Figure 7.

Inhibition of Ca²⁺ Release-activated Ca²⁺ Influx with Tyrphostin A9 Inhibited AngII-, but not Elevated [K⁺]_e-, elicited PLD Activation. (A) [³H]Oleate-prelabeled cells were incubated for 30 minutes with KRB+ containing 0.5% ethanol and vehicle (0.1% DMSO, control) or 10 nM AngII (+ 0.1% DMSO) in the presence or absence of 10 μM tyrphostin A9 (TA9) for 30 minutes. Reactions were terminated by the addition of 0.2% SDS containing 5 mM EDTA, and [ ³H]phosphatidylethanol was extracted, separated by thin-layer chromatography and quantified as in (Jung et al., 1998). Values are expressed as -fold over the control and represent the means (± SEM) of 5 separate experiments; **p<0.01, ***p<0.001 versus the control value, ††p<0.01 versus AngII alone. (B) [³H]Oleate-prelabeled cells were incubated for 30 minutes with 3.5 mM K⁺-KRB⁺ or 15 mM K⁺-KRB⁺ containing 0.5% ethanol and vehicle (0.1% DMSO, control) or 10 μM tyrphostin A9 (TA9) for 30 minutes, and PLD activity monitored as above. Values are expressed as -fold over the control and represent the means (± S.E.M.) of 4 separate experiments; *p<0.05, **p<0.01 versus the control value. (C) Bovine adrenal glomerulosa cells were incubated for 1 hour with KRB⁺ containing no additions (Con) or 10 μM 22(R)-hydroxycholesterol in the presence or absence of 10 μM tyrphostin A9 (TA9) or vehicle (0.1-0.2% DMSO). Supernatants were collected and assayed for aldosterone secretion by radioimmunoassay. Values represent the means ± S.E.M., expressed as the ng aldosterone/mL/60 minutes, of 3 separate experiments; *p<0.03 versus 22(R)-hydroxycholesterol alone, as determined using an unpaired Student’s t-test. Note that 22(R)-hydroxycholesterol induced a significant, approximately 11,000-fold increase over the control aldosterone secretory value.