JOURNAL OF CELLULAR PHYSIOLOGY 151512-518 (1992)
Elevated cAMP I s Required for Stimulation of Eicosanoid Synthesis by lnterleukin 1 and Bradykinin in BALB/c 3T3 Fibroblasts RONALD M. BURCH* AND IANE R. CONNOR Nova Pharmaceutical Corporation, Baltimore, Marylmd 2 7224 In Swiss 3T3. murine fibroblasts, interleukin 1 (IL-1) and bradykinin stimulate prostdglandin E, (PGE,) synthesis. However, in the present study, we found that neither agonisi stimulated PGE, synthesis in BALBic 3T3 murine fibroblasts, this in spite of expression of similar numbers of receptors tor rach agonist compared to Swiss 3T3 cells. When BALBic 3T3 cells were preincubated with CAMP analogs, both IL-1 and bradykinin stimulated PGE, synthesis to levels similar to those observed in Swiss 3T3 cells. Similarly, when the cells were preincubated with forskolin, which activates the catalytic subunit of adenylate cyclase directly, or NECA, which stimulates cellular cAMP accumulation by activating adenosine receptors, IL-l and bradykinin stimulated PGE, synthesis. Rp-CAMPS,an inhibitor of CAMP-dependent protein kinase, blocked the ability of CAMP or NECA to render cells responsive to IL-1 and bradykinin. In basal BALBIc 3T3 cells, bradykinin and I L - I stimulated arachidonate release in the absence of CAMP, but littlc conversion of released arxhidonate to PGE, occurred. CAMP, forskolin, and NECA all increased cyclooxygenase activity in the cells. SV-T2 is a clonal line originating from BALBlc 3T3 transformed with SV-40. In these cells, I L - l and bradykinin stimulated PGE, synthesis despite basal intracellular CAMPconcentrations similar to BALBic, and CAMP only modestly potentiated the response. In summary, cyclooxygenase expression appears to be regulated by CAMP in BALKic 3T3 cells, and SV-40 transformation results in increased cyclooxygenase expression, apparently independent of CAMP.
Stimulation of eicosanoid synthesis by hormones and growth factors has been studied extensively in murine 3T3 fibroblasts (Burch and Axelrod, 1987; Burch et al., 1988a,b 1989; Burch, 1989, 1990; Rozengurt et al., 1983; Habenicht et al., 1985). In 3T3 fibroblasts from Swiss Albino mice, bradykinin, thrombin, IL-1, tumor necrosis factor, and other hormones stimulate PGE, synthesis. In response to agonists such a s bradykinin and thrombin, stimulation is rapid, synthesis being maximum by 5 minutes (Burch and Axelrod, 1987; Burch, 1990). Stimulation by these agonists appears to be due largely to activation of phospholipase A,, coupled to the receptors through GTP-binding regulatory proteins (Burch and Axelrod, 1987). Certain growth factors, including IL-1 and tumor necrosis factor, stimulate PGE, synthesis after a time delay, significant increases not being detectable before 1 hour, and continuing for several hours to days (Burch et al., 1988a; Burch and Tiffany, 1989). Stimulation of PGE, synthesis by these agents may be secondary to induction of cyclooxygenase (Burch et al., 1988a; Burch and Tiffany, 1989; Raz et al., 1988) or other transduction proteins, perhaps including phospholipase-activating proteins (Lin et al., 1989). In the SV-T, cell line obtained by SV-40 transformation of BALBic 3T3 fibroblasts, hormones and growth factors stimulate eicosanoid synthesis to greater levels than in Swiss 3T3 fibroblasts (Burch et al., 1988a). 8 1992 WILEY-LISS, INC
However, we have observed that the parental BALBk 3T3 cells exhibit virtually no basal synthesis of PGE2, and neither bradykinin nor IL-1 stimulate PGE, synthesis. In the present study, we report that if BALBic 3T3 cells are first incubated with cell-permeable cAMP analogs or agents that increase intracellular CAMP, both bradykinin and IL-1 stimulate PGE, synthesis.
MATERIALS AND METHODS Cell cultures SV-T, (ATCC CCL 163.1), BALBic 3T3 (ATCC CCL 1631, and Swiss albino 3T3 (ATCC CCL 92) cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium containing glucose (4.5 giL), 10% calf serum, and penicillin (100 unitsiml) and streptomycin (100 pgiml) (Burch and Axelrod, 1987). Cells were grown in 100 mm dishes or 12-well or 24-well plates. For the experiments reported in the present study, 3 separate vials of BALB/c 3T3 cells were obtained from the ATCC over a period of 14 months.
ReceivedJune 17,1991; accepted January 30, 1992.
*To whom reprint requestsicorrespondence should be addressed.
cAMP IN IL-1 STIMULATED PGEz SYNTHESIS
513
says were terminated by the addition of 300 p1 isopropano1:n-heptane:acetic acid (40:lO:O.l) with vortexing. Then, 100 p1 n-heptane was added with vortexing, followed by microcentrifugation for 1minute to separate phases. A 150 pl aliquot of the upper phase was added to a Pasteur pipet containing 50 mg silica gel (Sigma) and fatty acid was eluted with 200 pl diethyl ether (Ulevitch et al., 1988). In several experiments [‘“C jcholine-labeled phosphatidylcholine was used as substrate, and lysophosphatidylcholine was isolated (Bjerve et al., 1974) to confirm that the enzyme activity being examined was phospholipase A,. Cyclooxygenase activity Confluent cultures in 24-well plates were rinsed once in growth medium minus serum. Then growth medium minus serum but containing 10 pM arachidonate was added for 5 minutes. Medium was collected for assay of PGE,. Using this protocol maximum PGE, synthesis occurred a t 3-5 pM arachidonate and was linear for 10-15 minutes. PGE, concentration in arachidonatecontaining media incubated with cells contained up to Arachidonate release 2,000 pgiml PGE,. Addition of 10 pM indomethacin to Confluent cultures in 24-well plates were incubated the arachidonate-containing medium incubated with overnight with 10 pCi/well I3Hjarachidonate plus any cells reduced PGE, synthesis greater than 95%. Arachiexperimental agents. Then each well was washed 4 donate-containing media incubated without cells contimes with Hanks’ balanced salt solution containing 2 tained too little PGE, (
Elevated cAMP I s Required for Stimulation of Eicosanoid Synthesis by lnterleukin 1 and Bradykinin in BALB/c 3T3 Fibroblasts RONALD M. BURCH* AND IANE R. CONNOR Nova Pharmaceutical Corporation, Baltimore, Marylmd 2 7224 In Swiss 3T3. murine fibroblasts, interleukin 1 (IL-1) and bradykinin stimulate prostdglandin E, (PGE,) synthesis. However, in the present study, we found that neither agonisi stimulated PGE, synthesis in BALBic 3T3 murine fibroblasts, this in spite of expression of similar numbers of receptors tor rach agonist compared to Swiss 3T3 cells. When BALBic 3T3 cells were preincubated with CAMP analogs, both IL-1 and bradykinin stimulated PGE, synthesis to levels similar to those observed in Swiss 3T3 cells. Similarly, when the cells were preincubated with forskolin, which activates the catalytic subunit of adenylate cyclase directly, or NECA, which stimulates cellular cAMP accumulation by activating adenosine receptors, IL-l and bradykinin stimulated PGE, synthesis. Rp-CAMPS,an inhibitor of CAMP-dependent protein kinase, blocked the ability of CAMP or NECA to render cells responsive to IL-1 and bradykinin. In basal BALBIc 3T3 cells, bradykinin and I L - I stimulated arachidonate release in the absence of CAMP, but littlc conversion of released arxhidonate to PGE, occurred. CAMP, forskolin, and NECA all increased cyclooxygenase activity in the cells. SV-T2 is a clonal line originating from BALBlc 3T3 transformed with SV-40. In these cells, I L - l and bradykinin stimulated PGE, synthesis despite basal intracellular CAMPconcentrations similar to BALBic, and CAMP only modestly potentiated the response. In summary, cyclooxygenase expression appears to be regulated by CAMP in BALKic 3T3 cells, and SV-40 transformation results in increased cyclooxygenase expression, apparently independent of CAMP.
Stimulation of eicosanoid synthesis by hormones and growth factors has been studied extensively in murine 3T3 fibroblasts (Burch and Axelrod, 1987; Burch et al., 1988a,b 1989; Burch, 1989, 1990; Rozengurt et al., 1983; Habenicht et al., 1985). In 3T3 fibroblasts from Swiss Albino mice, bradykinin, thrombin, IL-1, tumor necrosis factor, and other hormones stimulate PGE, synthesis. In response to agonists such a s bradykinin and thrombin, stimulation is rapid, synthesis being maximum by 5 minutes (Burch and Axelrod, 1987; Burch, 1990). Stimulation by these agonists appears to be due largely to activation of phospholipase A,, coupled to the receptors through GTP-binding regulatory proteins (Burch and Axelrod, 1987). Certain growth factors, including IL-1 and tumor necrosis factor, stimulate PGE, synthesis after a time delay, significant increases not being detectable before 1 hour, and continuing for several hours to days (Burch et al., 1988a; Burch and Tiffany, 1989). Stimulation of PGE, synthesis by these agents may be secondary to induction of cyclooxygenase (Burch et al., 1988a; Burch and Tiffany, 1989; Raz et al., 1988) or other transduction proteins, perhaps including phospholipase-activating proteins (Lin et al., 1989). In the SV-T, cell line obtained by SV-40 transformation of BALBic 3T3 fibroblasts, hormones and growth factors stimulate eicosanoid synthesis to greater levels than in Swiss 3T3 fibroblasts (Burch et al., 1988a). 8 1992 WILEY-LISS, INC
However, we have observed that the parental BALBk 3T3 cells exhibit virtually no basal synthesis of PGE2, and neither bradykinin nor IL-1 stimulate PGE, synthesis. In the present study, we report that if BALBic 3T3 cells are first incubated with cell-permeable cAMP analogs or agents that increase intracellular CAMP, both bradykinin and IL-1 stimulate PGE, synthesis.
MATERIALS AND METHODS Cell cultures SV-T, (ATCC CCL 163.1), BALBic 3T3 (ATCC CCL 1631, and Swiss albino 3T3 (ATCC CCL 92) cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium containing glucose (4.5 giL), 10% calf serum, and penicillin (100 unitsiml) and streptomycin (100 pgiml) (Burch and Axelrod, 1987). Cells were grown in 100 mm dishes or 12-well or 24-well plates. For the experiments reported in the present study, 3 separate vials of BALB/c 3T3 cells were obtained from the ATCC over a period of 14 months.
ReceivedJune 17,1991; accepted January 30, 1992.
*To whom reprint requestsicorrespondence should be addressed.
cAMP IN IL-1 STIMULATED PGEz SYNTHESIS
513
says were terminated by the addition of 300 p1 isopropano1:n-heptane:acetic acid (40:lO:O.l) with vortexing. Then, 100 p1 n-heptane was added with vortexing, followed by microcentrifugation for 1minute to separate phases. A 150 pl aliquot of the upper phase was added to a Pasteur pipet containing 50 mg silica gel (Sigma) and fatty acid was eluted with 200 pl diethyl ether (Ulevitch et al., 1988). In several experiments [‘“C jcholine-labeled phosphatidylcholine was used as substrate, and lysophosphatidylcholine was isolated (Bjerve et al., 1974) to confirm that the enzyme activity being examined was phospholipase A,. Cyclooxygenase activity Confluent cultures in 24-well plates were rinsed once in growth medium minus serum. Then growth medium minus serum but containing 10 pM arachidonate was added for 5 minutes. Medium was collected for assay of PGE,. Using this protocol maximum PGE, synthesis occurred a t 3-5 pM arachidonate and was linear for 10-15 minutes. PGE, concentration in arachidonatecontaining media incubated with cells contained up to Arachidonate release 2,000 pgiml PGE,. Addition of 10 pM indomethacin to Confluent cultures in 24-well plates were incubated the arachidonate-containing medium incubated with overnight with 10 pCi/well I3Hjarachidonate plus any cells reduced PGE, synthesis greater than 95%. Arachiexperimental agents. Then each well was washed 4 donate-containing media incubated without cells contimes with Hanks’ balanced salt solution containing 2 tained too little PGE, (
