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ABSTRACT

The present study deals with the effects of withdrawal of dopamine (DA) on the translocation of protein kinase C (PKC) isozymes and release of prolactin (Prl) in resting- and substance P (SP)-stimulated cultures of enriched rat pituitary lactotrophs.

Following a brief tonic input (10 min), DA withdrawal induced a redistribution of PKC α- and β-immunoreactivity (IR) to the particulate fraction with maximal levels, attained after 5 min, remaining translocated for 20 min. DA withdrawal prolonged the effect of SP-induced translocation of PKC α-and β-IR. Similar effects were detected when the catalytic activity of PKC in response to DA withdrawal was evaluated. Thus, DA washout redistributed PKC catalytic activity and prolonged the effect of SP on catalytical PKC translocation to the particulate fraction. Pretreatment of cells with the protein kinase A inhibitor, rp-adenosine-3′,5′-cyclic monophosphothionate (rp-cAMP), reduced the amount of PKC α- and β-IR redistributed after DA withdrawal. Furthermore, this treatment also reduced the DA withdrawal effect on SP-mediated translocation of PKC α- and β-IR. Methoxyverapamil, a blocker of voltage-gated Ca²⁺ channels, completely inhibited the redistribution of PKC isozymes after DA withdrawal, but also reduced the potentiating effect of DA withdrawal on SP-induced redistribution of PKC isozyme-IR.

In perifused enriched lactotrophs, DA withdrawal induced a release of Prl that lasted 45-55 min and prolonged the effect of SP on Prl secretion. rp-cAMP did not significantly affect Prl release due to DA removal, but the prolonging effect of DA withdrawal on SP-induced Prl secretion was abolished. Methoxyverapamil completely abolished the rebound release of Prl after DA withdrawal, and the potentiating effect of DA removal on SP-mediated Prl release was also diminished. Readdition of DA after DA withdrawal was able to suppress the translocation of PKC isozyme-IR and catalytic activity and to reduce the release of Prl to baseline levels. Moreover, readdition of DA reduced the potentiating effects of DA withdrawal on the same parameters after SP-stimulation of cells.

On the basis of these results it is concluded that in resting cells following DA withdrawal prolactin is released and specific PKC isozymes and concomitant catalytic activity are translocated to the particulate fraction in enriched lactotrophs. While cAMP/PKA and influx of Ca²⁺ seem to work in concert in translocating PKC, influx of Ca²⁺ is the primary mechanism responsible for the rebound release of Prl after DA withdrawal. DA withdrawal exerts a potentiating effect on SP-induced PKC translocation and Prl release. It is suggested that the biochemical events involved in these processes are cAMP/PKA and Ca²⁺ influx.

ABSTRACT

Translocation of protein kinase C (PKC) from the cytosol to the plasma membranes is believed to reflect activation of the enzyme. We have studied translocation of PKC in lactotroph-enriched anterior pituitary cell cultures by measuring the incorporation of γ-³²P from [γ-³²P]ATP into a synthetic peptide substrate, MBP_4–14, and by immunoblotting of PKC isozymes. Using cells permeabilized with digitonin the effects of PKC cofactors on the distribution of the enzyme were studied. Ca⁺ (50 nm) and dioctanoyl-sn-glycerol had no effect when tested alone, but in combination they caused a redistribution of PKC from the soluble to the particulate fraction. Arachidonic acid needed Ca⁺ to induce translocation of PKC, while being ineffective under Ca²⁺-free conditions. Western blot analysis of partly purified PKC from lactotroph-enriched pituitary cells revealed the presence of the α, β, δ, and ζ isozymes. 12-O-Tetradecanoylphorbol 13-acetate (TPA) and substance P displayed different patterns of redistribution of PKC isozyme immunoreactivity from soluble to membrane-attached forms. Thus, TPA induced time- and dose-dependent (mean effective concentration (EC₅₀)=1 nm) translocation of the α, β and δ species, while substance P stimulated time- and dose-dependent (EC₅₀=1 nm) redistribution of the α and β isozymes. ζ subtype immunoreactivity could not be translocated by either agonist; neither could the immunoreactivity of ζ be down-regulated by long-term treatment (24 h) with TPA.

The results indicate that simultaneous activation of phospholipases C and A₂ induces a synergistic activation of PKC. Finally it is suggested that substance P may exert some of its effects in lactotrophs by translocation of PKC isozymes α and β.