Method of stimulating ovulation rate in Merino ewes may affect conception but not embryo survival

We have recently shown, on young adult rat aorta rings, that elastin peptides induce a dose and endothelium-dependent vasodilation mediated by the 67 kDa subunit of the high affinity elastin-laminin receptor and, at least in part, by EDRF (NO). Here we have studied the effects of elastin peptides at circulating concentrations and below, on noradrenaline-contracted rat aortic rings, as a function of age. First, we have observed that, unlike 2-month-old (2M), 4-6-month-old (4M) and 12-month-old (12M) rat aorta rings, 30-month-old (30M) rat aorta rings were unable to maintain their contraction in long lasting experiments. Secondly, elastin peptides at physiological circulating concentrations (10(-6)-10(-3) mg/ml) induce a dose-dependent vasodilation on 4M rings. By contrast, only higher elastin peptide concentrations (10(-3) mg/ml) were effective on 12M rings, whereas rings from both younger (2M) and older animals (30M) did not respond to elastin peptides. Finally, using lactose and laminin as inhibitors, we have demonstrated that elastin peptide-induced vasodilation on 4M and 12M rings is mediated by the 67 kDa subunit of the elastin-laminin receptor. These experiments suggest that the functional availability of the 67 kDa subunit of the elastin-laminin receptor changes with age. It could be hypothesized that in young animals (0-2M) the reusable shuttle role recently demonstrated for the 67 kDa receptor subunit during elastic fiber formation leads to a major decrease in its availability for signal transduction. On the contrary, in adult animals. (4-12M), when developmental elastogenesis is completed, this subunit is essential for extracellular signal transduction. Inefficiency of this receptor in old animals (30M) can be attributed to its uncoupling from its transduction pathway, as previously shown on human cells. Finally, the age-dependent variations of circulating elastin peptide concentration and elastin-laminin receptor responsiveness to elastin peptides are two independent parameters which could influence the vascular tension regulation.     

Mechanisms of aging of the extracellular matrix: role of the elastin-laminin receptor

Aging of connective tissues is important for the understanding of aging mechanisms of tissues rich in extracellular matrix and of age-dependent diseases often affecting such tissues. Aging mechanisms of such tissues can be divided as follows: (1) age-dependent modifications of matrix biosynthesis; (2) postsynthetic modifications of extracellular matrix, and (3) modifications of cell-matrix interactions. Examples are discussed for all three aspects of tissue aging, with special emphasis on the role of epigenetic reactions. These reactions include the Maillard reaction, uncontrolled proteolytic degradation, and free radical release. Proteolytic fragments of fibronectin and of elastic fibers were shown to produce noxious effects and to be engaged in vicious circles of autoentertained and self-amplified mechanisms. We studied in particular the role of the elastin-laminin receptor in tissue aging and in atherogenesis. The presence of saturating concentrations of elastin peptides in the circulation results in a chronic overstimulation of the receptor with sustained free radical and lytic enzyme production. Other examples of age-dependent uncoupling of receptors also illustrate the importance of altered receptor function in tissue aging and related pathologies.     

Aorto-intestinal fistula as a possible cause of endoscopically undetermined gastrointestinal hemorrhage

Elastin peptides are present in human blood. As elastin receptors exist on several cell types, especially endothelial cells, this investigation was carried out to study the effect of elastin peptides on vascular tone. For this purpose, rat aortic rings were mounted in an organ bath for isometric tension measurements. Elastin peptides (kappa-elastin) were added in the concentration range of 0.1 ng/ml to 1 microgram/ml, concentrations similar to those found in the circulating blood. In rat aortic rings, precontracted or not with noradrenaline (10(-6) M), elastin peptides induced an endothelium-dependent vasodilation. The pretreatment of aortic rings with N-omega-nitro-L-arginine methyl ester (10(-5) M), an inhibitor of nitric oxide (NO) production, or with indomethacin (10(-5) M), an inhibitor of cyclooxygenase, prevented elastin peptide-induced vasodilation. These findings suggest that elastin peptides act through the synthesis of prostanoids, leading to the production of NO. Moreover, this relaxant effect of elastin peptides was decreased or inhibited when aortic rings were treated with lactose (10(-5) to 10(-2) M) or laminin (10(-6) to 10(-4) mg/ml) whereas lactose or laminin was unable to inhibit acetylcholine-induced vasodilation. These findings suggest that the inhibitory effects of lactose and laminin are specific for elastin peptide receptors and are in agreement with previous studies on these receptors. As there is evidence of the degradation of elastin in several vascular diseases, the concept that elastin peptides may contribute to the control of vascular tone is discussed.     

Binding of 92 kDa and 72 kDa progelatinases to insoluble elastin modulates their proteolytic activation

92 kDa and 72 kDa gelatinases, two neutral proteinases exhibiting elastinolytic activity and secreted as zymogens by aortic smooth muscle cells, were shown to bind to insoluble elastin. The active form of each enzyme interacted with substrate more avidly than latent form. Once bound to insoluble elastin, 92 kDa progelatinase was totally unaffected by any potential activators tested (tissue kallikrein, neutrophil elastase, plasmin, and stromelysin-1), except aminophenylmercuric acetate (APMA). Binding of 72 kDa progelatinase to insoluble elastin induced a fast autoactivation of the proenzyme followed by its inactivation. This process can be partly inhibited by tissue inhibitor of matrix metalloproteinases-2 (TIMP-2), EDTA and a synthetic inhibitor of matrix metalloproteinases (BB-94). Such an autoactivation process was also partially observed following adsorption of 72 kDa gelatinase to elastin-derived peptides but not to gelatin. Therefore, elastin can act as a template to direct its own proteolysis by 72 kDa gelatinase; such a mechanism could be relevant to the focal elastolysis in the arterial wall during arteriosclerosis.     

Presence of the elastin-laminin receptor on human activated lymphocytes

A variety of cells - fibroblasts, vascular smooth muscle cells, endothelial cells, monocytes and polymorphonuclear leukocytes (PMNs) - carry the elastin-laminin receptor. The activation of this receptor by elastin peptides triggers a variety of reactions as chemotactic movements to an elastin peptide gradient, release of lytic enzymes and oxygen-free radicals, modifications of ion fluxes. We now show that human lymphocytes also express this receptor. Membrane labelling of the receptor by specific antibodies shows capping. In the presence of elastin peptides lymphocytes show increased proliferation and increased production of an elastase type serine protease apparently identical to PMN-elastase, inhibited by cycloheximide and by anti-PMN elastase antibodies. T-lymphocytes are present in atherosclerotic plaques where elastin degradation occurs and could contribute to the chronicity of the lesion by the above mechanism.     

Evidence for production and functional activity of nitric oxide in seminiferous tubules and blood vessels of the human testis

Previous studies have demonstrated that nitric oxide (NO) influences Leydig cell function. Here we provide evidence for NO production and activity in seminiferous tubules and blood vessels of the human testis. By immunohistochemistry, the soluble guanylyl cyclase (sGC), the intracellular NO receptor, and the second messenger, cyclic guanosine monophosphate (cGMP), were detected in myofibroblasts of the peritubular lamina propria in Sertoli cells, as well as in endothelial and smooth muscle cells of testicular blood vessels. Performed with isolated tubules and blood vessels, the biological activity of sGC could be proved by cGMP generation in response to treatments with the NO donor, sodium nitroprusside. The endothelial and neuronal subtypes of NO synthase (NOS) were localized immunohistochemically to the same cell types that express sGC and cGMP. In isolated tubules and vessels, the presence of endothelial NOS and neuronal NOS was confirmed by immunoblotting, and NOS activity was demonstrated by decreased cGMP production upon incubation with the NOS inhibitor L-nitro arginine methylester. These findings show that peritubular cells, Sertoli cells, and testicular blood vessels may be sites of NO production and activity, possibly involved in relaxation of seminiferous tubules and blood vessels to modulate sperm transport and testicular blood flow, respectively.     

Astrocytoma cell interaction with elastin substrates: implications for astrocytoma invasive potential

Elastin has been identified within the meninges and the microvasculature of the normal human brain. However, the role that elastin plays in either facilitating astrocytoma cell attachment to these structures or modulating astrocytoma invasion has not been previously characterized. We have recently shown that astrocytoma cell lines and specimens produce tropoelastin, and express the 67 kDa elastin binding protein (EBP). In the present report, we have established that astrocytoma cells attach to elastin as a substrate in vitro. The U87 MG astrocytoma cell line demonstrated the greatest degree of adhesion. In addition, all astrocytoma cell lines examined were capable of penetrating and migrating through an intact elastin membrane, and of degrading tritiated-elastin, a process that could be prevented by the pre-incubation of astrocytoma cells with EDTA, but not with alpha1-antitrypsin. Astrocytoma cells were also capable of penetrating 1 mm sections of human brain tissue maintained as organotypic cultures. Interestingly, the invasive potential of cultured astrocytoma cells plated on organotypic cultures of human brain was significantly increased after exposure to elastin degradation products (kappa-elastin), which interact with astrocytoma cell surface EBP. Our data show that astrocytoma cells express a functional 67 kDa EBP, enabling them to potentially recognize and attach to elastin as a substrate. These data also suggest that this elastin receptor may be involved in processes which regulate regional astrocytoma invasion.     

Functional measurements of [Ca2+] in the endoplasmic reticulum using a herpes virus to deliver targeted aequorin

Nitric oxide (NO) is a free radical gas that is synthesized from L-arginine by NO synthase (NOS). Activation of NMDA, non-NMDA or metabotropic glutamate receptors causes NO formation through NOS activation. From data obtained in experiments performed by microdialysis together with nitrate assay, we have proposed that NO production in the cerebellum following non-NMDA and metabotropic glutamate receptor activation may be independent of NOS activity, while NMDA receptor-mediated NO production depends on its activity. Glial cells appear to play a role in modulating NO production by regulating L-arginine availability. Activation of NMDA receptors and the increase in intracellular calcium concentration is a trigger for the long-term potentiation (LTP). NO acts as a retrograde messenger in the hippocampal LTP to enhance glutamate release from presynaptic nerve terminal, in which cyclic GMP may be involved. Behavioral studies demonstrate that NO is involved in some forms of learning and memory. Our studies suggest that NMDA/NO/cyclic GMP signaling plays a role in spatial working memory. Further, it is suggested that NO production in the brain is altered by aging. These results support the hypothesis that NO plays a role in mechanism of synaptic plasticity.     

Aging and matrix biology

This introduction to a theme issue of Pathologie Biologie on the extracellular matrix starts with a brief overview of the advances made over the last few years and of the increasing specialization they have resulted in. A review is then presented of cell-matrix interactions, with emphasis on those mediated by the elastin-laminin receptor during physiologic processes and during aging and age-related diseases. The activated human lymphocyte expressing the elastin-laminin receptor is used as an example. When exposed to low levels of elastin peptides (kappa-elastin, 75 kappa D, 1-10 micrograms/ml, i.e., 1.4 to 14.10(-8)M), this receptor mediates increases in cell growth and in the production of serine-elastase. Levels of about 100 micrograms/ml are associated with cell death due to necrosis and to apoptosis. This example illustrates the key role played by epigenetic phenomena in aging of cells and tissues.     

Elastase derived elastin peptides: putative autoimmune targets in giant cell arteritis

OBJECTIVE: Histological analysis of giant cell arteritis (GCA) reveals a granulomatous reaction around the internal elastic lamina. Elastolysis by multinucleated giant cells has also been reported. We investigated elastin derived peptides as putative recall antigens for peripheral blood mononuclear cells (PBMC) from patients with GCA. METHODS: PBMC were collected from 17 patients with GCA (Group 1), 17 patients with vascular diseases, connective tissue diseases, or polymyalgia rheumatica without GCA (Group 2), and 17 healthy controls (Group 3). Cultures of PBMC with different elastin derived peptides or elastase were analyzed. RESULTS: A proliferative response was obtained only with elastate derived elastin peptides in 12/13 untreated patients with GCA. Steroid treatment was believed to abolish this proliferative response in 4 patients with GCA. PBMC from only 3/34 non-GCA subjects responded to these antigens. No proliferative response was obtained for other elastin derived peptides or elastase in any subject. CONCLUSION: Degradation of native elastin by leukocyte elastase can provide elastin derived peptides that act as autoimmune targets for T cells in GCA.     

Characterization of the elastin binding domain in the cell-surface 25-kDa elastin-binding protein of staphylococcus aureus (EbpS)

Our previous studies have established that a cell-surface 25-kDa elastin-binding protein of Staphylococcus aureus (EbpS) mediates binding of this pathogen to the extracellular matrix protein elastin. Results from binding assays examining the activity of various EbpS fragments suggested that the elastin recognition domain is contained within the first 59 amino acids. In this report, we have used functional analyses with synthetic peptides and recombinant truncated forms of EbpS to localize the elastin binding domain to a 21-amino acid region contained within residues 14-34 of EbpS. Further evidence for the importance of this domain was obtained by demonstrating that the inhibitory activity of anti-EbpS antibodies on staphylococcal elastin binding was neutralized when these antibodies were pre-absorbed with a truncated recombinant EbpS construct containing residues 1-34. Overlapping synthetic peptides corresponding to EbpS residues 14-36 were then generated and tested for elastin binding activity to define further the elastin binding domain, and results from these studies showed that sequences spanning amino acids Gln14-Asp23, Asp17-Asp23, and Thr18-Glu34 inhibit binding of Staphylococcus aureus to elastin. Our analyses indicate that the hexameric sequence Thr18-Asn-Ser-His-Gln-Asp23 is the minimal sequence common to all active synthetic peptides, proteolytic fragments, and recombinant constructs of EbpS. Furthermore, substitution of Asp23 with Asn abrogated the blocking activity of the synthetic peptides, demonstrating the requirement for a charged amino acid at this location. The composite data indicate that staphylococcal elastin binding is mediated by a discrete domain defined by short peptide sequences in the amino-terminal extracellular region of EbpS.     

Human cytomegalovirus increases modified low density lipoprotein uptake and scavenger receptor mRNA expression in vascular smooth muscle cells

Evidence suggests a possible role for human cytomegalovirus (HCMV) in the development of arteriosclerosis. One of the earliest events in plaque formation is the accumulation of lipid-laden foam cells, derived from macrophages and smooth muscle cells (SMCs). The lipid accumulation that occurs depends upon the uptake of oxidized LDL (Ox-LDL), a process in which the scavenger receptor (SR) has been postulated to play an important role. We therefore examined the effects of HCMV on this process. We demonstrate that HCMV infection of human SMCs increases modified LDL uptake and stimulates class A SR gene (SR-A) mRNA expression. In addition, infection of rat SMCs with HCMV, which causes immediate early gene expression (IE72/IE84), but no early or late HCMV gene products and no cytopathic effects, also increases SMC uptake of Ox-LDL and acetylated LDL, with either effect blocked by an excess of either cold Ox-LDL or acetylated-LDL, and by fucoidin, an SR competitor. Cotransfection of an IE72, but not an IE84, expression plasmid and a plasmid containing a Class A SR promoter/reporter gene construct enhances SR promoter activity. Since increased Ox-LDL uptake is believed to play an important role in arteriosclerosis, these results provide a link between HCMV infection and arteriosclerotic plaque formation.     

Nuclear and cytoplasmic free calcium level changes induced by elastin peptides in human endothelial cells

The extracellular matrix protein "elastin" is the major component of elastic fibers present in the arterial wall. Physiological degradation of elastic fibers, enhanced in vascular pathologies, leads to the presence of circulating elastin peptides (EP). EP have been demonstrated to influence cell migration and proliferation. EP also induce, at circulating pathophysiological concentrations (and not below), an endothelium- and NO- dependent vasorelaxation mediated by the 67-kDa subunit of the elastin-laminin receptor. Here, by using the techniques of patch-clamp, spectrofluorimetry and confocal microscopy, we demonstrate that circulating concentrations of EP activate low specificity calcium channels on human umbilical venous endothelial cells, resulting in increase in cytoplasmic and nuclear free calcium concentrations. This action is independent of phosphoinositide metabolism. Furthermore, these effects are inhibited by lactose, an antagonist of the elastin-laminin receptor, and by cytochalasin D, an actin microfilament depolymerizer. These observations suggest that EP-induced signal transduction is mediated by the elastin-laminin receptor via coupling of cytoskeletal actin microfilaments to membrane channels and to the nucleus. Because vascular remodeling and carcinogenesis are accompanied by extracellular matrix modifications involving elastin, the processes here described could play a role in the elastin-laminin receptor-mediated cellular migration, differentiation, proliferation, as in atherogenesis, and metastasis formation.     

Morphine-induced conformational changes in human monocytes, granulocytes, and endothelial cells and in invertebrate immunocytes and microglia are mediated by nitric oxide

We evaluated the contribution of nitric oxide (NO) to morphine-induced rounding of spontaneously activated (mobile) ameboid human monocytes, granulocytes, or arterial endothelial cells and invertebrate immunocytes and microglia. Morphine induced significant rounding and inactivation of ameboid cells within 20 min except for arterial endothelial cells, which became rounded 24 h after morphine exposure. The effects of morphine on cell conformation were blocked in the presence of N-nitro-L-arginine, a nitric oxide synthase inhibitor. Treatment of cells with the NO donor, sodium nitroprusside, induced cell rounding similar to that observed following morphine exposure, suggesting that NO release may mediate morphine-induced changes in cell conformation. The contribution of NO release to morphine-induced cell rounding was determined by direct evaluation of NO concentration in real-time using a NO-specific amperometric probe. Significant increases in NO concentration were observed 2 min after morphine stimulation, whereas morphine-induced NO release was markedly impaired by pretreatment with N-nitro-L-arginine or the opiate alkaloid antagonist, naloxone. In contrast, opioid peptides failed to induce NO release, consistent with our previous observations that demonstrated the failure of opioid peptides to promote cell rounding. Taken together, these data suggest that morphine-induced NO release may be mediated by activation of the opiate alkaloid-selective, opioid peptide-insensitive micro3 receptor, and that functional coupling of morphine to NO production has been conserved during evolution and may modulate cellular activation.     

Rhesus prophylaxis--history and current status]

The essential problem of the vicious circle leading to end-stage cardiovascular disease is atherosclerosis. This paper focuses on the functional changes centred on the endothelium that accompany the development of atherosclerosis, examining in particular pathological alterations in the L-arginine/nitric oxide (NO) pathway. Changes in the NO system are associated with altered platelet and monocyte interactions with the vessel wall, abnormal vasoconstriction and altered vascular structure. Diabetes, hyperglycaemia, hypertension and hypercholesterolaemia are all involved in this process. Endothelin is a vasoconstrictor peptide produced by endothelial cells which is upregulated under these conditions. Normalising endothelial function could involve platelet inhibition, lipid-lowering agents to prevent foam cell formation and decrease the lipid load of the blood vessel wall, and agents to interfere with some of the mechanisms involved in vasoconstriction, proliferation and migration, including ACE-inhibitors and angiotensin receptor antagonists, and possibly new tools such as endothelin receptor antagonists.     

Pharmacobehavioural evidence for nitric oxide and noradrenaline interactions with ryanodine receptors during memory formation in the young chick.

Impairment of nitric oxide (NO) production, ryanodine receptor (RyR) calcium channel function and adrenoceptor activation have been found to prevent the formation of the long-term memory stage in young chicks trained on a single-trial discrimination avoidance task. The current study investigated whether these three activities were linked, and if so, the sequence of activation. Young chicks were trained using either a strongly or weakly reinforced variant of the single-trial discrimination avoidance task, yielding either a persistent or labile memory trace, respectively. Following strongly reinforced training, retention loss induced by a RyR inhibitor was prevented by a NO donor or noradrenaline (NA). A RyR agonist also prevented retention loss induced by either NO synthase or β1+2-adrenoceptor inhibition. These findings were interpreted to reflect the capacity of NO, RyR-dependent calcium release and NA to modulate memory by preventing retention loss. A second set of studies used weakly reinforced training. Although the administration of a RyR agonist promoted long-term memory formation, this facilitation was compromised in the presence of a β1+2-adrenoceptor antagonist, but not a NO synthase inhibitor. Similarly, the inhibition of RyRs interfered with the facilitation of retention induced by a NO donor, but not NA. These differential findings with weakly reinforced training suggest that NO facilitates memory formation through mechanisms involving RyR-dependent calcium release. The findings also indicate that RyRs may promote memory formation through noradrenergic activation of β2-adrenoceptors. This study demonstrates an intricate role for RyRs underlying memory formation. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Cytokine-induced nitric oxide inhibits bone resorption by inducing apoptosis of osteoclast progenitors and suppressing osteoclast activity

Interferon-gamma (IFN-gamma) has been shown to inhibit interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) stimulated bone resorption by strongly stimulating nitric oxide (NO) synthesis. Here we studied the mechanisms underlying this inhibition. Osteoclasts were generated in 10-day cocultures of mouse osteoblasts and bone marrow cells and the effect of cytokine-induced NO on osteoclast formation and activity was determined. Stimulation of the cocultures with IL-1 beta, TNF-alpha and IFN-gamma markedly enhanced NO production by 50- to 70-fold, and this was found to be derived predominantly from the osteoblast cell layer. When high levels of NO were induced by cytokines during early stages of the cocultures, osteoclast formation was virtually abolished and bone resorption markedly inhibited. Cytokine stimulation during the latter stages of coculture also resulted in inhibition of bone resorption, but here the effects were mainly due to an inhibitory effect on osteoclast activity. At all stages, however, the inhibitory effects of cytokines on osteoclast formation and activity were blocked by the NO-synthase inhibitor L-NMMA. Further investigations suggested that the NO-mediated inhibition of osteoclast formation was due in part to apoptosis of osteoclast progenitors. Cytokine stimulation during the early stage of the culture caused a large increase in apoptosis of bone marrow cells, and these effects were blocked by L-NMMA and enhanced by NO donors. We found no evidence of apoptosis in osteoclasts exposed to high levels of cytokine-induced NO at any stage in the culture, however, or of apoptosis affecting mature osteoclasts exposed to high levels of NO, suggesting that immature cells in the bone marrow compartment are most sensitive to NO-induced apoptosis. In summary, these studies identify NO as a potentially important osteoblast-osteoclast coupling factor which has potent inhibitory effects on bone resorption. These actions, in turn, are mediated by inhibition of osteoclast formation probably due to NO-induced apoptosis of osteoclast progenitors and by inhibition of the resorptive activity of mature osteoclasts.