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J Comp Physiol B (1992) 162:436M39 Journal of Comparative Systemic, Biochemical, and Environ- Physiology B ~~ PhySiology Springer-Verlag 1992 Effects of eel atrial natriuretic peptide on NaCI and water transport across the intestine of the seawater eel Masaaki Ando l, Kyoko Kondo 1 and Yoshio Takei 2 1 Laboratory of Physiology, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshirna 730, Japan, and 2 Department of Physiology, Kitasato University School of Medicine, Kanagawa 228, Japan Accepted February 6, 1992 Summary. Eel atrial natriuretic peptide inhibited the serosa-negative transepithelial potential difference and short-circuit current, accompanied by a decrease in NaC1 and water absorption across the seawater eel intestine. Similar effects were obtained after treatment with N- terminally truncated eel atrial natriuretic peptide (5-27), indicating that N-terminal amino acids are not essential for the action of eel atrial natriuretic peptide. Although mammalian atrial natriuretic peptides also inhibited the short-circuit current, a 100-fold higher concentration was required to obtain the same effect as with eel atrial natriuretic peptide, indicating that eel atrial natriuretic peptide is 100 times as potent in eel intestine as the mammalian atrial natriuretic peptides. Similarly, in mammalian atrial natriuretic peptide, the four N-termi- nal amino acids had no significant effects. However, when the C-terminal tyrosine was removed, the potency of rat atrial natriuretic peptide was lowered. Compared with the effects of acetylcholine, serotonin and histamine, eel atrial natriuretic peptide was the most potent in- hibitor, with 100% inhibition at 10 -7 M; 50% inhibition was obtained at 10 -2 Min acetylcholine, and 30% inhibi- tion in serotonin (10-SM) and histamine (10 -3 M). These inhibitory effects of eel atrial natriuretic peptide were not diminished even in the presence of tetradoxin, and were mimicked by 8-bromoguanosine 3',Y-cyclic monophosphate. Based on these results, structure-activ- ity relationships of eel atrial natriuretic peptide and a possible mechanism of action of eel atrial natriuretic peptide are discussed. Key words: Eel atrial natriuretic peptide - NaC1 absorp- tion - Water absorption - Eel intestine - Structure-activ- ity relationship Abbreviations: 8BrcGMP, 8-bromoguanosine 3',5'-cyclic mono- phosphate; eANP, eel atrial natriuretie peptide; hANP, human atrial natriuretic peptide; 5-HT, 5-hydroxytryptamine creatine sulphate; Is~,short-circuit current; PD, transepithelial potential dif- ference; rANP, rat atrial natriuretic peptide; Rt, tissue resistance; TTX, tetrodotoxin Correspondence to: Masaaki Ando Introduction A previous study (Mori and Ando 1991) demonstrated that both acetylcholine and serotonin lower the serosa- negative PD and Isc , accompanied by a decrease in NaC1 and water absorption across the eel intestine. In the same paper the existence of other unknown regulators for ion and water transport across the intestine was proposed. ANP may be one such regulator. It has widespread actions in animals and humans, modulating the renal excretion of water and salts, decreasing blood pressure, and inhibiting renin and aldosterone secretion (Bailer- mann and Brenner 1985; Canfin and Genest 1985; Genest and Cantin 1988). Moreover, O'Grady et al. (1985) demonstrated that in the flounder intestine rat ANP inhibits Isc, net Na + and CI- fluxes, and Rb- uptake across the brush-border membrane. However, the same authors applied rat ANP to fish intestine but did not measure water flux. To elucidate a physiological role for ANP in fish it is important to use a homologous system and to measure water flux, since water absorption is a primary role of the intestine in marine teleosts. Takei et al. (1989) have recently isolated ANP from eel atria. Using the homologous eANP (H-Ser-Lys-Ser- Ser- Ser-Pro-Cys-Phe-Gly-Gly-Lys-Leu-Asp-Arg-Ile-Gly- Ser-Tyr- Ser- Gly-Leu-Gly- Cys-Asn-Ser-Arg-Lys- OH) the present study was performed to clarify the role of eANP in the seawater eel intestine. In addition, structure- activity relationships of various ANPs and the mode of action of eANP were examined. Materials and methods Japanese cultured eels, An#uilla japonica, weighing about 230 g, were kept in seawater aquaria (20 ~ for more than 1 week before use. After decapitation, the intestine was removed and external muscle layers were carefully stripped off using forceps according to And o and Kobayashi (1978). The stripped intestine (middle section) was opened by cutting longitudinally and mounted as a fiat sheet in an Ussing chamber with an exposed area of 0.785 cm2. Both sides of the intestine were bathed with Krebs' bicarbonate Ringer solu-

Effects of eel atrial natriuretic peptide on NaCl and water transport across the intestine of the seawater eel

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J Comp Physiol B (1992) 162:436M39 Journal of Comparative Systemic, Biochemical,

and Environ-

Physiology B ~ ~ PhySiology

�9 Springer-Verlag 1992

Effects of eel atrial natriuretic peptide on NaCI and water transport across the intestine of the seawater eel

Masaaki Ando l, Kyoko Kondo 1 and Yoshio Takei 2

1 Laboratory of Physiology, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshirna 730, Japan, and 2 Department of Physiology, Kitasato University School of Medicine, Kanagawa 228, Japan

Accepted February 6, 1992

Summary. Eel atrial natriuretic peptide inhibited the serosa-negative transepithelial potential difference and short-circuit current, accompanied by a decrease in NaC1 and water absorption across the seawater eel intestine. Similar effects were obtained after treatment with N- terminally truncated eel atrial natriuretic peptide (5-27), indicating that N-terminal amino acids are not essential for the action of eel atrial natriuretic peptide. Although mammalian atrial natriuretic peptides also inhibited the short-circuit current, a 100-fold higher concentration was required to obtain the same effect as with eel atrial natriuretic peptide, indicating that eel atrial natriuretic peptide is 100 times as potent in eel intestine as the mammalian atrial natriuretic peptides. Similarly, in mammalian atrial natriuretic peptide, the four N-termi- nal amino acids had no significant effects. However, when the C-terminal tyrosine was removed, the potency of rat atrial natriuretic peptide was lowered. Compared with the effects of acetylcholine, serotonin and histamine, eel atrial natriuretic peptide was the most potent in- hibitor, with 100% inhibition at 10 -7 M; 50% inhibition was obtained at 10 -2 Min acetylcholine, and 30% inhibi- tion in serotonin (10-SM) and histamine (10 -3 M). These inhibitory effects of eel atrial natriuretic peptide were not diminished even in the presence of tetradoxin, and were mimicked by 8-bromoguanosine 3',Y-cyclic monophosphate. Based on these results, structure-activ- ity relationships of eel atrial natriuretic peptide and a possible mechanism of action of eel atrial natriuretic peptide are discussed.

Key words: Eel atrial natriuretic peptide - NaC1 absorp- tion - Water absorption - Eel intestine - Structure-activ- ity relationship

Abbreviations: 8BrcGMP, 8-bromoguanosine 3',5'-cyclic mono- phosphate; eANP, eel atrial natriuretie peptide; hANP, human atrial natriuretic peptide; 5-HT, 5-hydroxytryptamine creatine sulphate; Is~, short-circuit current; PD, transepithelial potential dif- ference; rANP, rat atrial natriuretic peptide; Rt, tissue resistance; TTX, tetrodotoxin Correspondence to: Masaaki Ando

Introduction

A previous study (Mori and Ando 1991) demonstrated that both acetylcholine and serotonin lower the serosa- negative PD and Isc , accompanied by a decrease in NaC1 and water absorption across the eel intestine. In the same paper the existence of other unknown regulators for ion and water transport across the intestine was proposed.

ANP may be one such regulator. It has widespread actions in animals and humans, modulating the renal excretion of water and salts, decreasing blood pressure, and inhibiting renin and aldosterone secretion (Bailer- mann and Brenner 1985; Canfin and Genest 1985; Genest and Cantin 1988). Moreover, O'Grady et al. (1985) demonstrated that in the flounder intestine rat ANP inhibits Isc, net Na + and CI- fluxes, and Rb- uptake across the brush-border membrane. However, the same authors applied rat ANP to fish intestine but did not measure water flux. To elucidate a physiological role for ANP in fish it is important to use a homologous system and to measure water flux, since water absorption is a primary role of the intestine in marine teleosts.

Takei et al. (1989) have recently isolated ANP from eel atria. Using the homologous eANP (H-Ser-Lys-Ser- Ser- Ser-Pro-Cys-Phe-Gly-Gly-Lys-Leu-Asp-Arg-Ile-Gly- Ser-Tyr- Ser- Gly-Leu-Gly- Cys-Asn-Ser-Arg-Lys- OH) the present study was performed to clarify the role of eANP in the seawater eel intestine. In addition, structure- activity relationships of various ANPs and the mode of action of eANP were examined.

Materials and methods

Japanese cultured eels, An#uilla japonica, weighing about 230 g, were kept in seawater aquaria (20 ~ for more than 1 week before use. After decapitation, the intestine was removed and external muscle layers were carefully stripped off using forceps according to And o and Kobayashi (1978). The stripped intestine (middle section) was opened by cutting longitudinally and mounted as a fiat sheet in an Ussing chamber with an exposed area of 0.785 cm 2. Both sides of the intestine were bathed with Krebs' bicarbonate Ringer solu-

M. Ando et al.: Role of eel ANP in eel intestine 437

tion containing 5 mmol-1 - t glucose and alanine. The bathing solutions, 4 ml each, were kept at 20 ~ and circulated continuously by lifting with a 95% 02/5% CO2 gas mixture (pH 7.4).

The PD was recorded with a polyrecorder (EPR-100A, Toadempa, Tokyo, Japan) as the serosal potential with respect to the mucosa through a pair of calomel electrodes. Rectangular pul- ses, 50 gA for 1 s, were applied across the tissue every 5 min. Rt was calculated from the deflection of the PD. I~o was obtained from the ratio of PD to Rt.

The net water flux was calculated directly from the difference between the rates of effluent and perfusate flow in the perfusion system. Details of simultaneous measurements of net water flux and PD have been described elsewhere (Ando et al. 1986). Briefly, the serosal fluid was perfused through the everted intestine at a constant rate (around 173 pl. min-~) and the effluent was collected every 10 min. Therefore, the difference between the two rates (net water flux) was obtained every 10 rain. In some preparations, net Na § K § and CI- fluxes were also calculated simultaneously from the collected volume and ionic concentrations as described previously (Ando 1983). Na + and K + concentrations were measured by flame photometry (FPF-2A, Hiranuma, Mito, Japan), and CI- con- centration was determined with a chloride counter (CL-SM, Hir- anuma).

After the PD reached a steady state in the Ussing chamber or perfused preparation, one or more of the following agents were added to the serosal fluid : eANP (Peptide Institute, Osaka, Japan), hANP (Peninsula Lab., USA), rANP (Peninsula Lab.), acetylcho- line (Daiichi Seiyaku, Tokyo, Japan), carbamylcholine chloride (carbachol, Sigma, USA), acetyl-13-methylcholine bromide (metha- choline, Sigma), 5-HT (Sigma), histamine dihydrochloride (sigma), 8BrcGMP (Sigma), and TTX (Sankyo, Osaka, Japan).

Results

Effects on electrical parameters

W h e n e A N P was added to the serosal fluid, the serosa- negative P D and I~o decreased with a latent per iod o f abou t 5 rain and Rt tended to increase gradually. Even in the presence o f TTX, the effects o f e A N P were consis- tently observed (Fig. 1). T T X itself had no effect on PD, I~r and R t. In some preparat ions, however, the effects o f e A N P were no t mainta ined for long periods and recovery was spontaneous . These inhibi tory effects were dose- dependent with a threshold concent ra t ion o f 10-9 M and

maximal effect at 1 0 - 7 M . Figure 2 shows dose- response curves for the effects o f various A N P s on I~. W h e n the effects were transient the peak values were obtained. As shown in Fig. 2, a similar dose-response curve was obta ined for e A N P (1-27), e A N P (3-27) and e A N P (5-27), indicating that four amino acid residues at the N-terminal , Ser-Lys-Ser-Ser, are no t essential for e A N P action. Two different m a m m a l i a n ANPs , h A N P and rANP, also inhibited the I~o in a dose-dependent manner , bu t 100-fold higher concentra t ions were re- quired to obtain the same effects as with eANP. A m o n g m a m m a l i a n ANPs , h A N P (1-28), r A N P (1-28) and r A N P (5-28) showed similar dose-response curves, in- dicating tha t the four N-terminal amino acid residues, Ser-Leu-Arg-Arg, are also no t essential for m a m m a l i a n A N P action. However , the dose-response curve o f r A N P (5-27), which lacks Tyr at the C-terminal, shifted to the right (higher concentrat ions) . This indicates tha t Tyr at the C-terminal o f r A N P enhances binding affinity to a receptor in the eel intestine.

Effects on net water and ion fluxes

Since the ma jo r role o f the intestine o f the seawater eel is absorp t ion o f water, the effect o f e A N P on net water flux was examined. W h e n e A N P was added to the serosal fluid, the net water flux was reduced gradually, accompa- nied by a decrease in the P D (Fig. 3). After washing ou t eANP, bo th net water flux and P D returned gradual ly to their original levels. The relatively slow recovery suggests the involvement o f intracellular media tors in this inhibi- to ry act ion o f eANP. Dur ing depression of the net water flux with eANP, net N a + and C1- fluxes were also re- duced (Table 1).

Comparison of the effects of other inhibitors

Since it has been shown that NaC1 and water absorp t ion across the eel intestine are inhibited by acetylcholine, serotonin or histamine (Mori and A n d o 1991), the dose- response curves o f the effects o f these inhibitors were

q20 r ~

~ ~ Wash 100

1 0 ._E

0 1 s0 = = = = 225

�9 ,v-J~l-= = =

o, c~- 50

0 50 100 150

1 Time (rain) 2

Fig. 1. Effects of eANP on PD (O), I~o (e) and R t (m) in the presence ofTTX. TTX (10 -6 M) was added to the serosal fluid at 20 rain and washed out at 85 rain. Then eANP (10 -7 M) was applied to the serosal fluid at thefirst arrows. Tracing is representative of three ex- periments on tissues taken from three eels

eANP(1-27) �9 ~ hANP(1-28) �9 eANP(3-2?) o / ]'~" r ANP(1-28) ~ eANP(5-27) u t / ' r ANP(5-28) �9 /

/ / rANP(5-27)* / J

(3) (6) (6)

9 8 7 6 5

-log [ANP] (M/I)

F i g . 2 . D o s e - r e s p o n s e c u r v e s o f v a r i o u s a t r i a l n a t r i u r e t i c p e p t i d e s . The peak response in the Is~ was plotted against each concentration (log scale). A 100% decrease indicates complete inhibition of the I~c. Each point indicates a mean value; number of experiments in paren- theses. Vertical bars indicate SE of the mean. Prefixes e, h and r denote eel, human and rat, respectively. The sequences of amino acid residues are shown in Fig. 6

438 M. Ando et al. : Role of eel ANP in eel intestine

Table 1. Effects of eANP on the transepithelial PD, net ion fluxes (j~gv o,~,,rK -,~ulc~ ~ and net water flux (j~go) across the sea-water eel intestine

n PD Na [H2o dnet JnKe, "In (l` onet

(mV) (pEq- cm -z) (gi- cm -z - 10 min l)

Control 10 -8 .1 ___0.6" 2,1 +0.2 -0 .1 • 2.5_+0.2 14.4+ 1.2 eANP (10 -7 M) 10 - 1.0_+0.3"* 0,9___0.2** 0.1 • 1.1 _+0.2*** 5.0•

Mean + SE, n denotes number of eels *** P<0 .001 (paired t-test for difference from control values)

E (23

E o

23_ v

oJ

z

15 -8

Wash -4 0

0 lOO 200 3oo

Time (min)

Fig. 3. Effects of eel atrial natriuretic peptide (eANP) on PD (�9 and net water flux (e). At tbefirst arrows 10 -v M eANP was added to the serosal fluid, and washed out at the second arrows. Tracing is representative of ten experiments on tissues taken from ten eels

compared with those of ANPs (Fig. 4). Even carbachol and methacholine (acetylcholine agonists which are not hydrolysed by choline esterase) decreased the I,~ by only 50% at a concentration of 10 -s M. With serotonin and histamine, the maximal effect was only a 30% decrease in I~, and higher concentrations were required to obtain a significant effect; the maximal effects were observed with 10 .5 M serotonin and 10 .3 M histamine. In con- trast, eANP inhibited the l~ completely at a lower con-

centration (10 -7 m ) , indicating that eANP is the most potent inhibitor.

Figure 5 shows the effects of 8BrcGMP. In the presence of 10 -4 M 8BrcGMP, the PD and I~ started to decrease gradually and attained near-zero levels. Rt tended to increase. Comparison of these results with those shown in Fig. 1 suggests that 8BrcGMP mimics the effects of eANP. In the presence of 8BrcGMP, 10 -7 M eANP did not affect the PD, I~ or &. These results suggest that eANP and 8BrcGMP act on the same pathway regulating ion and water transport.

Discussion

The present study demonstrates that in a homologous system eANP inhibits the serosa-negative PD, I~r net Na § and C1- fluxes and net water fluxes across the seawater eel intestine. Similar effects of rANP have been demonstrated in heterologous flounder intestine (O'Grady et al. 1985). Therefore, it is likely that ANP inhibits NaC1 and water absorption across the intestine of seawater teleosts. In addition, the present study dem- onstrates that eANP has higher potency in the seawater eel intestine than hANP or rANP. In the flounder intes- tine, however, rANP (5-28) inhibits the I~ at a lower concentration (20% inhibition at 10-9 M) but maximal inhibition (90%) is obtained at 10 -6 M (O'Grady et al. 1985). Such differences in the dose-response curve for the effects of rANP (5-28) between eel and flounder intes-

100

50

23

e A N / h A N P

10-9 i0 ,8 I0 -7 10 -6 10 .5 10 -4 I0 -3 i0 -2

4 Concentration (M/I)

Fig. 4. Comparison of the effects of various inhibitors on 1~. The dose-response curves of acetylcholine (ACh, &), histamine (His, A) and serotonin (5-HT, S) show mean values obtained from three ex- periments, and those of carbaehol (CCh, I ) and methacholine (MCh, �9 from four experiments�9 The dose-response curves for eel (O) and human (O) ANPs are taken from Fig. 2

12~ 8

60t I \ l % I 10:4 - o[sBroGMp . . . . . .

70 f

5o o 50 loo

5 Time (win)

Fig. 5. Effects of 8BrcGMP on PD (O), I~ (o) and R, (R). After attainment of a steady state, 10 -~ M 8BrcGMP was added to the serosal fluid (first arrows). At the second arrows, ! 0 - v M eANP was applied to the serosal fluid

M. Ando et al. : Role of eel ANP in eel intestine 439

hANP

eANP

eANP(3-27)

eANP(5-27)

rANP

rANP(5-28)

rANP(5-27)

1 5 10 15 20 25 ; - : ; . . . . . . . . . . . . i ; . . . . . . . . . . ! ! ......................... J ;-!

S-L-R-R'S2S-:C-F-G-G:R-M~D-R-~-G; A-Q~S-G-L-G-C-N-S~F~R~V , :: iL i i i ] i i

S-K-S-S~-S~P;C-F-G-G~K-L~D-R-I-G~S-Y~S-G-L-G-C-N-S-: ' .R'K ! : ! L ', i : i ] ' !

S-S~S'~P~C-F-G-G~K -L-~D-R-I-G~S-Y ~:S-G-L-G-C-N-S~ +R,:-K i i l t : .: :' l i i : : ':S;P~C-F-G-G'~K-L-~D-R-I-G-S-Y-~S-G-L-G-C-N-S-: ,"-R'K i , ' i l : I i i !

S-L-R-R];S-,':S-~:-F-G-G ;R- - D-R-I-GaA-Q~S-G-L-G-C-N-S'F~RY ! it i ! ' '~ I i i

S-," S~C-F-G-G'R-I- b-R-I-G~A-Q~S-G-L-G-C-N-S'F~R~Y

: S~SJI~-F-G-G;R-I- ', ~)-R-I-G-~-Q-'S-G-L-G-?-N-S-:,'F~R : , , i ! : ' i i

. . . . . . . . . . . . . . . . . . . . i

Fig. 6. Amino acid sequences (single letter code) of hANP, eANP, and rANP. Common amino acid residues are boxed with d o t t e d l ine. Disulphide linkages between cysteine residues are conserved in all ANPs

tines may indicate that receptor(s) and/or transduction and effector systems differ between these species.

Figure 6 shows the pr imary structures of various ANPs used in this study, eANP and h A N P have 17 amino acid residues in common, including disulphide linkage of Cys residues. These common amino acids may contribute to the inhibitory action of ANP. However, eANP has 100-fold the potency of hANP. This dis- crepancy may be due to the difference in the residual 10 amino acids. Among these ten residues, however, four residues at the N-terminal may have no significant role, because eANP (1-27) and eANP (5-27) have the same effects. The 12th residue (Met and Leu) can also be excluded, since hANP (1-28) and r A N P (1-28) have the same effects. One of the most plausible sites attributable to the pontency seems to be the C-terminal (-Arg-Lys), since C-terminal-deficient rANP (5-27) has a lower potency than rANP (5-28). The significance of the C- terminal o f rANP has been recognized in rat renal N a § excretion (Thibault et al. 1984) and in binding capacity in rat jejunum (Bianchi et al. 1989).

Although acetylcholine and serotonin also inhibit NaC1 and water absorpt ion across the eel intestine (Mori and Ando 1991), their effects were less potent than eANP, which appears to be a powerful regulator in the seawater eel intestine. A N P may act to lower the plasma NaC1 level in the marine teleost by inhibiting NaC1 up- take through the intestine and by increasing NaC1 ex- trusion through the gill, since Scheide and Zadunaisky (1988) reported that rANP (3-28) stimulates Isc through modulat ion of C1- secretion across the opercular epithe- lium of seawater killifish. However, in the flounder oper- cular epithelium rANP (5-28) has no effect on the PD (O 'Grady et al. 1985). Ra t A N P may act differently in killifish and flounder. To clarify the role of ANP in these teleosts, homologous A N P must be used.

A N P has been reported to stimulate guanylate cyclase and to increase c G M P concentration in various tissues, such as mammal ian kidney (Murphy et al. 1985; Tremb- lay et al. 1985) and intestine (Waldman et al. 1984; Ito et al. 1988) and flounder intestine (O 'Grady et al. 1985). Recently, it has been shown that a membrane form of

guanylate cyclase is itself an A N P receptor (Chinkers et al. 1989). Similarly, eANP may also stimulate guanylate cyclase in the eel intestine, since the action of eANP is closely mimicked by treatment with 8BrcGMP and no effects of eANP are observed after t reatment with 8BrcGMP.

A c k n o w l e d o e m e n t s . This research was supported in part by Grant- in-Aid Nos. 02804062 and 01304027 from the Ministry of Educa- tion, Sciences and Culture, Japan.

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Ando M, Sasaki H, Huang KC (1986) A new technique for measur- ing water transport across the seawater eel intestine. J Exp Biol 122:257-268

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O'Grady SM, Field M, Nash NT, Rao MC (1985) Atrial natriuretic factor inhibits Na-K-C1 cotransport in teleost intestine. Am J Physiol 249: C531-C534

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Takei Y, Takahashi A, Watanabe TX, Nakajima K, Sakakibara S (1989) Amino acid sequence and relative biological activity of eel atrial natriuretic peptide. Biochem Biophys Res Commun 164:537-543

Thibault G, Garcia R, Carrier F, Seidah NG, Lazure C, Chr~tien M, Cantin M, Genest J (1984) Structure-activity relationships of atrial natriuretic factor (ANF). I. Natriuretic activity and relaxation of intestinal smooth muscle. Biochem Biophys Res Commun 125:938-946

Tremblay J, Gerger R, Vinay P, Pang SC, Beliveau R, Hamet P (1985) The increase of cGMP by atrial natriuretic factor cor- relates with distribution of particular guanylate cyclase. FEBS L e t t 181 : 17-22

Waldman SA, Rapoport RM, Murad F (1984) Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cGMP in rat tissues. J Biol Chem 259:14332-14334