Amiloride potentiates atrial natriuretic factor inhibitory action by increasing receptor binding in bovine adrenal zona glomerulosa

Life Sciences, Vol. 39, pp. 1109-1116 Printed in the U.S.A.

Pergamon Journals

AMILORIDE POTENTIATES ATRIAL NATRIURETIC FACTORINHIBITORY ACTION BY INCREASING RECEPTORBINDING IN BOVINE ADRENAL ZONAGLOMERULOSA Andr~ De L~an Laboratory of Molecular Pharmacology, Clinical Research Institute of MoNtreal, llO Pine Avenue West, Montreal, Quebec, Canada H2W IR7. (Received in final form June 24, 1986) Summary The interaction

of atrial natriuretic factor (ANF) with the

diuretic amiloride was studied in bovine adrenal zona glomerulosa. Amiloride enhances 2 to 3-fold high a f f i n i t y binding of [12511ANF to zona glomerulosa membrane receptor with an EDs0 of lO uM. This effect is due to a recruitement of high a f f i n i t y receptor sites and to an increase of their a f f i n i t y from a Kd of 23 to 8 pM. This enhancing effect is almost equipotently elicited by guanabenz, while clonidine is 20-fold less potent and arginine is inactive. ATP reduces by 30 to 50% [1251] ANF binding with an IC50 of 50 ~M. Amiloride and ATP opposite effects on [1251] ANF binding are mutually competitive. Low concentrations of amiloride (lO0 uM) directly inhibit aldosterone secretion with an ICs0 of 500 uM and a maximum of 80 to I00% reversal of stimulation by various secretagogues. These results indicate that amiloride synergistically potentiates ANF inhibitory action by altering ANF receptor binding properties. They also suggest a role for sodium transport and for phosphorylation-dephosphorylation mechanisms in the mode of action of ANF. Introduction Atrial natriuretic factor e l i c i t s vasorelaxant, natriuretic and diuretic effects through its interaction with specific high a f f i n i t y receptor sites (I-3). ANF inhibits adenylate cyclase activity (4) and stimulates particulate guanylate cyclase activity (5) in all target cell systems studied. Yet its cellular mode of action is not very well understood in target tissues e.g. adrenal zona glomerulosa where ANF potently inhibits aldosterone biosynthetic pathway (1,6). Interestingly, in vivo uptake of ANF is documented in epithelia e.g. kidney collecting tubule or intestinal mucosa involved in fluid and solute reabsorption (7). ANF also blocks sodium uptake in several systems through an amiloride-sensitive mechanism (8-I0). Amiloride is a potassium sparing diuretic with a wide spectrum of effects. In epithelia i t blocks sodium uptake occuring through a passive conductive pathway and also a Na/H exchange system ( l l ) . Higher concentrations of amiloride block growth factorinduced mitosis (12) and also inhibit tyrosine kinase (13), calcium and phospholipid-dependent kinase (14) and cyclic AMP dependent kinase (15). 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.

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The close association of ANF and amiloride action in epithelia suggested a need to test for t h e i r potential interaction in a mode] target cell system for ANF e.g. adrenal zona glomerulosa. We report that amiloride potentiates ANF action by regulating i t s receptor binding properties and at higher concentration mimics the inhibitory effects of ANF on corticosteroid production. The results are discussed in terms of potential molecular mechanisms of action of ANF and of perspectives for in vivo therapeutic synergisms. Methods Fresh bovine adrenals were obtained from a local abattoir. The zona glomerulosa layer was immediately dissected out and used both for membrane preparation and for primary cell culture as previously described (16). Membrane receptor preparation was aliquoted, frozen and stored at -70 C. Zona glomerulosa cells were plated at lO 6 cells/ml in F-12 medium supplemented with I0% horse serum and antibiotics. Rat (Ser99-TyrI26)ANF ( I n s t i t u t Armand Frappier, Montreal) was iodinated with Iodo-Beads (Pierce) and purified on octadesilyl reversed phase HPLC (17). A specific a c t i v i t y above 3000 CPM/fmol was t y p i c a l l y obtained. In the receptor binding assay 7 to 40 ~g of membrane receptor was incubated in duplicate tubes at I0 C for 4 hours with 8 pM (30,000 CPM) of [12si] ANF and various concentrations of native ANF in l ml of buffer containing 50 mM Tris-Cl pH 7.4, 5 mM MnCl2, O.l ~M EDTA, 0.5% BSA (Buffer A). Prolongating incubation of the receptor binding assay up to 18 hours s l i g h t l y increased overall binding without changing any of the observed properties of the system. Receptor-bound [12sI] ANF was separated by f i l t r a t i o n and washing with Buffer A over GF/C glass fiber f i l t e r s pretreated with I% polyethyleimine. Membrane protein was assayed using a k i t (Pierce) for Bradford method. Zona glomerulosa cells cultured for 3 days were washed 3 F-12 medium and were then stimulated in quadruplicate wells with agents diluted in F-12 medium supplemented with Aldosterone production in culture medium was then assayed radioimmunoassay (18).

times with fresh for three hours 0.1% lysosyme. in duplicate by

ANF was f i r s t prepared at O.l mM concentration in O.l N acetic acid and was then diluted to final concentration in F-12 medium with 0.1% lysosyme. Amiloride was f i r s t prepared at l M concentration in dimethylsulfoxide then diluted to final concentration in F-12 medium. No influence of dimethylsulfoxide alone after final dilution was observed on aldosterone secretion.

Dose-response curves were analyzed by weighted non l i n e a r regression analysis using a four parameter l o g i s t i c equation (19). Radioligand binding competition curves were s i m i l a r l y analyzed using a model based on the law of mass action (20). All experiment reported were repeated at least 2 or 3 times with reproducible r e s u l t s . Results Amiloride enhanced [12Sl] ANF binding to bovine adrenal zona glomerulosa membrane receptor. Figure l ( l e f t panel) shows that lO0 ~ amiloride e l i c i t e d a 2.4-fold increase in receptor-specific binding with no change in the nonspecific binding component. As i l l u s t r a t e d in the Scatchard plot transform (Fig. l , right panel) amiloride lead to a recruitement of high a f f i n i t y sites from 335 to 740 fmol/mg protein as well as to an increase in i t s a f f i n i t y from a Kd of 23 to 8 pM.

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Synergism of Amiloride on ANF Action

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FIG. 1

Representative effect of amiloride on ANF binding to adrenal membranes. {12Sl] ANF (8 pM) and varying concentrations of native hormone were incubated at lO C for 4 hours with 18 ug/m] of membrane protein. In the absence of amiloride (~) ANF binding involves high a f f i n i t y (4.4xlO1OM -I) and low a f f i n i t y (I.8xlOgM -I) components with binding capacities of 6.4 and 8.8 pM, respectively, while in the presence of O.l mM amiloride ( | ) a single component with high affinity (I.3xIOIIM -I) and capacity of 13.7 pM is observed, Right panel is a Scatchard plot of the same data.

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Representative dose-response curves of amiloride (e), guanabenz ( l ) , clonidine (A) and arginine (@) on ANF binding to adrenal membranes. [;2s I] ANF (8 pM) was incubated at lO C for 4 hours with 7 ~g/ml of membrane protein in the presence of the indicated concentration of agents. Dose-response curves were characterized by an EDso of 20 ~M f o r amiloride, of 40 ~M f o r guanabenz, of 2 mM f o r c l o n i d i n e , while arginine had no e f f e c t on ANF binding.

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Dose-response curves for amiloride on ANF binding were characterized by an EDs0 of 5 to 20 ~M and a maximum 4-fold stimulation at ] ~ (Fig. 2). Since amiloride structure is characterized by an unconstrained guanidium moiety connected to an aromatic ring, we then compared its effect with those of other structurally related compounds. Guanabenz which also features a guanidium moiety and an aromatic ring was 2-fold less potent than amiloride (Fig. 2), while clonidine which posesses an aromatic ring connected to a constrained guanidium group was lO0-fold less potent than amiloride. Arginine which features a guanidium moiety but no aromatic ring was t o t a l l y inactive. The s p e c i f i c i t y of the effect of amiloride on ANF receptor was also assessed by testing the effect of the diuretic on ANF radioimmunoassay and on angiotensin receptor. Amiloride failed to a l t e r binding of ANF to anti-ANF antibody, neither did i t modulate the binding of agonist or antagonist analogs of angiotensin II (data not shown).

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FIG. 3 Representative effect of ATP on ANF binding to adrenal membranes. Competitive binding curves were obtained with 8 pM [125I] ANF and 14 ~g of membranes incubated at lO C for 18 hours. In the absence of ATP (e) high a f f i n i t y (l.7xlO11M ~ ) and low a f f i n i t y (l.OxlOgM~ ) components with capacities of 4.4 and 15.8 pM were obtained while with l mM ATP (o) the high a f f i n i t y component decreased to 2.9 pM and the low a f f i n i t y component increased correspondingly, with l i t t l e change in the a f f i n i t y of either component. Right panel is a Scatchard plot of the same data.

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Since amiloride inhibits various protein kinases (13-15) and possibly stimulates phosphatases (21), we then tested the effect of ATP on ANF receptor binding properties. Figure 3 shows that l mM ATP inhibits by 36% [1251] ANF binding ( l e f t panel). ATP decreases the proportion of high a f f i n i t y binding sites from)319 to 210 fmol/mg with l i t t l e change in their a f f i n i t y (Fig. 3, right panel . A dose-response curve of ATP on [12Sl] ANF binding indicates an EDso of lO0 uM and a maximum decrease of 66% (Fig. 4, right panel). FIG. 4 Representative d o s e responsecurves of amiloride, ATP and their combination on ANF binding to adrenal z o n a glomerulosa membranes. [12sI] ANF (8 pM) was incubated at lO C for 18 hours with 14 ~g of membranes. Left panel shows dose-response curves of amiloride in the absence (e) or in the presence ( ~ of O.l mM ATP. Data analysis indicated an EDs0 of 6 and II uM without and with ATP, respectively. Right panel shows doses response-curves of ATP in the absence (0) or,the presence ) of 20~ M amiloride. These curves are characterized by an EDso of 51 and 56 uM without and with amiloride, respectively.

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Synergism of Amiloride on ANF Action

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The inhibitory effect of ATP on [12sI] ANF binding is completely reversed by amiloride (Fig. 4, l e f t panel). A comparison of the EDs0 of amiloride in the absence (lO uM) and in the presence (20 uM) of I00 uM ATP suggests that amiloride behaves as a competitive inhibitor of ATP, an observation formely reported in other systems (13). Figure 4, right panel however shows that ATP only p a r t i a l l y reverses the effect of 20 uM amiloride, revealing a partly noncompetitive behavior of the two modulatory agents. These results suggest that amiloride and ATP might compete for the same site but that they might also interact a l l o s t e r i c a l l y to regulate ANF receptor binding properties. #

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FIG. 5 Representative e f f e c t o f a m i l o r i d e on the potency o f ANF as inhibitor of PGE1-stimulated a l d o s t e r o n e s e c r e t i o n . Cultured zona glomerulosa cells were incubated for 3 hours with v a r y i n g doses o f ANF i n the

absence (o) or the presence (o) of lO ~M amiloride. The doseresponse curves of ANF inhibitory effect on mineralocorticoid production are characterized by an EDso of 63 and 37 pM in the absence and in the presence of amiloride, respectively.

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In order to test whether the potentiating action of amiloride on ANF binding to its receptor was reflected in the biological properties of the hormone, we assessed the effect of amiloride on the potency of ANF as an inhibitor of aldosterone secretion. Figure 5 shows that lO ~M amiloride potentiates the inhibitory effect of ANF, leading to a 3-fold decrease in the EDsp of ANF from 200 to 70 pM. This concentration of amiloride is also equal to i t s EDs0 in promoting ANF binding to its receptor sites (Fig. 2). 3 -4/

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FIG. 6 Representative effect of high concentrations of amiloride on al dosterone secretion. Primary cultures of zona glomerulosa cells w e r e stimulated for 3 hours with l uM PGEI and varying concentrations of amiloride (e), with PGE I and I0 nM ANF (ANF labeled bar) or without PGEI (basal labeled bar). The doseresponse curve for inhibition by amiloride was characterized by an ED so o f 50 ~M i n h i b i t i o n down to an e f f e c t nearly obtained w i t h I 0 nM

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In addition to i t s potentiating effect on ANF inhibition of adrenal steroidogenesis, higher doses of amiloride could d i r e c t l y i n h i b i t aldosterone secretion. Figure 6 shows a dose-response curve of amiloride as an i n h i b i t o r of PGE1-stimulated steroidogenesis. The inhibition curve was abrupt (from 100 uM to l mM) and reached a maximum I00% inhibition of aldosterone secretion at l mM. This inhibitory effect was nearly twice that obtained with lO pM ANF (Fig. 6). In a separate series of experiments, we observed that amiloride can also completely i n h i b i t aldosterone secretion stimulated by angiotensin, ACTH, forskolin and phorbol ester (data not shown). This general inhibitory effect of amiloride against a l l secretagogues is reminiscent of that observed for ANF (1). Maximally effective doses of ANF (lO pM) and submaximally effective concentra ions of amiloride (200 uM) inhibited aldosterone secretion to the same extent. Howevertheir effects were not additive when added simultaneousl y , suggesting that the two agents might share some common mechanism of inhibitory action (data not shown).

Discussion The mechanism of action of ANF in various target tissues e.g. kidney glomeruli, c o l l e c t i n g tubules, a r t e r i a l wall smooth muscle and adrenal cortex is s t i l l subject to debate. I t is widely recognized that ANF d i r e c t l y stimulates c y c l i c GMP production by increasing p a r t i c u l a t e guanylate cyclase a c t i v i t y (5). The role of c y c l i c GMP in mediating the vasorelaxant e f f e c t of ANF is supported by i t s analogy with that for the other vasorelaxant sodium nitroprusside (22). Cyclic GMP has also been shown to modulate ion fluxes and to mimic the e f f e c t of ANF in flounder i n t e s t i n e (9). However a role for c y c l i c GMP in the control of adrenal steroidogenesis has been generally denied. We have observed that 8-bromo-cyclic GMP f a i l s to mimic the i n h i b i tory e f f e c t of ANF on aldosterone secretion (unpublished data). We have also recently observed that pertussis toxin treatment which completely ~ d i f i e s by ADP-ribosylation of GTP binding regulatory proteins involved in hormoneinduced i n h i b i t i o n of adenylate cyclase a c t i v i t y does not a l t e r the i n h i b i t o r y properties of ANF on aldosterone production (unpublished data). Thus a role for c y c l i c nucleotides in the mode of action of ANF on adrenal cortex is unclear. With regards to calcium-dependent mechanims, Goodfriend has recently provided evidence to exclude any role of calcium fluxes or of phosphatidyl i n o s i t o l turnover in the mode of action of ANF in the adrenal cortex (6). In addition, we have previously shown that ANF potently i n h i b i t s aldosterone secretion e l i c i t e d by agents acting through c y c l i c AMP e.g. ACTH, PGEI, f o r s k o l i n , as well as by agents acting through i n o s i t o l trisphosphate production and calcium-phospholipid dependent C-kinase, e.g. angiotensin I f , phorbol ester (1). Taken together, these data strongly suggest that ANF i n h i b i t s zona glomerulosa cell a c t i v i t y through a s t i l l unknown mechanism at a crucial step e i t h e r downstream at the level of cholesterol mobilization to the mitochondria (23) or perhaps through a broad and general i n h i b i t i o n of plasma membrane effectors. The i n i t i a l structure-activity relationships of the effects of amiloride (Fig. 2) indicate a requirement for a free guanidium moiety and for an aromatic ring. The structural s p e c i f i c i t y of the effect of amiloride and i t s lack of effect on ANF binding to anti-ANF antibody or on angiotensin receptor indicate that amiloride probably does not a l t e r ANF binding by interacting with the hormone but rather by specific modification of ANF receptor conformation. The potency of amiloride in promoting ANF receptor binding and i t s structural requirements are reminiscent of i t s properties as an i n h i b i t o r of the Na/H exchange system for which i t displays an EDs0 of 7 uM and the same requirement for a free unsubstituted guanidium (24).

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The observation that both amiloride and guanabenz potentiate ANF interaction with its receptor suggest that these drugs might amplify its in vivo effects e.g. natriuresis and diuresis. Interestingly, amiloride is known as a potassium-sparing diuretic acting on collecting tubule to inhibit sodium reabsorption ( l l ) , one of the presumed sites of action of ANF (3). Guanabenz is a centrally acting anti-hypertensive agent with alpha2-adrenergic properties (25). Somediuretic properties of guanabenz have also been documented, especially after acute extracellular volume expansion (26-27), a condition known to promote ANF release from atria (28). Further studies with both agents will be required in order to assess whether or not they might potentiate vasorelaxant, natriuretic or diuretic effects of ANF. Besides its inhibitory effects on sodium uptake in various epithelia ( l l ) , amiloride is also an inhibitor of various kinases (12-15) and possibly of phosphoprotein phosphatases (21). It has been shown that amiloride and ATP mutually compete at the active site of tyrosine kinase of EGF receptor (13). Our observations of opposite and competitive properties of amiloride and of ATP also suggest that a phosphorylation-dephosphorylation mechanism might regulate ANF receptor a f f i n i t y . We might hypothesize that amiloride induces a high a f f i n i t y state of the receptor by either inhibiting its phosphorylation or by promoting its dephosphorylation. ANF receptor phosphorylation would reduce its interaction with the hormone, a mechanism which could be associated with receptor desensitization (29). Amiloride directly inhibits hormone-induced aldosterone secretion, an effect which is not additive with that of ANF. T h i s suggests that amiloride and ANF might share some common mode of action, perhaps through inhibition of protein phosphorylation. A potential target for their similar action might be the mobilization of cholesterol to mitochondria where i t serves as a substrate for the enzyme cholesterol side chain cleavage. Intracellular cholesterol mobilization appears to be an important regulatory mechanism in steroidogenesis (23) and seems to be altered by ANF (6). Further studies will be required in order to provide support for this proposed mechanism. We have documented potentiating effects of amiloride on ANF adrenal receptor functions. These effects which provide a basis for further insight into the mechanism of action of ANF are not unique to the adrenal cortex and we have recently observed identical results in cultured kidney tumor cells and in aorta. These results provide a novel approach to the study of the mode of action of ANF in various target tissues and suggest the assesment of potent i a l l y useful synergisms in the therapeutic applications of ANF. Ackowledgments We wish to thank Carmen Gagnon and Marjolaine Roy f o r t h e i r e x c e l l e n t technical assistance. A.D.L. is a Scholar of the Canadian Medical Research Council of Canada. This work was supported by a Grant from the MRC to the M u l t i d i s c i p l i n a r y Research Group on Hypertension. References I. A. DE LEAN, K. RACZ, J. GUTKOWSKA, T.T. NGUYEN, M. CANTIN and J. GENEST, Endocrinology 115 1636-1638 (1984). 2. M.A. NAPIER, R.T. VANDLEN, G. ALBERS-SCHONBERG, R.F. NUTT, S. BRADY, T. LYLE, R. WINQUIST, E.P. FAISON, L.A. HEINEL and E.H. BLAINE, Proc. Natl. Acad. Sci. (USA) 81 5946-5950 (1984). 3. A. DE LEAN, P. VINAY and M. CANTIN, FEBS Lett. 195 239-242 (1985).

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4. M.B. ARMAND-SRIVASTAVA, D.J. FRANKS, M. CANTIN and J. GENEST, Biochem. Biophys. Res. Commun. 121 855-862 (1984). 5. S.A. WALD~tAN, R.M. RAPOPORTand F. MURAD, J. Biol. Chem. 250 14332-14334 (1984). 6. T.L. GOODFRIEND, M.E. ELLIOT and S.A. ATLAS, Life Sci. 35 1675-1682 (1984). 7. C. BIANCHI, J. GUTKOWSKA, G. THIBAULT, R. GARCIA, J. GENEST and M. CANTIN, Histochemistry 82 441-452 (1985). 8. T.G. HAMMOND,A.N.K. YUSUFI, F.G. KNOX and T.P. DOUSA, J. Clin. Invest. 75 1983-1989 (1985). 9. S.M. O'GRADY, M. FIELD, N.T. NASHand M.C. RAO, Am. J. Physiol. 249 C531-534 (1985). lO. H.F. CANTIELLO and D.A. AUSIELLO, 134 852-860 (1986). I I . D.J. BENOS, Am. J. Physiol. 242 C13T-C145 (1982). 12. J.M. BESTERMAN, S.J. TYREY, E.J. CRAGOEand P. CUATRECASAS, Proc. Natl. Acad. Sci (USA) 81 6762-6766 (1984). 13. R.J. DAVIS and M.-]~. CZECH, J. Biol. Chem. 260 2543-2552 (1985). 14. J.M. BESTERMAN,W. STRATFORD-MAY, H, LEVINE, E.J. CRAGOEand P. CUATRECASAS, J. Biol. Chem. 260 I155-I159 (1985). 15. R.K. RALPH, J. SMART, S.J. WOJCIK and J. MC QUILLAN, Biochem. Biophys. Res. Commun, I04 I054-I059 (1982). 16. A. DELEAN, G. ThIBAULT, N.G. SEIDAH, C. LAZURE, J. GUTKOWSKA, M. CHRETIEN, J. GENEST, and M. CANTIN. Biochem. Biophys. Res. Commun 132 360-367 (1985). 17. A. DE LEAN, J. GUTKOWSKA, N. MC NICOLL, P.W. SCHILLER, M. CANTIN and J. GENEST, Life Sci. 35 2311-2318 (1984). 18. A. DE LEAN, K. RACZ, N. MC NICOLL and M.L. DESROSIER. Endocrinology l l 5 485-492 (1984). 19. A. DE LEAN, P.J. MUNSONand D. RODBARD. Am. J. Physiol. 235 E97-E102 (I 978). --20. A. DE LEAN, A. HANCOCKand R.J. LEFKOWITZ, Molec. Pharmacol. 21 5-16 (1982). 21. A. LE CAM, P. AURERGERand M. SAMSON, Biochem. Biophys. Res. Commun 106 1062-1070 (1982). 22. R.M. RAPOPORT, A.S. WALDFtAN, K. SCHWARTZ, R.J. WINQUIST and F. MURAD, Eur. J. Pharmacol. I15 219-229 (1985). 23. M.J..DI BARTOLOMEIS~d C.R. JEFCOATE, J. Biol. Chem. 259 10159-10167 (1984). 24. P. VIGNE, C. FRELIN, E.J. CRAGOEand M. LAZDUNSKI, Molec. Pharmacol. 25 131-136 (1984). 25. R. HOLMES, R.N. BRODGEN, R.C. HEEL, T.M. SPEIGHT and G.S. AVERY, Drugs 26 212-229 (I 983). 26. J. STEIN and R. OSGOOD, J. Cardiov. Pharmacol. 6 5787-5792 (1984). 27. J.W. STRANDHOY,M. MORRIS, B.D. STEGand W.M. BUCKALEW, J. Pharm. Exp. Ther. 226 419-424 (1983). 28. R.E. L ~ , H. THOLBEN, D. GANTEN, F.C. LUFT, H. RUSKOAHOand T.H. UNGER, Nature 314 264-266 (1985). 29. P. NAMB ,T~-D.R. SIBLEY, J.M. STADEL, T. MICHELL, J.R. PETERSand R.J. LEFKOWITZ, J. Biol. Chem. 259 4629-4633 (1984).