Cyclic GMP stimulates inositol phosphate production in cultured pituitary cells: possible implication to signal transduction

Addition of the stable and permeable analog 8-bromo cyclic GMP (8-BR-cGMP) to myo-[2-3H]inositol prelabeled cultured rat pituitary cells results in enhanced formation of [3H]-myo-inositol monophosphate (IP1). The stimulatory effect of the cyclic nucleotide analog is additive to the effect of Li+, which accumulates IP1 via inhibition of inositol 1-monophosphatase, and also to the effect of gonadotropin releasing hormone (GnRH) which stimulates the formation of IP1, as well as that of inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3) via enhanced hydrolysis of polyphosphoinositides. Many Ca2(+)-mobilizing hormones acting via phosphoinosite turnover also stimulate cGMP formation. The cyclic nucleotide might then serve as a modulator by further hydrolysis of phosphoinositides needed for protein kinase C activation.     

Differential expression of multiple protein kinase C subspecies in rat central nervous tissue

Protein kinase C from a number of areas of rat central nervous tissue was resolved into three distinct fractions upon hydroxyapatite column chromatography. One of the enzyme fractions, designated type II, could be further distinguished into two subspecies with polyclonal antisera, which were raised against synthetic peptides specific for the predicted amino acid sequences of two alternative cDNA clones encoding this enzyme type. Using a combination of these biochemical and immunological techniques, the relative activity of the multiple subspecies of protein kinase C was assessed for each brain area. A distinct regional pattern of expression was found, which per se may be an important factor in determining the response of different neuronal cell types to extracellular stimuli.     

Effect of phorbol ester on stimulus-secretion coupling mechanisms in gonadotropin releasing hormone-stimulated pituitary gonadotrophs

The feedback regulatory control mechanism exerted by activated Ca2+/phospholipid-dependent protein C kinase upon gonadotropin releasing hormone (GnRH) binding, stimulation of phosphoinositide turnover and gonadotropin secretion was investigated in cultured pituitary cells. Addition of the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), at concentrations which activate pituitary protein C kinase, to cultured pituitary cells resulted in up-regulation of GnRH receptors (155% at 4 h). The stimulatory effect of GnRH on [3H]inositol phosphates (Ins-P) production in myo-[2-3H]inositol prelabeled pituitary cells was not inhibited by prior treatment of the cells with TPA (10(-9)-10(-7) M). Higher concentrations of TPA (10(-6)-10(-5) M) inhibited the effect of GnRH on [3H]Ins-P production. Increasing concentrations of TPA or the permeable analog of diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) stimulated luteinizing hormone (LH) release from cultured pituitary cells with ED50 values of 5 x 10(-9) M and 10 micrograms/ml, respectively. No consistent inhibition or additivity of LH release was observed when increasing doses of TPA or OAG were added with a submaximal dose of GnRH. These results suggest that protein C kinase might mediate the known homologous up-regulation of GnRH receptors during the reproductive cycle. Protein C kinase is positively involved in mediating the process of gonadotropin secretion. Unlike many other systems, activation of protein C kinase in pituitary gonadotrophs is not involved in negative feed-back regulation of stimulus-secretion-coupling mechanisms in GnRH-stimulated gonadotrophs.     

Human cholinesterase genes localized by hybridization to chromosomes 3 and 16

Summary A cloned human cDNA for cholinesterase (ChE) was used as a probe for in situ hybridization to spread lymphocyte chromosomes to map the structural human CHE genes to distinct chromosomal regions. The recent genetic linkage assignment of the CHE1 locus of the CHE gene to chromosome 3q was confirmed and further refined to 3q21-q26, close to the genes coding for transferrin (TF) and transferrin receptor (TFRC). The CHE1 allele localizes to a 3q region that is commonly mutated and then associated with abnormal megakaryocyte proliferation in acute myelodysplastic anomalies. In view of earlier findings that ChE inhibitors induce megakaryocytopoiesis in culture, this localization may indicate that ChEs are involved in regulating the differentiation of megakaryocytes. A second site for ChEcDNA hybridization was found on chromosome 16q11-q23, demonstrating that the CHE2 locus of the cholinesterase gene, which directs the production of the common C5 variant of serum ChE, also codes for a structural subunit of the enzyme and is localized on the same chromosome with the haptoglobin (HP) gene, both genes being found on the long arm of chromosome 16. The finding of two sites for ChEcDNA hybridization suggests that the two loci coding for human ChEs may include nonidentical sequences responsible for the biochemical differences between ChE variants.     

Human acetylcholinesterase and butyrylcholinesterase are encoded by two distinct genes

1. Various hybridization approaches were employed to investigate structural and chromosomal interrelationships between the human cholinesterase genes CHE and ACHE encoding the polymorphic, closely related, and coordinately regulated enzymes having butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) activities. 2. Homologous cosmid recombination with a 190-base pair 5' fragment from BuChEcDNA resulted in the isolation of four overlapping cosmid clones, apparently derived from a single gene with several introns. The Cosmid CHEDNA included a 700-base pair fragment known to be expressed at the 3' end of BuChEcDNA from nervous system tumors and which has been mapped by in situ hybridization to the unique 3q26-ter position. In contrast, cosmid CHEDNA did not hybridize with full-length AChEcDNA, proving that the complete CHE gene does not include AChE-encoding sequences either in exons or in its introns. 3. The chromosomal origin of BuChE-encoding sequences was further examined by two unrelated gene mapping approaches. Filter hybridization with DNA from human/hamster hybrid cell lines revealed BuChEcDNA-hybridizing sequences only in cell lines including human chromosome 3. However, three BuChEcDNA-homologous sequences were observed at chromosomal positions 3q21, 3q26-ter, and 16q21 by a highly stringent in situ hybridization protocol, including washes at high temperature and low salt. 4. These findings stress the selectivity of cosmid recombination and chromosome blots, raise the possibility of individual differences in BuChEcDNA-hybridizing sequences, and present an example for a family highly similar proteins encoded by distinct, nonhomologous genes.     

Carbon dioxide-independent and -dependent components of light inhibition of the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in oat leaves

Light inhibits while carbon dioxide enhances the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene in oat ( Avena sativa L. cv. Victory) leaf segments. The possibility that the light inhibition is mediated through changes of carbon dioxide has been investigated. The level of CO₂ increases or decreases in the sealed incubation vial in darkness or in light, respectively, which can apparently account for the differences in ACC-dependent ethylene production between the dark and light treatments. However, although the evolution of ethylene from ACC in the dark is reduced upon depletion of CO₂, the difference between light and dark is still very noticeable. Moreover, the production of the ethylene in CO₂-free air in the dark was still higher than in the light, where the concentration of CO₂ was 0.01%. It is proposed that the light effect on the conversion of ACC to ethylene is composed of two distinguishable components: one CO₂-mediated and the other CO₂-independent.     

Isolated soluble fractions from the murine B16 melanoma induce primary in vitro syngeneic antitumor responses

Summary This paper extends our previous studies, which documented our ability to isolate immunogenic entities from nonimmunogenic or weakly immunogenic tumors.B16 melanoma cells failed, in our in vitro experimental system, to induce anti-B16 cytotoxic responses in spleen cells derived from normal syngeneic C57BL/6 mice. The B16 melanoma cellular homogenate was fractionated on an Ultrogel AcA 34 column, and the various fractions were tested for their ability to induce anti-B16 cytotoxic responses under the same conditions as those used for intact B16, the nonimmungenic tumor cells. Certain fractions, some of them with relatively low protein concentrations, induced anti-B16 cytotoxic responses in spleen cells of normal C57BL/6 mice, whereas others, some of them with relatively high protein concentrations, failed to induce such responses. One fraction (Fr.), designated Fr. 5/6, was examined in detail. It was found that in normal syngeneic spleen cells this fraction induced effector cells that efficiently killed (at various E : T ratios) the relevant B16 target cells and RBL5 syngeneic tumor cells, but not the YAC allogeneic tumor cells or C57BL/6 lymphoblasts. Furthermore, an excess of unlabeled B16 cells most efficiently blocked the ability of these anti-B16 effector cells to kill radiolabeled B16 target cells. RBL5 tumor cells, YAC tumor cells, or C57BL/6 lymphoblasts failed to block these effector cells efficiently. A significant fraction of the effector cells induced with Fr. 5/6 was characterized as thymus-derived cells (Thy-1⁺, Thy-2⁺3⁺ cells). It was suggested that another fraction of the cellular population was natural killer cells, which cytolyzed the RBL5 target cells. Various theoretical and practical aspects of these findings are discussed.     

The T-Cell factor specific for poly(Tyr,Glu)-Poly(Pro)-Poly(Lys) is an I-Region gene product

The nature of a T-cell factor specific for poly(Tyr,Glu)-poly(Pro)-poly(Lys) [(T,G)-pro-L] was established in the present study. The activity of the (T,G)-Pro-L-specific factor was not removed by anti-mouse immunoglobulin Sepharose columns, suggesting that it is not a classical immunoglobulin. On the other hand, the factor lost its activity after passage through immunoadsorbents prepared with anti-H-2 sera raised against theH-2 haplotypes of the mouse strains in which the factor was prepared. Furthermore, this factor was adsorbed byI region-specific antisera but not by antisera directed against theI-J andI-C subregions as well as theK andD regions of theH-2 complex. Thus, the (T,G)-Pro-L-specific T-cell factor is most probably anI-A subregion gene product.     

A possible role for cyclic GMP in mediating the effect of luteinizing hormone releasing hormone on gonadotropin release in dispersed pituitary cells of the female rat.

In continuing studies on cyclic nucleotide involvement in the regulation of gonadotropin release, we have measured the cyclic nucleotide content and rate of LH and FSH release during stimulation by LHRH of dispersed overnight cultured cells from the pituitaries of adult female rats. The minimal effective concentration of LHRH was 0.1 nM and half maximal stimulation of gonadotropin release was observed in the presence of 1.0 nM LHRH. Significant release of both LH and FSH was detectable after only 10 min in the presence of 5 nM LHRH. The presence of fetal calf serum (FCS) in the overnight culture medium increased basal cGMP levels significantly, whereas horse serum (HS) had no effect, therefore all experiments were conducted on cells cultured in the presence of HS. Treatment of the cultured cells with the phosphodiesterase inhibitors theophylline (TH) or isobutyl-methyl-xanthine (MIX) revealed a preferential stimulatory effect of TH on basal cAMP levels and of MIX on cGMP levels. Throughout these experiments, LHRH had no effect on cAMP levels. In the presence of MIX, concentrations of the releasing hormone as low as 1 nM induced a significant rise in the level of cGMP whereas in its absence, cGMP levels appeared to be unchanged by LHRH. The increase was detectable after 10 min of incubation. MIX alone slightly increased LH and FSH release and significantly potentiated the response of the cells to increasing doses of LHRH up to, but not beyond, 10 nM. The data support the possibility that cGMP may be involved in the mechanism of action of LHRH.