Interleukin-8 is a key mediator of FKBP51-induced melanoma growth, angiogenesis and metastasis.

BACKGROUND: FKBP51 is overexpressed in melanoma and impacts tumour cell properties. However, its comprehensive role in melanoma pathogenesis and underlying mechanism(s) remain elusive. METHODS: FKBP51 was stably silenced in aggressive melanoma cell lines and its effect examined in vitro and in mouse model. Histological/immunohistochemical analyses were performed to confirm metastasis, angiogenesis and neutrophil infiltration. Gene expression was analyzed by qRT-PCR, immunoblot and/or ELISA. NF-κB transcriptional activity and promoter binding were monitored by luciferase-based promoter-reporter and ChIP assays, respectively. Interleukin (IL)-8 inhibition was achieved by gene silencing or neutralising-antibody treatment. RESULTS: FKBP51 silencing reduced melanoma growth, metastasis, angiogenesis and neutrophil infiltration and led to IL-8 downregulation through NF-κB suppression in cell lines and tumour xenografts. IL-8 inhibition drastically decreased growth, migration and invasiveness of FKPB51-overexpressing cells; whereas its treatment partially restored the suppressed phenotypes of FKBP51-silenced melanoma cells. Interleukin-8 depletion in conditioned medium (CM) of FKBP51-overexpressing melanoma cells inhibited endothelial cell proliferation and capillary-like structure formation, whereas its treatment promoted these effects in endothelial cells cultured in CM of FKBP51-silenced melanoma cells. CONCLUSIONS: FKBP51 promotes melanoma growth, metastasis and angiogenesis, and IL-8 plays a key role in these processes. Thus, targeting of FKBP51 or its upstream or downstream regulatory pathways could lead to effective therapeutic strategies against melanoma.

Restasis for the treatment of 'dry eye' in Aotus nancymaae.

BACKGROUND: This case report describes the treatment of three male owl monkeys (Aotus nancymaae) diagnosed with chronic dry eye with a topical cyclosporine product, Restasis, approved for use in humans. These owl monkeys had ocular disease resulting from procedures performed at a biotechnology company. They were moved to the Center for Neotropical Primate Research and Resources at University of South Alabama to be incorporated into the breeding colony. MATERIALS AND METHODS: Schirmer tear testing was performed initially and during the course of treatment to monitor efficacy of twice daily administered Restasis. The goals of treatment were to reduce pain and/or distress and if possible to quantitatively increase tear production. RESULTS AND DISCUSSION: All animals had improvements in conjunctival inflammation and had an increase in tear production.

Overexpression of the FK506-binding immunophilin FKBP51 is the common cause of glucocorticoid resistance in three New World primates.

Many New World primates have high circulating levels of cortisol to compensate for the expression of glucocorticoid receptors (GRs) with low activity. Recent work in squirrel monkeys has suggested that this may be due to either the expression of GRs that are transcriptionally incompetent or the expression of an FK506-binding immunophilin that inhibits GR binding. The goal of this study was to resolve this controversy by determining the molecular basis of glucocorticoid resistance not only in species of squirrel monkeys but also in other glucocorticoid-resistant New World primates. First, the transcriptional activity of the GR from the Bolivian squirrel monkey was compared to that of the human GR. Incubation of COS-7 cells transfected with the squirrel monkey GR with 10 nM dexamethasone resulted in a robust stimulation of MMTV-luciferase activity (up to 260-fold), which was similar in magnitude to that achieved with the human GR. Second, the effect of FK506 on GR binding was determined in cytosol from cells from two species of squirrel monkeys as well as glucocorticoid-resistant cotton-top tamarins and owl monkeys. Incubation with 10 microM FK506 increased GR binding by at least 4-fold in cytosol from cells of each of the New World primates but had no effect on GR binding in cytosol from human WI-38 VA13 cells. Third, Western blots showed elevated expression of FKBP51 in New World primate cells and liver samples from two squirrel monkey species. On the other hand, the levels of FKBP52 were significantly lower in cells and liver from New World primates. The sequences of FKBP51 from the cotton-top tamarin, owl monkey and squirrel monkey are closely related and share differences from the human, rhesus monkey, mouse, and lemur FKBP51 sequences in the same 18 positions. Fourth, the relative activities of FKBP51 from the cotton-top tamarin, owl monkey and squirrel monkey were determined in cytosol mixing and GR transactivation studies and showed that FKBP51 from each of these primates was a potent inhibitor of GR activity. These results indicate that the elevated expression of FKBP51 contributes to glucocorticoid resistance in three New World primate genera.

The origin of four squirrel monkey cell lines established by karyotype analysis.

The squirrel monkey is a neotropical primate genus which is widely used in biomedical research but includes individual species and subspecies that respond differently to experimental perturbations. GTG-banding patterns of chromosomes 15 and 16, which are distinct among different squirrel monkey species and subspecies, were used to determine the origin of three lung fibroblast cell lines from squirrel monkeys of unknown genetic background (DPSO 114/74, SqMkLu/68, and 7603830) and to confirm the origin of a lymphoblast cell line (GSML) recently established from Guyanese squirrel monkey. DPSO 114/74 cells are from Peruvian squirrel monkey, SqMkLu/68 cells are Bolivian squirrel monkey, and 7603830 cells are from a Peruvian/Bolivian hybrid. Chromosome analysis of GSML cells confirmed that they are from Guyanese squirrel monkey.

Identification of an estrogen-inducible phosphatase (PP5) that converts MCF-7 human breast carcinoma cells into an estrogen-independent phenotype when expressed constitutively.

The proliferation of many estrogen receptor (ER)-positive breast cancer cells depends on estradiol, and tumors arising from these cells are often responsive initially to treatment with selective ER modulators, which produce an antiestrogen effect. However, tumors that are refractory to the antiestrogenic effects of selective ER modulators often reemerge, and the prognosis for these patients is poor because of the lack of additional effective therapy. Accordingly, deciphering the cellular events associated with estrogen-dependent growth and the subsequent outgrowth of tumors with an estrogen-independent phenotype is of considerable interest. Here we show that the expression of PP5, an evolutionarily conserved Ser/Thr phosphatase that functions as an inhibitor of glucocorticoid- and p53-induced signaling cascades leading to growth suppression, is responsive to 17beta-estradiol (E(2)) in ER-positive human breast carcinoma cells (MCF-7). Northern analysis revealed that E(2)-induced PP5 expression is blocked by treatment with tamoxifen, and a consensus ER recognition element was identified in the PP5 promoter. The PP5-ER recognition element associates with human ERs and confers E(2)-induced transcriptional activation to reporter plasmids. The specific inhibition of PP5 expression ablates E(2)-mediated proliferation in MCF-7 cells without having an apparent effect on E(2)-induced expression of c-myc or cyclin D1. Thus, although critical for cell growth, PP5 likely acts either downstream or independently of c-Myc and Cyclin D1. To further characterize the role of PP5 in E(2)-regulated growth control, we constructed stable MCF-7 cell lines in which the expression of PP5 was placed under the control of tetracycline-regulated transactivator and operator plasmids. Studies with these cells revealed that the constitutive overexpression of PP5 affords E(2)-dependent MCF-7 cells with the ability to proliferate in E(2)-depleted media. Together, these studies indicate that E(2)-induced PP5 expression functions to enhance E(2)-initiated signaling cascades leading to cell division and that aberrant PP5 expression may contribute to the development of MCF-7 cells with an estrogen-independent phenotype.

Squirrel monkey immunophilin FKBP51 is a potent inhibitor of glucocorticoid receptor binding.

Squirrel monkeys have high circulating cortisol to compensate for expression of low-affinity glucocorticoid receptors (GRs). We have demonstrated that the FK506-binding immunophilin FKBP51 is elevated in squirrel monkey lymphocytes (SML) and, in preliminary studies, have shown that squirrel monkey FKBP51 is inhibitory to GR binding. In this report, we have demonstrated that elevated FKBP51 is the unequivocal cause of glucocorticoid resistance in SML in the following ways: 1) FK506 increased GR binding in cytosol from SML in a concentration-dependent manner, an effect reproduced by rapamycin but not cyclosporin A. The apparent K6 (6.1 nM) and rank-order of steroid displacement of [3H]dexamethasone binding in FK506-treated SML cytosol are characteristic of high-affinity GR binding. 2) cytosol from COS-7 cells expressing squirrel monkey FKBP51 inhibited GR binding in cytosol from human lymphocytes by 74%. Cytosol from COS-7 cells expressing human FKBP51 inhibited GR binding by 23%. 3) expression of squirrel monkey FKBP51 increased the median effective concentration (EC50) for dexamethasone in GR transactivation studies in COS-7 cells by approximately 17-fold, compared with the EC50 in control cells. The expression of human FKBP51 increased the EC50 for dexamethasone in COS-7 cells by less than 3-fold, compared with control. Squirrel monkey FKBP51 shares 94% overall amino acid homology with human FKBP51, with 92% and 99% homology with human FKBP51 in the peptidyl-prolyl isomerase and the tetratricopeptide repeat domains, respectively. Amino acid differences in the more variable N- or C-terminal regions or in regions which join the highly homologous functional domains may be responsible for its more potent inhibitory activity.

Isolation and characterization of the human secretogranin II gene promoter.

The goal of this study was to isolate and functionally characterize the human secretogranin II (SgII) gene promoter. SgII is a member of the granin family of proteins which are selectively expressed in neurosecretory cells. The human SgII promoter contains a consensus TATA box and cyclic AMP response element (CRE) 35 and 74 bp upstream of the transcription start site, respectively, elements also found in the mouse and rat SgII gene promoters. Transfection studies showed that 869 bp of the human SgII promoter were sufficient to confer cell type-specific expression of an SgII promoter-luciferase reporter gene in neurosecretory PC-12, GH and BE(2)-M17 cells. The activity of the human SgII promoter was also compared in three N-type, human neuroblastoma cell lines [BE(2)-M17, SMS-KAN and SH-SY5Y], which differ markedly in the level of SgII expression. SgII promoter activities in the neuroblastoma cell lines correlated not only with the levels of SgII but also the levels of the cyclic AMP response element-binding protein CREB which were highest in BE(2)-M17 cells and lowest in SH-SY5Y cells. To establish that the activity of the human SgII promoter in these neuroblastoma cell lines is dependent on the level of CREB, rat CREB was overexpressed in SH-SY5Y cells. SgII promoter activity was up to 8-fold higher in SH-SY5Y cells overexpressing CREB. These results suggest that SgII expression is a marker for neuronal differentiation in human neuroblastoma cell lines and is dependent on the level of CREB expression.

Ser/Thr protein phosphatase type 5 (PP5) is a negative regulator of glucocorticoid receptor-mediated growth arrest.

Ligand-induced glucocorticoid receptor (GR) activation has recently been linked to the inhibition of cell proliferation via the transcriptional induction of p21(WAF1/Cip1), which functions as a universal inhibitor of cyclin-dependent protein kinases. Herein, we identify a Ser/Thr protein phosphatase (PP5) that promotes cellular proliferation by inhibiting both glucocorticoid and p53-mediated signaling pathways leading to p21(WAF1/Cip1)-mediated growth arrest. The suppression of PP5 expression (1) markedly increases the association of GR with its cognate DNA-binding sequence, (2) induces GR transcriptional activity without the addition of hormone, and (3) increases dexamethasone-mediated induction of GR reporter activity to a level that is approximately 10 times greater than the maximal response obtainable in the presence of PP5. PP5 has no apparent effect on the binding of hormone to the GR, and dexamethasone-mediated growth arrest correlates with an increase in p53 phosphorylation. Comparative studies in p53-wild-type, p53-defective, and p53-deficient cell lines indicate that either (1) p53 participates in GR-mediated induction of p21(WAF1/Cip1), with the hyperphosphorylation of basal p53 induced by glucocorticoids sufficient for the propagation of an antiproliferative response when PP5 expression is inhibited, or (2) PP5 acts where p53-mediated and GR-induced signaling networks converge to regulate the transcriptional induction of p21(WAF1/Cip1). Thus, aberrant PP5 expression may have an additive effect on the development of human cancers by promoting cell proliferation via the inhibition of a GR-induced antiproliferative signaling cascade, and facilitating neoplastic transformation via the inhibition of a growth-arresting p53-mediated response that guards against genomic instability.

Glucocorticoid resistance in the squirrel monkey is associated with overexpression of the immunophilin FKBP51.

Squirrel monkeys are neotropical primates that have high circulating cortisol to compensate for expression of glucocorticoid receptors (GRs) with reduced affinity. The low binding affinity of squirrel monkey GR does not result from substitutions in the receptor, because squirrel monkey GR expressed in vitro exhibits high affinity. Rather, squirrel monkeys express a soluble factor that, in mixing studies of cytosol from squirrel monkey lymphocytes (SML) and mouse L929 cells, reduced GR binding affinity by 11-fold. In an effort to identify this factor, the cellular levels of components of the GR heterocomplex in SML and human lymphocytes (HL) were compared. The immunophilin FKBP51 was 13-fold higher in SML than in HL cytosol; FKBP52 in SML was 42% of that in HL cytosol. A role for changes in immunophilins, causing glucocorticoid resistance in neotropical primates, is supported by the following: the changes in FKBP51 and FKBP52 were observed in cells from other neotropical primates with glucocorticoid resistance; the elevated level of FKBP51 was reflected in an abundance of FKBP51 in heat shock protein 90 complexes in SML; when cytosols of SML and L929 cells were mixed, the decrease in GR binding was associated with incorporation of FKBP51 into GR heterocomplexes; the effect of SML cytosol on GR binding was reproduced with cytosol from COS cells expressing squirrel monkey FKBP51; and both the effect of SML cytosol on GR binding and the incorporation of FKBP51 into GR heterocomplexes were blocked by FK506. Regulation of GR binding by FKBP51 represents a previously unrecognized mechanism for regulating glucocorticoid sensitivity.

PACAP colocalizes with luteinizing and follicle-stimulating hormone immunoreactivities in the anterior lobe of the pituitary gland.

Pituitary adenylate cyclase activating polypeptide (PACAP) and its close relative vasoactive intestinal polypeptide (VIP) were demonstrated in the anterior pituitary gland. The cells which exhibited PACAP immunoreactivity were oval or round shaped. Their distribution was similar to that of gonadotropes but the number of PACAP immunoreactive cells was less. Double labeling revealed that PACAP immunoreactivity partially colocalized with luteinizing and follicle-stimulating hormone; however, colocalization with other pituitary hormone immunoreactivities was not demonstrated. Our results suggest an autocrine or paracrine role of PACAP in the regulation of pituitary functions.

The cAMP-response element mediates induction of secretogranin II by CHX and FSK in GH4C1 cells.

The effect of cAMP on secretogranin II (SgII) gene transcription in GH4C1 (GH) cells is not observed unless protein synthesis is inhibited. We have defined elements in the SgII promoter that mediate regulation by cycloheximide (CHX) and forskolin (FSK) and characterized the nuclear proteins that interact with them. GH cells were transfected with p2774Luc, p351Luc, p242Luc, and p223Luc containing 2,612, 189, 80, and 61 bp of the SgII promoter upstream of the luciferase gene, respectively. Treatment with CHX and FSK increased promoter activity 8- to 12-fold in cells transfected with p2774Luc, p351Luc, and p242Luc but had not effect in cells transfected with p223Luc. The same 19-bp element (-80 to -62) mediates regulation by CHX alone, as CHX caused a 3.8-fold increase in activity in GH cells transfected with p242Luc but not p223Luc. Gel mobility shifts using sequences -84 to -53 resulted in three complexes, which contained cAMP response element-binding protein heterodimerized with cAMP response element modulator or activating transcription factor-1. No differences were observed in complex formation when cells were treated with either CHX, FSK, or CHX and FSK. Thus CHX affects the response to FSK in GH cells by inhibiting the synthesis of a protein, which does not itself interact with DNA or affect the binding of CRE-binding proteins with the SgII promoter, but likely interferes with the interaction of CRE-binding proteins with the general transcriptional machinery.

Glucocorticoid-resistant B-lymphoblast cell line derived from the Bolivian squirrel monkey (Saimiri boliviensis boliviensis).

The goal of the study reported here was to develop a continuous cell line from the squirrel monkey that expresses the species-specific phenotype of impaired sensitivity to glucocorticoids. Thirty milliliters of blood from a male Bolivian squirrel monkey (Saimiri boliviensis boliviensis) was fractionated, and the buffy coat was obtained and incubated in the presence of B95-8 cell-conditioned medium, an abundant source of Epstein-Barr virus (EBV), and 2 micrograms of cyclosporin A/ml. Cell growth was detected within 8 weeks, after which the cells were cloned by use of the limiting dilution method. One clone (4D8) was characterized in detail. The chromosomal count and G-banding pattern confirmed that the cells were of Bolivian squirrel monkey origin. The B-cell origin of these cells was indicated by electron microscopic analysis and was confirmed by expression of CD20. The cells stained strongly for LMP1, a marker of latent EBV infection, and occasionally for the lytic infection marker ZEBRA (BZLF1). The responsiveness of clone 4D8 cells to glucocorticoids was determined by comparing the effects of dexamethasone on cell growth and the induction of a glucocorticoid-inducible mRNA in 4D8 cells with the effects on a human EBV-transformed B-lymphoblast cell line (HL). Dexamethasone inhibited the growth of HL cells, with IC50 of approximately 9 nM, but had no effect on the growth of 4D8 cells. The induction of FK506-binding protein FKBP51 mRNA by dexamethasone was also significantly blunted in 4D8 cells. Thus, we have developed and characterized a squirrel monkey lymphoblastic cell line derived by transformation of B-lymphocytes with EBV; the cell line has diminished growth and transcriptional responses to glucocorticoids.

Epidermal growth factor reduces L-type voltage-activated calcium current density in GH4C1 rat pituitary cells.

Long-term treatment of rat pituitary tumor cells with epidermal growth factor (EGF) inhibits 45Ca2+ uptake, intracellular calcium levels and subsequent prolactin secretion in response to membrane depolarization. In the present study we have used whole-cell voltage-clamp and single-channel patch-clamp recording to determine directly the effects of EGF (10 nM for 48 h) on L-type calcium current density, the current-voltage relationship, single-channel amplitude, and opening and closing dwell times in rat GH4C1 pituitary tumor cells. Sustained, nimodipine-sensitive inward currents (barium as the carrier) with an activation threshold of approximately -30 mV were elicited in both control and EGF-treated GH4C1 cells by depolarization. Mean current density normalized to membrane capacitance was reduced to 45% of control after EGF treatment. There was no difference in the voltage-dependent activation of L-type channels between control and EGF-treated cells. Analysis of single-channel current recordings showed that EGF treatment had no effect on unitary current amplitude or channel open and close durations. These results suggest that EGF reduces the number of voltage-gated calcium channels in GH4C1 cell membranes, which likely contributes to the decreased calcium uptake.

Cloning and expression of the glucocorticoid receptor from the squirrel monkey (Saimiri boliviensis boliviensis), a glucocorticoid-resistant primate.

New World primates such as the squirrel monkey have elevated cortisol levels and glucocorticoid resistance. We have shown that the apparent binding affinity of the glucocorticoid receptor in squirrel monkey lymphocytes is 5-fold lower than that in human lymphocytes (apparent Kd, 20.9 +/- 1.8 and 4.3 +/- 0.2 nmol/L, respectively; n = 3), consistent with previous studies in mononuclear leukocytes isolated from the two species. As a first step in understanding the mechanism of decreased binding affinity in New World primates, we used reverse transcription-PCR to clone the glucocorticoid receptor from squirrel monkey liver and have compared the sequence to receptor sequences obtained from owl monkey liver, cotton-top tamarin B95-8 cells, and human lymphocytes. The squirrel monkey glucocorticoid receptor is approximately 97% identical in nucleotide and amino acid sequence to the human receptor. The ligand-binding domain (amino acids 528-777) of the squirrel monkey glucocorticoid receptor contains four amino acid differences (Ser551 to Thr, Ser616 to Ala, Ala618 to Ser, and Ile761 to Leu), all of which are present in owl monkey and cotton-top tamarin receptors. The DNA-binding domain (amino acids 421-486) is completely conserved among human, squirrel monkey, owl monkey, and cotton-top tamarin receptors. Twenty-two differences from the human sequence were found in the N-terminal region (amino acids 1-421) of the squirrel monkey receptor. None of the substitutions in the ligand-binding domain matched mutations known to influence binding affinity in other species. To determine whether the substitutions per se were responsible for decreased affinity, squirrel monkey and human glucocorticoid receptors were expressed in the TNT Coupled Reticulocyte Lysate System. Expressions of human and squirrel monkey glucocorticoid receptors and a squirrel monkey receptor in which Phe774 was mutated to Leu (F774L) were similar. When expressed in the TNT System, squirrel monkey and human glucocorticoid receptors had similar, high affinity binding for dexamethasone (apparent Kd, 5.9 +/- 1.2 and 4.3 +/- 0.5 nmol/L, respectively; n = 3), whereas the squirrel monkey F774L receptor had lower affinity binding (apparent Kd, 20.4 +/- 2.0 nmol/L; n = 3). Thus, substitutions within the ligand-binding domain of the squirrel monkey glucocorticoid receptor cannot account for the decreased binding affinity of these receptors in squirrel monkey cells. Rather, the binding affinity is probably influenced by the expression of cytosolic factors that affect glucocorticoid receptor function.

Cell-specific expression of the rat secretogranin II promoter.

Secretogranin II (SgII) is a member of the granin family of secretory proteins, which are selectively expressed in neuroendocrine cells. As a first step in understanding the molecular basis for cell type-specific expression of SgII, we isolated a 12-kb clone from a rat genomic library that contained the entire rat SgII coding region, the transcription initiation site, and approximately 3 kb of 5'-flanking region. Within 75 bp of the transcription start site (+1) we located a TATA box and a consensus cAMP responsive element. Within the 5'-flanking region, a number of potential cis-acting elements were identified, including 2 Pit-1 binding sites, 15 E box motifs, and near-perfect matches for AP-1 and AP-2 sites. To demonstrate cell type-specific expression the rat SgII gene, a plasmid containing 2.6 kb of the 5'-flanking region of the SgII gene fused to the luciferase reporter gene (p2774Luc) was transfected into rat pheochromocytoma PC-12 cells, rat pituitary GH4C1 (GH) cells, human BE(2)-M17 (M17) neuroblastoma cells, and mouse fibroblast NIH/3T3 cells. The promoter activity was 6- to 36-fold higher in neuroendocrine cells than in NIH/ 3T3 cells. Progressive deletions in the 5'-flanking region to 61 bp upstream of the start site (p223Luc) had no effect on promoter activity in PC-12 cells. On the other hand, a 5'-deletion in the SgII promoter to -1032 increased promoter activity 3.8-fold in GH cells. This level of expression was maintained when the SgII promoter was further truncated to -189, whereas truncation to -61 resulted in a 2.6-fold reduction in promoter activity. These results suggest that the sequence between -61 and +162 bp is sufficient for SgII promoter activity in PC-12 cells. However, other elements in the 5'-flanking region contribute to both positive and negative regulation of the rat SgII gene in GH cells.

Changes in bioactive prolactin-like activity in plasma and its relationship to incubation behavior in breeding ring doves.

Previous radioimmunoassay (RIA) data indicate that plasma prolactin (PRL) is elevated during the late incubation and the early posthatching periods of the ring dove breeding cycle. Although these changes are temporally associated with changes in PRL-dependent crop sac growth, the precise relationship between immunoreactive and bioactive PRL has not been directly examined. To investigate this question and to further explore the relationship between sitting behavior and PRL secretion, we used rat Nb2 lymphoma cell proliferation to estimate the concentration of bioactive PRL-like activity (PLA) in the plasma of breeding ring doves. Serial dilutions of dove pituitary homogenate and dove plasma stimulated mitogenic responses that were parallel to those observed with purified ovine PRL. Changes in plasma PLA during the breeding cycle closely resembled changes in PRL that have been previously reported by RIA, although the relative changes in PLA were more pronounced. In both sexes, PLA remained at basal levels prior to egg laying and during early incubation (Day 4-5) but then abruptly increased to reach peak values near the time of hatching (Day 14-15). Activity remained high for 3-4 days after hatching, declined gradually thereafter, and returned to baseline values by Posthatching Days 14-17. Plasma PLA levels of birds sampled at the end of incubation were correlated with those of their breeding partners. In the majority of pairs, females had higher PLA levels than their mates at this stage even though no significant overall sex differences in PLA levels were observed. Plasma PLA declined precipitously in birds that were nest deprived on the last day of the incubation period. Nevertheless, plasma PLA levels of normally breeding birds at the end of incubation were not correlated with the average time spent in the nest during the incubation period. However, day-to-day variability in time spent in the nest correlated negatively with plasma PLA in incubating males, and females exhibited a similar trend that approached significance. These data suggest (1) that published RIA estimates of PRL are reasonably accurate reflections of changes in bioactive PLA in dove plasma and (2) that while sitting duration itself is not strongly related to plasma PLA, large day-to-day fluctuations in nest occupation time are associated with reduced PLA levels in incubating doves.

Different ultrastructural localization of VIP and prolactin in anterior pituitary cells of rats chronically treated with estrogen.

In the present study we investigated the effect of a long-term estrogen treatment on the intracellular distribution of VIP immunoreactivity in pituitary prolactin cells using double-labeling immunocytochemistry. With the use of pre-embedding ABC method it was found that VIP immunoreactivity was associated with the outer surface of membrane-bound organelles, and was not found in secretory granules. However, prolactin immunoreactivity demonstrated by postembedding immunogold technique was mainly associated within the secretory granules of the same cells. The discrepancy between our and Hsu et al.'s results (1989), who observed VIP immunoreactivity in secretory granules of human anterior pituitary cells, may be owing to the overstimulation of VIP cells by estrogen. It is possible that estrogen treatment depleted the VIP content of the secretory granules and enhanced the cytosolic VIP. The appearance of an alternative form of VIP in estrogen-treated rats with preferential distribution in the cytosol cannot be excluded.

Regulation of secretogranin II mRNA in rat neuronal cultures.

The regulation of SgII mRNA expression was investigated in primary cultures of neurons prepared from the hypothalamus and brainstem of 1-day-old rats. The administration of forskolin (FSK) resulted in a time- and dose-dependent increase in SgII mRNA expression, a 9-fold effect within 6 h being achieved with 10 microM FSK, which maximally increased cellular cAMP levels. SgII mRNA levels remained elevated for 24 h. Activation of protein kinase C with 100 nM phorbol 12-myristate 13-acetate (PMA) also increased SgII mRNA expression, although induction with PMA was slower and more moderate (3.8-fold above control after 24 h). Neither 10 microM 1,9-dideoxyforskolin nor 100 nM 4 alpha-phorbol 12,13-didecanoate, inactive analogues of FSK and PMA respectively, had an effect on SgII mRNA. Depolarization of neuronal cultures with 50 mM KCl had a small and variable effect on SgII mRNA levels (1.8-fold above control) in neuronal cultures and did not influence induction with FSK. To investigate whether neuron-like regulation of SgII mRNA expression could be reproduced in PC12 cells, PC12 cells were treated with 100 nM nerve growth factor (NGF) for 7 days prior to challenge with FSK or PMA. Whereas NGF alone modestly increased SgII mRNA expression in PC12 cells (1.8-fold above control), it did not uncover a stimulatory effect of FSK or PMA. These studies indicate that SgII mRNA expression is enhanced by an increase in cellular cAMP and activation of protein kinase C in primary cultures of neurons and emphasize that SgII mRNA is regulated in a cell-specific manner.