▪ Abstract At the interface between the innate and adaptive immune systems lies the high-output isoform of nitric oxide synthase (NOS2 or iNOS). This remarkable molecular machine requires at least 17 binding reactions to assemble a functional dimer. Sustained catalysis results from the ability of NOS2 to attach calmodulin without dependence on elevated Ca2+. Expression of NOS2 in macrophages is controlled by cytokines and microbial products, primarily by transcriptional induction. NOS2 has been documented in macrophages from human, horse, cow, goat, sheep, rat, mouse, and chicken. Human NOS2 is most readily observed in monocytes or macrophages from patients with infectious or inflammatory diseases. Sustained production of NO endows macrophages with cytostatic or cytotoxic activity against viruses, bacteria, fungi, protozoa, helminths, and tumor cells. The antimicrobial and cytotoxic actions of NO are enhanced by other macrophage products such as acid, glutathione, cysteine, hydrogen peroxide, or superoxid...

Nitric oxide and macrophage function

The neurohypophysial peptide oxytocin (OT) and OT-like hormones facilitate reproduction in all vertebrates at several levels. The major site of OT gene expression is the magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. In response to a variety of stimuli such as suckling, parturition, or certain kinds of stress, the processed OT peptide is released from the posterior pituitary into the systemic circulation. Such stimuli also lead to an intranuclear release of OT. Moreover, oxytocinergic neurons display widespread projections throughout the central nervous system. However, OT is also synthesized in peripheral tissues, e.g., uterus, placenta, amnion, corpus luteum, testis, and heart. The OT receptor is a typical class I G protein-coupled receptor that is primarily coupled via Gq proteins to phospholipase C-β. The high-affinity receptor state requires both Mg2+ and cholesterol, which probably function as allosteric modulators. The agonist-binding region of the receptor has bee...

The Oxytocin Receptor System: Structure, Function, and Regulation

Eleven distinct isoforms of phosphoinositide-specific phospholipase C (PLC), which are grouped into four subfamilies (beta, gamma, delta, and epsilon), have been identified in mammals. These isozymes catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to inositol 1,4,5-trisphosphate and diacylglycerol in response to the activation of more than 100 different cell surface receptors. All PLC isoforms contain X and Y domains, which form the catalytic core, as well as various combinations of regulatory domains that are common to many other signaling proteins. These regulatory domains serve to target PLC isozymes to the vicinity of their substrate or activators through protein-protein or protein-lipid interactions. These domains (with their binding partners in parentheses or brackets) include the pleckstrin homology (PH) domain [PtdIns(3)P, beta gamma subunits of G proteins] and the COOH-terminal region including the C2 domain (GTP-bound alpha subunit of Gq) of PLC-beta; the PH domain [PtdIns(3,4,5)P3] and Src homology 2 domain [tyrosine-phosphorylated proteins, PtdIns(3,4,5)P3] of PLC-gamma; the PH domain [PtdIns(4,5)P2] and C2 domain (Ca2+) of PLC-delta; and the Ras binding domain (GTP-bound Ras) of PLC-epsilon. The presence of distinct regulatory domains in PLC isoforms renders them susceptible to different modes of activation. Given that the partners that interact with these regulatory domains of PLC isozymes are generated or eliminated in specific regions of the cell in response to changes in receptor status, the activation and deactivation of each PLC isoform are likely highly regulated processes.

Regulation of phosphoinositide-specific phospholipase C

Activation of Transcription Factor NF-κB Is Suppressed by Curcumin (Diferuloylmethane)

Phosphoinositide-specific phospholipase C (PLC) subtypes β, γ, and δ comprise a related group of multidomain phosphodiesterases that cleave the polar head groups from inositol lipids. Activated by ...

Structure, Function, and Control of Phosphoinositide-Specific Phospholipase C

N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), serine protease inhibitors, block many cytotoxic functions of immune cells including superoxide anion production, cytokine release, cell-mediated cytolysis, and nitric oxide (NO)-related macrophage functions. IFN-gamma/LPS-induced NO production from murine peritoneal macrophages was inhibited by TPCK and TLCK in a dose-dependent manner (EC50s: approximately 20 microM for TPCK and approximately 30 microM for TLCK). Viability exceeded 91% with 25 microM TPCK and with 80 microM TLCK. When TPCK treatment was delayed until 1 h of activation, the inhibitory effect was markedly reduced. After 2 h of the activation, TPCK was not effective anymore. Addition of either TNF-alpha or conditioned media from IFN-gamma/LPS-activated macrophage culture did not prevent the inhibitory effect of TPCK. Neither TPCK nor TLCK reduced enzymatic NO production from macrophage lysates. Lysates from TPCK-treated cells did not generate NO even after supplementing necessary cofactors for NO synthase. Immunoblotting analysis showed that simultaneous treatment of TPCK with IFN-gamma/LPS abolished the NO synthase expression, whereas delayed addition of TPCK was either partially effective or not effective at all. Furthermore, TPCK treatment reduced the concentration of mRNA for NO synthase without decreasing mRNA stability. Thus, the serine protease inhibitors directly blocked an early event in expression of NO synthase. Electrophoretic mobility shift assay indicated that TPCK blocked the activation of nuclear factor-kappa B, a transcription factor necessary for NO synthase induction. TPCK also blocked disappearance of I kappa B from cytosolic fraction, and nuclear translocation of NF-kappa B subunits p50 and p65. Delaying the addition of TPCK by 10 min partially prevented the inhibition of the NF-kappa B activation process and allowed partial resuming of NO production. Thus, TPCK inhibited NO synthase induction by blocking NF-kappa B activation.

Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by preventing activation of nuclear factor-kappa B.

Streptozotocin (STZ), a diabetogenic agent, is thought to damage pancreatic beta-cells by activating immune mechanisms and by alkylating DNA. In the present study, we demonstrated that STZ can prod...

Nitric oxide generation from streptozotocin.

Although the oxytocin receptor modulates intracellular Ca2+ ion levels in myometrium, the identities of signal molecules have not been clearly clarified Our previous studies on oxytocin receptor signalling demonstrated that 80 kDa Ghalpha is a signal mediator [Baek, Kwon, Lee, Kim, Muralidhar and Im (1996) Biochem J 315, 739-744] To elucidate the effector in the oxytocin receptor signalling pathway, we evaluated the oxytocin-mediated activation of phospholipase C (PLC) by using solubilized membranes from human myometrium and a three-component preparation containing the oxytocin receptor-Ghalpha-PLC-delta1 complex PLC-delta1 activity in the three-component preparation, as well as PLC activity in solubilized membranes, was increased by oxytocin in the presence of Ca2+ and activated Ghalpha (GTP-bound Ghalpha) Furthermore the stimulated PLC-delta1 activity resulting from activation of Ghalpha via the oxytocin receptor was significantly attenuated by the selective oxytocin antagonist desGly-NH2d(CH2)5[Tyr(Me)2,Thr4]ornithine vasotocin or GDP Consistent with these observations, co-immunoprecipitation and co-immunoadsorption of PLC-delta1 in the three-component preparation by anti-Gh7alpha antibody resulted in the PLC-delta1 being tightly coupled to activated Ghalpha on stimulation of the oxytocin receptor These results indicate that PLC-delta1 is the effector for Ghalpha-mediated oxytocin receptor signalling

Phospholipase C-delta1 and oxytocin receptor signalling: evidence of its role as an effector.

One of the primary functions of the oxytocin receptor is to modulate intracellular calcium levels in myometrium. The oxytocin receptor has been purified and cloned. Although it has been suggested that oxytocin receptor couples with a GTP-binding regulatory protein (G-protein), the identity of this G-protein remains unclear. To elucidate the mechanism of oxytocin receptor signalling, we used the oxytocin-receptor-G-protein ternary complex preparation from human myometrium, and evaluated oxytocin-mediated activation of [35S]guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[S]) binding and [alpha-32P]GTP photoaffinity labelling to a G-protein. Binding of [35S]GTP[S] and the intensity of the [alpha-32P]GTP photoaffinity labelled protein resulting from activation of the oxytocin receptor were significantly attenuated by the selective oxytocin antagonist, desGlyNH2d(CH2)5[Tyr(Me)2,Thr4]OVT. Furthermore, the molecular mass of the specific GTP-binding protein was approximately 80 kDa; homologous with the Gh alpha family, the new class of GTP-binding proteins first identified in rat liver that couples to the alpha 1B-adrenoceptor. Consistent with these observations, in co-immunoprecipitation and co-immunoadsorption of the oxytocin receptor in the ternary complex preparation by anti-Gh7 alpha antibody, the Gh alpha family protein tightly coupled to the oxytocin receptor. These findings demonstrate that oxytocin receptor couples with approximately 80 kDa Gh alpha in signal mediation.

Hee Sung Lee

Papers

Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by preventing activation of nuclear factor-kappa B.

Nitric oxide generation from streptozotocin.

Phospholipase C-delta1 and oxytocin receptor signalling: evidence of its role as an effector.

Oxytocin receptor couples to the 80 kDa Gh alpha family protein in human myometrium.

Distinct characteristic of Galpha(h) (transglutaminase II) by compartment: GTPase and transglutaminase activities.