Proc. Natl. Acad. Sci. USAVol. 84, pp. 1294-1298, March 1987Cell Biology

a2-Adrenergic receptor-mediated sensitization offorskolin-stimulated cyclic AMP production

(HT29 cells/UK14,304/desensitization)

SUSAN B. JONES, MYRON L. TOEWS, JOHN T. TURNER, AND DAVID B. BYLUNDDepartment of Pharmacology, University of Missouri-Columbia, Columbia, MO 65212

Communicated by James 0. Davis, November 17, 1986 (received for review August 28, 1986)

ABSTRACT Preincubation ofHT29 human colonic adeno-carcinoma cells with a2-adrenergic agonists resulted in a 10- to20-fold increase in forskolin-stimulated cyclic AMP productionas compared to cells preincubated without agonist. Similarresults were obtained using either a [3H1adenine prelabelingassay or a cyclic AMP radioimmunoassay to measure cyclicAMP levels. This phenomenon, which is termed sensitization,is a2-adrenergic receptor-mediated and rapid in onset andreversal. Yohimbine, an a2-adrenergic receptor-selective an-tagonist, blocked norepinephrine-induced sensitization, where-as prazosin (ol-adrenergic) and sotalol (,8-adrenergic) did not.The time for half-maximal sensitization was 5 min and thehalf-time for reversal was 10 min. Only a 2-fold sensitization ofcyclic AMP production stimulated by vasoactive intestinalpeptide was observed, indicating that sensitization is relativelyselective for forskolin. Sensitization reflects an increased pro-duction of cyclic AMP and not a decreased degradation of cyclicAMP, since incubation with a phosphodiesterase inhibitor andforskolin did not mimic sensitization. Increasing the levels ofcyclic AMP during the preincubation (using a phosphodies-terase inhibitor) had no effect on sensitization, indicating thatsensitization is not caused by decreased cyclic AMP levelsduring the preincubation. This rapid and dramatic sensitiza-tion of forskolin-stimulated cyclic AMP production is a previ-ously unreported effect that can be added to the growing list ofa2-adrenergic responses that are not mediated by a decrease incyclic AMP.

Activation of several hormone or neurotransmitter receptorsleads to inhibition of adenylate cyclase. Examples of these"negatively coupled" receptors include muscarinic cholin-ergic and somatostatin receptors in AtT-20 mouse pituitarytumor cells and a2-adrenergic receptors in human platelets,NG108-15 neuroblastoma x glioma cells, and HT-29 humancolonic adenocarcinoma cells (1-5). The inhibition of ade-nylate cyclase in these systems is mediated by the guaninenucleotide-binding regulatory protein Ni (6-8).

Agonist treatment of various cellular systems can result ina phenomenon called desensitization. Desensitization is acellular adaptation to agonist treatment that results in ablunting of the physiological response to a subsequent chal-lenge by the agonist. This phenomenon has been studiedextensively in several systems that are coupled to stimulationof adenylate cyclase, in particular the ,B-adrenergic receptorsystem (9, 10). Desensitization of the negatively coupledsomatostatin and muscarinic cholinergic systems in theAtT-20 cell line has also been reported (1, 11). Desensitiza-tion of epinephrine-initiated aggregation of human plateletscan occur without concomitant down-regulation or desensi-tization ofthe a2-adrenergic receptor/adenylate cyclase com-plex (12, 13).

In addition to desensitization, an increase in forskolin-stimulated adenylate cyclase activity following somatostatinor muscarinic agonist preincubation has been observed inAtT-20 cells (1, 11). Similar phenomena have been reportedfor the a2-adrenergic receptor system. Norepinephrine, act-ing through a2-adrenergic receptors in NG108-15 cells,causes an increased activity of adenylate cyclase after pro-longed exposure to the agonist (14). Epinephrine, partlythrough a2-adrenergic receptors, transiently potentiatesforskolin-stimulated cyclic AMP accumulation in isolated ratpancreatic islets but, with longer incubation, decreases cyclicAMP levels (15).We have examined the effects of agonist preincubation on

cyclic AMP synthesis in a human colonic adenocarcinomacell line, HT29. We report here a pronounced, rapid, andreversible sensitization of forskolin-stimulated cyclic AMPsynthesis following a2-adrenergic agonist preincubation.

MATERIALS AND METHODSCell Culture. HT29 cells, a human colonic adenocarcinoma

cell line, were obtained from J. Fogh (Sloan-KetteringInstitute, Rye, NY). Cells were grown routinely in Dulbec-co's modified Eagle's medium with high glucose supplement-ed with 5% (vol/vol) fetal bovine serum and 5% (vol/vol)newborn bovine serum in 75-cm2 disposable tissue cultureflasks in a humidified atmosphere of 5% C02/95% air (5).Cells were subcultured with 0.05% (wt/vol) trypsin/0.01%(wt/vol) Na2EDTA and were seeded at moderate density in35-mm tissue culture dishes with confluence being reached in=5 days.

Cyclic AMP Production Assay. Confluent cultures of HT29cells in 35-mm dishes were utilized for cyclic AMP assays bya modification of the method of Shimizu et al. (16). Allincubations and washes of cells were in serum-free Dulbec-co's modified Eagle's medium. Cells were washed twice with2 ml of medium and then prelabeled with [3H]adenine (5 p.Ciper dish; 1 Ci = 37 GBq) with and without agonist and otheradditions for 30 min at 37°C. The medium was then removedand the cells were washed twice with 2 ml of medium. Cellswere incubated for 2 min with 10 ,uM forskolin in 1 ml withand without further additions. At the end of the incubationperiod, the medium was aspirated and 1 ml of 5% trichloro-acetic acid was added. The samples were then passedsequentially over Dowex and alumina columns to isolatecyclic [3H]AMP and [3H]ATP. Each fraction was collected ina scintillation vial to which 10 ml of Budget Solve (ResearchProducts International, Mount Prospect, IL) was added. Theradioactivity of the samples was determined by standardscintillation spectroscopy with an efficiency of 35%. Valuesare expressed as percentage conversion of [3H]ATP to cyclic[3H]AMP {cyclic [3H]AMP/(cyclic [3H]AMP + [3H]ATP)}.

Cyclic AMP Radioimmunoassay. Cells were washed twicewith 2 ml of medium and then pretreated with and withoutagonist and other additions in 1 ml of medium for 30 min at37°C. Cells were washed as described above and then 1 ml of

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Proc. Natl. Acad. Sci. USA 84 (1987) 1295

medium with and without 10 /iM forskolin was added for a2-min stimulation. At the end of the stimulation, 1.5 ml of 0.3M perchloric acid was added to terminate the reaction. Thereaction mixture was neutralized with 270 tLI of 2 M KOH andthe samples were frozen. After centrifugation to remove theprecipitate, the cyclic AMP content of the supernatant wasmeasured by radioimmunoassay (17, 18). One of the threeradioimmunoassays was performed with a kit purchased fromNew England Nuclear and the other two were performedwith antibody kindly provided by Leonard Forte.

Materials. (-)-Norepinephrine bitartrate, isoproterenolbitartrate, and yohimbine hydrochloride were purchasedfrom Sigma. Vasoactive intestinal peptide was purchasedfrom Peninsula Laboratories (Belmont, CA). Forskolin wasobtained from Calbiochem-Behring. Dulbecco's modifiedEagle's medium was purchased from Kansas City Biologicals(Lenexa, KS), and sera and trypsin were obtained fromGIBCO. [3H]Adeninine (40 Ci/mmol) was purchased fromICN. The following drugs were graciously donated by therespective companies: prazosin hydrochloride and UK14,304-18 [5-bromo-6-(2-imidazolin-2-amino)quinoxaline] (Pfizer,Groton, CT), sotalol hydrochloride (Bristol-Myers, Evans-ville, IL), 1-propranolol hydrochloride (Ayerst Laboratories,New York), and Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinedione] (Hoffmann-La Roche Laboratories,Nutley, NJ).

RESULTSForskolin (10 ,uM) caused a 4-fold increase in cyclic [3H]AMPproduction by intact HT29 cells prelabeled with [3H]adenine(Figs. 1 and 2). Norepinephrine and UK14,304, an a2-adrenergic selective agonist, inhibited forskolin-stimulatedcyclic AMP production in a dose-dependent manner, indi-cating that the a2-adrenergic receptor in HT29 cells isnegatively coupled to adenylate cyclase (Fig. 1). The maxi-mal inhibition observed in these intact cell assays was >80%,in contrast to 32% maximal inhibition previously observed inmembrane adenylate cyclase assays (5). UK14,304, whichdecreased cyclic AMP production in intact cells with an IC50of 3 nM, was more potent than norepinephrine, which had anIC50 of 300 nM. These values are similar to the affinitiesobserved in membrane binding assays for UK14,304 (2 nM)and norepinephrine (47 nM) (5). UK14,304 and norepineph-rine inhibited vasoactive intestinal peptide-stimulated cyclicAMP production with potencies similar to those seen withforskolin (data not shown).

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Our initial interest was to study the effect of agonistpreincubation on desensitization of the a2-adrenergic recep-tor in HT29 cells. Cells were preincubated for 30 min with orwithout norepinephrine prior to assays of cyclic AMP pro-duction with or without 10 ,uM forskolin or 10 ,uM forskolinand 10 ,uM norepinephrine. To eliminate possible ,B-adren-ergic effects of norepinephrine, propranolol (10 ,uM) wasincluded with norepinephrine in both phases of the experi-ment. Agonist preincubation led to a remarkable 20-foldincrease in forskolin-stimulated cyclic AMP production (Fig.2). There was no apparent attenuation of the inhibition ofcyclic AMP production by norepinephrine when the cate-cholamine was included during the forskolin stimulation step,indicating a lack of desensitization under these conditions.Thus, further studies were performed to investigate therobust increase in forskolin-stimulated cyclic AMP produc-tion following agonist preincubation. This phenomenon isreferred to as sensitization.

Norepinephrine-mediated sensitization was dose depen-dent with maximal sensitization occurring at 10 ,uM (Table 1).To determine whether sensitization was unique to norepi-nephrine, cells were preincubated with UK14,304 (Table 1).The dose response of UK14,304 for mediating sensitizationwas biphasic, with a maximal 6-fold sensitization occurring at100 nM. Since both norepinephrine and UK14,304 have beenshown to be full agonists at the a2-adrenergic receptor inthese and other cells, an explanation was sought for theweaker biphasic sensitization observed with UK14,304. Alikely possibility was that UK14,304, because of its lipophilic-ity and high affinity relative to norepinephrine, was retainedby the cells following preincubation and that the retainedUK14,304 inhibited forskolin-stimulated cyclic AMP produc-tion during the stimulation step. To test this hypothesis,yohimbine, an a2-adrenergic selective antagonist, was includ-ed in the stimulation step to block any inhibition that mightoccur because of retained agonist. With yohimbine present inthe stimulation step, sensitization by UK14,304 preincuba-tion was similar to that seen with norepinephrine preincuba-tion (Table 2). These data indicate that norepinephrine andUK14,304 are equally efficacious in causing sensitization.One possibility to be considered was that the observed

increase in cyclic AMP production was a result of decreaseddegradation rather than increased synthesis of cyclic AMP. Ifsensitization were simply the result of phosphodiesteraseinhibition, then maximal inhibition of phosphodiesterase inthe incubation would produce the same (or greater) sensiti-zation as norepinephrine preincubation. To test this hypoth-esis, a maximally effective concentration (0.2 mM) of the

N o r e p n e p h r n e FIG. 1. Agonist inhibition of forskolin-stimulat-ed cyclic AMP production. Cells were prelabeledwith 5 ,uCi of [3H]adenine (in the presence of 100

\uM propranolol for norepinephrine inhibition).This medium was removed and 1 ml of 10 uM(UK14,304) or 30 ,uM (norepinephrine) forskolinwith or without the indicated concentrations of

I UK14,304 or norepinephrine (plus propranolol) wasadded. Following a 2-min stimulation, cyclic[3H]AMP content was determined. Values aremeans ± SEM for five experiments. The 100%

_______.______.______.______._____ value for UK14,304 experiments was 0.12% con-7 6 5 4 3 version, and for norepinephrine experiments it was

0.39% conversion. Basal activity (0.03% conver-)N S T] M sion) was subtracted from all values.

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Cell Biology: Jones et al.

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Proc. Natl. Acad. Sci. USA 84 (1987)

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FIG. 2. Effect of norepinephrine (NE) preincubation on forskolin(FSK)-stimulated cyclic AMP production. Cells were prelabeledwith [3H]adenine with or without 10 ,uM norepinephrine (plus 10 AMpropranolol). Cells were then incubated with or without 10 ,uMforskolin with and without 10 ,.uM norepinephrine plus propranololfor 2 min and cyclic [3H]AMP content'was determined. Values shownare from a representative experiment. Ba, basal.

cyclic AMP phosphodiesterase inhibitor Ro 20-1724 wasincluded during the stimulation step. The inclusion of Ro20-1724 in the stimulation step resulted in only 0.25% +0.06% conversion as compared to 1.28% ± 0.13% conversion(n = 3) following norepinephrine preincubation, indicatingthat sensitization is not simply a result of decreasedphosphodiesterase activity.The onset and reversal of norepinephrine-induced sensiti-

zation were determined as a function of time ofpreincubation(Fig. 3). Sensitization occurred rapidly, with half-maximaleffect after =5 min of preincubation. The maximal responsewas reached in 10-20 min and was stable for at least 60 minin the continued presence of agonist. Sensitization wasrapidly and completely reversible upon removal of theinducing agonist, with a half-time for reversal of =10 min.The observation that the a2-adrenergic agonist UK14,304,

Table 1. Effect of preincubation with a2-adrenergic agonists onforskolin-stimulated cyclic AMP production in HT29 cells

Forskolin-stimulatedcyclic AMP production

Concentration Norepinephrine UK14,30410 nM 3.34 ± 0.15

100 nM 5.96 ± 0.761 ,uM 4.0 ± 1.5 1.30 ± 0.12

10 ,uM 19.3 ± 2.3 0.61 ± 0.09100 JLM 20.0 ± 4.4

Cells were prelabeled with [3H]adenine with or without theindicated concentrations of norepinephrine or UK14,304. Propran-olol (1 gM) was included in norepinephrine pretreatments. Cells werethen incubated for 2 min with 10 ,uM forskolin and cyclic [3H]AMPcontent was determined. Values are expressed as -fold increase overforskolin stimulation in the absdnce of agonist preincubation (0.17%conversion). Maximal forskolin-stimulated cyclic AMP productionfollowing norepinephrine preincubation was 3.55% conversion. Val-ues are means ± SEM for three experiments.

Table 2. Effect of including yohimbine during forskolinstimulation of cyclic AMP production in cellspreincubated with UK14,304

Cyclic AMP productionUK14,304 Forskolin +

concentration Forskolin yohimbine

100 nM 30 ± 9 100 ± 91 jaM 7 ± 2 107 ± 10

10 ,UM 3 ± 1 90 ± 16

Cells were prelabeled with [3H]adenine with or without theindicated concentrations of UK14,304. Values are percent of re-sponse observed with cells preincubated with 10 AtM norepinephrine(2.55% conversion). Forskolin-stimulated cAMP production in cellspreincubated without agonist was 0.21% conversion. Values aremeans ± SEM for three experiments. Yohimbine was 10 AtM.as well as norepinephrine (plus propranolol), caused sensi-tization suggested that the effect is mediated by a2-adrenergicreceptors. To evaluate further the a2-adrenergic selectivity ofthe sensitization phenomenon, receptor-selective antagonistswere included in the preincubation (Fig. 4). Sotalol, aP-adrenergic antagonist, and prazosin, an a1-adrenergic an-tagonist, both had little effect on the sensitization producedby 10 gM norepinephrine. Yohimbine, an a2-adrenergicantagonist, inhibited sensitization completely and in a dose-dependent manner. These results confirm that sensitization ismediated by a2-adrenergic receptors.Dose-response curves to forskolin without or with agonist

preincubation are presented in Fig. 5. Agonist preincubationresulted in an apparent shift to the left in the dose-responsecurve to forskolin. However, due to the limited solubility offorskolin, we were unable to determine whether the maximalresponse to forskolin was altered by agonist preincubation.To determine whether or not sensitization is unique to

forskolin stimulation, the effects of a2-adrenergic agonist

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FIG. 3. Time course for induction and reversal of sensitization offorskolin-stimulated cyclic AMP production. For induction, cellswere prelabeled with [3H]adenine and at appropriate times, eitherbefore or during prelabeling, norepinephrine (10 ALM) was added.Following prelabeling (30 min) and preincubation (indicated times),10 ,uM forskolin was added for 2 min and then cyclic [3H]AMPcontent was determined. For reversal of sensitization, cells werepreincubated for 30 min with 10 ,uM norepinephrine. At appropriatetimes during or after the preincubation, medium was removed and[3H]adenine was added for 30 min of prelabeling. Prior to and/orduring the prelabeling, cells were incubated for the indicated timesin the absence of norepinephrine prior to addition of 10 ,uM forskolinfor 2 min. Cyclic [3H]AMP content was then determined. Results aremeans SEM for three experiments. Forskolin stimulation in theabsence of agonist pretreatment (0.17% conversion) has been sub-tracted. The 100% value was 1.91% conversion.

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Proc. Natl. Acad. Sci. USA 84 (1987) 1297

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FIG. 4. Effect of antagonists on sensitization of forskolin-stimulated cyclic AMP production. Cells were prelabeled with[3H]adenine in the presence of 10 ,uM norepinephrine with or withoutantagonists at the indicated concentrations. Cells were then incu-bated with 10 MuM forskolin for 2 min and cyclic [3H]AMP contentwas determined. Values are means ± SEM for three experiments.Forskolin-stimulated cyclic AMP production in the absence ofagonist preincubation was 0.21% conversion. Percentage conversionfor preincubation with norepinephrine in the absence of antagonist(100% value) was 2.23%.

preincubation on cyclic AMP production stimulated by otheragents were tested. Stimulation by vasoactive intestinalpeptide following norepinephrine preincubation was only 1.9times the stimulation in the absence of norepinephrinepreincubation (0.72% ± 0.05% vs. 0.38% ± 0.06% conver-

sion; n = 3). There was no apparent change in isoproterenol-stimulated (,-adrenergic receptor-mediated) cyclic AMPproduction with norepinephrine preincubation (0.02% ±

0.01% in both control and norepinephrine preincubated cells;n = 3).

Since a2-adrenergic agonist effects traditionally have beenthought to result from inhibition of cyclic AMP synthesis, theeffects on sensitization of altered levels of cyclic AMP duringthe preincubation phase were examined. Table 3 shows theresults from experiments in which the phosphodiesteraseinhibitor Ro 20-1724 was included during the preincubationstep. In these experiments, cyclic AMP levels were deter-mined by radioimmunoassay in addition to the standard[3H]adenine prelabeling assay. Cyclic AMP levels were

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FIG. 5. Dose-response curves to forskolin following preincuba-tion without or with an a2-adrenergic agonist. Cells were prelabeledwith [3H]adenine in the absence or presence of 10 MiM norepinephrinewith propranolol. Cells were then incubated with the indicatedconcentrations of forskolin for 2 min and cyclic [3H]AMP contentwas determined. Values are means ± SEM for six experiments.

increased 3-fold in cells preincubated with norepinephrineand Ro 20-1724. Sensitization occurred in these cells in spiteof the fact that cyclic AMP levels were increased rather thandecreased, indicating that sensitization does not result simplyfrom decreased levels of cyclic AMP during the preincuba-tion. The data in Table 3 also demonstrate excellent agree-ment between the results obtained with the two differentassay systems.

DISCUSSION

a2-Adrenergic agonist preincubation of cells could be expect-ed to cause desensitization of the a2-adrenergic receptor,resulting in an attenuation of inhibition of cyclic AMPproduction upon subsequent challenge with agonist. How-ever, exposure of HT29 cells to a2-adrenergic agonists ledinstead to a marked sensitization of forskolin-stimulatedcyclic AMP accumulation in intact cells with no indication ofdesensitization. This sensitization phenomenon may repre-sent an attempt by the cells to maintain cyclic AMPhomeostasis under conditions of prolonged inhibition ofadenylate cyclase by the a2-adrenergic agonist.

This sensitization phenomenon differs from those reportedin AtT-20 cells in at least three ways. First, the magnitude of

Table 3. Effect of including Ro 20-1724 during preincubation with a2-adrenergic agonist on sensitization of forskolin-stimulated cyclicAMP production

Radioimmunoassay,[3H]Adenine prelabeling assay, % conversion pmol of cyclic AMP per dish

Preincubation Basal Forskolin Basal Forskolin

0 0.022 ± 0.004 (0.8) 0.28 ± 0.12 (9) 4.9 ± 0.3 (0.8) 18.6 ± 2.1 (3)Ro 0.032 ± 0.010 (1.0) 0.70 ± 0.26 (22) 5.6 ± 0.7 (1.0) 83 ± 5 (14)NE 0.030 ± 0.006 (1.1) 2.20 ± 0.49 (73) 6.7 ± 0.9 (1.1) 366 ± 41 (62)

NE + Ro 0.084 ± 0.025 (2.7) 2.90 ± 0.49 (100) 12.4 ± 1.6 (2.1) 591 ± 46 (100)Cells were preincubated in the absence or presence of 0.25 mM Ro 20-1724 (Ro) and/or 10 MM norepinephrine (NE) as indicated. Cells were

then incubated for 2 min with or without 10 MM forskolin prior to determination of cyclic AMP content by the indicated assays. Values are themeans ± SEM for three experiments. Numbers in parentheses are percent of the value for NE + Ro pretreatment followed by forskolinstimulation.

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Proc. Natl. Acad. Sci. USA 84 (1987)

the sensitization response in HT29 cells was 10- to 20-fold,compared to only a 2- to 4-fold sensitization seen in AtT-20cells following either somatostatin or muscarinic cholinergicagonist preincubation (1, 11). Second, the time course forsensitization in HT29 cells was rapid, with half-maximalsensitization occurring in -5 min. Muscarinic cholinergicsensitization requires at least 3 hr of agonist preincubationbefore any effect is observed. The half-maximal effect couldnot be determined, since at the longest time studied (8 hr) theeffect did not appear to be maximal (1). Preincubation withsomatostatin causes maximal sensitization of forskolin-stim-ulated cyclic AMP production at 16 hr with half maximaleffects seen at 4 hr (11). Third, the sensitization phenomenonobserved in AtT-20 cells is blocked by cycloheximide,suggesting that protein synthesis is required for sensitizationin these cells. Because of the rapidity of the sensitizationresponse, involvement of protein synthesis in HT29 cellsensitization seems unlikely. However, cycloheximide ex-periments will be necessary to show that protein synthesis isnot required for sensitization.The pharmacological data clearly indicate that sensitiza-

tion is mediated by a2-adrenergic receptors. Yohimbine, ana2-adrenergic-selective antagonist, prevented sensitizationwhile a1- and p-adrenergic antagonists were without effect.UK14,304, an a2-adrenergic-selective agonist, was consider-ably more potent than norepinephrine in causing both sen-sitization and inhibition of forskolin-stimulated cyclic AMPproduction. Furthermore, 100 nM UK14,304 was maximallyeffective for both processes, indicating a similar potency ofUK14,304 in eliciting both effects.

It seemed possible that sensitization might result from adecrease in cyclic AMP degradation (decreased phosphodi-esterase activity) rather than from an increase in cyclic AMPsynthesis, as previously demonstrated in rat submandibulargland (19). However, the increase in cyclic AMP levels whenphosphodiesterase was maximally inhibited with Ro 20-1724during the stimulation step was only 15-20% of that observedin cells preincubated with a2-adrenergic agonist, indicatingthat phosphodiesterase inhibition is not the mechanism ofsensitization. Furthermore, the relative lack of sensitizationof cyclic AMP production stimulated by vasoactive intestinalpeptide and isoproterenol indicates that phosphodiesterase isnot an important factor in forskolin sensitization. The pos-sibility that sensitization is an artifact of the [3H]adenineprelabeling procedure can be rejected since similar resultswere obtained when cyclic AMP levels were measured byradioimmunoassay.The mechanism of a2-adrenergic receptor-mediated sensi-

tization of forskolin-stimulated cyclic AMP production isunknown. We tested the hypothesis that decreased levels ofcyclic AMP during the a2-adrenergic agonist preincubationwere responsible for sensitization. Our data indicate that thisis not the mechanism, since increased cyclic AMP levels didnot prevent a2-adrenergic agonist-induced sensitization.However, the inhibitory guanine nucleotide regulatory pro-tein (Ni), or other components of the receptor/cyclasesystem may be involved in sensitization even if cyclic AMP

itself is not involved. Our results are consistent with recentliterature reviewed by Limbird and Sweatt (20), whichindicates that although decreasing cyclic AMP levels may besufficient to elicit a2-adrenergic receptor-mediated responsesin some tissues, in other tissues such a signal may be neithernecessary nor sufficient. Systems in which additional oralternative mechanisms are implicated include the secondaryaggregation response in human platelets, chloride and watersecretion in endothelial cells, and the a2-adrenergic inhibitionof glucose-stimulated insulin release from the pancreas (15,20, 21). Our results suggest that sensitization can now beadded to this list.

In summary, we have demonstrated that preincubation ofHT29 cells with an a2-adrenergic agonist causes a markedsensitization of forskolin-stimulated cyclic AMP production,which is rapid in onset and reversal and is mediated via thea2-adrenergic receptor. Sensitization does not appear to bemediated by decreased cyclic AMP levels and hence anothermechanism remains to be elucidated.

This work was supported by Grant HL32931 from the NationalInstitutes of Health.

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1298 Cell Biology: Jones et al.