Effects of bryostatin 1, a novel anticancer agent, on intestinal transport and barrier function: Role of protein kinase C

Farokhzad, Omid C.; Mun, Edward C.; Sicklick, Jason K.; Smith, Jeremy; Matthews, Jeffrey B.

doi:10.1067/msy.1998.90574

Cited by 6 publications

(14 citation statements)

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“…This early transient activation is generally followed by a prolonged and profound inhibition of transepithelial Cl Ϫ secretion. PMA treatment inhibits NKCC1 activity (14,30) and causes a decrease in bumetanide binding, indicating a decrease in NKCC1 at the cell surface (30). We have shown that both the transient stimulatory effect and the longterm inhibitory effect of PMA are exerted primarily at the level of basolateral transporters (29,30) with specific effects on NKCC1 (14,15).…”

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confidence: 77%

“…Our laboratory previously reported that PMA treatment inhibits NKCC1 activity (14,30) and markedly reduces the binding of [ 3 H]bumetamide, an NKCC1 inhibitor, to the basolateral membrane of polarized T84 cells (30). Inhibition of Fig.…”

Section: Discussionmentioning

confidence: 98%

“…Activation of PKC by phorbol ester induces a transient increase in electrogenic Cl Ϫ secretion, a response that is much smaller in magnitude than that elicited by stimulation of the cAMP-PKA pathway in various epithelial tissues, including intestinal (12,46), tracheal (3), and conjunctival epithelium (45), as well as in cultured cell lines (30,43). We have shown that both the transient stimulatory effect and the longterm inhibitory effect of PMA are exerted primarily at the level of basolateral transporters (29,30) with specific effects on NKCC1 (14,15). For example, in the intestinal epithelial cell lines T84 and HCT29cl.19A, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (PMA) inhibits cAMP-dependent Cl Ϫ secretion in a time-and dose-dependent manner (29,30,37,43).…”

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confidence: 87%

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Dynamic regulation of Na⁺-K⁺-2Cl⁻ cotransporter surface expression by PKC-ε in Cl⁻-secretory epithelia

Castillo

Fedor-Chaiken²,

Song³

et al. 2005

American Journal of Physiology-Cell Physiology

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In secretory epithelia, activation of PKC by phorbol ester and carbachol negatively regulates Cl(-) secretion, the transport event of secretory diarrhea. Previous studies have implicated the basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) as a target of PKC-dependent inhibition of Cl(-) secretion. In the present study, we examined the regulation of surface expression of NKCC1 in response to the activation of PKC. Treatment of confluent T84 intestinal epithelial cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA) reduced the amount of NKCC1 accessible to basolateral surface biotinylation. Loss of cell surface NKCC1 was due to internalization as shown by 1) the resistance of biotinylated NKCC1 to surface biotin stripping after incubation with PMA and 2) indirect immunofluorescent labeling. PMA-induced internalization of NKCC1 is dependent on the epsilon-isoform of PKC as determined on the basis of sensitivity to a panel of PKC inhibitors. The effect of PMA on surface expression of NKCC1 was specific because PMA did not significantly alter the amount of Na(+)-K(+)-ATPase or E-cadherin available for surface biotinylation. After extended PMA exposure (>2 h), NKCC1 became degraded in a proteasome-dependent fashion. Like PMA, carbachol reduced the amount of NKCC1 accessible to basolateral surface biotinylation in a PKC-epsilon-dependent manner. However, long-term exposure to carbachol did not result in degradation of NKCC1; rather, NKCC1 that was internalized after exposure to carbachol was recycled back to the cell membrane. PKC-epsilon-dependent alteration of NKCC1 surface expression represents a novel mechanism for regulating Cl(-) secretion.

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confidence: 77%

Section: Discussionmentioning

confidence: 98%

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confidence: 87%

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Dynamic regulation of Na⁺-K⁺-2Cl⁻ cotransporter surface expression by PKC-ε in Cl⁻-secretory epithelia

Castillo

Fedor-Chaiken²,

Song³

et al. 2005

American Journal of Physiology-Cell Physiology

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“…In addition, PKC influences the barrier function of epithelial cells. In T84 cells, we have shown that PMA impairs junctional integrity and slowly but profoundly decreases transepithelial electrical resistance (TER) (7,20). Hecht and coworkers (10) showed that extended PMA treatment induces disassembly of T84 epithelial monolayers and causes cellular multilayering.…”

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confidence: 99%

Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms

Song

Hanson

Tsai

et al. 2001

American Journal of Physiology-Cell Physiology

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The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits Cl(-) secretion (short-circuit current, I(sc)) and decreases barrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes in this response, we compared PMA with two non-phorbol activators of PKC (bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozyme selectivity profiles. PMA sequentially inhibited cAMP-stimulated I(sc) and decreased TER, as measured by voltage-current clamp. By subcellular fractionation and Western blot, PMA (100 nM) induced sequential membrane translocation of the novel PKC epsilon followed by the conventional PKC alpha and activated both isozymes by in vitro kinase assay. PKC delta was activated by PMA but did not translocate. By immunofluorescence, PKC epsilon redistributed to the basolateral domain in response to PMA, whereas PKC alpha moved apically. Inhibition of I(sc) by PMA was prevented by the conventional and novel PKC inhibitor Gö-6850 (5 microM) but not the conventional isoform inhibitor Gö-6976 (5 microM) or the PKC delta inhibitor rottlerin (10 microM), implicating PKC epsilon in inhibition of Cl(-) secretion. In contrast, both Gö-6976 and Gö-6850 prevented the decline of TER, suggesting involvement of PKC alpha. Bryostatin-1 (100 nM) translocated PKC epsilon and PKC alpha and inhibited cAMP-elicited I(sc). However, unlike PMA, bryostatin-1 downregulated PKC alpha protein, and the decrease in TER was only transient. Carbachol (100 microM) translocated only PKC epsilon and inhibited I(sc) with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1 and carbachol inhibition of I(sc). We conclude that basolateral translocation of PKC epsilon inhibits Cl(-) secretion, while apical translocation of PKC alpha decreases TER. These data suggest that epithelial transport and barrier function can be modulated by distinct PKC isoforms.

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“…With the recent molecular characterization of the two major isoforms of the Na ϩ /K ϩ /2Cl Ϫ cotransporter (Gamba et al, 1994;Payne et al 1995), it has become possible to examine the factors that control expression (Farokhzad et al, 1998;Matthews et al, 1998;Topper et al 1997) and function (Isenring et al, 1997(Isenring et al, , 1998a(Isenring et al, , 1998b of the cotransporter. The more widely distributed NKCC1 is the so-called secretory or basolateral isoform present in numerous epithelial and nonepithelial cell types.…”

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Protein kinase C activation downregulates the expression and function of the basolateral Na+/K+/2Cl? cotransporter

et al. 1999

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The basolateral Na+/K+/2Cl(-) cotransporter (NKCC1) has been shown to be an independent regulatory site for electrogenic Cl(-) secretion. The proinflammatory phorbol ester, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), inhibits basal and cyclic adenosine monophosphate (cAMP)-stimulated NKCC1 activity in T84 intestinal epithelial cells and decreases the steady state levels of NKCC1 mRNA in a time- and dose-dependent manner. The levels of NKCC1 protein also fall in accordance with the NKCC1 mRNA transcript and these levels are unaffected by 4alpha-phorbol, which does not activate PKC. Inhibition of maximal (cAMP-stimulated) NKCC1 functional activity by PMA was first detected by 1 h, whereas decreases in the steady state levels of NKCC1 mRNA were not detectable until 4 h. NKCC1 mRNA expression recovers toward control levels with extended treatment of cells with PMA suggesting that the PMA effects on NKCC1 expression are mediated through activation of PKC. Although NKCC1 mRNA and protein levels return to control values after extended PMA exposure, NKCC1 functional activity does not recover. Immunofluorescence imaging suggest that the absence of functional recovery is due to failure of newly synthesized NKKC1 protein to reach the cell surface. We conclude that NKCC1 has the capacity to be regulated at the level of de novo expression by PKC, although decreased NKCC1 expression alone cannot account for either early or late loss of NKCC1 function.

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Effects of bryostatin 1, a novel anticancer agent, on intestinal transport and barrier function: Role of protein kinase C

Cited by 6 publications

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Dynamic regulation of Na⁺-K⁺-2Cl⁻ cotransporter surface expression by PKC-ε in Cl⁻-secretory epithelia

Dynamic regulation of Na⁺-K⁺-2Cl⁻ cotransporter surface expression by PKC-ε in Cl⁻-secretory epithelia

Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms

Protein kinase C activation downregulates the expression and function of the basolateral Na+/K+/2Cl? cotransporter

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Effects of bryostatin 1, a novel anticancer agent, on intestinal transport and barrier function: Role of protein kinase C

Cited by 6 publications

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Dynamic regulation of Na+-K+-2Cl− cotransporter surface expression by PKC-ε in Cl−-secretory epithelia

Dynamic regulation of Na+-K+-2Cl− cotransporter surface expression by PKC-ε in Cl−-secretory epithelia

Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms

Protein kinase C activation downregulates the expression and function of the basolateral Na+/K+/2Cl? cotransporter

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Dynamic regulation of Na⁺-K⁺-2Cl⁻ cotransporter surface expression by PKC-ε in Cl⁻-secretory epithelia

Dynamic regulation of Na⁺-K⁺-2Cl⁻ cotransporter surface expression by PKC-ε in Cl⁻-secretory epithelia