TABLE 1

Effects of incubation of NIH 3T3 cells with forskolin, H89, and cycloheximide on PDE4 activity

Wild-type or PKI-transfected NIH 3T3 cells were incubated with forskolin with or without H89 and/or cycloheximide for 4 h. After the incubations, the cells were lysed and processed, and total cAMP PDE and PDE4 activity were determined as outlined under Materials and Methods. Calculation of % increases: [(PDE4 activity_treatment - PDE activity_DMSO)/PDE activity_DMSO] × 100. Values are mean ± S.E.M. from three determinations.

Additions (n = 3)	cAMP PDE Activity	PDE4 Activity	PDE4 Activity
	pmol/min/mg		%
NIH 3T3
DMSO [0.1% (v/v)]	111.4 ± 9.0	89.6 ± 6.5
H89 (10 μM) + Chex (100 μM)	93.5 ± 3.5	77.2 ± 4.8
Fsk (10 μM)	275.3 ± 2.2^a	236.9 ± 1.4^a	164
Fsk (10 μM) + H89 (10 μM)	146.8 ± 10.7^a^b^c	120.1 ± 8.6^a^b^c	34
Fsk (10 μM) + Chex (100 μM)	174.1 ± 5.2^a^b	154.0 ± 3.3^a^b	72
Fsk (10 μM) + H89 (10 μM) + Chex (100 μM)	115.6 ± 3.8^b^c	95.6 ± 4.3^b^c
NIH 3T3
DMSO [0.1% (v/v)]	257.1 ± 3.7	154.1 ± 20.6
Fsk (1 μM)	412.2 ± 18.4^a	307.7 ± 11.7^a	100
Fsk (1 μM) + H89 (10 μM)	318.8 ± 5.5^a^b	228.0 ± 8.9^a^b	48
NIH 3T3/PKI
DMSO [0.1% (v/v)]	273.2 ± 9.2	206.9 ± 9.4
Fsk (1 μM)	319.6 ± 9.6^a	237.3 ± 12.6	15

Fsk, forskolin; Chex, cycloheximide.
↵ a P < 0.05 in comparison with DMSO.
↵ b P < 0.05 in comparison with forskolin.
↵ c P < 0.05 in comparison with forskolin + cycloheximide.