May 27, 1987 - Inc. Vol. 263, No. 2, Issue of January 15, pp. 714-721,1988. Printed in U. S. ..... dependence for SRIF inhibition of VIP-stimulated cAMP ac- cumulation. ..... that SMS 201-995 continues to lower plasma growth hormone levels in ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 16
Vol. 263, No. 2, Issue of January 15, pp. 714-721,1988 Printed in U.S.A.
19843 by The American Society for Biochemistry and Molecular Biology. Inc
Somatostatin Pretreatment Increases the Number of Somatostatin Receptors inGH& Pituitary Cells and Does Not ReduceCellular Responsiveness to Somatostatin* (Received for publication, May 27, 1987)
David H. PreskyS andAgnes SchonbrunnQ From the Departmentof Pharmacolom, HarvardMedical School and Laboratory of Toxicology, Harvard School of Public Health, Boston, Masiachusetts02115 "
The GH4C1 pituitary cell linecontains specific Somatostatin(SRIF)'is a 14-amino acid peptide which plasma membrane receptors for the inhibitory neuro- inhibits secretion from many different target cells (1).We peptidesomatostatin(SRIF).Unlikeotherpeptides have used theGH4Cl clonalstrain of rat pituitary tumorcells which bind to cell surface receptors on these cells,asSRIF a homogeneous systemto investigate the biochemical is not rapidly internalized via receptor-mediated enmechanisms of SRIF action (2). In these cells SRIF inhibits docytosis. Here we examined the effects of chronic both prolactin andgrowth hormone release, and thebiological SRIF pretreatmenton the subsequent abilityof GH4Cl effects of SRIF are mediated by a specific, saturable, plasma cells to bind and respond to this hormone. Treatment membrane receptor (3).SRIF inhibitionof hormone secretion of cells with 100 nM SRIF increased ['2sI-Tyr']SRIF involves two mechanisms: inhibition of secretagogue-stimubinding to a maximum value of 220% of control after lated adenylyl cyclase activity and cAMP accumulation and 20 h. Scatchard analysis demonstrated that the num- a CAMP-independent reduction in intracellularfree Ca2+conber, but not the affinity, of the receptors was altered. centrations (2, 4-8). The effect of SRIF was dose-dependent (EDSO= 2.3 f We have recently demonstrated that, unlike most peptide 0.4 nM), was not mimicked by an inactive analog, and ligands, receptor-bound ['251-Tyr']SRIF is not rapidly interwas specific for the SRIF receptor. Furthermore, prenalized by either GH4Cl pituitarycells (9) or RINm5F insutreatment of cells withotheragents,which mimic SRIF's action to decrease intracellularcAMP and free linoma cells (10). After a 2-h binding incubation at 37 "C, Ca2+ concentrations, did not mimic the SRIF-induced over 80% of specifically bound ['251-Tyr']SRIF is associated increase in receptor number. Thus, occupancy of the with the cell surface. Furthermore, during subsequent chase SRIF receptor was required for SRIF receptor up- incubations a t 37 "C, there is no increase in the amount of '251-labeled regulation. Inhibition of protein synthesis with cyclo- intracellular ['251-Tyr']SRIF. Incontrast,both growth factor and ['251-Tyr4]bombe~in are interheximide did not prevent the SRIF-induced increase epidermal in receptors, consistent with an effect of SRIF to either nalizedby GH4CI cells via receptor-mediated endocytosis reduce receptor degradation or causeslow redistribu- within10min at 37 "C (9, 11). Thus,the unusually slow tion of preexisting receptors to the plasma membrane. processing of ['251-Tyr']SRIF reflects a specific property of In contrast to the effects on receptor binding, pretreat- the SRIFreceptor and not the GH4Clcell line. Prolonged treatment of cells with hormoneswhich undergo ing cells with SRIF did not alter either basal cAMP levels or the potency of SRIF to inhibit cAMP accu- receptor-mediated endocytosis often causes a decrease in the mulation (EDSO= 0.5 +: 0.2 nM). However, the maxi- steady-state level of their cell surface receptors as a consemum cAMP produced by stimulators of adenylyl cy- quence of the internalization of the receptor along with the clasewasincreased. The observation thatchronic bound ligand (12). In the absence of receptor recycling, the SRIF exposure did not causehomologous desensitiza- higher rate of internalization of the ligand-receptorcomplex tion inGH4Clcells and increased rather than decreased compared to the unoccupied receptor leads to a decrease in SRIF receptor number is consistent with the fact that the number of cell surface receptors, i.e. receptor down-reguthis neuropeptide is not rapidly internalizedby recep- lation. In addition, chronic exposure to saturating concentrator-mediated endocytosis. tions of agonistsoften desensitizescells to a subsequent hormonal challenge (12, 13). Inthisstudy we have taken advantage of the unusual properties of the SRIF receptor to determine theeffect of chronic peptide treatment on a recep* This investigation was supported by Research Grant DK 32234 tor which does not mediate rapidendocytosis of bound ligand. from the National Institute of Arthritis, Diabetes, Digestive and ' The abbreviations used are: SRIF, somatostatin; TRH, thyrotroKidney Diseases. The costs of publication of this article were defrayed L-prolylin part by the payment of page charges. This article must therefore pin-releasing hormone; [3H-Me]TRH, [~-histidinyl-4-~H, be hereby marked "advertisement" in accordancewith 18 U.S.C. 3,4-3H]TRH;VIP, vasoactive intestinal peptide;EGF,epidermal growth factor; PIA, (-)-P-(R)-(2-phenylisopropyl)adenosine;[3H] Section 1734 solely to indicate this fact. IBMX, 3-isobutyl-1-methylxantbine; $ Partially supportedby National Research Service Award Train- leucine, [4,5-3H]~-leucine; ing Grant 5 T32 GM07306 and a fellowship from the Albert J. Ryan HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid F-10Foundation, Cincinnati, OH. Present address: Laboratory of Immu- lh, Ham's F-10 medium without bicarbonate, supplemented with 5 mg/ml lactalbumin hydrolysate; HEPES-F-10-lh, F-10-lhwith 20 nology, NIAID, Bldg. 10, Rm. llN311, Bethesda, MD 20892. mM HEPES, pH 7.4; HBSS-AcOH, Hanks' balanced salt solution f To whom reprint requests should be addressed Dept. of Phar20708, with 20 mM acetic acid [Ca'+],, the intracellular free calcium ion macology,University of Texas MedicalSchool,P.O.Box concentration; RIA, radioimmunoassay. Houston, TX 77225.
714
Homologous Up-regulation of SRIF Receptors
715
Chronic incubations with SRIF were carried out in serumfree F-10-lh to prevent degradation of the peptide by serum proteases. Analysis of the stability of SRIF in this medium RESULTS by a radioreceptor assay(22) showed that, under the pretreatDevelopment of a NondenaturingProcedure to Rapidly Dis- ment conditionsemployed, SRIF activitydecayed with a halfsociate the SRIF-Receptor Complex-We have previously time of 16 & 2 h at 37 "C. Thus, a single addition of 100 nM shown that at 37 "C receptor-bound ['251-Tyr']SRIF remains SRIF provided a saturating concentration of peptide during on the cell surface until it slowly dissociates (9). Therefore, pretreatment incubations in F-10-lhfor over 2 days. to determine theeffect of SRIF pretreatment on the binding To determine theeffect of SRIF pretreatment on the SRIF and functional propertiesof the SRIF receptor,a method was receptor, GH4C1 cells were incubated at 37 "C with 100 nM required to rapidly release bound SRIF without damaging theSRIF for various periods, washed with HBSS-AcOH, pH 5 , receptor. As is true for many peptides(18-21) the binding of to dissociate preboundSRIF,andthenincubated with ['251-Tyr']SRIF to its receptor inGH4C1 cellsis strongly pH- ['*'I-Tyr']SRIF according to the binding protocol described sensitive (9). Treatment of cells with 0.2 M acetic acid, 0.5 M under "ExperimentalProcedures." SRIF pretreatmentcaused sodium chloride, pH 2.5, at 4 "C releases greater than 80% of a time-dependent increase in ['*'I-Tyr']SRIF binding which specifically bound [1251-Tyr']SRIFwithin 30 s (9). However, reached a maximum value of 200% of untreated cultures after although this acid/salt dissociationprotocol did not cause 20 h (Fig. 2). The level of ['251-Tyr']SRIF binding remained leakage of cytoplasmic contents (9), it significantly inhibited constant between 24 and 48 h of SRIF pretreatment (data subsequent cAMP production andresponsiveness of the cells not shown). In 22 experiments, incubation of cells with 100 to SRIF (data not shown). To avoid the detrimental effects nM SRIF for 24 h increased ['251-Tyr']SRIF binding to 221 f of such a harsh acid/salt treatment, we determined the least 8% of control values. This increase in ['251-Tyr1]SRIFbinding acidic p H which would stillcauserapid dissociation of occurred inthe absence of anysignificant effect on cell [1251-Tyr']SRIF from its receptor. Treatment of cells for 5 proliferation: cultures pretreated with 100 nM SRIF for 24 h min at 37 "C with Hanks' balanced salt solution (HBSS) had 97 & 2% ( n = 24) of the numberof cells present in control buffered with 20 mM sodium acetate to different pH values cultures. Whereas treatmentof cells with 100nM SRIF for 24 showed that solutions of pH 5 , or less, enhanced the release h increased ["T-Tyr']SRIF binding2-fold, it did not alter the of specifically bound ['251-Tyr']SRIF (data not shown). After amount of ['251-Tyr4]bombesin, 9 - E G F , '251-insulin, or incubation in HBSS-AcOH, pH 5.0, for 10 min at 37 "C, the [3H-Me]TRHboundto cells during a subsequent60-min same amount of ['251-Tyr']SRIF was released from the recep- incubation (Fig. 3). Thus, chronic SRIF pretreatment appears tor as after treatment with 0.2 M acetic acid, 0.5 M sodium to specifically affect the SRIFreceptor. chloride, pH 2.5 (Fig. 1). Concentration Dependence and Specificity of the SRIF-inSince HBSS-AcOH,pH 5.0, was able todissociate surface- duced Increase in f*'I-Tyr']SRIF Binding-The concentrabound ['251-Tyr']SRIF from GH4Cl cells, we next examined tion dependence for the SRIF-induced increase in [lZ5I-Tyr'] the effect of this treatment on the subsequent binding prop- SRIF binding isshown in Fig. 4. A half-maximal increase in erties of theSRIFreceptor(TableI).Theamount of binding was produced by 2.5 f 0.4 nM SRIF. In three inde[12'II-Tyr']SRIFbound tocells treated with HBSS-AcOHwas pendent experiments, the EDso for SRIF was 2.3 k 0.4 nM, not significantly different from that bound to cells washed similar to the EDS0 for SRIF inhibition of prolactin release with F-10-lh. Since receptor bindingwas measured at a con- which is about 1 nM (3). Pretreatment of cells with 100 nM centration of ['*'I-Tyr']SRIF ( ~ 3 PM) 0 at least 10-fold lower dicarboxymethyl-SRIF, a linear, inactive SRIF analog,did than itsKd for the SRIF receptor(0.5 nM) (3), the observation not significantly alter the bindingof ['"I-Tyr'ISRIF (Fig. 4). that['2sI-Tyr']SRIFbinding was unaltered indicated that Both the specificity for the cyclic structure of SRIF and the neither the number nor the affinity of the SRIF receptorwas dose-response characteristicsindicatedthattheSRIF-inaffected by the pH5 wash. The data in Table I also show that preincubation of cells witha saturating concentrationof SRIF markedly reduced the subsequent bindingof ['251-Tyr']SRIF. However, treatment with HBSS-AcOH, pH 5.0, restored ["'I-Tyr'ISRIF binding to control values. Thus, after incubation with SRIF, most of the receptors remained occupied on the cell surface but could be induced to rapidly release prebound SRIF by exposure tolow pH. The pH 5 wash protocol did not alter either basal cAMP levels, the stimulatory effect of VIP on cAMP accumulation, or the effect of SRIF to inhibit VIP stimulation (Table 11). These hormonal effects on cAMP concentrations are caused by changes in adenylyl cyclase activity (4, 6). Together our results demonstrate that the pH 5 wash protocol does not perturb either the binding properties of the SRIF receptor or its coupling to adenylyl cyclase. We used this wash in all subsequent experiments torapidly dissociate the SRIF-recepHours tor complex afterchronicpretreatment of cells withthis peptide. FIG. 2. Time course of the SRIF-induced increase in [ Y Effects of Chronic SRIF Treatment on Receptor BindingTyr'ISRIF binding. GH4Cl cells were incubated in F-10-lh for 24 EXPERIMENTAL PROCEDURES~
* Portions
of this paper (including "Experimental Procedures," Tables 1-111, and Figs. 1 and 3 ) are presented in miniprint at theend of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
h at 37 "C.At the indicated times before the completion of this 24-h incubation, 100 nM SRIF was added. At the end of the pretreatment, all cells were rinsed with HBSS-AcOH, pH 5.0, for 10 min End [1*51-Tyr']SRIFbinding was subsequently determined as described under "Experimental Procedures." Error burs represent the S.E. of triplicate determinations.
716
Homologous Up-regulation of SRIF Receptors
treated cells (Fig.5 ) . Nonlinear regression analysis of the data gave the ED,, values for SRIF as 0.39 f 0.05 nM and 0.30 f 0.04 nM in control and SRIF-pretreated cells, respectively. The values for B,,, in control and SRIF-treated cultures were 7,900 & 1,000 sites/cell and 14,700 f 1,900 sites/cell, respectively. In three experiments there was no significant difference in the ED50for SRIF inhibitionof ["51-Tyr1]SRIF binding between control and SRIF-treatedcells, whereas the B,,, was increased in pretreated cells to 240 f 20% of control values. Therefore, SRIF treatment increased the number of SRIF receptors percell without altering the affinity of these receptors for SRIF. The Mechanism of SRIF Receptor Up-regulation-The steady-state level of a protein is determined both by its rate 0 10-11 IO-IO 10-9 10-8 10" of synthesis and by its rate of degradation. To investigate whether SRIF pretreatment increased the number of SRIF [Peptide] ( M I FIG. 4. Concentration dependence for the SRIF-induced in- receptors by increasing receptor synthesis, we examined the crease in ['261-Tyr']SRIFbinding. GH& cells were incubated in effect of cycloheximide on SRIF receptor regulation (Table F-10-lh for 24 h at 37 "C with the indicated concentration of SRIF IV). As reported previously (28), chronic incubation of cells (a)or dicarboxymethyl SRIF (0).The cells were then washed with with concentrations of cycloheximide which reduced protein HBSS-AcOH, pH 5.0, for 10 min and ['251-Tyr']SRIFbinding was synthesis also decreased ['251-Tyr']SRIF binding, presumably determinedas described under "Experimental Procedures."Error bars represent the S.E. of triplicate determinations. The line represents by inhibiting the synthesisof new receptors. However, cycloheximidedid not affect the percent increasein ['*'I-Tyr'] the computer-fitted regressionlinedetermined as described under SRIF binding produced by SRIF pretreatment (Table IV). "Experimental Procedures." Thus, theincrease in SRIF receptor number could not be due ducedincreasein['251-Tyr']SRIF binding was a receptor- to enhanced receptor synthesis. To determine whether SRIF pretreatment altered the total mediated process. number of intracellular plus extracellular SRIFreceptors, we SRIF reduces the concentrations of two intracellular messengersin GH4Cl cells: cAMP and [Ca2+li (2,4, 7, 8). To measured the binding of ['251-Tyr']SRIF to membranes predetermine whether either of these mediatorscaused the SRIF- pared from control and SRIF-treatedcells (Table V). Recovinduced increase in ["sI-Tyr']SRIF binding, we characterized ery of membranes was maximized by using ultracentrifugation the effects of other agentswhich alter thelevel of intracellular to prepare membranes from cell homogenates and by using cAMP or [Ca2+Ii (Table111). Although both carbamylcholine filtration through 0.2-pm filters to collect membrane-bound and PIA inhibit cAMP production and reduce [Ca2+Ii in GH ['2'I-Tyr']SRIF following the binding incubation. In contrast to the increased binding observed in intact cells after SRIF cells (23-25),3these neurotransmitter analogs did increase not pretreatment, theamount of ['251-Tyr1]SRIF specifically ['T-Tyrl]SRIF binding (Table 111). Furthermore, even bound to membranes prepared from SRIF-treated cultures though pretreatment of cells with VIP decreased ['*'I-Tyr'] was slightly lessthan that bound to control membranes (Table SRIF binding, pretreatment with forskolin did not, despite the fact that both agents increase cAMP accumulation and V).In four experiments, ['"I-Tyr'ISRIF bindingtomem[Ca2+Ii in GH cells (4, 25).4TRH, which increases [Ca2+Ii(26, branes prepared from SRIF-pretreated cells was 88 f 5% of 27) without altering intracellular cAMP levels (4), reduced that from untreated cells. ['"I-Tyr'ISRIF binding, consistent with the previously reported effect of chronic T R H pretreatment to decrease SRIF receptor number in GH,C, cells (28). Only VIP and TRH reproducibly altered ['2sI-Tyr']SRIF binding: in three independentexperiments,VIPand T R H decreased [1251-Tyr'] SRIF binding by 12 f 4% and 20 f 3%, respectively. In the same experiments, pretreatment with SRIF caused a 2-fold increase in ['"I-Tyr'ISRIF binding. The absence of any cor\ 0. Bound (frnoles) relation between the effects of different agents on [1251-Tyr1] SRIF binding and on cAMP levels or [Ca2+Ii demonstrated that theincrease in SRIF binding produced by SRIF pretreatment required occupancy of the SRIFreceptor and could not be mimicked by changing the intracellular levels of these messengers. Iz_r Comparison of the Binding Propertiesof the S R I F Receptor OL 4 1 ' in Control and SRIF-treatedCells-The increase in[1251-Tyr'] 0 lo-" lO"0 10'~ 10'8 10" SRIF binding caused by SRIF pretreatment could be due to [SRIF] (MI an increase in receptor affinity, an increase in receptor numFIG. 5. Effect of SRIF Pretreatment on the binding propber, or a combination of the two. To determine which of these erties of the SRIF receptor. GH4Clcells were incubated in F-10parameters were altered, ['2'I-Tyr1]SRIF binding was deter- lh with or without 100 nM SRIF for 24 h at 37 "C. The cells were mined under steady-state conditions in the presence of in- rinsed with HBSS-AcOH, pH 5.0, to remove receptor-bound SRIF creasing concentrations of SRIF in both control and SRIFwas then determined to control (0) and total ['251-Tyr']SRIF binding and pretreated (0) cultures in the presence of the indicated concenB. D. Koch and A. Schonbrunn, unpublished observations. trations of SRIF. Error bars represent the S.E. of triplicate determiKoch, B. D., Blalock, J. B., and Schonbrunn, A. (1988) J. Biol. nations and the lines represent computer-fittedregression lines. Inset, Chem. 263,216-225. saturable binding data were plotted by the method of Scatchard (29). I
"
I
I
Homologous Up-regulation
of SRIFReceptors
717
TABLE IV Effect of cycloheximide on SRZF receptor modulation GH,C, cells were incubated in F-10-lh containing the indicated concentration of cycloheximide for 60 min at 37 "C. SRIF (100 nM) was then added to the appropriate dishes and the incubation was continued for an additional 24 h at 37 "C. The cells were subsequently rinsed with HBSS-AcOH, pH 5.0, to remove receptor-bound SRIF, and [1251-Tyr1]SRIF binding was determined as described under "Experimental Procedures." In parallel dishes, proteinsynthesis was measured after a1-h pretreatment with cycloheximideby determining the incorporation of [3H]leucine into trichloroacetic acid-insoluble material during a subsequent 60-min incubation a t 37 "C as described under "Experimental Procedures." Treatment
['Z61-Tyr']SRIF bound
Inhibition of protein synthesis
Control
a
SRIF I
%
cprn/&gprotein"
Control Cycloheximide 1d m 1
21.1 48.7 f 0.5
f 1.4 (231%)b
16.0 79
f 0.7
82
9.1 f 0.3
37.3 f 0.7 (234%) 19.4 f 0.5 (213%)
10
Mean f S.E. of triplicate determinations.
' % of control value.
TABLE V Comparison of the effect of SRIF pretreatment on P2'I-Tyr11SRIF binding to intact cells and cell membranes GH,C, cells were incubated in F-10-lh with or without 100 nM SRIF for 24 h at 37 "C. The cells were then rinsed with HBSS-AcOH, pH 5.0, to remove receptor-bound SRIF as described under "Experimental Procedures" and splitinto two groups. In one group, [12SI-Tyr1]SRIF binding was measured to intact cells, whereas in the other group binding was measured to membranes prepared from the Dretreated cells as described under "Experimental Procedures."
0
IO
1
20
Hours FIG. 6. Time course of the SRIF-induced changes in cellular responsiveness to VIP and forskolin. GH,C, cells were incubated in F-10-lh for 24 h at 37 "C. At the indicated times before the completion of this 24-h incubation, 100 nM SRIF was added. IBMX was then added to a final concentration of 250 p~ and thecells were incubated for 30 min at 37 "C. Following a 10-min wash with HBSSAcOH, pH 5.0, containing 250 p M IBMX, cyclic AMP accumulation was determined during a 30-min, 37 "C incubation in fresh HEPESF-10-lh medium containing 250 p M IBMX alone (0)or IBMX plus either 100 nM VIP (0),or 1 p M forskolin (W). Error burs represent the S.E. of triplicate determinations.
o-+""?
['261-Tyr']SRIF bound Pretreatment
Control SRIF
Cells
Membranes
cprn/106 cells"
cprn/pgproteinb
2933 f 169 7352 f 236 60.4
70.5 f 4.6 f 6.0
Mean f S.E. of duplicate determinations. Mean f S.E. of five determinations.
5b Oz
4
0
Since SRIF pretreatment did notincrease total membrane SRIF receptors, we determined whether it increased the receptor-mediated internalizationof ['2'I-Tyr']SRIF during the binding assay with whole cells. Following incubation of GH4C1 cells with ["sI-Tyr']SRIF for 60 min at 37 "C, the fractionof the total radiolabeled peptide which was removed by an acid wash was the same in control and SRIF-pretreated cultures (data not shown). Therefore, an alteration in receptor-mediated ligand processing cannot explain the observation that SRIF pretreatment did not significantly change the binding of ["T-Tyr*]SRIFtomembranesunderconditions where binding tocells was markedly increased. Effect of SRIF Pretreatmenton the Biological Responses of Gff4c1 Cells-SRIF does notaffectbasalcAMP levels in GH,C, cells but reduces stimulation of adenylyl cyclase by hormones and forskolin (4, 5 ) . Therefore, we examined the effects of SRIF pretreatment both on the actions of agents that stimulate adenylyl cyclase and on the abilityof SRIF to inhibit this stimulation. These experiments focusing on receptor coupling included the same pH 5 wash protocol after pretreatment with o r without SRIF as the experiments examining ['"51-Tyr1]SRIF binding. As shown in Table 11, this mild acid wash did not affect basal cAMP levels or VIP and SRIF regulation of cAMP accumulation. SRIF pretreatment
1
' IO"'
I
10-l~
I
IO-^
I
I
10-8
IO-^
[SRIF] (MI FIG. 7. Effect of SRIF pretreatment on the concentration dependence for SRIF inhibition of VIP-stimulated cAMP accumulation. GH,C, cells were incubated in F-10-lh with (0)or without (0)100 nM SRIF for 24 h at 37 "C. After the addition of 250 p~ IBMX, the cells were incubated for 30 min at 37"C and then rinsed with HBSS-AcOH, pH 5.0, containing 250 p~ IBMX. The cells were subsequently incubated for 30 min at 37 "C in HEPES-F10-lh containing 250 p~ IBMX, 100 nM VIP, and the indicated concentrations of SRIF, and the cAMP accumulated in the medium was determined as described under "Experimental Procedures.'' The amount of cAMP accumulated in the absence of any added hormone was 6.48 f 0.95 pmol/106 cells for control and 6.52 f 0.23 pmol/106 cells for SRIF-pretreated cells. Error burs represent the S.E. of triplicate determinationsandthe lines represent computer-fitted regression lines determined as described under "Experimental Procedures." did not alter unstimulated cAMP levels but caused a timedependent increase in the stimulatory effects of VIP and forskolin to 167 and 136% of controls, respectively (Fig. 6). However, thepotency of SRIF t o inhibitVIP-stimulated cAMP accumulation was unaffected by SRIF pretreatment. In the experimentshown in Fig. 7, the ED,, for SRIF inhibition of VIP-stimulated cAMP accumulation in control and
718
Homologous Up-regulation of SRIF Receptors
SRIF-treated cells was 0.19 k 0.04 nM and 0.37 f 0.07 nM, hormone release in GH4Cl cells (5, 14), to uniformly increase respectively (p > 0.05). In four independent experiments, the ['251-Tyr']SRIF binding contrasts with the reported correlaED50 for SRIF was 0.46 f 0.19 nM in control cultures and tion between hormone secretion and SRIF receptor levels in 0.54 k 0.12 nM in SRIF-pretreated cultures. Furthermore, thepancreatic @-cells(35) and indicates that it isreceptor occuamount of inhibition producedby maximal concentrations of pancy and not alteredsecretory activity which leads to SRIF SRIF was not reduced (Fig. 7). Therefore, SRIF pretreatment receptor up-regulation in GH4Clcells. did not desensitize GHICl cells to subsequent SRIF chalThe ability of chronic SRIF exposure to modulate peptide lenges. binding to GH4Cl cells was specific for the SRIF receptor. The association of '251-insulin, ['251-Tyr4]bombe~in, 12,1-EGF, DISCUSSION and [3H-Me]TRH with cells was unaffected by SRIF pretreatThe SRIF receptor in GH4Cl cells is known to be regulated ment. However,because these peptidesundergoreceptorby several heterologous hormones.T R H causes an acute(560 mediated endocytosis and degradation at the temperatures min) increase in SRIF receptor number per cell (28). Chronic (22 or 37 "C) at which binding incubations were performed, treatment (210 h) with either T R H (28) or dexamethasone our protocol cannot distinguish between effects on receptor of internalization and/or process(30) decreases the number of SRIF receptors. However, the binding and on the kinetics ing. Nonetheless, since these peptides areprocessed at differeffect of SRIFonits own receptor (homologousreceptor modulation) had not been investigated previously and is of ent rates by GH4Cl cells (ll),it is very unlikely that changes particular interest since receptor-bound ['251-Tyr']SRIF does in receptor number were always completely obscuredby comnot undergo rapid receptor-mediated internalization (9). In pensating changes in processing parameters. Several mechanisms could result in homologous SRIF recontrast to the receptor down-regulation produced by peptides the measured increase in which are rapidly endocytosedsubsequent toreceptor binding, ceptor up-regulation. First, ['251-Tyr']SRIF binding could have been caused by increased chronic treatment of GH4C1 cells with SRIF resulted in an receptor-mediated uptake of ['251-Tyr']SRIF rather than by increase in SRIF receptor number. The extent of up-regulation of SRIF receptor number was true changes in receptor number. However, examination of dependent on the concentration of SRIF in the pretreatment the cellular distribution of specifically bound [lZ5I-Tyr1]SRIF ligand medium. The observed ED5, value of 2.3 nM for this chronic revealed that SRIF pretreatment did not stimulate effect correlated with both theknown binding affinity of the uptake. Two other potential mechanisms known to regulate SRIF receptor for SRIF ( K d = 0.6 nM) and the potency of the level of cell surface receptors are changes in the intracellulardistribution of receptors and changes in the rate of SRIF to acutely inhibit cAMP accumulation and prolactin release (ED,, E 1 nM) (3-5). In addition, the inactive, linear receptor turnover. Homologous receptor down-regulation has been shown to SRIF analogdicarboxymethyl SRIF did not alter SRIF recepof the tor number. Thus, the homologous receptor up-regulation occur due to the transferof receptors from the surface producedby SRIF pretreatment must be initiated by the cell to intracellular membrane compartments without any accompanying changesin receptor turnover.For example, binding of SRIF to itsreceptor. Occupancy of the SRIF receptor inGH4C1 cells leads to a treatment of chick liver cells with insulin leads to a decrease decrease in the concentration of two intracellular messengers: in insulin receptor number on the plasma membrane and a recepcAMP and Ca2+ (2, 4, 7, 8). SRIF inhibition of cAMP accu- concomitant increase in intracellular membrane-bound mulation results from the inhibition of stimulated adenylyl tors (36). This translocation of insulin receptors into thecell cyclase activity (4, 6). Reduction of [Ca"+Ii occurs as a con- has been linked toa higher rate of internalization of occupied sequence of the action of SRIF to increase a potassium con- receptors than of unoccupied receptors, a process which reductance, which hyperpolarizes GH4C1 cells and thereby in- sults in the rapid endocytosis of the bound ligand (13). Behibits Caz+ influx through voltage-dependent calcium chan- cause receptor-boundSRIF is not rapidly internalized by not GH4C1 cells(9), it was not surprising that SRIF did cause nels (2): SRIF does not affect diacylglycerol levels in GH cells (31). Changes in intracellular cAMP andCa2+levels are down-regulation of its receptor. In contrast to homologous receptor down-regulation, hoknown t o regulate membranereceptors. For example, folliclestimulatinghormone-inducedup-regulation of luteinizing mologous receptor up-regulationhasnot been reported to hormone receptors in rat granulosa cellsis mimicked by cyclic occur as a consequence of the redistribution of intracellular AMP analogs or activators of adenylyl cyclase (32, 33). Sim- receptors to the plasma membrane. However, some growth ilarly, depolarizing concentrations ofKC1 and the calcium factors have been shown to cause the translocation of heterionophore A23187 mimic the slow up-regulation of gonadotro- ologous receptors. For example, insulin increases cell surface insulin-like growth factor I1 receptors andglucose transportpin-releasing hormone receptors in pituitary cells produced ers by increasing the rate of transit of intracellular receptors by gonadotropin-releasinghormone (34). Incontrast,upregulation of SRIF receptorswas not due to SRIF's ability to to thecell surface (37,38). These effects occurwithin 60 min. alter cAMP production or[Ca2+Ii.Pretreatment of cells with However, homologous up-regulation of SRIF receptors was either carbamylcholine or the adenosine receptor agonist PIAnot maximal until 20 h of SRIF treatment. The requirement for prolonged exposure of cells to SRIF suggested that the didnot increase['251-Tyr']SRIF bindingalthoughthese agents mimic the effect of SRIF todecrease both cAMPlevels mechanism leading to anincrease in receptor number did not and [Ca2+Ii(23-25).3 Furthermore, pretreatment of cells with involve redistribution of a preexisting pool of intracellular forskolin, an agent which stimulates adenylyl cyclase and SRIF receptors to thecell surface. Rather, a decrease in the markedly increases cyclic AMP accumulation in GH4Cl cells turnover of SRIF receptors appearedto be a more attractive ( 5 ) ,did not reproducibly alter [1251-Tyr']SRIF binding.Since possibility. To determine whether the observed increase inSRIF recepheterologous agents whichregulate cAMPand[Ca2+Iiin GH4Cl cells did not modulate SRIF receptors appropriately, tor number could be due to an increasein SRIF receptor the action of SRIF to increase its receptors is unlikely to be synthesis, we examined the effects of the protein synthesis been shown to inhibit mediated by these intracellular messengers. Further, the ina- inhibitor, cycloheximide. This drug has bility of VIP, TRH, and forskolin, all of which stimulate agonist induced up-regulation of gonadotropin-releasing hor-
Homologous Up-regulation of SRIF Receptors
719
mone receptors in pituitarycells, demonstrating that changes the potency or efficacy of SRIF to inhibit VIP-stimulated efficacy of SRIF in receptor synthesis can result inhomologous receptor mod- cAMP production. Similarly, the potency and release were also unulation (34).However, theaction of SRIFto increase toinhibitVIP-stimulatedprolactin [‘251-Tyr’]SRIF binding was not affected when protein syn- changed by chronic re treatment.^ The lack of sensitization whichincrease SRIF thesis was markedly reduced. These results indicate that SRIFto SRIF under treatment conditions receptor up-modulation was not a consequence of increased receptor number to 220% of control was consistent with the conclusion discussedabove that the additional SRIF receptors receptor synthesis. Alternatively, SRIF treatmentcould have reduced the deg- induced by SRIF treatmentwere functionally uncoupled. In agreement with our findings, SRIF treatment has been radation rateof the SRIFreceptor. In fact the most commonly observed mechanism of homologous receptor regulation in- reported to sensitize AtT-20/D16-16 mouse pituitary tumor of hormone release and cAMP volves stimulation of receptor degradation. Although inhibi- cells to subsequent stimulation tion of receptor degradation by agonists is much less common, production, whereas basal hormonerelease and cAMP levels a decrease in the degradation ofrate the acetylcholine receptor are unaffected (40, 43). However, in contrast to our observahas been shown in chick muscle cells when the receptor is tions, chronic SRIF treatment hasbeen reported to decrease occupied by a-bungarotoxin (39). Both the observed slow time the potency of SRIF to inhibit cAMP production in AtT-20/ course for theinduced increase in SRIF receptor number and D16-16 cells (40, 44, 45). Nonetheless, the observation that theinability of cycloheximide toinhibitthis process are SRIF inhibition of 8-bromo-CAMP and K’-stimulated hornot undergoa consistent with aneffect of SRIF todecrease the degradation mone release in AtT-20/D16-16cellsdoes of the SRIF receptor. However, a decreased rate of receptor similar desensitization (44, 45) suggests that in this cell line, degradation, leading to an increase in SRIF receptor number as in GH4CIcells, desensitization does not occur at the level in cells, should have resulted in increased receptor density in of the receptor. The recent availability of a long-acting SRIF analog, SMS membrane preparations. Therefore, the absenceof a signifi201-995, has generated increased interest in the therapeutic cant change in [‘251-Tyr’]SRIF binding t o membranes preuses of SRIF (46,47).Chiodini et al. (48) have recently shown paredfrom cellswhichshoweda %fold increase in SRIF receptors was unexpected. We believe that the most likely that SMS201-995 continues tolower plasma growth hormone explanation for this finding results from the different forms levels in humans afterover 200 days of continuous treatment in vivo. Long-term effects on inhibitionof gastric secretionin of the somatostatin receptor measured in binding assays with vivo have also been reported (49). These observationssuggest whole cells and membranes. The SRIF receptor in GH4C, that the responses to SRIF do not desensitize i n uiuo. In cells is coupled to a guanine nucleotide-binding protein and the affinity of the receptor is decreased in the presence of addition, patients with SRIF-secreting tumors (somatostatiG T P analogs (6). In intact cells, the binding assay measures nomas) and elevated serum SRIF levels have reduced levels evidence that, the low affinity form of the receptor due to the high G T P of plasma growth hormone and insulin, further levels present intracellularly. In contrast, membrane binding as in GHICl cells, the cellularresponses toSRIFdonot assays preferentially measure the high affinity form of the desensitize (50, 51). The parallel between the lack of desenSRIF receptor present in the absence of G T P analogs (6). sitization to SRIF i n vivo and in GH&, cells makes these Thus, binding of [‘251-Tyr1]SRIFto membranes is markedly cells an excellent i n vitro model system for further examining decreased after uncoupling of the SRIF receptor from its the consequences of chronic SRIF treatment. associated G protein by treatment with Bordetella pertussis REFERENCES toxin, whereas binding t o whole cells is unaffected by this 1. Reichlin, S. (1983) in BrainPeptides (Krieger, D. T., Brownstein, toxin (8). Therefore, it is possible that SRIF treatment inM. J., and Martin, J. B., eds) pp. 711-752, Wiiey Interscience creased the numberof SRIF receptorsby decreasing receptor Publications, New York degradation but that these “additional” receptors are uncou- 2. Schonbrunn, A., and Koch, B. D. (1987) in Somatostatin: Basic pled and, asa consequence, are not detectedin the membrane and Clinical Status (Reichlin, S., ed) pp. 121-135, Plenum binding assays. Publishing Cop., New York 3. Schonbrunn, A., and Tashjian, A.H., Jr. (1978) J. Biol. Chem. In contrast to our inability to measure significant differ253,6473-6483 ences in[‘251-Tyr’]SRIFbinding to membranes prepared from 4. Dorflinger, L. J., and Schonbrunn, A. (1983) Endocrinology 1 1 3 , SRIF-treated GH4Cl cells, Heisler and Srikant (40)found 1541-1550 that membranes prepared from SRIF-pretreated AtT-20/Dl6 5. Dorflinger, L. J., and Schonbrunn, A. (1983) Endocrinology 1 1 3 , cells show a 30% decrease in SRIF receptordensity. However, 1551-1558 6. Koch, B. D., and Schonbrunn, A. (1984) Endocrinology 1 1 4 , we removed prebound SRIF from the cells by treatment at 1784-1790 pH 5.0 before preparing membrane fractions whereas this 7. Schlegel, W., Wuarin, F., Wollheim, C. B., and Zahnd, G . R. acidic washwas not performed in the previous studies. There(1984) Cell Calcium 5 , 223-236 fore, the reported reduction in SRIF binding to AtT-20/D16 8. Koch, B. D., Dorflinger, L. J., and Schonbrunn, A. (1985) J.Bid. membranes may be duetoprior occupancy of the SRIF Chem. 260, 13138-13145 9. Presky, D. H., and Schonbrunn, A. (1986) J. Cell Biol. 102,878receptors during pretreatment which would reduce the num888 ber of unoccupied receptors available during the binding as10. Sullivan, S. J., and Schonbrunn, A. (1986) J. Biol. Chem. 2 6 1 , say. 3571-3577 For manyreceptors, chronic treatment with saturating con11. Westendorf, J. M., and Schonbrunn, A. (1983) J. Biol. Chem. centrations of agonists causes desensitization to subsequent 258, 7527-7535 hormonal challenge. The desensitization process can occur 12. Catt, K. J., Hanvood, J. P., Aguilera, G., and Dufau, M. L. (1979) Nature 280, 109-116 prior to any change in receptor number as with the turkey erythrocyte P-adrenergic receptor (41, 42). Chronic preincu- 13. Gorden, P., Carpentier, J.-L., Fan, J.-Y., and Orci, L. (1982) Metabolism 31 , 664-669 bation of cells with SRIF increased the subsequent accumu14. Tasbjian, A. H., Jr. (1979) Methods Enzymol. 58, 527-535 lation of cAMP in response to both VIP and forskolin without 15. Peterson, G. L. (1977) Anal. Biochem. 83, 346-356 affecting basal cAMP levels. However, SRIF pretreatment did not alter either the affinityof the SRIFreceptor for SRIF or D. H. Presky and A. Schonbrunn, unpublished observations.
Homologous Up-regulation Receptors of SRIF
720
16. Carpenter, G., and Cohen, S. (1976) J. Cell Biol. 7 1 , 159-171 17. Wilson, M.A., and Miles, L. E. M. (1977) in Handbook of Radioimmunoassay (Abraham, G. E., ed) pp. 275-297, Marcel Dekker, Inc., New York 18. Haigler, H. T., Maxfield, F. R., Willingham, M. C., and Pastan, I. (1980) J. Biol. Chem. 255,1239-1241 19. Willingham, M. C., and Pastan, I. (1980) Cell 21,67-77 20. Marshall, S., Podlecki, D. A., and Olefsky, J. M. (1983) Endocrinology 113,37-42 21. DiPaola, M., and Maxfield, F. R. (1984) J. Biol. Chern. 2 5 9 , 9163-9171 22. Rorstad, 0.P., Schonbrunn, A., and Martin, J. B. (1983) Can. J. Biochem. Cell Biol. 6 1 , 532-537 23. Onali, P., Eva, C., Olianas, M. C., Schwartz, J. P., and Costa, E. (1983) Mol. Phurmacol. 2 4 , 189-194 24. Dorflinger, L. J., and Schonbrunn, A. (1985) Endocrinology 1 1 7 , 2330-2338 25. Schlegel, W., Wuarin, F., Zbaren, C., Wollheim, C. B., and Zahnd, G. R. (1985) FEBS Lett. 1 8 9 , 27-32 26. Albert, P. R., and Tashjian, A. H., Jr. (1984) J. Biol. Chern. 2 5 9 , 5827-5832 27. Gershengorn, M. C., Geras, E., Purrello, V. S., and Rebecchi, M. J. (1984) J. B i d . Chern. 259,10675-10681 28. Schonbrunn, A., and Tashjian, A. H., Jr. (1980) J. Biol. Chern. 255,190-198 29. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51,660-672 30. Schonbrunn, A. (1982) Endocrimlogy 1 1 0 , 1147-1154 31. Yajima, K., Akita, Y., and Saito, T. (1986) J. Biol. Chem. 261, 2684-2689 Wang, C., Casper, R. F., Mattson, G., and 32. Erickson, G.F., Hofeditz, C. (1982) Mol. Cell Endocr. 2 7 , 17-30 33. Knecht, M., and Catt, K. J. (1982) Endocrinology 1 1 1 , 11921200
EXPERIMENTALPROCEDURES
-
Materials
*ere o b t a i n e df r o mt h ef o l l o w i n g
~ o u ~ c e i :s y n t h e t i c SRIF, [ T y r l I S R I F ,
I T R H ) , v a s o a c t i v ei n t e s t i n a lp e p t i d e( V I P 1a n db a m b e r i nf m m
t h y r o t r o p i n - r e l e a r i n gh a m n e
Bachem, Torrance. CA: e p i d e r n a l . p r a t h
fwtm IEGFIfrom
z i n e - e t h a n e r u l f o n i ca c i d
MA:
KOR Chemicals.Cambridge,
insulin
3 - i ~ o b ~ f y l - l - ~ e t h y l x d n t h i(LBMXI. ne f o m k o l i n .b a C i t T a C i n ,
f r o mE l iL l i l y .I n d i a n a p o l i s .I N :
c y c l o h e x i m i d e . C M P I t d l l d l r d . CaTbbdmyl c h o l i n e . N o n i d e t IHEPESl fromSigmaChemical
P-40.and
Co..
Redium and sera from Grand island B i o l o g i c a l t o . .
4(2-hydroly-ethylI-l-pipera-
S t . L o u i s . MD; Ham's F I OC u l t u r e
Grand I s l a n d , NY: c u l t u r e p l a t e r
from F a l c o n
Labware. Oxnard. CA: [3HlMe-TRH ( 1 6 . 1 C i / m o l j , [ 3 H l l e u c i n e Kintillation fluid
(54 C i / m o l l and Aquaral MA: Na1251 (2200 C i l m o l l , l a c t a l b u m i n
frm New EnglandNuclear,Boston,
ester frm I.C.N.,
h y d r o l y s a t e . and [1251JsuccinylCAMP-tyrosineRethyl
wi:
frm EnZyRe Center, Boston,
a n t i s e r ar a i s e dt o
I r v i n e . CA: IgGsorb
~ ' - D - I u c c ~ ~ ~ ~ - c A Serum M P - ~albumin ~ ~ ~ ~ ~
MA:
frm M i l e s - Y e d d .L t d . ,R e h o v o t ,I s r a e l ;c e l l o t a t ef i l t e r sf r o mM i l l i p o r e .B e d f a r d , ( - l N 6 - l R - 2 - p h e n y l - i r ~ p r ~ p y l l a d e n o r i n e( P I A 1f m mB o e h r i n g e r
was k l n d l gp r o v i d e d
poli3,IN.OiCarboxynethylSRlF
He* England
Mannheim B i a c h e m l c a l r ,I n d i a n a -
by
D r s . P. B K h d and S. R e i c h l i n ,T u f t s
Rh.
MedicalCenter.Boston,
Methodl
"
-
C e l lC u l t u r e methodof
2-3 x
lo5
The p r o p e r t i e so ft h e
GH4C 1 c l o n a l r a t p i t u i t a r y t u m m c e l l s t r a i n
i t s C u l t u r e havebeendescribedpreviously
a i r a t 31-C
I"
c a l f rerum.
1141.
or 2-3 x
3-4 daysandexperimntr
C u l t u r e medium was replacedevery
weeks a f t e r p l a t i n g .
d e s c r i b e dp r e v i o u s l y
(31.
liquidchrmatographyuring 0.1'/.
I
lo6 c e l l r / 3 5 mm 1151 u s i n g
a s a standard.
R d d i o i o d i n a t r o no fP e p t i d e s
flow rite of
2.5-1. f e t a l
number was determined r i c h a C a u l t e rC o u n t e r
and c e l l p r o t e i n was measured b yt h eP e t e r s o nm o d i f i c a t i o no ft h eL o w r ym e t h o d bovinealbumin
tO2/95~/~
were Conducted 2-3
vas 1-2
The c e l l d e n s i t y a t t h e t i m e o f e x p e r i m e n t s
d i s h or 2.5-3.0 r IO5 c e l l r l l 6 m w e l l . C e l l
andthe
C e l l s were p l a t e d a t a d e n s i t yo f
lo4
c e l l r / l 6 - m w e l l and g r o w i n 5'10 Ham's F1D m d i u m r u p p l e m n t e d w i t h 1 2 . 5 ' 1 - horse rerum and
cellr/35-mdish
0.5 m l / m i n w i t h
-
[1251-Tyrl]SRIF
a S u p e l c oS u p e l C o l i l
was p u r i f i e db ym v e w e - p h d s eh i g hp e r f o r m a n c e
LC-18 column ( 2 5 cm
a 120 n i n l i n e a r g r a d i e n t f l o m
t r i f l u o r m c e t i c acid i n
and[12sl.Tyr11SRlF
was p r e p a r e d by c h l o r a m i n e 1 i o d i n a t i o n a s
The r e a c t i o nm i x t u r e
eo'/.
were 11 mi0and
0.1'1.
)I
4.6 n m l e l u t e d It a
triflUoTOaCetiCacidto
acetonitrile. The r e t e n t i o n timr for[TyrllSRIF 17 .in,
r e s p e c t i v e l y .T h e r e f o r e .t h l lp U l i f i C d t i o n
pwtwO1completelyseparatedradioiodinated[T~F~ISRIFfromunredctedpeptideandproduced [12sl.lyr1]snlF [Tyr'lbomberin
w i t h a s p e c i f i ca c t i v i t yo f
Ill1 were P d d i o l a b e l l e d
2200 C l l m o l .
w ~ t hlZ51
EGF 1161, i n s u l i n I l l 1 and
usingprOcedureedescribedPreViouSlY.
34. Loumaye, E., and Catt, K. J. (1983) J. Biol. Chern. 2 5 8 , 1200212009 35. Sussman, K. E., Mehler, P. S., Leitner, J. W., and Draznin, B. (1982) Endocrinology 1 1 1 , 316-323 36. Krupp, M., and Lane, M.D. (1981) J. Biol. Chern. 2 5 6 , 16891694 37. Wardzala, L. J., Simpson, I. A., Rechler, M. M., and Cushman, S. W. (1984) J. Biol. Chem. 259,8378-8383 38. Oka, Y., and Czech, M. P. (1984) J. Bwl. Chern. 259,8125-8133 39. Gardner, J. M., and Fambrough, D. M. (1979) Cell 16,661-674 40. Heisler, S., and Srikant, C. B. (1985) Endocrinology 1 1 7 , 217225 41. Sibley, D. R., and Lefkowitz, R. J. (1985) Nature 3 1 7 , 124-129 42. Stadel, J. M.,Nambi, P., Lavin, T. N., Heald, S. L., Caron, M. G., and Lefkowitz, R. J. (1982) J. Biol. Chern. 257,9242-9245 43. Reisine, T. D., and Takahashi, J. S. (1984) J. Neurosci. 4 , 812819 44. Reisine, T. (1984) J. Phurmacol. Exp. Ther. 2 2 9 , 14-20 45. Reisine, T. (1985) Endocrinology 1 1 6 , 2259-2266 46. Pless, J., Bauer, W., Briner, U., Doepfner, W., Marbach, P., Maurer, R., Petcher, T. J., Reubi, J.-C., and Vonderscher, J. (1986) Scand. J. Gastroenterol. 2 1 , Suppl. 119,54-64 47. Williams, G., Ball, 3. A., Burrin, J. M., Joplin, G. F., and Bloom, S. R. (1986) Lancet 2 ( 8 6 1 0 ) , 774-778 48. Chiodini, P. G., Cozzi, R., Dallabonzana, D., Oppizzi, G., Verde, G., Petroncini, M., Liuzzi, A., and Del Pozo, E. (1987) J. Clin. Endocrinol. Metab. 64,447-453 49. Gyr, K. E., Whitehouse, I., Beglinger, C., Kohler, E., Dettwiler, S., and Fried, M. (1986) Scand. J. Gastroenterol. 2 1 , Suppl. 119,96-102 50. Krejs, G. J., Orci, L., Conlon, J. M., Ravazzola, M., Davis, G. R., Raskin, P., Collins, S. M., McCarthy, D.M., Baetens, D., Rubenstein, A., Aldor, T. A. M., and Unger, R. H.(1979) N . Engl. J. Med. 3 0 1 , 285-292 51. Axelrod, L., Bush, M. A., Hirsch, H. J., and Loo, S. W. H. (1981) J. Clin. Endocrinol. Metab. 52,886-896
721
Homologous Up-regulationof S R I F Receptors r e a c t i o n were f l l t e r e d t h r o u g h M i l l i p o r e C e l l o t a t e f i l t e r s w i t h 0.2 m pole r i l e . The f i l t e r s were washed t h r e e t i n e l H I t h 5 m1 o f 4'C s a l i n e and t h e amount o f r d d i o x t l w t y r e t a i n e d on t h e filters
WII
Tyr'lSRIFbound
d e t e r m i n e d .S p e c i f i cb i n d i n g .C a l c u l a t e d i nt h ep r e s e n c ea n da b s e n c eo f
a s t h ed i f f e r e n c eb e t w e n
50*/- o f t o t a l
100 nH SRlF.representedabout
TABLE 11 Effectof
pH 5 wash on c e l l u l a r r e r p a n r i v e n e r r
blnding.
were I n c u b a t e d f o r 20 mln a t 37'C i n HEPES-F10-lh containing 100 Y M IBMX. The c e l l s were t h e n r i n s e d and i n c u b a t e d a t 37.C w i t h e i t h e l 1 m1 HBSS-AcOH.pH 5.0 Or I m1 GH4Cl
cells
IBHX.
F l u - l h b, o t hc o n t a i n i n g1 W
-
Mearvrenent o f c M P A c c u m u l a t i o n
c o n t a i n i n g 0.25 Rhl IBMX f o r 20 .in
previously publishedprocedures
O f
in 2 4 - w e l lC u l t u r ep l a t e s
B r i e f l y , CH4C1 c e l l s
agents on e x t r a c e l l u l a r c u l P
The e f f e c t o f v a r i o u s
aCCUmUlDtion was determined by 1 m O d l f i C F t i o n
The c e l l s were t h e ni n c u b a t e di n0 . 5
a t 37'C.
5W g
HEPES-F10-10 c o n t a i n i n g
141.
1W
A f t e r 10 .in.
t h eb u f f e r s
*ere r e p l a c e dw i t hf l e s h
uM IBMX and 1 W nH o f t h e i n d i c a t e d h o m n e r .
The C u l P dCCUmYla-
r e d I n t h e medium duping a 20 w i n i n c u b a t i o n a t 37'C was determined a s d e s c r i b e d i n E x p e r i -
were i n c u b a t e d i n 0.5 mlHEPES-FIO-lh
0.25 Rhl IBMX a n dt h ei n d i c a t e d
HEPES-Flu-lhcontaining
to h a m n e l
[1251-
mentalPPocedures. ml O ff r e s h
agents f o r 30 m i " a t 37°C.
Themedia
t o remove any f l o a t r n g ~ ~ 1 1and s . the c o n c e n t r a t i o n I n t h e supernatants was marurcd by RIA ( 4 1 . Under t h e s ec o n d i t i o n s .
were c o l l e c t e d . c e n t r i f u g e d a t
cA'P
4'C
f a r 1 0m i na t
Changes ~n e x t r a c e l l u l a r c u l P c o n c e n t r a t i o n s p a r a l l e l e f f e c t s
37'C f o r 60min
4-C HEPES-FIO-lh a n d t r e a t e d
(31.
- SRlF
4'C
5'1-
4.74
CDntrDl VIP
tTiCh1orOaCetlCacid.the
o f " o n - i n t e r a c t i n g receptarr i n CH4CI
b i n d s t o a s i n g l eC l a s s
t d n g y l a rh y p e r b o l ab y
rpno1er/lo6
cc1l~la
The c e l l s were t h e n
5'1. t r i c h l o r O d C e t i C a c i d
YIP
+
SRlF
4.32
* 0.84
f
0.88
46.8
f
2.66
41.8
14.08
23.0
f
1.98
22.4
f
1.62
20't.. cells
were C O W u t e r - f i t t e d t o a rec-
T h e r e f a r e ,d o s e - r e r p o n r ea n de q u i l i b r i u mb i n d i n gd a t a
n o n l i n e a r regression program ( p r o c e d u r e WLlN, S t a t i s t i c a l A n a l y s i s
I
System. SA8 I n s t i t u t e .
Of
rerldue was di1101ved i n 1 m1 0 . 1 N n e VIP TRH
3371 7708 3012 3723 2873 2695
contra1 SRIF Fomuolin
3638 6611 3931
f
t
* * I
50 122 102 65 76 65
89 167 44
