Regulation of insulin-like growth factor binding protein synthesis and secretion in human retinal pigment epithelial cells

Cultured human retinal pigment epithelial cells (RPE) secrete insulin-like growth factor binding proteins (IGFBPs), a family of polypeptides which modulate the actions of the insulin-like growth factors. RPE cells secrete two IGFBPs with Mr estimates of 34,000 and 46,000, respectively. Treatment of RPE cells with IGF-I markedly stimulated the secretion of the 46,000 Mr form. This stimulation occurred via an IGF-I receptor independent mechanism because both [QAYL]IGF-I (an IGF-I analogue with decreased affinity for the IGFBPs but normal affinity for the IGF-I receptor) and Α-IR 3 (a blocking monoclonal antibody against the IGF-I receptor) had no effect on IGF-I stimulated increases in IGFBPs. Additionally, [QAYL]IGF-I enhanced RPE cell proliferation to the same magnitude as IGF-I. Treatment with IGF-I, [QAYL]IGF-I, or Α-IR 3 had no effect on steady-state levels of the 2.5 kb IGFBP-3 or the 1.3 kb IGFBP-6 mRNA transcripts as measured by Northern blotting and quantitative autoradiography. Forskolin and a group of candidate growth factors, including platelet-derived growth factor, epidermal growth factor, and acidic and basic fibroblast growth factor, modestly increased IGFBP secretion when compared to untreated cells, but these effects were small when compared to IGF-I treatment. Fetal calf serum enhanced the presence of the 2.5 kb IGFBP-3 mRNA transcript in a dose-dependent fashion but had no effect on the 1.3 kb IGFBP-6 mRNA transcript. IGF-I, forskolin, and the candidate growth factors had no effect on either IGFBP-3 or IGFBP-6 mRNA. These data suggest that the production of IGFBPs in human RPE cells is regulated by distinct mechanisms which include (1) an IGF-I receptor independent interaction of IGF-I with secreted IGFBPs and (2) de novo synthesis of IGFBPs by serum-containing factors. © 1994 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49887/1/1041580124_ftp.pd

Expanding the role of tachykinins in the neuroendocrine control of reproduction

Reproductive function is driven by the hormonal interplay between the gonads and brain–pituitary axis. Gonadotropin-releasing hormone (GnRH) is released in a pulsatile manner, which is critical for the attainment and maintenance of fertility; however, GnRH neurons lack the ability to directly respond to most regulatory factors, and a hierarchical upstream neuronal network governs its secretion. We and others proposed a model in which Kiss1 neurons in the arcuate nucleus (ARC), called as KNDy neurons, release kisspeptin (a potent GnRH secretagogue) in a pulsatile manner to drive GnRH pulses under the coordinated autosynaptic action of its cotransmitters, the tachykinin neurokinin B (NKB, stimulatory) and dynorphin (inhibitory). Numerous genetic and pharmacological studies support this model; however, additional regulatory mechanisms (upstream of KNDy neurons) and alternative pathways of GnRH secretion (kisspeptin independent) exist, but remain ill defined. In this aspect, attention to other members of the tachykinin family, namely substance P (SP) and neurokinin A (NKA), has recently been rekindled. Even though there are still major gaps in our knowledge about the functional significance of these systems, substantial evidence, as discussed below, is placing tachykinin signaling as an important pathway for the awakening of the reproductive axis and the onset of puberty to physiological GnRH secretion and maintenance of fertility in adulthood

Histoplasma capsulatum Encodes a Dipeptidyl Peptidase Active against the Mammalian Immunoregulatory Peptide, Substance P

The pathogenic fungus Histoplasma capsulatum secretes dipeptidyl peptidase (Dpp) IV enzyme activity and has two putative DPPIV homologs (HcDPPIVA and HcDPPIVB). We previously showed that HcDPPIVB is the gene responsible for the majority of secreted DppIV activity in H. capsulatum culture supernatant, while we could not detect any functional contribution from HcDPPIVA. In order to determine whether HcDPPIVA encodes a functional DppIV enzyme, we expressed HcDPPIVA in Pichia pastoris and purified the recombinant protein. The recombinant enzyme cleaved synthetic DppIV substrates and had similar biochemical properties to other described DppIV enzymes, with temperature and pH optima of 42°C and 8, respectively. Recombinant HcDppIVA cleaved the host immunoregulatory peptide substance P, indicating the enzyme has the potential to affect the immune response during infection. Expression of HcDPPIVA under heterologous regulatory sequences in H. capsulatum resulted in increased secreted DppIV activity, indicating that the encoded protein can be expressed and secreted by its native organism. However, HcDPPIVA was not required for virulence in a murine model of histoplasmosis. This work reports a fungal enzyme that can function to cleave the immunomodulatory host peptide substance P

Purification and Characterization of a Sperm Motility Inhibiting Factor from Caprine Epididymal Plasma

Several studies have been reported on the occurrence of sperm motility inhibiting factors in the male reproductive fluids of different mammalian species, but these proteins have not been adequately purified and characterized. A novel sperm motility inhibiting factor (MIF-II) has been purified from caprine epididymal plasma (EP) by Hydroxylapatite gel adsorption chromatography, DEAE-Cellulose ion-exchange chromatography and chromatofocusing. The MIF-II has been purified to apparent homogeneity and the molecular weight estimated by Sephacryl S-300 gel filtration is 160 kDa. MIF-II is a dimeric protein, made up of two subunits each having a molecular mass of 80 kDa as shown by SDS-PAGE. The isoelectric point of MIF-II is 5.1 as determined by chromatofocusing and isoelectric focusing. It is a heat labile protein and maximal active at the pH 6.9 to 7.5. The sperm motility inhibiting protein factor at 2 µg/ml (12.5 nM) level showed maximal motility-inhibiting activity. The observation that the epididymal plasma factor lowered the intracellular cAMP level of spermatozoa in a concentration-dependent manner suggests that it may block the motility of caprine cauda spermatozoa by interfering the cAMP dependent motility function. The results revealed that the purified protein factor has the potential of sperm motility inhibition and may serve as a vaginal contraceptive. The antibody raised against the MIF-II has the potential for enhancement of forward motility of cauda-spermatozoa. This antibody may thus be useful for solving some of the problems of male infertility due to low sperm motility

Recent Progress in the Use of Glucagon and Glucagon Receptor Antagonists in the Treatment of Diabetes Mellitus

Glucagon is an important pancreatic hormone, released into blood circulation by alpha cells of the islet of Langerhans. Glucagon induces gluconeogenesis and glycogenolysis in hepatocytes, leading to an increase in hepatic glucose production and subsequently hyperglycemia in susceptible individuals. Hyperglucagonemia is a constant feature in patients with T2DM. A number of bioactive agents that can block glucagon receptor have been identified. These glucagon receptor antagonists can reduce the hyperglycemia associated with exogenous glucagon administration in normal as well as diabetic subjects. Glucagon receptor antagonists include isoserine and beta-alanine derivatives, bicyclic 19-residue peptide BI-32169, Des-His1-[Glu9] glucagon amide and related compounds, 5-hydroxyalkyl-4-phenylpyridines, N-[3-cano-6- (1,1 dimethylpropyl)-4,5,6,7-tetrahydro-1-benzothien-2-yl]-2-ethylbutamide, Skyrin and NNC 250926. The absorption, dosage, catabolism, excretion and medicinal chemistry of these agents are the subject of this review. It emphasizes the role of glucagon in glucose homeostasis and how it could be applied as a novel tool for the management of diabetes mellitus by blocking its receptors with either monoclonal antibodies, peptide and non-peptide antagonists or gene knockout techniques

Chemogenomic Analysis of G-Protein Coupled Receptors and Their Ligands Deciphers Locks and Keys Governing Diverse Aspects of Signalling

Understanding the molecular mechanism of signalling in the important super-family of G-protein-coupled receptors (GPCRs) is causally related to questions of how and where these receptors can be activated or inhibited. In this context, it is of great interest to unravel the common molecular features of GPCRs as well as those related to an active or inactive state or to subtype specific G-protein coupling. In our underlying chemogenomics study, we analyse for the first time the statistical link between the properties of G-protein-coupled receptors and GPCR ligands. The technique of mutual information (MI) is able to reveal statistical inter-dependence between variations in amino acid residues on the one hand and variations in ligand molecular descriptors on the other. Although this MI analysis uses novel information that differs from the results of known site-directed mutagenesis studies or published GPCR crystal structures, the method is capable of identifying the well-known common ligand binding region of GPCRs between the upper part of the seven transmembrane helices and the second extracellular loop. The analysis shows amino acid positions that are sensitive to either stimulating (agonistic) or inhibitory (antagonistic) ligand effects or both. It appears that amino acid positions for antagonistic and agonistic effects are both concentrated around the extracellular region, but selective agonistic effects are cumulated between transmembrane helices (TMHs) 2, 3, and ECL2, while selective residues for antagonistic effects are located at the top of helices 5 and 6. Above all, the MI analysis provides detailed indications about amino acids located in the transmembrane region of these receptors that determine G-protein signalling pathway preferences