The role of calcium influx pathways in phospholipase D activation in bovine adrenal glomerulosa cells

Abstract

The steroid hormone aldosterone maintains sodium homeostasis and is therefore important in the control of blood volume and pressure. Angiotensin II (AngII) and elevated extracellular potassium concentrations ([K(+)](e)), the prime physiologic regulators of aldosterone secretion from adrenal glomerulosa cells, activate phospholipase D (PLD) in these cells. The role of Ca(2+) in the activation by these agents is unknown, although nitrendipine, a voltage-dependent Ca(2+) channel antagonist, does not inhibit AngII-elicited PLD activation, despite the fact that this compound blocked elevated [K(+)](e)-stimulated PLD activity. PLD activation triggered by AngII was also unaffected by the T-type calcium channel inhibitor nickel. Nevertheless, Ca(2+) influx was required for AngII-induced PLD activation in both primary cultures of bovine adrenal glomerulosa cells and a glomerulosa cell model, the NCI H295R adrenocortical carcinoma cell line. The involvement of store-operated Ca(2+) (SOC) influx and Ca(2+) release-activated Ca(2+) (CRAC) influx pathways in PLD activation was investigated using thapsigargin, an endoplasmic reticulum Ca(2+) pump inhibitor that empties the store to induce SOC influx, and the SOC inhibitor YM-58483 (BTP2), as well as a CRAC inhibitor, tyrphostin A9. In bovine glomerulosa cells, tyrphostin A9 inhibited AngII-induced PLD activation without affecting elevated [K(+)](e)-stimulated enzyme activity. On the other hand, differences were observed between the bovine adrenal glomerulosa and H295R cells in the involvement of Ca(2+) influx pathways in PLD activation, with the involvement of the SOC pathway suggested in the H295R cells. In summary, our results indicate that Ca(2+) entry only through certain Ca(2+) influx pathways is linked to PLD activation.

Figure 1

Inhibition of Ca²⁺ Influx with Nitrendipine Completely Inhibited Elevated [K⁺]_e-elicited PLD Activation. [³H]Oleate-prelabeled cells were incubated for 30 minutes with KRB⁺ containing 3.5 mM KCl (3.5 mM K⁺-KRB⁺, control), 1 μM nitrendipine (NTR) in 3.5 mM K⁺-KRB⁺, 15 mM K⁺-KRB⁺ or 1 μM NTR in 15 mM K⁺-KRB⁺ in the presence of 0.5% ethanol. [Note that NaCl in the medium was substituted for by KCl to maintain osmolarity in the 15 mM K⁺-KRB⁺ as in (Betancourt-Calle et al., 2001).] 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<0.05 versus the control value.

Figure 2

Inhibition of alpha1H T-type Ca²⁺ Channels with Nickel Inhibited Elevated [K⁺]_e-, but Not AngII-, elicited PLD Activation. (A) [³H]Oleate-prelabeled cells were incubated for 30 minutes with KRB+ containing 0.5% ethanol and with and without 10 nM AngII in the presence or absence of 50 μM nickel (Ni) 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 4 separate experiments; *p<0.05 versus the control value. (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 in the absence or presence of 50 μM nickel (Ni) 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<0.01 versus the control value.

Figure 3

AngII-induced PLD Activation was Modulated by Ca²⁺ in Primary Bovine Adrenal Glomerulosa Cells. Bovine adrenal glomerulosa cells were prelabeled for 20-24 hours with 2.5-5 μCi/mL [³H]oleate in serum-free medium. Cells were then stimulated with or without 10 nM AngII in the presence of 0.5% ethanol in medium containing 1.2 mM Ca²⁺ or no added Ca²⁺ for 30 minutes. Lipids were extracted and separated by thin-layer chromatography. PLD was measured as the radioactivity in phosphatidylethanol (PEt) relative to the control, and values are expressed as the means ± SEM of 4 separate experiments; ***p<0.001 versus the control value; ††p<0.01 versus the value in the presence of Ca²⁺.

Figure 4

Thapsigargin Had Little or No Effect on AngII-Induced PLD Activation, but Enhanced the Effect of Elevated [K⁺]_e on PLD Activity in Bovine Adrenal Glomerulosa Cells. (A) Bovine adrenal glomerulosa cells were prelabeled for 20-24 hours with 2.5-5 μCi/mL [³H]oleate in serum-free medium. Cells were then stimulated with or without 10 nM AngII in the presence or absence of 2 μM thapsigargin (Thaps) or vehicle (0.1-0.2% DMSO) in the presence of 0.5% ethanol for 30 minutes. Lipids were extracted and separated by thin-layer chromatography. Values represent the radioactivity in phosphatidylethanol relative to the control and are expressed as the means ± S.E.M. of 9 separate experiments; *p<0.05 versus the control value. (B) [³H]Oleate-prelabeled bovine adrenal glomerulosa cells were incubated with 3.5 mM K⁺-KRB⁺ or 15 mM K⁺-KRB⁺ in the presence or absence of 2 μM thapsigargin (Thaps) or vehicle (0.1-0.2% DMSO) in the presence of 0.5% ethanol for 30 minutes. Lipids were extracted and separated by thin-layer chromatography. Values represent the radioactivity in phosphatidylethanol relative to the control and are expressed as the means ± S.E.M. of 5 separate experiments; **p<0.01 versus the control value; †p<0.05 versus K⁺ alone.

Figure 5

Thapsigargin Had Little or No Effect Alone or on AngII-Stimulated Aldosterone Secretion but Enhanced Steroidogenesis in Response to Elevated [K⁺]_e and PMA in Primary Cultures of Bovine Adrenal Glomerulosa Cells. (A) Bovine adrenal glomerulosa cells were incubated for 1 hour with a bicarbonate-buffered Kreb’s Ringer solution with 2.5 mM sodium acetate (KRB⁺) containing no additions (Con), 15 mM KCl (K⁺; iso-osmotic substitution with Na⁺) or 10 nM AngII in the presence or absence of 2 μM thapsigargin (Thaps) or vehicle (0.1-0.2% DMSO). Supernatants were collected and assayed for aldosterone secretion by radioimmunoassay. Values represent the means ± S.E.M. of 4-5 separate experiments; *p<0.05, **p<0.01 versus control by ANOVA and a Student-Newmann-Keuls post-hoc test. (B) Bovine adrenal glomerulosa cells were incubated for 1 hour with a bicarbonate-buffered Kreb’s Ringer solution with 2.5 mM sodium acetate (KRB⁺) containing no additions (Con) or 100 nM PMA in the presence or absence of 2 μM thapsigargin (Thaps) or vehicle (0.1-0.2% DMSO). Supernatants were collected and assayed for aldosterone secretion by radioimmunoassay. Values represent the means ± S.E.M. of 4-5 separate experiments, expressed as the percentage of the maximal aldosterone secretory response; *p<0.05, **p<0.01 versus control by ANOVA and a Student-Newmann-Keuls post-hoc test.

Figure 6

Inhibition of Store-operated Ca²⁺ Influx with BTP2 (YM-58483) Had No Effect on AngII-induced, and Increased 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) or 10 nM AngII in the presence or absence of 1 μM BTP2 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 4 separate experiments; *p<0.05 versus the control value. (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 with vehicle (0.1% DMSO) or 1 μM BTP2 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 versus the control value.

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.

Figure 8

AngII-induced PLD Activation was Modulated by Ca²⁺ in NCI H295R Adrenocortical Carcinoma Cells. H295R cells were prelabeled for 20-24 hours with 2.5-5 μCi/mL [³H]oleate in serum-free medium. Cells were then stimulated with or without 100 nM AngII in KRB⁺ containing 1.2 mM Ca²⁺ or no added Ca²⁺ in the presence of 0.5% ethanol for 5 minutes. Lipids were extracted and separated by thin-layer chromatography. Values represent the radioactivity in phosphatidylethanol (PEt) relative to the control and are expressed as the means ± S.E.M. of 4 separate experiments; **p<0.01, ***p<0.01 versus the control value, †p<0.05 versus the value in the presence of Ca²⁺.

Figure 9

Thapsigargin Enhanced the Effect of AngII on PLD Activation Cells but Had No Effect on AngII- (or Elevated [K⁺]_e-) Stimulated Aldosterone Secretion in H295R. (A) H295R cells were pre-labeled for 20-24 hours with 2.5-5 μCi/mL [³H]oleate in serum-free medium. Cells were then stimulated with or without 10 or 100 nM AngII in the presence or absence of 2 μM thapsigargin (Thaps) or vehicle (0.2% DMSO) in the presence of 0.5% ethanol. Lipids were extracted and separated by thin-layer chromatography. Values represent the radioactivity in phosphatidylethanol relative to the control and are expressed as the means ± S.E.M. of 3 separate experiments; *p<0.05 versus the control value. (B) H295R cells were incubated for 5 hours with medium containing no additions (Con), 15 mM KCl (K⁺) or 100 nM AngII in the presence or absence of 2 μM thapsigargin (Thaps) or vehicle (0.1-0.2% DMSO) as indicated. Supernatants were collected and assayed for aldosterone secretion by radioimmunoassay. Values represent the means ± S.E.M. of 4-5 separate experiments; *p<0.05, **p<0.01, ***p<0.001 versus the control value.