Synthesis and Release of Luteinizing Hormone by Rat Anterior Pituitary Cells: Effects of Cytochalasins B and D*

Abstract

We determined the role of microfilaments in regulating LH synthesis (translation or glycosylation) and release from cultured rat anterior pituitary cells under basal and GnRH-stimulated conditions. Cells were pretreated for 2 h with microfilament-disrupting drugs, cytochalasin B (CB; 2 and 20 microM) or cytochalasin D (CD; 1 and 10 microM). LH synthesis and release were measured after 4 h of incubation with or without 1 nM GnRH and drugs. LH translation and glycosylation were monitored by measuring the incorporation of [14C]alanine and [3H]glucosamine, respectively, into total (cell and medium) immunoprecipitable LH. Immunoreactive LH (IRLH) in medium and cells was measured by RIA. GnRH at 1 nM significantly (P less than 0.01) increased the release of IRLH and total [3H]LH (glycosylation), but had no effect on total [14C]LH (translation), uptake, or incorporation of precursors into total protein. Neither CB (2 and 20 microM) nor CD (10 microM) altered basal or GnRH-stimulated IRHL release. Neither drug altered basal medium concentrations of [3H]LH or [14C]LH. In contrast, both CB and CD reduced (P less than 0.01) GnRH-stimulated [3H]LH in the medium and total system (LH glycosylation). CB reduced (P less than 0.01) [3H]glucosamine uptake, total [3H]protein synthesis, and basal level of total [3H]LH, while CD had no effects on these parameters. Thus, CD exerted a more specific inhibitory effect on GnRH-stimulated LH glycosylation than CB. CB (2 and 20 microM) increased (P less than 0.01), while CD (10 microM) decreased (P less than 0.01) [14C]alanine uptake, total [14C]LH, and [14C]protein under both basal and GnRH-stimulated conditions. These results demonstrated that while the cytochalasins did not inhibit either basal or GnRH-stimulated IRLH release, they did inhibit GnRH-stimulated LH glycosylation, although the effect of CB was due partially to reduced [3H]glucosamine uptake. Integrity of microfilaments appears to be important for GnRH-enhanced LH glycosylation, but not for GnRH-enhanced LH release.

Differential Actions of Arachidonic Acid and Melittin on Luteinizing Hormone Release and Synthesis

“…We also found that both AA and melittin preferentially released stored IR-LH,whereas GnRH released both stored and newly glycosylated and translated LH. These findings support our previous observations [42] that release o f stored LH appears to be regulated independently of newly syn thesized LH.…”

Changes in actin network during calcium-induced exocytosis in permeabilized GH3 cells: calcium directly regulates F-actin disassembly

“…Values are means + or S.E.M. and the effect of cytochalasin has been investigated (Liu & Jackson 1986). However, only a few reports have shown changes in the cytoskeletons of pituitary cells during cell excitation (van de Moortele et al 1991, Kiley et al 1992, Carbajal & Vitale 1997.…”

Regulation of Luteinizing Hormone Subunit Biosynthesis in Cultured Male Anterior Pituitary Cells: Effects of Gonadotropin-Releasing Hormone and Testosterone*

“…The goals of the first experiment were 1) to establish that male pituitary cells cultured under the present conditions remained functionally viable, as measured by incorporation of 35 S-Met and 3 H-Gln into total protein as a function of time, and 2) to determine the effects of continuous exposure to GnRH for various periods of time on LH release and radiolabeled precursor incorporation into immunoprecipitable LH subunits. Pituitary cells, cultured for 4 days in AMEM containing 10% CHS, were preincubated for 1 h in 10 ml glucosamine-and methionine-free Krebs-Ringer-I bicarbonate buffer (KRB-I) to reduce the endogenous pool of synthetic precursors.…”

“…Pituitary cells, cultured for 4 days in AMEM containing 10% CHS, were preincubated for 1 h in 10 ml glucosamine-and methionine-free Krebs-Ringer-I bicarbonate buffer (KRB-I) to reduce the endogenous pool of synthetic precursors. The cells were then transferred to 1.5-cm (diameter) culture dishes containing 1.0 ml KRB-I, 30 fid 3 H-Gln (43 Ci/mmol), and 15 /iCi 35 S-Met (1116 Ci/mmol), with or without 1 nM GnRH. The cells were incubated for 2, 4, 6, 8, or 12 h at 37 C under a humidified air-CO 2 mixture.…”

“…In Exp 2, GnRH enhanced total 3 H-Gln (but not 36 S-Met) incorporation into both LH subunits. GnRH stimulated the release of 35 S-Met LHa and 3 H-Gln LH subunits and increased the relative glycosylation of secreted LH subunits without altering the relative glycosylation of intracellular LH subunits. T inhibited radioimmunoassayable LH release and incorporation of both precursors into total and secreted LH subunits (with or without GnRH).…”