Modulation of cAMP-dependent protein kinase by derivatives of chondroitin sulfate

Here, we report investigations about the direct effect of glycosaminoglycans, such as dermatan sulfate, chondroitin 4- and 6-sulfate upon cAMP-dependent protein kinase activity. The results indicate that glycosaminoglycans strongly influence the phosphorylation activity of this enzyme against histone type IIa and [Val6, Ala7]-kemptide. While chondroitin 4-sulfate and dermatan sulfate exhibit inhibitory effects, chondroitin 6-sulfate shows a stimulating effect. In addition, the chondroitin 6-sulfate is also able to reduce the chondroitin 4-sulfate and dermatan sulfate specific inhibition.     

Direct inhibitory effect of adrenomedullin and guanylin on isolated caecal circular smooth muscle cells of guinea pig

Smooth muscle cells isolated from the caecal circular smooth muscle layers of the guinea pig were used to determine whether adrenomedullin and guanylin can inhibit the contractile response produced by 10(-9) M cholecystokinin octapeptide (CCK-8). In addition, to elucidate each intracellular mechanisms, we examined the effects of an inhibitor of cAMP-dependent protein kinase, an inhibitor of particulate guanylate cyclase, and an inhibitor of soluble guanylate cyclase on the adrenomedullin- or guanylin-induced relaxation of the caecal circular smooth muscle cells. Both adrenomedullin and guanylin inhibited the contractile response produced by CCK-8 in a dose-dependent manner, with IC50 values of 0.12 nM and 2.4 pM, respectively. An inhibitor of cAMP-dependent protein kinase significantly inhibited the relaxation produced by adrenomedullin. In contrast, an inhibitor of particulate guanylate cyclase and an inhibitor of soluble guanylate cyclase did not have any significant effect on the relaxation produced by adrenomedullin. On the other hand, an inhibitor of particulate guanylate cyclase significantly inhibited the guanylin-induced relaxation, although an inhibitor of cAMP-dependent protein kinase and an inhibitor of soluble guanylate cyclase did not have any significant effect on the guanylin-induced relaxation. In this study, we first demonstrated the direct inhibitory effects of adrenomedullin via cAMP system and guanylin via particulate guanylate cyclase system on the isolated caecal circular smooth muscle cells.     

Identification of a signaling pathway activated specifically in the somatodendritic compartment by a heparan sulfate that regulates dendrite growth

In two earlier reports we demonstrated that natural heparan sulfate, but not dermatan or chondroitin sulfate glycosaminoglycans, stimulate axonal elongation and inhibit dendrite growth in vitro (Lafont et al., 1992). The latter specific effect on dendrite elongation was reproduced by chemically synthesized heparan sulfates and by SR 80037A, a purified sulfated and hexanoylated heparin fragment (Lafont et al., 1994). Adding radioactive SR 80037A to purified neurons demonstrated the existence, at the neuronal surface, of heparan sulfate-specific and saturable binding sites, suggesting that SR 80037A activates specific signal transduction pathways. In the present study, using rat or mouse neurons from the embryonic cortex, we show that SR 80037A signaling involves one or several G-coupled receptor or receptors, small GTPases rhoA and/or rhoC, and one or several PKCs. We also demonstrate that the rapid soma rounding elicited by SR 80037A does not require protein synthesis but that the long-term effect on dendrite initiation requires protein synthesis in a short period after the addition of the heparan sulfate. Finally, by preparing membranes from the somatodendritic or axonal compartments we demonstrate that the identified signaling pathway is activated by SR 80037A primarily in the somatodendritic compartment and is not sensitive to the addition of a dermatan sulfate glycosaminoglycan that does not induce the axonal phenotype by impairing dendrite initiation and elongation.     

Inhibition of T lymphocyte activation by cAMP is associated with down-regulation of two parallel mitogen-activated protein kinase pathways, the extracellular signal-related kinase and c-Jun N-terminal kinase

The induction of T cell proliferation requires signals from the TCR and a co-receptor molecule, such as CD28, that activate parallel and partially cross-reactive signaling pathways. These pathways are disrupted by agonists that utilize adenylate cyclase and cAMP-dependent protein kinase A (PKA). We found that the adenylate cyclase activator, forskolin, inhibits anti-CD3-induced shift in Lck electrophoretic mobility, suggesting an intervention at the TCR-coupled phosphoinositide turnover that precedes the activation of PKC. The shift of Lck following direct PKC activation by 12-O-tetradecanoyl phorbol 13-acetate, which bypasses early receptor-triggered biochemical events, is insensitive to forskolin. Nevertheless, forskolin also inhibits PKC downstream events, such as c-jun expression, which is critical for the activation process of T cells. To further analyze potential cross points between positively and negatively regulating signaling pathways in T cells, we tested the effects of activators of the adenylate cyclase or PKA on two parallel mitogen-activated protein kinase signaling pathways mediated by extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase. Using a PKC-specific inhibitor, GF109203X, or PKC-depleted T cells, we found that a large part of the anti-CD3-induced ERK activation is PKC dependent. Both PKC-dependent and -independent activation of ERK were sensitive to inhibition by forskolin or a cell-permeable cAMP analogue, dbcAMP. Furthermore, the effect of 12-O-tetradecanoyl phorbol 13-acetate and ionomycin, which synergized to fully activate c-Jun N-terminal kinase, was also sensitive to inhibition by forskolin. Our results suggest that PKA inhibits T cell activation by interfering with multiple events along the two signaling pathways operating downstream of the TCR and the CD28 co-receptor molecules.     

Stimulative effect of high-level hypergravity on differentiated functions of osteoblast-like cells

The exposure of freshly isolated osteoblasts and osteoblast-like cells to high-level hypergravity caused the inhibition of cell growth, elevation of cAMP content, and the stimulation of differentiated functions such as alkaline phosphatase activity, collagen synthesis, and osteocalcin synthesis. Blockage of elevation of cAMP by SQ22536, an inhibitor of adenylate cyclase, resulted in the inhibition of the hypergravity-stimulated alkaline phosphatase activity, indicating that cAMP is the intracellular mediator of this action of hypergravity. H89, an inhibitor of cAMP-dependent protein kinase (PKA), further inhibited the cell growth that was already inhibited by the hypergravity, and further stimulated the alkaline phosphatase activity that was already stimulated by hypergravity. If cAMP acts through the PKA system, H89 should have blocked the changes in cell function effected by the exposure to hypergravity. Therefore the elevated intracellular cAMP by the exposure of hypergravity caused the changes in cell function by a PKA-independent pathway.     

Transmission imaging for nonuniform attenuation correction using a three-headed SPECT camera

ACTH (1-24) induces cell shape changes in the immunocytes of the bivalve mollusc, Mytilus galloprovincialis. Using computer-assisted microscopic image analysis, we have found that the G protein antagonist suramin sodium, the adenylate cyclase inhibitor 2',5'-dideoxyadenosine, and the protein kinase inhibitor staurosporine inhibit this effect. The highly specific inhibitors H-89 (for protein kinase A) and calphostin C (for protein kinase C) only inhibited partially the morphological alterations. In contrast, the simultaneous action of H-89 and calphostin C completely blocked these changes. The above findings indicate that ACTH (1-24) induces cell shape changes in molluscan immunocytes via adenylate cyclase/cAMP/protein kinase A pathway, as well as the activation of protein kinase C.     

Catabolism of adipose tissue by a tumour-produced lipid-mobilising factor

Induction of lipolysis in murine white adipocytes by a tumour lipid-mobilising factor (LMF) was associated with stimulation of adenylate cyclase in adipocyte plasma membrane preparations. Induction of lipolysis was attenuated by the adenylate cyclase inhibitor MDL12330A and the protein kinase A inhibitor H8, suggesting that cAMP was the intracellular mediator of induction. The effect of LMF on adenylate cyclase was responsive to GTP, with low concentrations (0.1 microM) causing stimulation and high concentrations (10 microM) causing inhibition, suggesting the involvement of both stimulatory (Gs) and inhibitory (Gi) guanine nucleotide-binding proteins. At a concentration of 10 microM, propranolol noncompetitively reduced the induction of lipolysis by LMF. Thus, lipolysis in white adipose tissue during the process of cancer cachexia is mediated by a tumour factor which stimulates cAMP production, possibly through a beta-adrenergic receptor.     

Influence of aluminum on the regulation of PTH- and 1,25(OH)2D3-dependent pathways in the rat osteosarcoma cell line ROS 17/2.8

The role of hormonal status in the development of aluminum (Al)-dependent renal osteodystrophy, which is characterized by reduced bone matrix deposition, still remains largely unknown. To address this question, we used the osteoblast-like osteosarcoma cell line ROS 17/2.8 to evaluate the role of Al on parathyroid hormone (PTH)- and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent activities in these cells. Al (1 microM) caused an inhibition of basal and 1,25(OH)2D3-induced alkaline phosphatase, but only at low doses (< 1 nM) of the steroid. Al partly inhibited basal osteocalcin (OC) secretion in ROS cells (p < 0.001), and the dose-dependent increase in 1,25(OH)2D3-induced OC release by these cells was also reduced by 1 microM Al at low concentrations of the steroid (< or = 1 nM), whereas high doses of 1,25(OH)2D3 (> or = 5 nM) totally prevented the inhibiting effects of Al. Al also had strong inhibitory actions on PTH-dependent cAMP production by ROS cells over the concentration range tested (0.5-50 nM). This inhibitory action of Al was also observed for PTH-related peptide- (PTHrp, 50 nM) but not for Isoproterenol-dependent (100 nM) cAMP formation. To evaluate more fully the mechanism of this inhibition of cAMP formation, we investigated the effect of Al on toxin-modulated, G protein-dependent regulation of cAMP formation and on the activation of adenylate cyclase by Forskolin. Cholera toxin (CT, 10 micrograms/ml), applied to cells for 4 h prior to PTH challenge, enhanced cAMP production about 2-fold above PTH alone (p < 0.001), a process that was further stimulated by Al. Pertussis toxin (PT, 1 microgram/ml, 4 h) did not modify basal PTH-dependent cAMP formation by ROS cells. However, PT treatment prevented the inhibitory effect of Al on cAMP formation by these cells (p < 0.025). The stimulation of adenylate cyclase by Forskolin (0.1 and 1 microM), which bypasses G protein regulation, was not modified by Al, indicating that Al does not affect adenylate cyclase directly. Northern blot analysis of PTH receptor mRNA levels showed that Al did not modify PTH receptor message in ROS cells. Likewise, Western blot analyses of G protein subunits showed that Al did not significantly alter Gs alpha subunit levels, in accordance with the results obtained for cAMP-dependent formation in response to CT. In contrast, Gi alpha-1 and Gi alpha-2 subunits were decreased by Al treatment, consistent with PT-restricted increases in cAMP formation in Al-treated ROS cells. Taken together, these results suggest that Al has multiple actions in osteoblast-like ROS cells. The effects of Al are modulated by hormonal control of the pathways investigated. Al affects 1,25(OH)2D3-regulated functions only when this steroid is low. Al has large inhibitory effects on PTH- and PTHrp-dependent cAMP formation. This last feature is related to the ability of Al to alter the G protein transducing pathway for PTH/PTHrp-dependent formation of cAMP since it does not affect adenylate cyclase activity directly and does not affect the PTH receptor message level. Thus, Al has stronger deleterious effects in osteoblast-like cells with an already compromised 1,25(OH)2D3 status and can modulate specifically PTH/PTHrp-mediated cAMP formation at the postreceptor level.     

Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea

cAMP is involved in signaling appressorium formation in the rice blast fungus Magnaporthe grisea. However, null mutations in a protein kinase A (PKA) catalytic subunit gene, CPKA, do not block appressorium formation, and mutations in the adenylate cyclase gene have pleiotropic effects on growth, conidiation, sexual development, and appressorium formation. Thus, cAMP signaling plays roles in both growth and morphogenesis as well as in appressorium formation. To clarify cAMP signaling in M. grisea, we have identified strains in which a null mutation in the adenylate cyclase gene (MAC1) has an unstable phenotype such that the bypass suppressors of the Mac1(-) phenotype (sum) could be identified. sum mutations completely restore growth and sexual and asexual morphogenesis and lead to an ability to form appressoria under conditions inhibitory to the wild type. PKA assays and molecular cloning showed that one suppressor mutation (sum1-99) alters a conserved amino acid in cAMP binding domain A of the regulatory subunit gene of PKA (SUM1), whereas other suppressor mutations act independently of PKA activity. PKA assays demonstrated that the catalytic subunit gene, CPKA, encodes the only detectable PKA activity in M. grisea. Because CPKA is dispensable for growth, morphogenesis, and appressorium formation, divergent catalytic subunit genes must play roles in these processes. These results suggest a model in which both saprophytic and pathogenic growth of M. grisea is regulated by adenylate cyclase but different effectors of cAMP mediate downstream effects specific for either cell morphogenesis or pathogenesis.     

An EGF receptor-mediated signal attenuates the inhibitory effect of LPA on an adenylate cyclase activity

A tyrosine kinase receptor-mediated and a heterotrimeric G protein-coupled receptor-mediated signals have been shown to evoke distinct intracellular signaling events. There has been increasing evidence that cross-talk exists between a tyrosine kinase receptor-mediated and a heterotrimeric G protein-coupled receptor-mediated signal transduction pathways. In the present study, we have studied effects of EGF receptor activation on activities of inhibitory G protein (Gi). We show that the amounts of Gi/Go ADP-ribosylated by islet-activating protein (IAP) increased by 30-40% in the membranes of Rat 1 fibroblast cells pretreated with EGF compared with those without pretreatment. When an effect of lysophosphatidic acid (LPA) stimulation on an adenylate cyclase activity was examined, LPA partly attenuated forskolin-stimulated adenylate cyclase activity via Gi because IAP pretreatment blocked the inhibitory effect of LPA. Pretreatment with EGF reduced the ability of LPA to inhibit the forskolin-stimulated adenylate cyclase activity, while the pretreatment did not have any effects on the forskolin-stimulated activity. Thus, the EGF receptor-mediated signal appears to cause the impairment of Gi function in Rat 1 fibroblast cells.     

Glycosaminoglycan specificity of a heparin-binding peptide

Glycosaminoglycans are complex sulfated polysaccharides with a diverse range of biological functions. Three glycosaminoglycan standards--chondroitin sulfate, dermatan sulfate and heparin--were characterized during this study. The interaction of the heparin binding site of protein C inhibitor, represented by the peptide sequence 264-283, in solution with the above glycosaminoglycan standards was studied. Circular dichroism spectroscopy was used to determine the dominant secondary structure induced in the peptide upon binding the relevant glycosaminoglycans. The various glycosaminoglycans induced different secondary structures. The level of induced secondary structure by dermatan sulfate and heparin was approximately twice that induced by chondroitin sulfate. For chondroitin sulfate and heparin, alpha-helix was the dominant ordered secondary structure, whereas for dermatan sulfate the beta-strand conformation dominated. The order of secondary structure induction of the protein C inhibitor peptide by the glycosaminoglycans paralleled the reported biological activities of these glycosaminoglycans for mediation of the biological activity in the intact protein. The strength of the interaction of dermatan sulfate and heparin with the protein C inhibitor peptide was measured by determining the concentration of salt required to inhibit 50% of the interaction. The values determined were 0.1 and 0.3 M salt for dermatan sulfate and heparin, respectively. These results show that different glycosaminoglycans can support different secondary structures in the protein C inhibitor peptide.     

The human and dog 5-HT1D receptors can both activate and inhibit adenylate cyclase in transfected cells

The cloned human serotonin 1D (5-HT1D) receptor has been shown to inhibit adenylate cyclase while the corresponding cloned dog receptor has been characterized by its enhancement of cAMP accumulation. To resolve this apparent discrepancy, the human 5-HT1D receptor has been cloned and expressed in Chinese hamster ovary (CHO) cells and the corresponding dog receptor expressed in mutant Y1 adrenal (Y1 Kin-8) cells. It is shown that both receptors when activated by sumatriptan depress forskolin induced adenosine 3'5'-cyclic monophosphate (cAMP) accumulation by a pertussis toxin sensitive mechanism, presumably involving Gi (the adenylate cyclase inhibitory GTP transducing protein). In the absence of forskolin, the dog receptor enhances cAMP accumulation, thus activating Gs (the adenylate cyclase stimulatory GTP transducing protein). When its overriding action on Gi is blocked by pertussis toxin pretreatment, the human receptor also enhances cAMP accumulation. Thus both 5-HT1D receptors activate markedly Gi and to a lesser extent Gs and can exert opposite effects on the same effector system, adenylate cyclase.     

An agarose gel electrophoretic method for analysis of sulfated glycosaminoglycans of cultured cells

Agarose disc gel electrophoresis has been adapted to achieve the separation of the major sulfated glycosaminoglycans produced by cells in culture. By use of buffers containing barium ion, mixtures of chondroitin sulfate, dermatan sulfate, and heparan sulfate are well resolved into discrete bands. The technique can be used preparatively as well as analytically to separate quantities of glycosaminoglycans up to a milligram in a 6-mm diameter gel.     

Complexing of fibronectin glycosaminoglycans and collagen

Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20-40% of radioactivity in [35S]heparin. Fibronectin attached directly to Sepharose also bound [35S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [35S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit. The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfate heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads. It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells.     

Activation of protein kinase C by oxytocin inhibits the biological activity of the human myometrial corticotropin-releasing hormone receptor at term

The role of placental CRH in human pregnancy is currently unknown. The myometrium expresses CRH receptors that during pregnancy become coupled to adenylate cyclase. Oxytocin (OT) is one of the main regulators of uterine activity, acting via activation of the inositol triphosphate pathway. In view of the possible cross-talk between the CRH and OT signal transduction pathways we have sought to examine in more detail the second messenger mechanisms involved. CRH receptor binding affinity for CRH and activation of adenylate cyclase were reduced in the presence of OT in pregnant (at term, but not preterm) human myometrium. OT action was mediated via pertussis toxin-sensitive G proteins, which directly inhibit adenylate cyclase and, via activation of protein kinase C, phosphorylate the CRH receptor, leading to desensitization. Activation of protein kinase C by OT could be partially inhibited in human pregnant myometrial cells by OT antagonists (F327 and CAP476; 1 microM) or phospholipase C inhibitors (U73122; 10 microM). These results suggest that in term myometrium, CRH receptor function is modulated by OT, leading to reduced biological activity, lower cAMP levels, and a subsequent shift in favor of contractility rather than relaxation.     

Characterization of recombinant perlecan domain I and its substitution by glycosaminoglycans and oligosaccharides

Recombinant mouse perlecan domain 1(173 residues) was produced in transfected embryonic kidney cells and purified from the culture medium on DEAE-cellulose. It was shown to be modified by glycosaminoglycans and could be partially separated into two protein pools which were either substituted with heparan sulfate (fragment IA) or, to a smaller extent (20%), with chondroitin/dermatan sulfate or a mixture of both glycosaminoglycans (fragment IB). The average molecular mass of the glycosaminoglycans was about 8-10 kDa and, thus, smaller than in tissue-derived perlecans. Sequence and carbohydrate analyses localized the heparan sulfate attachment site to three Ser residues within SGD consensus sequences. Furthermore, the N-terminal part of fragment IA contained six Thr/Ser residues substituted by branched galactosamine-containing oligosaccharides and an N-substituted Asn residue. Fragment I was also shown to contain unique immunological epitopes which are not dependent on glycosaminoglycans and are shared by tissue-derived perlecan. Circular dichroism demonstrated a distinct alpha helix (20%) and beta structure (60%) in fragment IA, consistent with predictions of a novel SEA protein module located in the C-terminal part of domain I.     

The biosynthesis of glycosaminoglycans in normal rat liver and in response to experimental hepatic injury

The synthesis of glycosaminoglycans in slices from normal and acutely injured rat liver was studied. The rates of incorporation of [14C]-glucosamine into specific types of glycosaminoglycans varied markedly; nearly 90% was incorporated into a fraction containing predominantly heparan sulfate and far less if any heparin; about 9.5% was incorporated into chondroitin 4-and 6-sulfate, and only 0.2% of the radioactivity was found in hyaluronic acid. The rate of synthesis of a fraction having several of the characteristics of keratan sulfate comprised only 0.3% of the synthesis of total glycosaminoglycans. No [14C]hexosamine was incorporated into dermatan sulfate. Following acute hepatic injury, the synthesis of glycosaminoglycans was stimulated by 80 to 100%, and the proportions of various types changed. If calculated on the basis of the specific activity of the precursors of glycosaminoglycans, which was found to be strongly reduced in injured liver, the maximum enhancement of total glycosaminoglycan synthesis was 6.6-fold 5 days after onset of liver injury.     

Genetic evidence that heparin-like glycosaminoglycans are involved in wingless signaling

We have identified the Drosophila UDP-glucose dehydrogenase gene as being involved in wingless signaling. Mutations in this gene, called kiwi, generate a phenotype identical to that of wingless. UDP-glucose dehydrogenase is required for the biosynthesis of UDP-glucuronate, which in turn is utilized in the biosynthesis of glycosaminoglycans. By rescuing the kiwi phenotype with both UDP-glucuronate and the glycosaminoglycan heparan sulfate, we show that kiwi function in the embryo is crucial for the production of heparan sulfate in the extracellular matrix. Further, injection of heparin degrading enzyme, heparinase (and not chondroitin, dermatan or hyaluronic acid degrading enzyme) into wild-type embryos leads to the degradation of heparin-like glycosaminoglycans and a 'wingless-like' cuticular phenotype. Our study thus provides the first genetic evidence for the involvement of heparin-like glycosaminoglycans in signal transduction.     

Endothelins inhibit cyclic-AMP induced renin gene expression in cultured mouse juxtaglomerular cells

We have recently described that endothelins-1 to -3 equipotently inhibit cAMP stimulated renin secretion from cultured mouse juxtaglomerular cells by a process involving phospholipase C activation. This study examined the influence of endothelin-2 on renin gene expression in renal juxtaglomerular cells. To this end we semiquantitated renin mRNA levels by competitive RT-PCR in primary cultures of mouse renal juxtaglomerular cells after 20 hours of incubation. We found that endothelin-2 (0.1 to 100 nmol/liter) did not change basal renin gene expression. The adenylate cyclase activator forskolin (3 mumol/ liter) increased renin mRNA levels to 400% of the controls and this stimulation was dose-dependently attenuated by ET-2 to 250% of the control value. The effect of ET-2 was mimicked by the ETB-receptor agonist sarafotoxin S6c. The kinase inhibitor staurosporine (100 nmol/ liter) increased renin secretion and renin mRNA levels. Combination of staurosporine with forskolin produced the same effects on renin secretion and renin mRNA levels as did staurosporine alone. In the presence of both forskolin and staurosporine ET-2 had no significant effect on renin secretion and renin gene expression. The phorbol ester PMA (30 nmol/ liter), which was used to stimulate protein kinase C activity, attenuated cAMP stimulated renin secretion and renin mRNA levels. Lowering the extracellular concentration of calcium by the addition of 1 mmol/liter EGTA did not inhibit the effect of ET-2 on cAMP induced renin secretion and renin gene expression. These findings suggest that endothelins inhibit cAMP stimulated renin gene expression by an event that is mediated via ETB receptors. This inhibitory effect may in part involve protein kinase C activation.