α1-Adrenergic stimulation differentially regulates ether-linked diacylglycerols in airway epithelial cells from normal and cystic fibrosis patients

Biochi~ic~a et Biophysica AEta

ELSEVIER

Biochimica et Biophysica Acta 1302 (1996) 264-270

Rapid report

ot 1-Adrenergic stimulation differentially regulates ether-linked diacylglycerols in airway epithelial cells from normal and cystic fibrosis patients Mark Kester a,c,*, Carole M. Liedtke b.c a Department of Medicine, The Cystic Fibrosis Center, Case Western Reserte Unit,ersity, School of Medicine, Cleveland, OH 44106, USA b Department of Pediatrics, The Cystic Fibrosis Center, Case Western Reserze Uniz,ersiO', School of Medicine, Cleveland, OH 44106, USA c Departments of Physiology and Biophysics, The Crstic Fibrosis Center, Case Western Reser~,e Unit,ersity, School of Medicine, Clet:eland, OH 44106, USA

Received 22 May 1996;accepted 23 May 1996

Abstract

ctt-Adrenergic stimulation of human airway epithelial cells induces a transient increase in polyphosphoinositide turnover coincident with augmented Na+CI-(K +) cotransport activity. This activation of airway epithelial cells also results in a biphasic elevation of diacylglycerols. To better understand the significance of these distinct diacylglycerol pools, we now characterize the mass of ether- and ester-linked diacylglycerol species. We demonstrate that the relative mass of ether-linked diacylglycerols is reduced in airway epithelium from cystic fibrosis patients in the presence or absence of ct t-adrenergic stimulation. This reduction in ether-linked diacylglycerol mass may represent a compensatory mechanism to help maintain normal chloride influx in cystic fibrosis patients. Keywords: Diacylglycerol;Methoxamine; Cystic fibrosis; Signal transduction

Classical transmembrane signaling theory suggests that some receptors are linked to a pbosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2)-specific phospholipase C (PLC) generating inositol phosphates and diacylglycerols (DG). However, distinct PLC and phospholipase D (PLD)-activities that hydrolyze ester- and ether-linked phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEth) may also release molecular species of DG and phosphatidic acids (PA). Previous studies demonstrate that a ladrenergic-receptor (AR) signaling pathways are coupled to a PtdIns-4,5-P2-dependent PLC that generates inositol phosphates [1] and diacylglycerols [2] in both human tracheal epithelial cells and nasal polyp epithelial cells. In these studies, subtle differences in the kinetics of total DG formation between airway epithelial cells (AEC) from cystic fibrosis (CF) and normal patients were noted. The generation of inositol bis- and trisphosphates was transient

Abbreviations: DG, diacylglycerols; diacylglycerol, 1,2-diacyl-snglycerol; alkyl,acyl-glycerol,1-O-alkyl-2-acyl-sn-glycerol. * Corresponding author. Fax: + 1 (216) 3681249.

with peak levels observed before 1 min. The formation of DG was biphasic with peak levels observed at less than 1 min and between 6 and 8 min [2]. The initial DG peak corresponds to the time-course of Ins-l,4,5-P 3 generation and suggests a common PtdIns-4,5-P 2 substrate. The second peak of DG has not been characterized and is a focus of the present study. Although DG can be generated from different phospholipid precursors, little information is currently available correlating agonist-induced DG structures with physiological functions. The formation of DG independent of PtdIns hydrolysis can be a result of either a direct PLCmediated mechanism hydrolyzing alternate phospholipids including PtdCho or an indirect mechanism that sequentially forms DG through a combined phospholipase D ( P L D ) / P A phosphohydrolase activity [3,4]. PtdCho-derived DG have been associated with mitogenesis, nuclear signaling and regulation of ion transport [5,6]. The role of DG molecular species is still controversial as PtdCho-derived DG have been shown to be effective [7] or ineffective [8,9] activators of protein kinase C activity. The characterization of molecular species of DG derived from

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M. Kester, C.M. Liedtke / Biochimica et Biophysica Acta 1302 (1996) 264-270

different phospholipid pools is especially important as ether-linked DG (alkyl,acyl- or alkenyl,acyl-DG species), in contrast to ester-linked (diacylglycerol) species, may inhibit PKC, not affect PKC, or only activate PKC in the presence of elevated intracellular calcium levels [10-14]. Furthermore, ether-linked DG inhibit PKC activation induced by diacylglycerol species [14,15]. Even a modest change in ether-linked DG concentration may be physiologically significant as 1 nM alkenyl,acyl-glycerol inhibits 67% of 100 nM diacylglycerol-stimulated total PKC activity [14], Ether-linked DG species have been observed in interleukin-l-treated mesangial cells, as well as in PMAand FMLP-stimulated neutrophils [ 14,16]. We are particularly interested in the species of DG generated by ot~-AR agonists as we have previously demonstrated a critical role for PKC in the regulation of N a + C I - ( K +) cotransport in A E C [2]. In AEC, N a + C I - ( K +) cotransport is crucial during secretion because it supplies chloride to the epithelial cell from the serosal side for secretion into the lumen. Cotransport is localized to the basolateral plasma membrane domain, whereas channels mediating secretory chloride exit are localized to the apical membrane. Cellular events leading to activation of N a ÷ C I - ( K ÷) cotransport and initiation of chloride secretion are not well understood. PMA was shown to stimulate cotransport activity while staurosporin, a PKC inhibitor, blunted both et~-AR-stimulated and okadaic acid-stimulated cotransport activity [2]. Interestingly, et~-AR-stimulation of chloride transport corresponded only to the initial DG peak and not the secondary, non-Ptdlns-specific, DG peak. These results are similar to studies in HT29 cells where PMA exposure transiently elevates cotransport activity [17]. Thus, DG species in the late peak may differentially regulate Na + C1-(K ÷) cotransport activity through either PKC-dependent or -independent mechanisms. In the present study, we extend our original findings that assess 3H-labeled DG flux measurements with studies that assess DG-mass directly. DG mass was quantitated as DG-[taC]acetates. This sensitive method is not affected by questions concerning specific activity of labeling or unlabeled pools. DG-acetates can be separated in terms of their s n - 1 bonding angle and, thus, ether-linked DG species can be separated and quantified separately from diacylglycerol species. The goal of the present study is to define the molecular species of DG generated in human AEC derived from normal and CF patients. This includes deter-

265

mining whether et ~-AR signaling generates ether- as well as ester-linked DG species resulting from either PLC- or PLD-hydrolysis of PtdCho. Tissue was obtained from 8 CF nasal polyp specimens obtained at time of polypectomy and 10 normal tracheae at time of autopsy through the Cystic Fibrosis Center at Rainbow Babies and Children Hospital. AEC were isolated and grown in in vitro cell cultures by previously described methods [1]. We compare DG mass from CF nasal epithelium with non-CF tracheal epithelial cells as we have previously shown that DG radioactive flux responses are identical between epithelial cells derived from human tracheal epithelial cells and nasal turbinate epithelium [2]. For analysis of phospholipid metabolites, cultured AEC from normal trachea were prelabeled with 1.0 IxCi (45 C i / m m o l ) 3H- l - O - h e x a d e c y l - 2 - 1 y s o - s n - g l y c e r o l p h o s p h o rylcholine ( 3 H - G P C ) / m l culture media for 3 h in nominally serum-free media at 37°C in a humidified atmosphere in 95% a i r / 5 % CO 2. Alkyl-lyso-GPC is rapidly reacylated and intercalates into the PtdCho pool and can be utilized as an indicator of PtdCho-derived lipid mediators [4]. Specifically, 505 361 5- 14418 dpm were incorporated into n = 4 normal trachea, equivalent to 5.1 fmol of GPC. Basal levels of lipid metabolites in these cells were 2290 5- 269, 1105 + 4 7 6 , 1097 5- 681 dpm for DG, PA and phosphatidylethanol (PEt), respectively. Just prior to experimental measurements, culture medium containing radiolabel was removed and cells were washed twice with prewarmed HPSS. To stimulate cells, drugs or vehicle were added as a 1 txl aliquot of concentrated stock solution in the presence or absence of 0.5% ethanol. PEt is a nonphysiological transphosphatidylation product and is a measure of specific PLD activation. Reactions were terminated at time points as indicated and lipids were extracted as previously described [ 1,2,4]. Neutral and polar lipids were developed in two separate TLC solvent systems The elution system for DG was petroleum ether/diethyl ether/acetic acid (70:30:2, v / v ) while PA and PEt were separated using a developing system of ethyl acetate/isooctane/acetic acid (90:50:20, v / v ) . This solvent system allowed good separation of PA (Rf = 0.24) and PEt ( g f = 0.36) from each other and neutral lipids, fatty acids and phospholipids. DG was visualized by exposure to iodine vapors and P A / P E t were resolved by staining with 0.03 g Coomassie brilliant blue in 100 ml of 100 mM NaC1 and 30% methanol followed by destaining in 100 mM NaC1 in 30% methanol [18]. Phospholipid metabolites that comi-

Fig. 1. Generation of ester and ether-linked DG species in AEC from CF and non-CF patients. AEC were stimulated with 10 IxM methoxamine or epinephrine for either 40 sec (Fig. 1A) or 6 rain (Fig. 1B). Methoxamine-HCl was supplied by Burroughs Wellcome (Research Triangle Park, NC) and L-epinephrine - HCI was purchased from Sigma (St. Louis, MO). Isolated DG from treated cells were reacted with [J4C]acetic anhydride and diacyl-, alkyl,acyl- and alkenyl,acyl-DG-[laC]acetateswere resolved on TLC and quantified as a measure of DG mass. Independent t-tests were used to establish significant differences between groups. The P-values of the individual comparisons were adjusted for multiple comparisons by the Bonferroni method. Values are mean _+S.E.M. for n = 4 experiments. * P < 0.05 compared to control values.

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M. Kester, C.M. Liedtke / Biochimica et Biophysica Acta 1302 (1996) 264-270

grated with authentic standards were scraped and radioactivity determined by liquid scintillation spectrophotometry. Diacylglycerols are composed of two long chain hydro-

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M. Kester, CM. Liedtke / Biochimica et Biophysica Acta 1302 (1996) 264-270

while alkyl, acyl-glycerol species are composed of an ether ( C - O - C ) bond at the s n - 1 position. Alkenyl,acylglycerols are ether-linked diacylglycerols that are composed of a vinyl ether linkage ( C - O - C = C ) at the s n - 1 position. Diacylglycerols generated in mammalian systems always have an ester bond in the s n - 2 position. The formation of DG-[14C]acetates can be used as a quantitative assay for DG mass after TLC separation into etherand ester-linked DG-acetate species. To determine the mass of diacyl-, alkyl,acyl- and alkenyl,acyl-DG species, DG were extracted from treated AEC as described above except that neutral lipids were eluted from the origin of silica gel 60 TLC plates with a mobile phase consisting of benzene/diethyl ether/ammonia (100:80~0.2, v / v ) . 1,2DG were well separated from 1,3-DG, monoacylglycerols, triacylglycerols, phospholipids and free arachidonate. AEC were prelabeled for 2 h with [3H]arachidonic acid (0.5 ~ C i / w e l l ) as an internal control. Lipids were visualized with toluidino-2-naphthalene-sulfonic acid and UV light. After extraction of the separated DG from the silica gel, the DG was acetylated with 1 ~Ci [14C]acetic anhydride and pyridine for 3 h at 37°C. The DG-acetates were rechromatographed by TLC or HPLC and then subjected to autoradiography for identification of DG-acetates and subsequent liquid scintillation analysis as previously described [4,14]. The DG-acetates (0.48 + 0.0039 R e) are well resolved from underivatized DG (0.09 + 0.01 R f ) using a sequential hexane/diethyl ether/methanol/acetic acid (90:20:3:2, v / v ) and toluene (100, V) TLC solvent system. Derivatizing authentic DG species to DG-t4C-acetates yielded products that comigrate with the corresponding DG-acetates prepared from phospholipid precursors [14]. Incorporation of 14C-acetate into DG was complete, linear with respect to DG mass, and showed no preference for ester- or ether-linked DG species and thus was used as a quantitative assay for DG mass. To demonstrate that the DG-acetate assay was complete, we utilized [3H]glycerollabeled DG to follow 14C-DG-acetic anhydride incorporation and noted that the tritium label did not remain at the TLC position for underivatized DG. Calibration curves were constructed for each experiment and revealed linear formation of DG-acetate from 0.05 to 100 Ixg of DG substrate utilized. Nonradioactive DG substrate was always used as an external standard after derivatization and was run on the outer and middle TLC lanes. DG-acetate extraction efficiency values were determined and were always greater than 80%. DNA mass was utilized as the denominator for these data and was determined fluorometrically using Hoechst dye. Similar data was obtained when we utilized total [3H]phospholipid as a denominator. Based upon our previous results, utilizing [3H]arachidonate-labeled AEC, in which an txl-AR-agonist stimulates 3H-DG formation in a biphasic manner, we measured DG mass as 14C-DG-acetate at time points corresponding to p e a k 3 H - D G formation. Normal and CF AEC were

Table 1 Ether/ester DG ratios are reduced in both unstimulated and eq-ARactivated CF cells Time

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stimulated with 10 ixM methoxamine, an txl-AR agonist, or the endogenous hormone epinephrine (10 p,M) for either 40 sec (Fig. 1A) or 6 min (Fig. 1B). HBSS served as vehicle control. The doses of methoxamine and epinephrine used elicit maximal cotransporter activity [1,2]. At both 40 sec and 6 min incubations, methoxamine and epinephrine significantly elevated diacylglycerol concentrations in both normal and CF cells. The most apparent difference between AEC from normal and CF patients was the decrease in basal ether-linked DG mass observed in CF cells. This observation can be best depicted when these data are re-analyzed as ether/ester-DG ratios (Table 1). Even though methoxamine elevated ether-linked DG mass in CF cells to concentrations observed under basal conditions in normal cells, the proportionally greater concomitant increase in diacylglycerol mass did not appreciably change (40 sec) or further reduced (6 min) the ether/ester DG ratio in CF cells. Moreover, the ether/ester DG ratio was further accentuated between CF and normal cells with the endogenous hormone, epinephrine. Both methoxamine or epinephrine maximally diminished ether/ester DG ratios after a 6 min stimulation. These results illustrate that the relative mass of ether-linked DG species were reduced in airway epithelium from CF patients in the presence or absence of et 1-adrenergic stimulation. Epinephrine-induced stimulation of diacylglycerol mass could be mediated though et- a n d / o r 13-adrenergic receptors. Thus, we next asked if a 13-AR agonist, isoproterenol, mimicked the effects of epinephrine upon diacylglycerol accumulation. (Fig. 2). At both early and late time points, isoproterenol (10 IxM) did not elevate any molecular species of DG in AEC from non-CF patients. Similar negative findings were observed in CF AEC and the diminished ether/ester DG ratios were still maintained (data not shown). These results suggest that elevated DG species in AEC are a result of ct- but not [3-adrenergic receptor signaling cascades. Confirming our results, propranolol, a ~-AR antagonist had no effect upon isoproterenol-treated AEC (data not shown). To test the specificity of the response to methoxamine through an Ctl-AR signaling pathway, we used prazosin,

268

M. Kester, C.M. Liedtke / Biochimica et Biophysica Acta 1302 (1996) 264-270

an o~~-AR antagonist. Prazosin blocks methoxamine stimulated cotransport activity and PtdIns-4,5-P 2 hydrolysis in AEC [1,2]. In this set of experiments, prazosin (10 I~M, 40 sec) partially decreased both methoxamine-stimulated ester- and ether-linked DG species (Fig. 3A). It should be noted that, in these series of experiments, there is a slight but insignificant elevation in ether-linked DG species from normal AEC. Again, the elevation in diacylglycerol mass is proportionally greater than the increase in ether-linked DG mass in CF cells reflecting a decrease in the ether/ester DG ratio in these CF cells. We next investigated the contribution of ct-and [3-AR receptors to the epinephrine response (Fig. 3B). AEC were pretreated for 30 sec with either prazosin, an e~-AR antagonist, or propranolol, a [3-AR antagonist, before a stimulation with epinephrine. Prazosin reduced epinephrine-induced ester-linked DG in AEC from CF patients. Propranolol also reduced ester-linked DG in both CF and normal cells. In contrast, propranolol induced an elevated ether-linked DG generation in CF and in non-CF cells. Antagonism of [3-AR signaling pathways may diminish a negative feedback pathway down-regulating ether-linked DG production via ct ~-AR signaling. Alternatively, the decrease in ester-linked DG formation induced by propranolol may be the result of inhibition of phosphatidic acid phosphohydrolase activity, an enzyme that catalyzes the formation of DG from PA. The finding that propranolol diminishes epinephrine-stimulated ester-linked DG species while augmenting basal ether-linked DG species further supports the notion that DG species are formed from separate phospholipid pools that are differentially metabolized or regulated by adrenergic receptor-coupled signaling cascades. To specifically find out whether ctl-AR hydrolyses PtdCho, we treated [3H]alkyl-lyso-GPC-labeled AEC from non-CF trachea with 10 ~ M methoxamine for either 40 sec or 6 min in the absence or presence of 0.5% ethanol

(Fig. 4). 3H-DG were elevated by methoxamine predominantly at 6 rain reflecting hydrolysis of a PtdCho pool. The magnitude of this methoxamine response is less than the DG mass data reported in Fig. 1. One explanation for this response is that exogenous ether-linked phospholipids are not preferred substrates for non-CF cells. PtdCho-derived DG species can be formed through both a direct PLC activity or via a sequential P L D / P A phosphohydrolase mechanism. As PEt levels were not augmented by exogenous ethanol in the presence of methoxamine (Fig. 4), the role of a PLD pathway to generate DG species can be ruled out. Thus, the methoxamine-induced elevation in PA levels suggests a regulated DG kinase activity in these cells. Even though o~j-AR do not activate PLD, AEC possess PLD activity since the phorbol ester PMA, an established PLD agonist, elevated PEt levels 3-fold over baseline in both non-CF and CF cells (data not shown). These results suggest that o~I-AR stimulation induces DG formation, in part, through activation of PtdCho-dependent PLC. A PtdCho-directed PLC has been described in endothelin-stimulated mesangial cells, complement-activated glornerular epithelial cells and acetylcholine-stimulated astrocytoma cells [4,19,20]. The observation that prazosin diminishes both methoxamine and epinephrine-stimulated DG as well as the observation that isoproterenol does not stimulate DG formation suggest in AEC that o~I-AR signaling results in sustained DG formation. Indeed, initial c~j-AR stimulation of PtdIns4,5-P2-specific PLC may be sequentially followed by activation of a PtdCho-directed PLC. The physiological role of PtdCho-dependent PLC may involve generation of unique species of DG that differentially regulate distinct PKC isotypes. Moreover, the reduction in the ether/ester DG ratio in CF cells even during ~l-adrenergic stimulation may reflect the recent observation that the activities of selected PKC isotypes were reduced in normal cells compared to CF cells [21]. Thus, the reduction in the relative

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M. Kester, C.M. Liedtke / Biochimica et Biophysica Acta 1302 (1996) 264-270

optimal chloride influx across the epithelium of CF patients. We, as well as others, have shown that the physio-

mass of ether-linked DG species in basal and a l-adrenergic-stimulated CF cells may reflect a compensatory mechanism by which augmented PKC activity maintains

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Fig. 3. Adrenergic antagonists prazosin and propranolol differentially regulated hormone stimulated ester- and ether-linked DG formation. (A) AEC from normal or CF patients were pretreated for 30 sec with 10 IxM prazosin before a subsequent 40 sec incubation with 10 IxM methoxamine. Tukey-Kramer multiple comparison tests were used to establish significant differences between groups. Mean values + S.E.M. from triplicate determinations. Significantly different than AEC treated with methoxamine alone: P < 0.05 except for ether-linked DG derived from CF cells where P < 0.07. (B) AEC from normal or CF patients pretreated with either 10 p.M prazosin or propranolol for 30 sec before a subsequent stimulation with 10 p~M epinephrine for 6 min. We chose a 6 min stimulation as epinephrine responses are maximal at this time point. However, in data not shown, similar responses were noted with a 40 sec epinephrine stimulation. Mean values _+ S.E.M. from duplicate determinations of n = 3 separate experiments for 6 min data. Ether-linked DG values for propranolol-treated cells are significantly different from cells treated with epinephrine alone: P < 0.01 (non-CF) and P < 0.08 (CF).

270

M. Kester, C.M. Liedtke / Biochimica et Biophysica Acta 1302 (1996) 264-270 3.C

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The authors thank Ms. Angela Musial, Patricia Vanah and Krista Viola for excellent technical assistance. This work was supported by National Institutes of Health grants AR40225 (MK), HL43967 (CL) and SCOR grant HL50160.

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Fig. 4. Generation of lipid metabolites by methoxamine in AEC derived from normal trachea. Cells were incubated with 10 IxM methoxamine in the absence or presence of 0.5% ethanol for either 40 sec or 6 min. Total lipids were recovered and analyzed by TLC as described in the text. Data were calculated as a ratio of recovered lipid (dpm)/total applied lipid (dpm) and are reported as per cent total applied lipid dprn. For sample populations with significantly different standard deviations, levels of significance were determined by nonparametric analysis using the Mann-Whitney U-test. Values are means + S.E.M. for n = 4 tissue specimens. * P < 0.05.

logical role of ether-linked DG may be down-regulation of receptor-mediated PKC activity [14,15]. We have further demonstrated that ether-linked DG may competitively bind to PKC without activating PKC activity [14]. It has been suggested that the carboxyl groups of the ester bond at the sn1 position as well as the resulting bond angle are essential for PKC activation [22,23] Alternatively, etherlinked DG may exert other effects independent of PKC. This is particularly intriguing in terms of Na+CI-(K +) cotransport as PKC-independent actions of DG including regulation of neuronal calcium ion current [24] and K+-in duced calcium ion influx have been reported [25]. Thus, optimal chloride flux may be maintained in ct l-adrenergic receptor-stimulated CF tissue by a further reduction in ether-linked DG species. The fact that bumetanide-sensitive Na+CI-(K +) cotransport in CF and normal AEC is elevated by methoxamine during the early DG peak but not the late DG peak [2], may suggest that additional chloride influx pathways, including the cAMP-dependent chloride channel, may be regulated by DG. In conclusion, a reduction in the relative mass of etherlinked DG species was observed in the presence or absence of ctl-AR activation in CF cells. The putative role ether-linked DG play in direct regulation of cotransport activity or in the regulation of PKC activity in human AEC is currently being investigated.

[1] Liedtke, C.M. (1992) Am. J. Physiol. 262, L183-L191. [2] Liedtke, C.M. (1995) Am. J. Physiol. 268, L414-L423. [3] Billah, M.M., Eckel, S., Mullman, T.J., Egan, R.W. and Siegel, M.J. (1989) J. Biol. Chem. 264, 17069-17077. [4] Baldi, E., Musial, A. and Kester, M. (1994) Am. J. Physiol. 266, F957-F965. [5] Larrodera, P., Cornet, M.E., Diaz-Meco, M.T., Lopez-Barahova, M., Guddac, P.H., Johansen, T. and Moscat, J. (1990) Cell 61, 11131120. [6] Price, B.D., Morris, J.D.H. and Hall, A. (1989) Biochem. J. 264, 509-515. [7] Slivka, S.R., Meier, K.E. and Insel, P.A. (1988) J. Biol. Chem. 263, 12242-12246. [8] Martin, T.W., Hsieh, K.P. and Porter, B.W. (1990) J. Biol. Chem. 265, 7623-7631. [9] Lin, P., Fung, W.J.C. and Gilfillan, A.F. (1992) Biochem. J. 287, 325-331. [10] Cabot, M.C. and Jaken, S. (1984) Biochem. Biophys. Res. Commun. 125, 163-169. [1 I] Heymans, F., DaSilva, C., Marrec, N., Godfroid, J.J. and Castagna, M. (1987) FEBS Lett. 218, 35-40. [12] Ganong, B.R., Loomis C.R., Hannun, Y.A. and Bell, R.M. (1986) Proc. Natl. Acad. Sci. USA 83, 1184-1188. [13] Ford, D.A., Miyake R., Glaser, P.E. and Gross, R.W. (1989) J. Biol. Chem. 264, 13818-13824. [14] Musial, A., Mandal, A., Coronets, E. and Kester, M. (1995) J. Biol. Chem. 270, 21632-21638. [15] Daniel, L.W., Small, G.W. and Schmidt, J.D. (1988) Biochem. Biophys. Res. Commun. 151,291-297. [16] Rider, L.G., Dougherty, R.W. and Niedel, J.E. (1988) J. Immunol. 140, 200-207. [17] Bajnath, R.B., Van Hoeve, M.H., DeJonge, H.R. and Grott, J.A. (1992) Am. J. Physiol. 263, C2037-C2047. [18] Xie, M. and Dubyak, G.R. (1991) Biochem. J. 278, 81-89. [19] Cybulsky, A.V. and Cyr, M.D. (1993) Am. J. Physiol. 265, F551F560. [20] Martinson, E.A., Goldstein, D. and Brown, J.H. (1989) J. Biol. Chem. 264, 14748-14754. [21] Liedtke, C.M. and Cole, T.S. (1996) FASEB J. 10, A91. [22] Rando, R.R. and Kishi, Y. (1992) Biochemistry 31, 2211-2218. [23] Leli, U., Hauser, G. and Rroimowitz, M. (1990) Mol. Pharm. 37, 286-295. [24] Hockberger, P., Toselli, M., Swandulla, D. and Lux, H.D. (1989) Nature 338, 340-342. [25] Thomas, T.P. and Belbez-Pek, S. (1992) Endocrinology 131, 19851992.