Ca(2+)-evoked serotonin secretion by parafollicular cells: roles in signal transduction of phosphatidylinositol 3'-kinase, and the gamma and zeta isoforms of protein kinase C

Abstract

Parafollicular (PF) cells secrete 5-HT in response to stimulation of a G-protein-coupled Ca(2+) receptor (CaR) by increased extracellular Ca(2+) (upward arrow[Ca(2+)](e)). We tested the hypothesis that protein kinase C (PKC) participates in stimulus-secretion coupling. Immunoblots from membrane and cytosolic fractions of isolated PF cells revealed conventional (alpha, betaI, and gamma), novel (delta and epsilon), and atypical (iota/lambda and zeta) PKCs. Only PKCgamma was found to have been translocated to the membrane fraction when secretion of 5-HT was evoked by upward arrow[Ca(2+)](e) or phorbol esters. Although phorbol downregulation caused PKCgamma to disappear, secretion was only partially inhibited. A similar reduction of upward arrow[Ca(2+)](e)-evoked secretion was produced by inhibitors of conventional and/or novel PKCs (Gö 6976, calphostin C, and pseudoA), and these compounds did not inhibit secretion at all when applied to phorbol-downregulated cells. In contrast, the phorbol downregulation-resistant component of secretion was abolished by pseudoZ, which inhibits the atypical PKCzeta. Stimulation of PF cells with upward arrow[Ca(2+)](e) increased the activity of immunoprecipitated PKCzeta (but not PKCiota/lambda), and the activity of this PKCzeta was inhibited by pseudoZ. PF cells were found to express regulatory (p85) and catalytic (p110alpha and p110beta) subunits of phosphatidylinositol 3'-kinase (PI3'-kinase). upward arrow[Ca(2+)](e) increased the activity of immunoprecipitated PI3'-kinase; moreover, PI3'-kinase inhibitors (wortmannin and LY294002) antagonized secretion. We suggest that PKC isoforms mediate secretion of 5-HT by PF cells in response to stimulation of the CaR. PKC involvement can be accounted for by PKCgamma and an isoform sensitive to inhibition by pseudoZ, probably PKCzeta, which is activated via PI3'-kinase.

Fig. 1.

Many isoforms of PKC are found in PF cell membrane and cytosolic fractions. Cytosol (Cyt) and membrane (Mem) fractions were obtained from nonstimulated PF cells. Proteins in the fractions (100 μg) were separated by SDS-PAGE, blotted onto nitrocellulose membranes, and incubated with monospecific antibodies to the indicated isoforms of PKC. Bound antibodies were visualized by chemiluminescence. PKC isoforms are indicated at theleft, and the molecular weight is shown at theright. The standards (Std) used for comparison were derived from HC11 cells that overexpress PKCα and PKCβ1 or from a commercial source. For PKCα, PKCβI, and PKCγ (Peptide, top right), the specificity of the antibodies used for identification of PKC isoforms was verified by blocking immunostaining with the corresponding peptide antigen at 10 μg/ml.

Fig. 2.

Phorbol ester (PMA) activates and downregulates PKCγ. Isoforms of PKC were investigated by Western blot analysis as in Figure 1. A, PF cells were treated with PMA (300 nm) for 0, 0.5, or 6.0 hr before collection and fractionation. The relative amount of PKCγ decreased in the cytosol and increased in the membrane fraction as a function of time after exposure to PMA. B, PF cells were treated with PMA (1.0 μm) for 0 or 24 hr before collection and fractionation. After the prolonged exposure to PMA, PKCγ could no longer be detected in either the cytosol or membrane fractions.

Fig. 3.

Effect of inhibitors of the regulatory and catalytic domains of PKC on Ca²⁺- and PMA-induced secretion. The natural PF secretogogue ↑[Ca²⁺]_e (5 mm, 10 min) and PMA (300 nm, 30 min) were used to evoke 5-HT secretion. Data are presented as a percentage of 5-HT released induced by either Ca²⁺ (A) or PMA (B) of resting release (assigned control; mean ± SE). The numbers of determinations are indicated in each bar. The effects of PKC inhibitors on the secretion elicited by each secretogogue were then assessed. In addition, PKC was downregulated by overnight exposure to PMA (1.0 μm). The regulatory site inhibitors calphostin C (5 μm) and Gö 6976 (0.5 μm), as well as PKC downregulation, all partially and weakly antagonized the secretion of 5-HT evoked by ↑[Ca²⁺]_e. In contrast, the catalytic inhibitor chelerythrine (5.0 μm) strongly inhibited secretion of 5-HT evoked by ↑[Ca²⁺]_e from both downregulated and nondownregulated cells (85 ± 6%; p < 0.001). All of the inhibitors (calphostin C, chelerythrine, and Gö 6976) and phorbol ester downregulation fully inhibited the secretion evoked by PMA (90 ± 5% inhibition;p < 0.001).

Fig. 4.

Secretion evoked by receptor stimulation is dependent on phorbol ester-sensitive and -insensitive forms of PKC. The effects of ↑[Ca²⁺]_e (5 mm) on 5-HT-induced secretion were assayed in the presence and absence of PKC inhibitors. Downregulation of cPKCγ was achieved by exposing cells overnight to 1 μm PMA. PseudoA and pseudoZ were added 30 min before the addition of the secretogogue. The resting release of 5-HT was taken as a control and the data for the evoked release of 5-HT are presented as a percent of control, which was assigned a value of 100% (mean ± SE). In the absence of phorbol ester-downregulation of PKC, the ↑[Ca²⁺]_e-evoked released of 5-HT was inhibited by both pseudoA (35 ± 4%; p < 0.005) and pseudoZ (65 ± 6%; p < 0.005). After the phorbol ester downregulation of PKC, the residual secretion of 5-HT could no longer be inhibited by pseudoA but now was blocked by pseudoZ (90 ± 5%; p < 0.001). The combination of pseudoA and pseudoZ abolished secretion regardless of whether cells had previously been phorbol ester-downregulated. These data are consistent with the idea that an aPKC, such as PKCζ, contributes to stimulus–secretion coupling and is responsible for mediating the downregulation-resistant component of the ↑[Ca²⁺]_e-evoked release of 5-HT.

Fig. 5.

Isoforms of PI3′-kinase are present in PF cells. Proteins from the total lysate of isolated PF cells (100 μg) were solubilized and separated by SDS-PAGE, blotted onto nitrocellulose membranes, and incubated with monospecific antibodies directed against subunits of PI3′-kinase. Bound antibodies were visualized by chemiluminescence. Lane 1, p85; lane 2, p110α; lane 3, p110β; lane 4, control preparation showing that p110β immunoreactivity is removed by absorption with the corresponding peptide antigen at a ratio of 2:1.

Fig. 6.

PI3′-kinase activity in PF cells is increased by ↑[Ca²⁺]_e and is antagonized by PI3′-kinase inhibitors. PF cells were incubated for 30 min in the absence or presence of wortmannin (100 nm). ↑[Ca²⁺]_e (5.0 mm) was used to stimulate the CaR in both sets of cells. Extracts were prepared, and equivalent amounts of protein (750 μg) were used for immunoprecipitation with antibodies to the p85 subunit of PI3′-kinase. The kinase activity of the immune complex was measured using phosphatidylinositol as a substrate. Lipids were extracted and analyzed by TLC and radioautography (A). Spots corresponding to phosphatidylinositol 3-phosphate (PI3-P) were cut out and analyzed by liquid scintillation (B). Radioactivity observed at the origin reflects residual, water-soluble ³²P-labeled material in the samples, the amount of which is not relevant to the results. PI3′-kinase activity is expressed as a percentage of that found in control (Cont) cells (average of 5 independent experiments). The elevation of PI3′-kinase induced by ↑[Ca²⁺]_e is significant (50 ± 5%; p < 0.001 vs the null hypothesis of 100%), as is its inhibition by wortmannin (p < 0.001, ↑[Ca²⁺]_e vs ↑[Ca²⁺]_e + wortmannin).

Fig. 7.

↑[Ca²⁺]_e-induced secretion of 5-HT by PF cells is antagonized by PI3′-kinase inhibitors. PF cells were chronically treated with PMA to downregulate cPKCγ. Cells were pretreated for 30 min with wortmannin (Wort; 100 nm) or LY294002 (LY; 100 μm). Secretion was measured after the addition of ↑[Ca²⁺]_e (5.0 mm). Data are presented as a percentage of control (no secretogogue; mean ± SE). Both wortmannin and LY294002 inhibited the ↑[Ca²⁺]_e-induced secretion of 5-HT by PF cells. Note that the magnitude of inhibition by 100 nm wortmannin is approximately equal to that caused by 100 μm LY294002. Wortmannin is therefore more potent than LY294002, but both are effective inhibitors of the secretion of 5-HT. The inhibition of 5-HT induced by ↑[Ca²⁺]_e is significant (60 ± 5%; p < 0.001 vs the null hypothesis of 100%), as is its inhibition by LY294002 (p < 0.001, ↑[Ca²⁺]_e vs ↑[Ca²⁺]_e + LY294002).

Fig. 8.

PKC_ζ but not PKCι/λ activity is increased in PF cells stimulated by ↑[Ca²⁺]_e and is antagonized by PI3′-kinase inhibitors or pseudoZ. PF cells were incubated for 30 min in the absence or presence of wortmannin (100 nm) or pseudoZ (40 μm). ↑[Ca²⁺]_e (5.0 mm) was used to stimulate the CaR in both sets of cells. Extracts were prepared, and equivalent amounts of protein (750 μg) were used for immunoprecipitation with antibodies to PKCζ. PKC activity of the immune complex was measured using myelin basic protein (MBP) as a substrate. Proteins were separated by electrophoresis, dried gels were exposed to film (A), and the films were developed and quantified densitometrically (B). Note in the Western blots (A) that an equal amount of protein was immunoprecipitated for assay of PKCζ under each of the experimental conditions studied. In contrast, the activity of PKCζ was inhibited by wortmannin and pseudoZ. PKCζ activity is expressed as a percentage of that found in control cells. The elevation of PKCζ induced by ↑[Ca²⁺]_e is significant (40%;p < 0.001 vs the null hypothesis of 100%), as is its inhibition by wortmannin (p < 0.001, ↑[Ca²⁺]_e vs ↑[Ca²⁺]_e + wortmannin). PseudoZ completely abolished the PKCζ activity. To investigate the possible activation of PKCι/λ by ↑[Ca²⁺]_e, cells were activated as above, but extracted protein was immunoprecipitated (IP) with antibodies to PKCι/λ instead of PKCζ (C). Note that the basal activity of PKCι/λ was unchanged by exposing cells to ↑[Ca²⁺]_e (87.8–89.8% of control).