forskolin-stimulated cyclic AMP production

Proc. Natl. Acad. Sci. USA Vol. 84, pp. 1294-1298, March 1987 Cell Biology a2-adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production (HT29 cells/uk14,34/desensitization) SUSAN B. JONES, MYRON L. TOEWS, JOHN T. TURNER, AND DAVD B. BYLUND Department of Pharmacology, University of Missouri-Columbia, Columbia, MO 65212 Communicated by James. Davis, November 17, 1986 (received for revie August 28, 1986) ABSTRACT Preincubation of HT29 human colonic adenocarcinoma cells ith a2-adrenergic agonists resulted in a 1- to 2-fold increase in forskolin-stimulated cyclic AMP production as compared to cells preincubated ithout agonist. Similar results ere obtained using either a [3H1adenine prelabeling assay or a cyclic AMP radioimmunoassay to measure cyclic AMP levels. This phenomenon, hich is termed sensitization, is a2-adrenergic receptor-mediated and rapid in onset and reversal. Yohimbine, an a2-adrenergic receptor-selective antagonist, blocked norepinephrine-induced sensitization, hereas prazosin (ol-adrenergic) and sotalol (,8-adrenergic) did not. The time for half-maximal sensitization as 5 min and the half-time for reversal as 1 min. Only a 2-fold sensitization of cyclic AMP production stimulated by vasoactive intestinal peptide as observed, indicating that sensitization is relatively selective for forskolin. Sensitization reflects an increased production of cyclic AMP and not a decreased degradation of cyclic AMP, since incubation ith a phosphodiesterase inhibitor and forskolin did not mimic sensitization. ncreasing the levels of cyclic AMP during the preincubation (using a phosphodiesterase inhibitor) had no effect on sensitization, indicating that sensitization is not caused by decreased cyclic AMP levels during the preincubation. This rapid and dramatic sensitization of forskolin-stimulated cyclic AMP production is a previously unreported effect that can be added to the groing list of a2-adrenergic responses that are not mediated by a decrease in cyclic AMP. Activation of several hormone or neurotransmitter receptors leads to inhibition of adenylate cyclase. Examples of these "negatively coupled" receptors include muscarinic cholinergic and somatostatin receptors in AtT-2 mouse pituitary tumor cells and a2-adrenergic receptors in human platelets, NG18-15 neuroblastoma x glioma cells, and HT-29 human colonic adenocarcinoma cells (1-5). The inhibition of adenylate cyclase in these systems is mediated by the guanine nucleotide-binding regulatory protein Ni (6-8). Agonist treatment of various cellular systems can result in a phenomenon called desensitization. Desensitization is a cellular adaptation to agonist treatment that results in a blunting of the physiological response to a subsequent challenge by the agonist. This phenomenon has been studied extensively in several systems that are coupled to stimulation of adenylate cyclase, in particular the,b-adrenergic receptor system (9, 1). Desensitization of the negatively coupled somatostatin and muscarinic cholinergic systems in the AtT-2 cell line has also been reported (1, 11). Desensitization of epinephrine-initiated aggregation of human platelets can occur ithout concomitant don-regulation or desensitization ofthe a2-adrenergic receptor/adenylate cyclase complex (12, 13). The publication costs of this article ere defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance ith 18 U.S.C. 1734 solely to indicate this fact. 1294 n addition to desensitization, an increase in forskolinstimulated adenylate cyclase activity folloing somatostatin or muscarinic agonist preincubation has been observed in AtT-2 cells (1, 11). Similar phenomena have been reported for the a2-adrenergic receptor system. Norepinephrine, acting through a2-adrenergic receptors in NG18-15 cells, causes an increased activity of adenylate cyclase after prolonged exposure to the agonist (14). Epinephrine, partly through a2-adrenergic receptors, transiently potentiates forskolin-stimulated cyclic AMP accumulation in isolated rat pancreatic islets but, ith longer incubation, decreases cyclic AMP levels (15). We have examined the effects of agonist preincubation on cyclic AMP synthesis in a human colonic adenocarcinoma cell line, HT29. We report here a pronounced, rapid, and reversible sensitization of forskolin-stimulated cyclic AMP synthesis folloing a2-adrenergic agonist preincubation. MATERALS AND METHODS Cell Culture. HT29 cells, a human colonic adenocarcinoma cell line, ere obtained from J. Fogh (Sloan-Kettering nstitute, Rye, NY). Cells ere gron routinely in Dulbecco's modified Eagle's medium ith high glucose supplemented ith 5% (vol/vol) fetal bovine serum and 5% (vol/vol) neborn bovine serum in 75-cm2 disposable tissue culture flasks in a humidified atmosphere of 5% C2/95% air (5). Cells ere subcultured ith.5% (t/vol) trypsin/.1% (t/vol) Na2EDTA and ere seeded at moderate density in 35-mm tissue culture dishes ith confluence being reached in =5 days. Cyclic AMP Production Assay. Confluent cultures of HT29 cells in 35-mm dishes ere utilized for cyclic AMP assays by a modification of the method of Shimizu et al. (16). All incubations and ashes of cells ere in serum-free Dulbecco's modified Eagle's medium. Cells ere ashed tice ith 2 ml of medium and then prelabeled ith [3H]adenine (5 p.ci per dish; 1 Ci = 37 GBq) ith and ithout agonist and other additions for 3 min at 37 C. The medium as then removed and the cells ere ashed tice ith 2 ml of medium. Cells ere incubated for 2 min ith 1,uM forskolin in 1 ml ith and ithout further additions. At the end of the incubation period, the medium as aspirated and 1 ml of 5% trichloroacetic acid as added. The samples ere then passed sequentially over Doex and alumina columns to isolate cyclic [3H]AMP and [3H]ATP. Each fraction as collected in a scintillation vial to hich 1 ml of Budget Solve (Research Products nternational, Mount Prospect, L) as added. The radioactivity of the samples as determined by standard scintillation spectroscopy ith an efficiency of 35%. Values are expressed as percentage conversion of [3H]ATP to cyclic [3H]AMP {cyclic [3H]AMP/(cyclic [3H]AMP + [3H]ATP)}. Cyclic AMP Radioimmunoassay. Cells ere ashed tice ith 2 ml of medium and then pretreated ith and ithout agonist and other additions in 1 ml of medium for 3 min at 37 C. Cells ere ashed as described above and then 1 ml of

Cell Biology: Jones et al. medium ith and ithout 1 /im forskolin as added for a 2-min stimulation. At the end of the stimulation, 1.5 ml of.3 M perchloric acid as added to terminate the reaction. The reaction mixture as neutralized ith 27 tl of 2 M KOH and the samples ere frozen. After centrifugation to remove the precipitate, the cyclic AMP content of the supernatant as measured by radioimmunoassay (17, 18). One of the three radioimmunoassays as performed ith a kit purchased from Ne England Nuclear and the other to ere performed ith antibody kindly provided by Leonard Forte. Materials. (-)-Norepinephrine bitartrate, isoproterenol bitartrate, and yohimbine hydrochloride ere purchased from Sigma. Vasoactive intestinal peptide as purchased from Peninsula Laboratories (Belmont, CA). Forskolin as obtained from Calbiochem-Behring. Dulbecco's modified Eagle's medium as purchased from Kansas City Biologicals (Lenexa, KS), and sera and trypsin ere obtained from GBCO. [3H]Adeninine (4 Ci/mmol) as purchased from CN. The folloing drugs ere graciously donated by the respective companies: prazosin hydrochloride and UK14,34-18 [5-bromo-6-(2-imidazolin-2-amino)quinoxaline] (Pfizer, Groton, CT), sotalol hydrochloride (Bristol-Myers, Evansville, L), 1-propranolol hydrochloride (Ayerst Laboratories, Ne York), and Ro 2-1724 [4-(3-butoxy-4-methoxybenzyl)- 2-imidazolidinedione] (Hoffmann-La Roche Laboratories, Nutley, NJ). RESULTS Forskolin (1,uM) caused a 4-fold increase in cyclic [3H]AMP production by intact HT29 cells prelabeled ith [3H]adenine (Figs. 1 and 2). Norepinephrine and UK14,34, an a2- adrenergic selective agonist, inhibited forskolin-stimulated cyclic AMP production in a dose-dependent manner, indicating that the a2-adrenergic receptor in HT29 cells is negatively coupled to adenylate cyclase (Fig. 1). The maximal inhibition observed in these intact cell assays as >8%, in contrast to 32% maximal inhibition previously observed in membrane adenylate cyclase assays (5). UK14,34, hich decreased cyclic AMP production in intact cells ith an C5 of 3 nm, as more potent than norepinephrine, hich had an C5 of 3 nm. These values are similar to the affinities observed in membrane binding assays for UK14,34 (2 nm) and norepinephrine (47 nm) (5). UK14,34 and norepinephrine inhibited vasoactive intestinal peptide-stimulated cyclic AMP production ith potencies similar to those seen ith forskolin (data not shon). Proc. Natl. Acad. Sci. USA 84 (1987) 1295 Our initial interest as to study the effect of agonist preincubation on desensitization of the a2-adrenergic receptor in HT29 cells. Cells ere preincubated for 3 min ith or ithout norepinephrine prior to assays of cyclic AMP production ith or ithout 1,uM forskolin or 1,uM forskolin and 1,uM norepinephrine. To eliminate possible,b-adrenergic effects of norepinephrine, propranolol (1,uM) as included ith norepinephrine in both phases of the experiment. Agonist preincubation led to a remarkable 2-fold increase in forskolin-stimulated cyclic AMP production (Fig. 2). There as no apparent attenuation of the inhibition of cyclic AMP production by norepinephrine hen the catecholamine as included during the forskolin stimulation step, indicating a lack of desensitization under these conditions. Thus, further studies ere performed to investigate the robust increase in forskolin-stimulated cyclic AMP production folloing agonist preincubation. This phenomenon is referred to as sensitization. Norepinephrine-mediated sensitization as dose dependent ith maximal sensitization occurring at 1,uM (Table 1). To determine hether sensitization as unique to norepinephrine, cells ere preincubated ith UK14,34 (Table 1). The dose response of UK14,34 for mediating sensitization as biphasic, ith a maximal 6-fold sensitization occurring at 1 nm. Since both norepinephrine and UK14,34 have been shon to be full agonists at the a2-adrenergic receptor in these and other cells, an explanation as sought for the eaker biphasic sensitization observed ith UK14,34. A likely possibility as that UK14,34, because of its lipophilicity and high affinity relative to norepinephrine, as retained by the cells folloing preincubation and that the retained UK14,34 inhibited forskolin-stimulated cyclic AMP production during the stimulation step. To test this hypothesis, yohimbine, an a2-adrenergic selective antagonist, as included in the stimulation step to block any inhibition that might occur because of retained agonist. With yohimbine present in the stimulation step, sensitization by UK14,34 preincubation as similar to that seen ith norepinephrine preincubation (Table 2). These data indicate that norepinephrine and UK14,34 are equally efficacious in causing sensitization. One possibility to be considered as that the observed increase in cyclic AMP production as a result of decreased degradation rather than increased synthesis of cyclic AMP. f sensitization ere simply the result of phosphodiesterase inhibition, then maximal inhibition of phosphodiesterase in the incubation ould produce the same (or greater) sensitization as norepinephrine preincubation. To test this hypothesis, a maximally effective concentration (.2 mm) of the - 1 4 O 8,- a -J 2 W 6 4 yw 2 LL C' -. UK 1 4,34-18 N o r e p n e p h r n e FG. 1. Agonist inhibition of forskolin-stimulated cyclic AMP production. Cells ere prelabeled ith 5,uCi of [3H]adenine (in the presence of 1 \um propranolol for norepinephrine inhibition). This medium as removed and 1 ml of 1 um (UK14,34) or 3,uM (norepinephrine) forskolin ith or ithout the indicated concentrations of.... UK14,34 or norepinephrine (plus propranolol) as added. Folloing a 2-min stimulation, cyclic [3H]AMP content as determined. Values are means ± SEM for five experiments. The 1% value for UK14,34 experiments as.12% con- 1 1 1 9 8 7 6 5 4 3 version, and for norepinephrine experiments it as.39% conversion. Basal activity (.3% conver- -log [AGC)N S T] M sion) as subtracted from all values. _,. ȧ 2

1296 Cell Biology: Jones et al. a- a. +CL -t a. z C,) cn z 2.1L 1.1 ncubation Preincubation _m n. Ba FSK FSK+ NE NE Ba FSK FSK+ NE 1 um NE FG. 2. Effect of norepinephrine (NE) preincubation on forskolin (FSK)-stimulated cyclic AMP production. Cells ere prelabeled ith [3H]adenine ith or ithout 1,uM norepinephrine (plus 1 AM propranolol). Cells ere then incubated ith or ithout 1,uM forskolin ith and ithout 1,.uM norepinephrine plus propranolol for 2 min and cyclic [3H]AMP content'as determined. Values shon are from a representative experiment. Ba, basal. cyclic AMP phosphodiesterase inhibitor Ro 2-1724 as included during the stimulation step. The inclusion of Ro 2-1724 in the stimulation step resulted in only.25% +.6% conversion as compared to 1.28% ±.13% conversion (n = 3) folloing norepinephrine preincubation, indicating that sensitization is not simply a result of decreased phosphodiesterase activity. The onset and reversal of norepinephrine-induced sensitization ere determined as a function of time ofpreincubation (Fig. 3). Sensitization occurred rapidly, ith half-maximal effect after =5 min of preincubation. The maximal response as reached in 1-2 min and as stable for at least 6 min in the continued presence of agonist. Sensitization as rapidly and completely reversible upon removal of the inducing agonist, ith a half-time for reversal of =1 min. The observation that the a2-adrenergic agonist UK14,34, Table 1. Effect of preincubation ith a2-adrenergic agonists on forskolin-stimulated cyclic AMP production in HT29 cells Forskolin-stimulated cyclic AMP production Concentration Norepinephrine UK14,34 1 nm 3.34 ±.15 1 nm 5.96 ±.76 1,uM 4. ± 1.5 1.3 ±.12 1,uM 19.3 ± 2.3.61 ±.9 1 JLM 2. ± 4.4 Cells ere prelabeled ith [3H]adenine ith or ithout the indicated concentrations of norepinephrine or UK14,34. Propranolol (1 gm) as included in norepinephrine pretreatments. Cells ere then incubated for 2 min ith 1,uM forskolin and cyclic [3H]AMP content as determined. Values are expressed as -fold increase over forskolin stimulation in the absdnce of agonist preincubation (.17% conversion). Maximal forskolin-stimulated cyclic AMP production folloing norepinephrine preincubation as 3.55% conversion. Values are means ± SEM for three experiments. Table 2. Proc. Natl. Acad. Sci. USA 84 (1987) Effect of including yohimbine during forskolin stimulation of cyclic AMP production in cells preincubated ith UK14,34 Cyclic AMP production UK14,34 Forskolin + concentration Forskolin yohimbine 1 nm 3 ± 9 1 ± 9 1 jam 7 ± 2 17 ± 1 1,UM 3 ± 1 9 ± 16 Cells ere prelabeled ith [3H]adenine ith or ithout the indicated concentrations of UK14,34. Values are percent of response observed ith cells preincubated ith 1 AtM norepinephrine (2.55% conversion). Forskolin-stimulated camp production in cells preincubated ithout agonist as.21% conversion. Values are means ± SEM for three experiments. Yohimbine as 1 AtM. as ell as norepinephrine (plus propranolol), caused sensitization suggested that the effect is mediated by a2-adrenergic receptors. To evaluate further the a2-adrenergic selectivity of the sensitization phenomenon, receptor-selective antagonists ere included in the preincubation (Fig. 4). Sotalol, a P-adrenergic antagonist, and prazosin, an a1-adrenergic antagonist, both had little effect on the sensitization produced by 1 gm norepinephrine. Yohimbine, an a2-adrenergic antagonist, inhibited sensitization completely and in a dosedependent manner. These results confirm that sensitization is mediated by a2-adrenergic receptors. Dose-response curves to forskolin ithout or ith agonist preincubation are presented in Fig. 5. Agonist preincubation resulted in an apparent shift to the left in the dose-response curve to forskolin. Hoever, due to the limited solubility of forskolin, e ere unable to determine hether the maximal response to forskolin as altered by agonist preincubation. To determine hether or not sensitization is unique to forskolin stimulation, the effects of a2-adrenergic agonist J < O - X Ul <: o 8( 6 M 4 zce, 21 cz: z TME, min FG. 3. Time course for induction and reversal of sensitization of forskolin-stimulated cyclic AMP production. For induction, cells ere prelabeled ith [3H]adenine and at appropriate times, either before or during prelabeling, norepinephrine (1 ALM) as added. Folloing prelabeling (3 min) and preincubation (indicated times), 1,uM forskolin as added for 2 min and then cyclic [3H]AMP content as determined. For reversal of sensitization, cells ere preincubated for 3 min ith 1,uM norepinephrine. At appropriate times during or after the preincubation, medium as removed and [3H]adenine as added for 3 min of prelabeling. Prior to and/or during the prelabeling, cells ere incubated for the indicated times in the absence of norepinephrine prior to addition of 1,uM forskolin for 2 min. Cyclic [3H]AMP content as then determined. Results are means SEM for three experiments. Forskolin stimulation in the absence of agonist pretreatment (.17% conversion) has been subtracted. The 1% value as 1.91% conversion. 1 8 6 4 2

Cell Biology: Jones et al. Proc. Natl. Acad. Sci. USA 84 (1987) 1297-5. * +NOREPNEPHRNE 1 2 4. - NOREPNEPHRNE c O _j O W 6\ YOHMBNE O PRAOSN SOTALOL ) 3. t (L 4 > 1. O 2 8 7 6 5 -log [ANTAGONST], M FG. 4. Effect of antagonists on sensitization of forskolinstimulated cyclic AMP production. Cells ere prelabeled ith [3H]adenine in the presence of 1,uM norepinephrine ith or ithout antagonists at the indicated concentrations. Cells ere then incubated ith 1 MuM forskolin for 2 min and cyclic [3H]AMP content as determined. Values are means ± SEM for three experiments. Forskolin-stimulated cyclic AMP production in the absence of agonist preincubation as.21% conversion. Percentage conversion for preincubation ith norepinephrine in the absence of antagonist (1% value) as 2.23%. preincubation on cyclic AMP production stimulated by other agents ere tested. Stimulation by vasoactive intestinal peptide folloing norepinephrine preincubation as only 1.9 times the stimulation in the absence of norepinephrine preincubation (.72% ±.5% vs..38% ±.6% conversion; n = 3). There as no apparent change in isoproterenolstimulated (,-adrenergic receptor-mediated) cyclic AMP production ith norepinephrine preincubation (.2% ±.1% in both control and norepinephrine preincubated cells; n = 3). Since a2-adrenergic agonist effects traditionally have been thought to result from inhibition of cyclic AMP synthesis, the effects on sensitization of altered levels of cyclic AMP during the preincubation phase ere examined. Table 3 shos the results from experiments in hich the phosphodiesterase inhibitor Ro 2-1724 as included during the preincubation step. n these experiments, cyclic AMP levels ere determined by radioimmunoassay in addition to the standard [3H]adenine prelabeling assay. Cyclic AMP levels ere 7 6 5 4 - log [FORSKOLN], M FG. 5. Dose-response curves to forskolin folloing preincubation ithout or ith an a2-adrenergic agonist. Cells ere prelabeled ith [3H]adenine in the absence or presence of 1 MiM norepinephrine ith propranolol. Cells ere then incubated ith the indicated concentrations of forskolin for 2 min and cyclic [3H]AMP content as determined. Values are means ± SEM for six experiments. increased 3-fold in cells preincubated ith norepinephrine and Ro 2-1724. Sensitization occurred in these cells in spite of the fact that cyclic AMP levels ere increased rather than decreased, indicating that sensitization does not result simply from decreased levels of cyclic AMP during the preincubation. The data in Table 3 also demonstrate excellent agreement beteen the results obtained ith the to different assay systems. DSCUSSON a2-adrenergic agonist preincubation of cells could be expected to cause desensitization of the a2-adrenergic receptor, resulting in an attenuation of inhibition of cyclic AMP production upon subsequent challenge ith agonist. Hoever, exposure of HT29 cells to a2-adrenergic agonists led instead to a marked sensitization of forskolin-stimulated cyclic AMP accumulation in intact cells ith no indication of desensitization. This sensitization phenomenon may represent an attempt by the cells to maintain cyclic AMP homeostasis under conditions of prolonged inhibition of adenylate cyclase by the a2-adrenergic agonist. This sensitization phenomenon differs from those reported in AtT-2 cells in at least three ays. First, the magnitude of Table 3. Effect of including Ro 2-1724 during preincubation ith a2-adrenergic agonist on sensitization of forskolin-stimulated cyclic AMP production Radioimmunoassay, [3H]Adenine prelabeling assay, % conversion pmol of cyclic AMP per dish Preincubation Basal Forskolin Basal Forskolin.22 ±.4 (.8).28 ±.12 (9) 4.9 ±.3 (.8) 18.6 ± 2.1 (3) Ro.32 ±.1 (1.).7 ±.26 (22) 5.6 ±.7 (1.) 83 ± 5 (14) NE.3 ±.6 (1.1) 2.2 ±.49 (73) 6.7 ±.9 (1.1) 366 ± 41 (62) NE + Ro.84 ±.25 (2.7) 2.9 ±.49 (1) 12.4 ± 1.6 (2.1) 591 ± 46 (1) Cells ere preincubated in the absence or presence of.25 mm Ro 2-1724 (Ro) and/or 1 MM norepinephrine (NE) as indicated. Cells ere then incubated for 2 min ith or ithout 1 MM forskolin prior to determination of cyclic AMP content by the indicated assays. Values are the means ± SEM for three experiments. Numbers in parentheses are percent of the value for NE + Ro pretreatment folloed by forskolin stimulation.

1298 Cell Biology: Jones et al. the sensitization response in HT29 cells as 1- to 2-fold, compared to only a 2- to 4-fold sensitization seen in AtT-2 cells folloing either somatostatin or muscarinic cholinergic agonist preincubation (1, 11). Second, the time course for sensitization in HT29 cells as rapid, ith half-maximal sensitization occurring in -5 min. Muscarinic cholinergic sensitization requires at least 3 hr of agonist preincubation before any effect is observed. The half-maximal effect could not be determined, since at the longest time studied (8 hr) the effect did not appear to be maximal (1). Preincubation ith somatostatin causes maximal sensitization of forskolin-stimulated cyclic AMP production at 16 hr ith half maximal effects seen at 4 hr (11). Third, the sensitization phenomenon observed in AtT-2 cells is blocked by cycloheximide, suggesting that protein synthesis is required for sensitization in these cells. Because of the rapidity of the sensitization response, involvement of protein synthesis in HT29 cell sensitization seems unlikely. Hoever, cycloheximide experiments ill be necessary to sho that protein synthesis is not required for sensitization. The pharmacological data clearly indicate that sensitization is mediated by a2-adrenergic receptors. Yohimbine, an a2-adrenergic-selective antagonist, prevented sensitization hile a1- and p-adrenergic antagonists ere ithout effect. UK14,34, an a2-adrenergic-selective agonist, as considerably more potent than norepinephrine in causing both sensitization and inhibition of forskolin-stimulated cyclic AMP production. Furthermore, 1 nm UK14,34 as maximally effective for both processes, indicating a similar potency of UK14,34 in eliciting both effects. t seemed possible that sensitization might result from a decrease in cyclic AMP degradation (decreased phosphodiesterase activity) rather than from an increase in cyclic AMP synthesis, as previously demonstrated in rat submandibular gland (19). Hoever, the increase in cyclic AMP levels hen phosphodiesterase as maximally inhibited ith Ro 2-1724 during the stimulation step as only 15-2% of that observed in cells preincubated ith a2-adrenergic agonist, indicating that phosphodiesterase inhibition is not the mechanism of sensitization. Furthermore, the relative lack of sensitization of cyclic AMP production stimulated by vasoactive intestinal peptide and isoproterenol indicates that phosphodiesterase is not an important factor in forskolin sensitization. The possibility that sensitization is an artifact of the [3H]adenine prelabeling procedure can be rejected since similar results ere obtained hen cyclic AMP levels ere measured by radioimmunoassay. The mechanism of a2-adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production is unknon. We tested the hypothesis that decreased levels of cyclic AMP during the a2-adrenergic agonist preincubation ere responsible for sensitization. Our data indicate that this is not the mechanism, since increased cyclic AMP levels did not prevent a2-adrenergic agonist-induced sensitization. Hoever, the inhibitory guanine nucleotide regulatory protein (Ni), or other components of the receptor/cyclase system may be involved in sensitization even if cyclic AMP Proc. Natl. Acad. Sci. USA 84 (1987) itself is not involved. Our results are consistent ith recent literature revieed by Limbird and Seatt (2), hich indicates that although decreasing cyclic AMP levels may be sufficient to elicit a2-adrenergic receptor-mediated responses in some tissues, in other tissues such a signal may be neither necessary nor sufficient. Systems in hich additional or alternative mechanisms are implicated include the secondary aggregation response in human platelets, chloride and ater secretion in endothelial cells, and the a2-adrenergic inhibition of glucose-stimulated insulin release from the pancreas (15, 2, 21). Our results suggest that sensitization can no be added to this list. n summary, e have demonstrated that preincubation of HT29 cells ith an a2-adrenergic agonist causes a marked sensitization of forskolin-stimulated cyclic AMP production, hich is rapid in onset and reversal and is mediated via the a2-adrenergic receptor. 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