Effect of protein kinase C activation on the glycine evoked Cl(-) current in spinal cord neurons.

We investigated whether the effect of phorbol-12-myristate-13-acetate (PMA) was altered by a kinase inhibitor and by down-regulation of protein kinase C (PKC) in order to determine if glycine receptors in mouse spinal neurons, unlike those in hippocampal and trigeminal neurons, can be inhibited by PKC. To examine the above, electrophysiological and immunofluorescence studies were carried out in mouse spinal neurons kept in culture for up to 3 weeks. The inhibition of the glycine activated current by PMA (1 microM) increased from 12+/-3% during week 1 to 27+/-6% during week 3. The effect of PMA was completely blocked by the PKC selective inhibitor RO 31-8220 (1 microM). After culturing the cells with 1 microM PMA for 24 h, the inhibitory effect of acute application of PMA disappeared altogether, suggesting that the effect of PMA was via PKC. Immunofluorescence studies showed that a short stimulation with PMA translocated the enzyme to the periphery whereas longer term stimulation (24 h) down regulated the PKC signal. These results indicate that activation of PKC by PMA inhibits the glycine receptor in cultured spinal neurons and that its sensitivity changes during neuronal development.

Cellular mechanisms of estrogen- and dopamine-induced control of glandular kallikrein in the anterior pituitary of the rat.

Glandular kallikrein (GK, a trypsin-like serine protease) exhibits estrogen induction and dopamine repression in rat pituitary lactotrophs. Steroid induction may reflect primary actions to increase selectively the synthesis of specific proteins, or may be part of broad cellular responses secondary to steroid-induced phenotype transitions. This study examined the cellular mechanisms underlying estrogen and dopaminergic control of lactotroph GK using a quantified immunocytochemical approach. Pituitaries from ovariectomized rats exhibited little GK staining. Estradiol treatment for 10 days produced dose-dependent increases in pituitary mass, the percentage of lactotrophs (indicating lactotroph proliferation) and the percentage of GK-positive cells. Also, GK staining intensity was dependent upon estradiol dose, increasing 4-fold between 5 micrograms and 50 micrograms/48 h. Dopamine receptor blockade with haloperidol (2.5 mg/kg/24 h) elicited weak GK immunostaining in 46% of the lactotrophs in the absence of estradiol, and markedly potentiated GK staining intensity elicited with low but not high doses of estradiol. The results suggest that GK induction is a primary estrogen effect, and is not secondary to a phenotype transition: the induction is enhanced by estrogen-induced lactotroph proliferation. Dopaminergic systems strongly inhibit GK induction by low estradiol levels. This dopaminergic modulation may shift the induction of lactotroph GK to physiological events associated with high estradiol levels or low dopaminergic tone.

Hormonal regulation of pituitary glandular kallikrein: a morphometric study.

We have previously identified the lactotrophs as the Glandular Kallikrein (GK) containing cells in the rat anterior pituitary using immunocytochemistry, this localization has been independently confirmed with similar methods by other groups. The purpose of the present work was to evaluate the estrogen and dopaminergic control of the GK-containing cells using a morphometric analysis. Female (200-250 g) ovariectomized rats (n = 40) were treated with estradiol (5, 10, 50 micrograms/rat) in the presence or absence of haloperidol (2.5 mg/Kg). The pituitaries were fixed by perfusion with Bouin's and immunostained for prolactin (PRL) or GK. The number of cells and the intensity of the staining were determined by morphometric analysis. Little GK staining was observed in pituitaries from ovariectomized rats, whereas estradiol treatment produced a marked increase in GK staining; GK-positive lactotrophs increased from 4% in control to 75% with 5 micrograms of estradiol, higher doses produced little further increase. However, GK staining intensity in lactotrophs was markedly dependent upon estradiol dose increasing 4-fold between 5 micrograms and 50 micrograms. Haloperidol (2.5 mg/Kg) elicited weak GK staining in 46% of the lactotrophs in the absence of estradiol, and potentiated GK staining intensity elicited with low doses of estradiol. Estradiol also produced a dose-dependent increase in pituitary mass and % lactotrophs indicating lactotroph proliferation. Estradiol produced a dose dependent increase in pituitary wet weight, % PRL-positive cells and % GK-positive cells. Pituitary weight was correlated with % lactotrophs (r = 0.992), and % GK cells (r = 0.874), and % lactotrophs was correlated with % GK cells (r = 0.978).(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of immunoreactive glandular kallikrein in lactotrophs of the rat anterior pituitary.

Glandular kallikrein (a trypsin-like serine protease) is an estrogen-induced and dopamine-repressed protein in the rat anterior pituitary which predominantly exists as a latent zymogen (prokallikrein). Its regulation, presence in estrogen-induced pituitary tumors in F344 rats, and expression in GH3 cells has suggested a localization in lactotrophs (prolactin-producing cells). This study examined the cellular origin of glandular kallikrein using immunocytochemical techniques. Anterior pituitaries from estrogen-treated rats were fixed and embedded in paraffin (for preparation of semi thick sections; 5 microns) or methacrylate (for preparation of thin sections; 1 micron). Glandular kallikrein immunostaining was readily detected in the perinuclear (Golgi) region of parenchymal cells of the anterior pituitary in both thin and semi thick sections. Two-color double immunoperoxidase staining of thin and semi thick sections indicated that glandular kallikrein was localized in cells containing prolactin (PRL) but not other pituitary hormones. Immunoperoxidase staining of consecutive serial thin sections with alternating antisera confirmed a localization of glandular kallikrein in lactotrophs. The results establish that glandular kallikrein is colocalized with PRL in lactotrophs of the rat anterior pituitary. This is consistent with the hypothesis that the function of anterior pituitary glandular kallikrein is linked to PRL in some fashion--possibly as a PRL-processing protease.