R1 receptors in bovine adrenal zona glumerulosa membranes [42].

We were interested to examine if the ANF-R2-receptor-mediated inhibition of adenylate cyclase is also affected by such treatment. As shown in Fig. 2B, ANF, inhibited adenylate cyclase activity in aorta membranes in a concentration dependent manner, with an apparent Ki between 0.1-0.5mM, however this inhibition was completely abolished in trypsin-treated membranes. These data suggest that ANF-R2 receptors coupled to adenylate cyclase system through Gi-regulatory protein could also be modulated by trypsin treatment. The attenuation of ANF-mediated inhibition by trypsin may be due to the uncoupling effect. A similar attenuation of epinephrine-mediated inhibition of adenylate cyclase has also been reported previously [41].

Effect of N-ethyhnalernide (NEM) Since, NEM, a sulfydryl alkylating agent has been shown to decrease agonist binding to inhibitory receptors and attenuates the agonist-stimulated inhibition of adenylate cyclase [27, 43]; it was of great interest to investigate if the inhibitory effect of ANF on adenylate cyclase could also be blocked by NEM treatment. Figure 3 shows that ANF (10-~M) inhibited adenylate cyclase by about 40% in control aorta membranes, how-

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NEM Fig. 3. Effect of N-ethylmaleimide (NEM) treatment on ANF-R2 receptor-mediated inhibition of adenylate cyclase in rat aorta membranes. Aorta membranes were treated without (control) or with (treated) NEM as described under 'Materials and methods'. Adenylate cyclase activity was determined in the absence (D) or in the presence (N), N) of 0.1 p.M ANF as described under 'Materials and methods'. Values are mean +_S.E.M of three separate experiments. The basal enzyme activities in control and NEM-treated membranes were 129 ± 18 and 58 + 3pmol cAMP (mg protein, ll) rain) -~ respectively. e v e r , this i n h i b i t o r y effect of A N F was c o m p l e t e l y att e n u a t e d in N E M - t r e a t e d m e m b r a n e s . T h e a t t e n u a t i o n of A N F - R 2 r e c e p t o r - m e d i a t e d inh i b i t i o n of a d e n y l a t e cyclase by N E M m a y b e d u e to the p o s s i b i l i t y t h a t N E M t r e a t m e n t has a l t e r e d the r e c e p t o r f u n c t i o n at t h e A N F r e c e p t o r b i n d i n g d o m a i n o r / a n d at t h e level o f the A N F - R 2 r e c e p t o r c o u p l i n g to a d e n y l a t e cyclase t h r o u g h G i p r o t e i n . T h e i n h i b i t i o n of A N F m e d i a t e d g u a n y l a t e cyclase a c t i v a t i o n by N E M has also b e e n d e m o n s t r a t e d [44]. Since A N F - R 1 r e c e p t o r has intrinsic g u a n y l a t e cyclase activity a n d d o e s not r e q u i r e any c o u p l i n g p r o t e i n to c o u p l e t h e A N F - R 1 r e c e p t o r to g u a n y l a t e cyclase, it is p o s s i b l e t h a t sulfhydryl g r o u p s m a y b e i m p o r t a n t for t h e b i n d i n g d o m a i n of A N F - R 1 r e c e p t o r . H o w e v e r , t h e i n a c t i v a t i o n of a d e n y l a t e cyclase as well as t h e G i - r e g u l a t o r y p r o t e i n by N E M has also b e e n r e p o r t e d [27]. T a k e n t o g e t h e r , it can be sugg e s t e d t h a t s u l f h y d r y l g r o u p s m a y b e i m p o r t a n t for the b i n d i n g d o m a i n of A N F - r e c e p t o r s a n d / o r for the cou-

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Fig. 4. Effect of phospholipase A,_ (PHL-A2) and neuraminidase on ANF-R2 receptor-mediated inhibition of adenylate cyclase in rat aorta membranes. (A) Aorta membranes were treated without (control) or with PHLA2 (treated) as described under "Materials and methods'. Adenylate cyclase activity was determined in the absence (El) or presence of 0.1 p~M ANF in control ([]) and treated ([]) membranes as described under 'Materials and methods'. Values are means ___S.E.M. of three separate experiments. The basal enzyme activities in control and PHL-A2-treated membranes were 62 + 8 and 49 _+ 8 pmol cAMP (rag protein. 10 rain) ~respectively. (B) Aorta membranes were treated without (control) or with neuraminidase (treated) as described under 'Materials and methods'. Adenylate cyclase activity was determined in the absence (UI), or presence of 10 v M ANF in control (IN) and treated (N) membranes as described under 'Materials and methods'. Values are mean ± S.E.M. of three separate experiments. The basal enzyme activities in control and neuraminidase treated membranes were 46 ± 7 and 45 ± 4 pmol cAMP (rag protein. 10 rain) J respectively. piing of A N F - R 2 r e c e p t o r s to a d e n y l a t e cyclase t h r o u g h Gi-regulatory protein.

Effect of neurarninidase and phospholipase-A2 treatmen ts M e m b r a n e i n t e g r i t y has b e e n s h o w n to p l a y an i m p o r tant role in t h e e x p r e s s i o n of h o r m o n e - s e n s i t i v e a d e n y late cyclase activity. T h e n a t u r e of A N F - R 1 c o u p l e d g u a n y l a t e cyclase r e c e p t o r as a g l y c o p r o t e i n has b e e n d e m o n s t r a t e d by its sensitivity to n e u r a m i n i d a s e t r e a t m e n t [45]. H o w e v e r , the lack of n e u r a m i n i d a s e effect on A N F - r e c e p t o r b i n d i n g p r o p e r t i e s i n d i c a t e t h a t sialic acid r e s i d u e s are not i m p o r t a n t for h o r m o n e b i n d i n g

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Fig.5. (A) Effect of PMA treatment on ANF-medi~tted inhibition of adenylate cyclase in rat aorta homogenates. Aorta homogenates were treated without (control) or with PMA (treated) as described under 'Materials and methods'. Adenylate cyclase activity was determined in the absence (basal, [], it) or presence of I0 -7 M ANF (N, N), as described undcr "Materials and methods'• Values are means + S.E.M. of three separate experiments. The basal enzyme activities in control and PMA-treated membranes were 50 _+ 1 and 22 + 2 pmol cAMP (rag protein• 10min) ' respectively• (B) Effect of C-kinase on ANF-mediated inhibition of adenylate cyclase in rat aorta membranes, Adenylate cyclase activitywas determined in the absence (basal, [~) or presence of C-kinase (Jill),0.1/xM ANF (iN), or C-kinase + ANF ([]) as described under 'Materials and methods'• Values are mean _+S.E.M. of three separate experiments• domain [42]. We investigated, if the ANF-R2-receptormediated inhibition of adenylate cyclase could be modulated by such treatment. Figure 4 shows that A N F inhibited adenylate cyclase activity by about 30% which was completely attenuated by neuraminidase treatment. These results indicate that the sialic acid residues may be important for the expression of A N F - m e d i a t e d inhibition of adenylate cyclase. Since A N F binding activity is not affected by neuraminidase treatment [42], it may be possible that the observed attenuation of ANFmediated inhibition by neuraminidase may be due to its interaction at the site distal to the A N F - r e c e p t o r and possibly at the level of Gi-protein interaction with receptor. M e m b r a n e phospholipids have also been reported to be essential c o m p o n e n t s for the expression of stimulatory responses of several h o r m o n e s [46, 47]. We have previously shown the requirement of phospholipids in the coupling of d o p a m i n e receptors to adenylate cyclase in brain striatum [48]. To investigate, if A N F -

R2 receptor-coupled adenylate cyclase inhibition also requires the presence of m e m b r a n e phospholipids, we examined the effect of phospholipase-A2 treatment on A N F - m e d i a t e d adenylate cyclase inhibition and the results are shown in Fig. 4. A N F inhibited adenylate cyclase activity by about 40% in control m e m b r a n e s , and phospholipase-Az treatment completely abolished the ANF-induced inhibition of adenylate cyclase. These results indicate that A N F - R 2 receptor-mediated inhibition is dependent on the presence of m e m b r a n e phospholipids. The effect of phospholipase-A2 was probably not due to the detergent effect of lysophospholipids or fatty acids, the degradation products of phospholipase-A2 digestion, since the inclusion of fatty acid p o o r serum albumin during the treatment (data not shown) or extensive washing of the treated m e m b r a n e s with serum albumin which binds both products [49] did not prevent or reverse the attenuation of A N F - m e d i a t e d inhibition of adenylate cyclase (data not shwon). The data suggest that the observed effects of phospholi-

90 pase-A2 were due to the hydrolysis of the membrane phospholipids, and that the phospholipids are important for the expression of ANF-R2-receptor mediated inhibition of adenylate cyclase.

Effect of phorbol ester (PMA) and Ca2+-phospholipid dependent protein kinase (C-kinase) PMA and C-kinase have also been reported to modulate adenylate cyclase activity by interacting with Gi regulatory protein [25]. We have recently demonstrated that C-kinase which is believed to be a major cellular receptor for PMA [50] augmented the stimulatory responses of N-ethylcarboxamideadenosine (NECA), dopamine and forskolin to adenylate cyclase activation, and attenuated the inhibition exerted by oxotremorine at muscarinic receptors [26]. It was of interest to examine if ANF-R2 receptor-mediated inhibition could also be affected by PMA as well as C-kinase. As shown in Fig. 5, ANF inhibited adenylate cyclase by about 30% in control membranes whereas this inhibition was completely blocked in membranes treated with PMA (Fig. 5A). Similarly, ANF inhibited the adenylate cyclase activity in the absence of C-kinase, however in the presence of C-kinase, (which itself stimulated enzyme activity) the inhibition by ANF was not observed (Fig. 5B). These data indicate, that the attenuation of ANFR2 receptor-mediated inhibition by PMA or C-kinase may be due to the inactivation of Gic~ which can no more exert its inhibitory input on enzyme activity [25]. The phosphorylation and thereby inactivation of Gic~ by C-kinase has been demonstrated recently [25]. In this regard, it is like pertussis toxin (PT) which also inactivates the Gic~ by ADP-ribosylation [51] by a mechanism different than that of C-kinase. The inactivation of Gi(* results in the attenuation of inhibitory responses of hormones on adenylate cyclase. These results thus suggest that like other inhibitory hormone receptors, the ANF-R2 receptor mediated inhibition could also be modulated by PMA and/or C-kinase at the level of Gi regulatory protein. In summary, by using various modulators and membrane disrupting agents, which can affect receptor-effector coupling, we have shown that in addition to the inhibitory guanine nucleotide regulatory protein (Gi), membrane phospholipids and glycoproteins may also play an important role in the expression of ANF-R2 receptor-mediated inhibition of adenylate cyclase.

Acknowledgements I would like to thank Sylvie Picard, for her excellent technical assistance and Christiane Laurier for her valuable secretarial help. This work was supported by grants from the Quebec Heart Foundation and the Medical Research Council of Canada. Part of this work was presented in Third World Congress on Biologically Active Peptides in New York, 1988.

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