Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

HBV X protein alters the DNA binding specificity of CREB and ATF-2 by protein-protein interactions.

The hepatitis B virus (HBV) X gene product trans-activates viral and cellular genes. The X protein (pX) does not bind independently to nucleic acids. The data presented here demonstrate that pX entered into a protein-protein complex with the cellular transcriptional factors CREB and ATF-2 and altered their DNA binding specificities. Although CREB and ATF-2 alone did not bind to the HBV enhancer element, a pX-CREB or pX-ATF-2 complex did bind to the HBV enhancer. Thus, the ability of pX to interact with cellular factors broadened the DNA binding specificity of these regulatory proteins and provides a mechanism for pX to participate in transcriptional regulation. This strategy of altered binding specificity may modify the repertoire of genes that can be regulated by transcriptional factors during viral infection.

Cyclic AMP-responsive DNA-binding protein: structure based on a cloned placental cDNA.

Cyclic AMP (cAMP) is an intracellular second messenger that activates transcription of many cellular genes. A palindromic consensus DNA sequence, TGACGTCA, functions as a cAMP-responsive transcriptional enhancer (CRE). The CRE binds a cellular protein of 38 kD in placental JEG-3 cells. A placental lambda gt11 library was screened for expression of specific CRE-binding proteins with the CRE sequence as a radioactive probe. A cDNA encoding a protein of 326 amino acids with the binding properties of a specific CRE-binding protein (CREB) was isolated. The protein contains a COOH-terminal basic region adjacent to a sequence similar to the "leucine zipper" sequence believed to be involved in DNA binding and in protein-protein contacts in several other DNA-associated transcriptional proteins including the products of the c-myc, c-fos, and c-jun oncogenes and GCN4. The CREB protein also contains an NH2-terminal acidic region proposed to be a potential transcriptional activation domain. The putative DNA-binding domain of CREB is structurally similar to the corresponding domains in the phorbol ester-responsive c-jun protein and the yeast transcription factor GCN4.

Structural determinants outside of the leucine zipper influence the interactions of CREB and ATF-2: interaction of CREB with ATF-2 blocks E1a-ATF-2 complex formation.

Dimerization of leucine zipper-containing proteins has been associated characteristically with the formation of a coiled-coil structure between two compatible leucine zipper motifs. In the present study we demonstrate the association of the leucine zipper of cAMP response element-binding protein (CREB) with a zinc finger motif of ATF-2. The association of the CREB leucine zipper with the ATF-2 zinc finger is stabilized if the ATF-2 leucine zipper is intact, implying that the preferred interactive structure of ATF-2 juxtaposes the amino-terminal zinc finger motif of this protein with the carboxy-terminal leucine zipper of this same protein. Furthermore, we demonstrate that the association of the CREB leucine zipper with the ATF-2 zinc finger in vitro blocks the association of the adenoviral E1a protein with ATF-2. Similarly, overexpression of full-length CREB, or a truncated version of this protein corresponding to the carboxy-terminal 74 amino acids that make up the DNA-binding and dimerization domains, can block the ATF-2-mediated transcriptional stimulation by E1a in vivo. Mutation of the ATF-2 zinc finger motif stimulates DNA binding of this protein, and abolishes interactions with E1a and CREB proteins. These results demonstrate that the structural conformation of ATF-2 is critical for DNA binding and protein-protein interactions and, further, that leucine zippers can mediate protein-protein interactions with structural motifs other than leucine zippers.

Characterization of a cyclic AMP regulatory element DNA-binding protein.

Signal transduction pathways converge ultimately at the level of transcriptional activation to produce specific patterns of gene expression in response to environmental stimuli. The initiation of transcription mediated by these signaling pathways is regulated by the coordinate expression and/or activation of specific transcription factors that bind to the control regions of eukaryotic genes. Specific insights into the mechanisms underlying transcriptional activation have arisen from the studies of the structure and functions of eukaryotic transcription factors. One of these factors, a cyclic (c)AMP response element binding protein (CREB), has only recently been discovered and appears to play a key role in the regulation of gene expression in response to the activation of the cAMP-dependent protein kinase A. The transcription factors related to CREB, c-jun and c-fos, mediate transcriptional responses to activators of protein kinase C.

Upstream stimulatory factor, a basic-helix-loop-helix-zipper protein, regulates the activity of the alpha-glycoprotein hormone subunit gene in pituitary cells.

In an effort to determine whether basic-helix-loop-helix (bHLH) proteins are important in pituitary-specific expression of the alpha-glycoprotein hormone subunit gene, we examined the effect of the dominant negative HLH protein, Id, on the activity of the alpha-subunit gene promoter in pituitary cells. Id over-expression reduces the expression of alpha-subunit reporter genes in either alpha T3-1 gonadotrope-derived or alpha TSH thyrotrope-derived cells. A deletion fragment containing nucleotides from -131 to +44 of the human alpha-subunit promoter is inhibited to a similar degree as a -244 to +44 fragment in alpha T3-1 cells. Nuclear proteins in alpha T3-1 cells bind two potential bHLH protein binding sites (E-boxes, alpha EB1 and alpha EB2) present in this fragment but not to mutations that specifically alter only these sequences. An antibody-specific for upstream stimulatory factor, a widely expressed bHLH-leucine zipper protein, is able to inhibit factor binding to the alpha EB2 sequences but not the alpha EB1 site. Mutating the alpha EB1 element of the alpha-subunit promoter decreases basal activity of this promoter to about 42% of control levels in alpha T3-1 cells. A mutation that abolishes upstream stimulatory factor binding, either alone or in combination with the alpha EB1 mutation, reduces basal activity of the promoter to approximately 21% of control levels in alpha T3-1 cells and abolishes the decrease in promoter activity seen when Id is overexpressed. These results demonstrate that the bHLH family of proteins are important regulators of alpha-subunit gene expression in pituitary cells.

Distinct adenosine 3',5'-monophosphate and phorbol ester-responsive signal transduction pathways converge at the level of transcriptional activation by the interactions of DNA-binding proteins.

Evidence is presented that distinct cellular signal transduction pathways involving cAMP-dependent protein kinase-A and phorbol ester-stimulated protein kinase-C coordinately modulate gene transcription through common as well as distinct cis-acting elements and DNA-binding proteins. When transfected and expressed in HeLa and placental JEG-3 cells, fusion reporter plasmids that differ only by a single base deletion or addition to interconvert the octameric cAMP-responsive element TGACGTCA (CRE) to form the heptameric phorbol ester-responsive element TGACTCA (TRE) are differentially regulated by cAMP and phorbol esters [12-O-tetradecanoyl phorbol-14-acetate (TPA)]. Transcription directed by the CRE is stimulated by cAMP and not TPA, although the basal expression mediated by this element in JEG-3 and HeLa cells is augmented by endogenous protein kinase-C activity. In contrast, TRE mediates transcriptional responses to both cAMP and TPA, and the two agents together give synergistic responses. Inhibition of cAMP-dependent protein kinase-A by expression of a minigene encoding a peptide inhibitor of A-kinase abolishes the response of TRE to cAMP alone as well as the cAMP-induced component of the synergistic response to treatment with both TPA and cAMP. Desensitization of the protein kinase-C dependent pathway by prolonged exposure of cells to phorbol esters eliminates the TPA-induced transcription by TRE and inhibits the TPA-induced component of the synergistic response to both cAMP and TPA. Therefore, both protein kinases, A and C, are involved in transcriptional activation by the TRE; the function of either kinase alone results in a moderate level of activity, but the combined results of both functionally stimulated kinases are synergistically positive. Electrophoretic mobility shift assays using whole extracts of JEG-3 cells indicate that a common factor(s) binds both TRE and CRE; however, another factor(s) that binds to the CRE will not bind to the TRE. Further, a latent regulatory enhancer element (URE) located upstream of the CRE's in the human alpha gonadotropin gene, although inactive when paired alone with the alpha 100 promoter, induces basal and stimulated transcriptional activity of both CRE and TRE on the average of 10- to 20-fold. The data support the existence of a gene regulatory network consisting of related cis-acting elements and DNA-binding proteins whose transcriptional activities are regulated by the convergent actions of protein kinases-C and -A.

Role of the cyclic adenosine 3',5'-monophosphate response element in efficient expression of the rat thyrotropin receptor promoter.

The "minimal" promoter region of the TSH receptor gene, -195 to -39 basepairs (bp), exhibits basal promoter activity, thyroid specificity, and negative regulation by TSH via its cAMP signal. In FRT thyroid cells and by comparison to pTRCAT5'-199, 5'-deletion mutants of chloramphenicol acetyltransferase (CAT) constructs from -199 to -150 bp of the minimal promoter decrease basal CAT activity by 50%, whereas continued deletion to -146 bp increases activity more than 4-fold. Continued deletion to -131 bp results in basal activity less than that of the -199 bp construct. An octameric cAMP response element (CRE)-like sequence, TGAGGTCA, is within -146 to -131 bp and starts at -139 bp. Its mutation to a consensus CRE (TGACGTCA) or AP1 (TGAGTCA) site or mutation of several residues flanking its 3'-terminus can improve promoter activity as much as 8-fold compared to pTRCAT5'-199. A nonpalindromic mutation to CGAGGACA decreases basal promoter activity to the level of the 199-bp minimal promoter. The CRE-like sequence between -139 and -132 bp is a constitutive enhancer of promoter activity in FRT thyroid cells, since, ligated to a simian virus-40-promoter-driven CAT gene, it increases CAT activity in the absence of forskolin in proportion to copy number and independent of direction or position. It can, however, function as a cAMP-responsive CRE, as evidenced by the fact that forskolin increases the activity of the same simian virus-40-promoter-driven CAT gene constructs in Buffalo rat liver (BRL) cells. DNAase-I footprinting shows that the CRE region is protected by a purified binding region peptide of the CRE-binding protein, activating transcription factor-2, and recombinant AP1 (human c-jun) as well as by BRL, FRT, and FRTL-5 rat thyroid cell nuclear extracts. Gel mobility shift analyses show that multiple CRE-binding proteins in the BRL, FRT, and FRTL-5 cell nuclear extracts form complexes with the CRE-like site, that one of these is CRE-binding protein, and that all form complexes with mutant sequences of the CRE-like site in a manner that exactly parallels their effects on constitutive enhancer function in FRT thyroid cells. We show, therefore, that the CRE-like site in the minimal TSH receptor promoter functions as a constitutive enhancer of promoter activity in FRT thyroid cells yet is a cAMP-responsive CRE.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of multiple nuclear factors that interact with cyclic adenosine 3',5'-monophosphate response element-binding protein and activating transcription factor-2 by protein-protein interactions.

Understanding the nature and importance of protein-protein interactions in the mechanisms of eukaryotic gene expression is essential to understanding the normal and aberrant regulation of gene transcription. Using 125I-labeled cAMP response element-binding protein (CREB) and activating transcription factor-2 (ATF-2) recombinant peptides to probe Western blots of HeLa nuclear extracts, we have identified multiple separate nuclear factors that form specific protein-protein interactions with these leucine zipper-containing transcriptional regulatory proteins. The interaction is specific because preincubation of blots with cold homologous protein blocks the binding of labeled protein, whereas preincubation of blots with cold heterologous protein has no effect on labeled protein interactions. Although these studies focus on two specific transactivators, CREB and ATF-2, the approach is of general use for the study of other leucine zipper-containing mammalian transcription factors. Furthermore, in addition to allowing the detection of protein-protein interactions of CREB and ATF-2 with nuclear factors, we have used this strategy to isolate cDNA clones expressing these nuclear proteins. We demonstrate that CREB will form heterodimers with ATF-1, but not ATF-2, Jun, Fos, or C/EBP whereas, ATF-2 will form heterodimers with Jun and Fos, but not with C/EBP or ATF-1. This strategy, therefore, allows a systematic approach to identifying, characterizing, and cloning proteins involved in the control of eukaryotic transcriptional regulation. The identification and characterization of the components of eukaryotic transcription complexes will allow studies that address the molecular mechanisms of normal and abnormal control of cellular gene expression.

Multiple adenosine 3',5'-cyclic [corrected] monophosphate response element DNA-binding proteins generated by gene diversification and alternative exon splicing.

The protein sequence deduced from the open reading frame of a human placental cDNA encoding a cAMP-responsive enhancer (CRE)-binding protein (CREB-327) has structural features characteristic of several other transcriptional transactivator proteins including jun, fos, C/EBP, myc, and CRE-BP1. Results of Southwestern analysis of nuclear extracts from several different cell lines show that there are multiple CRE-binding proteins, which vary in size in cell lines derived from different tissues and animal species. To examine the molecular diversity of CREB-327 and related proteins at the nucleic acid level, we used labeled cDNAs from human placenta that encode two different CRE-binding proteins (CREB-327 and CRE-BP1) to probe Northern and Southern blots. Both probes hybridized to multiple fragments on Southern blots of genomic DNA from various species. Alternatively, when a human placental c-jun probe was hybridized to the same blot, a single fragment was detected in most cases, consistent with the intronless nature of the human c-jun gene. The CREB-327 probe hybridized to multiple mRNAs, derived from human placenta, ranging in size from 2-9 kilobases. In contrast, the CRE-BP1 probe identified a single 4-kilobase mRNA. Sequence analyses of several overlapping human genomic cosmid clones containing CREB-327 sequences in conjunction with polymerase chain reaction indicates that the CREB-327/341 cDNAs are composed of at least eight or nine exons, and analyses of human placental cDNAs provide direct evidence for at least one alternatively spliced exon. Analyses of mouse/hamster-human hybridoma DNAs by Southern blotting and polymerase chain reaction localizes the CREB-327/341 gene to human chromosome 2. The results indicate that there is a dichotomy of CREB-like proteins, those that are related by overall structure and DNA-binding specificity as well as those that are related by close similarities of primary sequences.

Antagonist-occupied human progesterone B-receptors activate transcription without binding to progesterone response elements and are dominantly inhibited by A-receptors.

When antagonist-occupied steroid receptors have agonist-like effects, the clinical consequences are grave. We present evidence that human progesterone B-receptors (hPRB) when occupied by progesterone antagonists, inappropriately activate transcription by an unusual mechanism that does not require the canonical progesterone response element (PRE). In HeLa cells cotransfected with a PRE-tk-chloramphenicol acetyltransferase reporter and a hPRB expression vector, strong transcription is seen not only when receptors are activated by the agonist R5020, but also in the presence of the three antiprogestins, RU486, ZK112993, and ZK98299. Human PRB occupied by ZK98299 do not bind to a PRE, suggesting that the transcriptional stimulation is independent of DNA binding. Indeed, a tk-chloramphenicol acetyltransferase promoter-reporter lacking the PRE loses transcriptional activation by the agonist, but retains transactivation by the three antagonists. The PRE-independent antagonist-induced transcription requires that hPRB have an intact DNA-binding domain, but hPR target gene specificity is not required, because a hPRB mutant that binds an estrogen response element still activates transcription. It appears that antagonist-occupied hPR activate transcription without binding to a PRE, perhaps by interacting with tethering proteins instead. Even a gene that is not a normal progesterone target could be aberrantly activated. Human cells contain equimolar amounts of hPRB and the N-terminally truncated natural isotype, hPRA. Unlike hPRB, hPRA are not transcriptionally activated by progesterone antagonists. We, therefore, tested the effects of antagonists when the two receptor isotypes are coexpressed and found that A-receptors can annul the inappropriate transcription by B-receptors. Thus, when both receptor forms are present, the hPRA phenotype is dominant. Moreover, pure hPRB/hPRA heterodimers, produced by fos/jun leucine zipper domain-hPR chimeras, also have the inactive transcriptional phenotype of hPRA. Our studies suggest not only that the two hPR isotypes are functionally quite different, but also that some of the agonist-like transcriptional effects of antagonist-occupied B-receptors proceed through novel mechanisms.

Modification of DNA topoisomerase II activity via direct interactions with the cyclic adenosine-3',5'-monophosphate response element-binding protein and related transcription factors.

DNA topoisomerase II (topo II) is an essential nuclear enzyme which catalyzes the interconversions of various forms of DNA. As predicted from the human topo II cDNA, the enzyme contains a potential leucine zipper protein dimerization motif. We therefore tested whether topo II could enter protein-protein interactions with other better characterized leucine zipper-containing proteins and determined if these interactions could modify topo II enzymatic activity in vitro. By far Western analyses, a large C-terminal fragment of human topo II was shown to interact with the DNA binding and dimerization regions of either cAMP response element binding protein (CREB) or the activating transcription factor-2. The C-terminal topo II fragment also interacted with full-length c-Jun, but not with full-length c-Fos. Using CREB as a prototype, the effect of this interaction on various topo II catalytic activities was assessed in vitro. CREB, at a 1- to 10-fold molar excess relative to topo II, inhibited site-specific DNA cleavage activity on a 242-base pair fragment of the human alpha-glycoprotein hormone subunit gene promoter. Very high CREB concentrations (400-fold excess) apparently inhibited topo II DNA relaxation activity, but this result was likely a direct effect of CREB on the topology of the DNA substrate. More interestingly, a 10-fold molar excess of CREB stimulated topo II decatenation activity, the essential function of this enzyme in cell division. This stimulatory effect could also be elicited by c-Jun, which interacts with topo II, but not by c-Fos, which does not bind topo II in our in vitro assay. Since similar amounts of CREB reduced the abundance of topo II DNA cleavage products from the human alpha-CG promoter yet also stimulated decatenation activity, it can be concluded that either: 1) CREB stimulated the religation rate of topo II; or 2) CREB directed topo II to a new cleavage site present on the decatenation substrate but not present on the limited alpha-CG promoter. The structural requirements for topo II protein-protein interactions were also investigated. Site-directed mutations which destroyed the putative topo II leucine zipper did not disrupt topo II protein-protein interactions. Since the putative leucine zipper in topo II does not appear to mediate protein-protein interactions, we propose that an alternate as yet uncharacterized structure is involved in the association of topo II with itself and other regulatory proteins.

Activating transcription factor-2 DNA-binding activity is stimulated by phosphorylation catalyzed by p42 and p54 microtubule-associated protein kinases.

Recent studies have detailed the ability of activating transcription factor-2 (ATF-2) to mediate adenoviral E1a stimulation of gene expression; however, an endogenous regulator for the transcriptional activity of this protein has not been described. To characterize the regulation of ATF-2 activity, we have expressed full-length and truncated peptides corresponding to various regions of the ATF-2 protein in bacteria and the baculovirus insect cell system. Bacterially expressed truncated (350-505) but not full-length ATF-2, was able to bind a consensus cAMP response element-containing oligonucleotide, suggesting the N-terminal moiety may serve as a negative regulator of DNA-binding activity. In contrast, the full-length ATF-2 protein expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus was fully competent to bind DNA. Protein phosphatase 2A reversed the DNA-binding activity by dephosphorylating the ATF-2 polypeptide. Microtubule-associated protein kinase catalyzed the phosphorylation and stimulated the DNA-binding activity of bacterially expressed full-length ATF-2. Phosphopeptide mapping of phosphorylated ATF-2 proteins identified a single peptide in the N-terminal moiety of ATF-2 phosphorylated by p42 or p54 microtubule-associated protein kinase. Therefore, we propose that phosphorylation of this regulatory site is sufficient to induce an allosteric structural change in the ATF-2 protein, which allows dimerization and subsequent DNA binding.

Structure/function relationships of CREB/ATF proteins.

Signal-transduction pathways converge ultimately at the level of transcriptional activation to produce specific patterns of gene expression in response to environmental stimuli. The initiation of transcription mediated by these signaling pathways is regulated by the coordinate expression and/or activation of specific transcription factors that bind to the control regions of genes. Specific insights into the mechanisms underlying transcriptional activation have recently arisen from studies of the structure and functions of these transcription factors. The CREB/ATF family of transcriptional transactivating proteins has only recently been discovered and appears to provide a link between the regulation of gene expression in response to activators of cellular signaling pathways and the regulation of gene expression by viral transactivating proteins. In addition, these proteins may be involved in the normal regulation of growth and differentiation. Understanding the nature and importance of the role(s) of these proteins in the normal regulation of growth and differentiation will have profound influences on the understanding of the aberrant regulation of these processes during oncogenesis.

Ras mutations in human melanoma: a marker of malignant progression.

In this study we address whether there is an association between ras mutations and disease progression in malignant melanoma. DNA was extracted from 100 paraffin-embedded melanomas and sequences around the 12th, 13th and 61st codons of N-, H-, and K-ras were amplified using the polymerase chain reaction and probed for single base pair mutations using synthetic oligonucleotide probes. Thirty-six melanomas contained mutations, which in 25 cases (69%) occurred at the 61st codon of N-ras. The results from dot blot hybridizations were confirmed by subcloning and sequencing the polymerase chain reaction products from two tumors. No ras mutations were found in Clark's level I melanomas, whereas 19% of level II and 45% of the more advanced primary tumors contained ras mutations (Chi squared test: p < 0.05). The median Breslow thickness of primary melanomas with ras mutations was 0.72 mm, significantly thicker than the 0.42 mm of melanomas without mutations (Mann-Whitney U test, p = 0.042). Ras mutations were found more frequently in primary tumors from continuously exposed skin (56%) than tumors from intermittently or non-sun exposed sites (21%). Fifty percent of locally recurrent and 47% of metastatic melanomas had ras mutations. We conclude that ras mutations occur in a subset of melanomas from sun-exposed skin as a feature of tumor progression.

Liver tissue produces a potent lactogen that partially mimics the actions of prolactin.

An in vitro bioassay (based on the detection of casein release from isolated mammary cells by reverse hemolytic plaque assay) was used to detect a lactogenic factor secreted by liver tissue from suckled rats. This material stimulates casein release in the absence of added PRL and is actually more potent than PRL in this regard. The substance(s) possesses the following additional characteristics: it is released from liver tissue of lactating but not virgin female or male rats; when tested together with PRL, its effects are additive rather than synergistic; and unlike PRL, it does not increase the proportion of mammary cells committed to casein release. These findings are consistent with the possibility that a Liver Lactogenic Factor may mediate, at least in part, the lactogenic component of PRL's action. This functional relationship may be similar to that which exists between GH and the somatomedins.

Capacity of individual somatotropes to release growth hormone varies according to sex: analysis by reverse hemolytic plaque assay.

A reverse hemolytic plaque assay was used to investigate the mechanism(s) underlying sexual differences in GH release which evolve at puberty in rats. The percentages of GH-secreting cells in 24-h pituitary cultures from each sex were similar for pituitary donors up to 30 days of age (range = 38.9% to 41.7% of all cells in culture, n = 3 separate experiments) but decreased by day 50. The decrease was more striking for females (to 24.1 +/- 0.3% mean +/- SE) than for males (to 33.2 +/- 1.1%). However, owing to the greater increase in total pituitary cell number exhibited by female rats at this time, the absolute numbers of somatotropes recovered from male and female pituitaries were almost identical on 50 and 100 days of age. To assess the secretory capacities of individual somatotropes, we measured the sizes of plaques formed. In prepubertal rats (days 10-30), the plaque areas under basal conditions were comparable for males and females at each age studied, and treatment with GH-releasing factor increased plaque sizes to approximately the same degree (10-fold) for both sexes at each age. However, by day 100, plaques that formed under both basal and stimulated conditions were consistently larger (P less than 0.01) for male than for female donors. Taken together, our results demonstrate that sexual differences in GH release are attributable to the secretory capacities of individual somatotropes rather than to differences in the numbers of GH cells in pituitaries of male and female rats.

Hypothalamic factors differentially affect the proportions of cells that secrete growth hormone or prolactin.

Hypothalmic factors have been shown to regulate acutely the synthesis and release of adenohypophyseal hormones, yet few studies have investigated the long term effects of these agents on adenohypophyseal cell types. In the present study, we assessed the chronic influence of selected hypothalamic factors on the relative proportions of GH- and PRL-secreting cells in pituitary cultures derived from 5-day-old rats. Primary cultures were established and incubated for 6 days in the presence or absence of 0.1-microM doses of GH-releasing factor, LHRH, CRF, TRH, or vasoactive intestinal polypeptide and then subjected to reverse hemolytic plaque assays for analysis of the percentages of cells that released GH or PRL. In cultures from males, GH-releasing factor and LHRH treatment caused an increase in the proportion of PRL secretors and a commensurate decrease in the GH population. CRF increased PRL cells without affecting the GH secretors, while TRH reduced the percentages of both cell types, and vasoactive intestinal polypeptide had no effect. Virtually identical results were obtained for cells isolated from females. These results demonstrate that hypothalamic factors have the capacity to induce differential effects on the proportions of GH- and PRL-secreting cells. Surprisingly, our findings also show that hypothalamic factors that do not normally influence the acute release of GH or PRL can exert a chronic effect on the proportion of cells that secrete these hormones.

Existence of somatotrope subpopulations which are differentially responsive to insulin-like growth factor I and somatostatin.

It is generally accepted that hypothalamic somatostatin and hepatic insulin-like growth factor I (IGF-I)/somatomedin-C act directly on the pituitary to inhibit GH release, but it is not known whether all somatotropes are responsive to these agents. In the present study, we used a reverse hemolytic plaque assay to compare the acute (8 h) effects of somatostatin and IGF-I on the release of GH from individual cells in 24-h cultures of male rat pituitaries. Treatment with these factors caused comparable dose-dependent decreases in both the rate of plaque formation and the percentage of cells which released GH. In 8-h incubations, maximal (10(-8) M) doses of IGF-I or somatostatin alone decreased the percentage of GH-releasing cells to approximately the same degree (from 34.4% in controls to 29.7% and 28.4%, respectively), yet the effects of these factors were additive when both agents were applied to the same cells (to 24.5%). When we analyzed the sizes of plaques (an index of the amount of hormone released per cell) which resulted from these treatments, we noted that somatostatin was a much greater suppressor (to 11% of control value) of GH release than IGF-I (60% of controls). Coincubation with 10(-8) M GH-releasing factor had no effect on the percentage of GH-releasing cells at 8 h but completely overrode the inhibitory effect of IGF-I on plaque size without affecting the somatostatin-induced decrease in this regard. Taken together, these data suggest that IGF-I and somatostatin act, at least in part, on separate subpopulations of rat somatotropes. Somatostatin is a much more effective inhibitor of total GH release than IGF-I and appears to affect most, if not all, somatotropes. In contrast, IGF-I acutely inhibits GH release (prevents plaque formation) from some somatotropes, but does not seem to affect the remaining GH cells.

Functional maturation of somatotropes in fetal rat pituitaries: analysis by reverse hemolytic plaque assay.

Immunocytochemistry (ICC) and a reverse hemolytic plaque assay for GH were used to investigate the temporal relationships between the initiation of hormone storage and release by developing somatotropes and the onset of responsiveness of these cells to stimulatory and inhibitory secretagogues. Anterior pituitaries obtained from rats on days 18-21 of fetal development (pups were generally delivered on fetal day 22, which is equivalent to day 0 of neonatal life) were monodispersed with trypsin, cultured for 24 h, and then subjected to reverse hemolytic plaque assay and/or ICC for GH. GH-containing cells (determined by ICC) were extremely rare (less than 1%) in cultures derived from day 18 fetuses, but accounted for 22.4%, 25.2%, and 24.5% of all cells in cultures from day 19-21 fetuses, respectively. The proportion of GH-releasing cells, as determined in a long term (120-min incubation with antibody) plaque assay, was less than 1%, 22.4%, and 22.9% for days 18, 20, and 21, respectively, but only 13.6% for day 19 cells. Thus, many pituitary cells from day 19 fetuses contained, but did not release, GH. While GH-releasing factor (1-44) (1 X 10(-7) M) had no effect on the percentage of GH plaque-forming cells in long term incubations, it enhanced (by approximately the same degree in day 19-21 groups) the percentage of cells that formed plaques and the size of the plaques in short term (45-min) incubations with antibody. Somatostatin (1 X 10(-7) M) exerted inhibitory effects on these variables when tested in long term incubations, and age of the donor rats did not influence pituitary responsiveness to this secretagogue. These results suggest that the capacities of fetal somatotropes to store GH and release it under basal and regulated conditions are attained, in large part, within an extremely narrow time frame between days 18 and 19 of fetal development.