Monocyte CD147 is induced by advanced glycation end products and high glucose concentration: possible role in diabetic complications.

CD147 is a highly glycosylated transmembrane protein that is known to play a role in regulation of many protein families. It has the unique ability to maintain functional activity in both the membrane bound state and in the soluble form. CD147 is known to play a role in regulation of matrix metalloproteinase (MMP) expression, but whether its expression is affected by the diabetic milieu is not known, and its role in regulation of monocyte MMPs in this environment has not been investigated. Therefore, in this study we investigated the effect of advanced glycation end products (AGEs) and high glucose (HG; 25 mM), on monocyte CD147 expression. Culture of THP-1 monocytes in the presence of AGEs or HG significantly increased CD147 at the gene and protein level. THP-1 cell results were confirmed using freshly isolated monocytes from human volunteers. The effect of AGEs and HG on CD147 expression was also mimicked by addition of proinflammatory cytokines. Addition of AGEs or HG also increased expression of monocyte MMP-1 and MMP-9 but not MMP-2. This increase in MMPs was significantly attenuated by inhibition of CD147 using either a small interfering RNA or an anti-CD147 antibody. Inhibition of NF-κB or addition of antibodies to either TNF-α or the receptor for AGE (RAGE) each significantly prevented in a dose-dependent manner the induction of CD147 gene and protein by AGE and also decreased MMP-1 and MMP-9. This novel result shows that AGEs can induce monocyte CD147 expression, an effect mediated by inflammatory pathways and RAGE. Because MMPs play a role in monocyte migration, inhibition of their regulator CD147 may assist in the prevention of diabetic complications, particularly those where monocyte infiltration is an early initiating event.

Metabolic effects of low dose angiotensin converting enzyme inhibitor in dietary obesity in the rat.

BACKGROUND AND AIMS: Given the recent observation of a local renin-angiotensin system (RAS) in adipose tissue, and its association with obesity-related hypertension, the metabolic effects of treatment with a low dose angiotensin converting enzyme inhibitor (ACEI) were investigated in a rodent model of diet-induced obesity. METHODS AND RESULTS: Male Sprague Dawley rats were exposed to either standard laboratory chow (12% calories as fat) or palatable high fat (30% calories as fat) diet for 12 weeks. A subset from both dietary groups was given low dose ACEI in drinking water (perindopril, 0.3 mg/kg/day) throughout the study. The high fat diet increased body weight, adiposity, circulating leptin and insulin and in the liver we observed fat accumulation and increased tissue ACE activity. Treatment with perindopril decreased food intake and circulating insulin in both diet groups, and hepatic ACE activity in high fat fed animals only. Decreased plasma leptin concentration with ACE inhibition was only evident in chow fed animals. These effects were independent of any blood pressure lowering effect of ACE inhibition. CONCLUSION: Chronic low dose ACEI treatment reduced circulating insulin and leptin levels with some reduction in food intake in chow fed rats. Fewer beneficial effects were observed in obesity, and further work is required to investigate higher ACEI doses. Our data suggest a reduction in hepatic ACE activity may affect lipid accumulation and other inflammatory responses, as well as improving insulin resistance. Our findings may have implications for maximizing the clinical benefit of ACEI in patients without overt cardiovascular complications.

Chronic liver injury in rats and humans upregulates the novel enzyme angiotensin converting enzyme 2.

BACKGROUND: Angiotensin converting enzyme (ACE) 2 is a recently identified homologue of ACE that may counterregulate the actions of angiotensin (Ang) II by facilitating its breakdown to Ang 1-7. The renin-angiotensin system (RAS) has been implicated in the pathogenesis of cirrhosis but the role of ACE2 in liver disease is not known. AIMS: This study examined the effects of liver injury on ACE2 expression and activity in experimental hepatic fibrosis and human cirrhosis, and the effects of Ang 1-7 on vascular tone in cirrhotic rat aorta. METHODS: In sham operated and bile duct ligated (BDL) rats, quantitative reverse transcriptase-polymerase chain reaction was used to assess hepatic ACE2 mRNA, and western blotting and immunohistochemistry to quantify and localise ACE2 protein. ACE2 activity was quantified by quenched fluorescent substrate assay. Similar studies were performed in normal human liver and in hepatitis C cirrhosis. RESULTS: ACE2 mRNA was detectable at low levels in rat liver and increased following BDL (363-fold; p < 0.01). ACE2 protein increased after BDL (23.5-fold; p < 0.05) as did ACE2 activity (fourfold; p < 0.05). In human cirrhotic liver, gene (>30-fold), protein expression (97-fold), and activity of ACE2 (2.4 fold) were increased compared with controls (all p < 0.01). In healthy livers, ACE2 was confined to endothelial cells, occasional bile ducts, and perivenular hepatocytes but in both BDL and human cirrhosis there was widespread parenchymal expression of ACE2 protein. Exposure of cultured human hepatocytes to hypoxia led to increased ACE2 expression. In preconstricted rat aorta, Ang 1-7 alone did not affect vascular tone but it significantly enhanced acetylcholine mediated vasodilatation in cirrhotic vessels. CONCLUSIONS: ACE2 expression is significantly increased in liver injury in both humans and rat, possibly in response to increasing hepatocellular hypoxia, and may modulate RAS activity in cirrhosis.

Membrane-associated zinc peptidase families: comparing ACE and ACE2.

In contrast to the relatively ubiquitous angiotensin-converting enzyme (ACE), expression of the mammalian ACE homologue, ACE2, was initially described in the heart, kidney and testis. ACE2 is a type I integral membrane protein with its active site domain exposed to the extracellular surface of endothelial cells and the renal tubular epithelium. Here ACE2 is poised to metabolise circulating peptides which may include angiotensin II, a potent vasoconstrictor and the product of angiotensin I cleavage by ACE. To this end, ACE2 may counterbalance the effects of ACE within the renin-angiotensin system (RAS). Indeed, ACE2 has been implicated in the regulation of heart and renal function where it is proposed to control the levels of angiotensin II relative to its hypotensive metabolite, angiotensin-(1-7). The recent solution of the structure of ACE2, and ACE, has provided new insight into the substrate and inhibitor profiles of these two key regulators of the RAS. As the complexity of this crucial pathway is unravelled, there is a growing interest in the therapeutic potential of agents that modulate the activity of ACE2.

Angiotensin-converting enzyme-2: a molecular and cellular perspective.

Angiotensin-converting enzyme-2 (ACE2) is the first human homologue of ACE to be described. ACE2 is a type I integral membrane protein which functions as a carboxypeptidase, cleaving a single hydrophobic/basic residue from the C-terminus of its substrates. ACE2 efficiently hydrolyses the potent vasoconstrictor angiotensin II to angiotensin (1-7). It is a consequence of this action that ACE2 participates in the renin-angiotensin system. However, ACE2 also hydrolyses dynorphin A (1-13), apelin-13 and des-Arg(9) bradykinin. The role of ACE2 in these peptide systems has yet to be revealed. A physiological role for ACE2 has been implicated in hypertension, cardiac function, heart function and diabetes, and as a receptor of the severe acute respiratory syndrome coronavirus. This paper reviews the biochemistry of ACE2 and discusses key findings such as the elucidation of crystal structures for ACE2 and testicular ACE and the development of ACE2 inhibitors that have now provided a basis for future research on this enzyme.

Structure-activity relationships of neurokinin A (4-10) at the human tachykinin NK(2) receptor: the role of natural residues and their chirality.

A structure-activity study of neurokinin A (NKA) (4-10) was performed to investigate the importance of residue and chirality for affinity and efficacy at the NK(2) receptor in human colon circular muscle. Two series of NKA(4-10) analogues were produced with either L-alanine or the D-enantiomer substituted. Their activities were determined in vitro by means of radioligand binding and isolated smooth muscle pharmacology. NKA was more potent than NKA(4-10) at the human, unlike the rabbit, NK(2) receptor. The contractile response of NKA(4-10) was unaffected by N-terminal acetylation. L-Ala substitution of Asp(4), Val(7), Leu(9), and Met(10) caused an 8- to 80-fold decrease, and substitution of Phe(6) caused a 5000-fold decrease in binding affinity (P < 0.01). Positions Ser(5) and Gly(8) were not significantly affected. In functional studies, a similar pattern was observed. The replacement of residues with their respective D-enantiomer drastically reduced binding affinity and functional potency, particularly at positions 6 and 7 (P < 0.05). NKA(4-10) analogues L-Ala(6), L-Ala(8), D-Phe(6), D-Val(7), and D-Met(10) were partial agonists. An excellent correlation was observed between binding and functional data (r = 0.95). A retro-inverso analogue of NKA(4-10) was inactive. In conclusion, the side chains of Asp(4), Phe(6), Val(7), Leu(9), and Met(10) are structurally important features of NKA(4-10) for agonist activity, and changes in amino acid chirality are detrimental to binding affinity and functional activity. Overall, our data are broadly similar to those of previous studies in the rat. However, at the human NK(2) receptor, unlike the rat, [Ala(8)]NKA(4-10) was an antagonist.

Circular muscle contraction, messenger signalling and localization of binding sites for neurokinin A in human sigmoid colon.

1. Neurokinin (NK)A is the endogenous ligand for the tachykinin NK2 receptor. In the present study, tachykinins and selective receptor agonists were tested as contractile agonists in human colon circular muscle and [125I]-NKA was used to localize binding sites in human colon. 2. In strips of circular muscle, removal of mucosa and submucosa significantly (P < 0.05) increased the potency and the maximum response achieved by NKA. 3. The rank order of potency of tachykinin and selective receptor agonists in contracting circular muscle strips was NKA > or = [Lys5,MeLeu9,Nle10]NKA(4-10) > or = neuropeptide (NP)gamma > or = [betaAla8]NKA(4-10) >> NKB > substance P (SP) >> senktide approximate to [Pro9]SP. 4. Specific binding sites for [125I]-NKA were densely localized over circular muscle and muscularis mucosae. Weak specific binding was seen on longitudinal muscle and taenia coli, whereas no binding sites were seen on mucosa, ganglia or blood vessels. 5. In circular muscle, the selective NK2 receptor agonist [LysS,MeLeu9,Nle10]NKA(4-10) produced weak increases (maximum 37%) in inositol monophosphate formation with a pD2 of 6.8+/-0.51 (n = 3). Carbachol (100 micromol/L) was also a weak stimulant (maximum 45%). These agonists were over 10-fold more efficacious in stimulation of inositol monophosphate in rat urinary bladder. 6. In conclusion, [125I]-NKA binding sites localized on human colon circular muscle were characterized as NK2 receptors. Functionally, the tachykinin NK2 receptor is mediating circular smooth muscle contraction. Although the human NK2 receptor is coupled to the phosphatidylinositol pathway, other second messenger mechanisms may also operate in this tissue.

Pharmacological and biochemical investigation of receptors for the toad gut tachykinin peptide, bufokinin, in its species of origin.

This is the first report of the development of a new radioligand [125I]Bolton-Hunter bufokinin ([125I]BH-bufokinin) and its use in the characterisation of tachykinin receptors in the small intestine of the cane toad, Bufo matrinus. The binding of [125I]BH-bufokinin to toad intestinal membranes was rapid, saturable, of high affinity and to a single population of binding sites with KD 0.57 nM and Bmax 3.1 fmol mg wet weight tissue(-1). The rank order of affinity of tachykinins to compete for [125I]-BH bufokinin binding revealed similarities with that of the mammalian NK1 receptor, being bufokinin (IC50, 1.7 nM)>physalaemin (6.7 nM)>substance P (SP, 10.7 nM)> or =neuropeptide gamma (NPgamma, 12.4 nM)> or =kassinin (17.8 nM)>scyliorhinin I (35.3 nM)> or =eledoisin (40.6 nM)> or =carassin (43.2 nM)> or =neurokinin A (NKA, 57.8 nM)> or =neurokinin B (NKB, 77.5 nM)>scyliorhinin II (338 nM). The mammalian NK3-selective agonist senktide was a very weak competitor. The radioligand [125I]neurokinin A showed no specific binding to toad intestinal membranes. In the toad isolated small intestine, the maximum contractile response to bufokinin was over 150% greater than that to acetylcholine in longitudinal muscle, whereas responses to bufokinin and acetylcholine were similar in circular muscle. Bufokinin was the most potent agonist (EC501 0.34 nM) and produced a long-lasting contraction. Other tachykinins such as physalaemin, SP and kassinin were also potent contractile agents. The potency values of mammalian and amphibian tachykinins derived from functional studies (pD2) correlated significantly with those from binding assays (pKi). The data for fish and molluscan tachykinins, however, showed poor correlation. Contractions to bufokinin and SP were unaffected by atropine, indomethacin and tetrodotoxin. The highly selective NK1 receptor antagonists CP 99994, GR 82334 and RP 67580 were ineffective in both binding and functional studies. Bufokinin increased inositol monophosphate formation in a concentration-dependent manner with an EC50 value of 10.7 nM, suggesting that the tachykinin receptor may be coupled to phosphoinositol hydrolysis. In summary, this study provides evidence for a high-affinity, bufokinin-preferring, NK1-like tachykinin receptor in the toad small intestine. This is probably not the receptor which mediates contraction to carassin, scyliorhinin II and eledoisin. The study also provides evidence that bufokinin and its receptor play an important physiological role in regulating intestinal motility.

Characterization of the [125I]-neurokinin A binding site in the circular muscle of human colon.

Neurokinin A (NKA) is a potent contractile agonist of human colon circular muscle. These responses are mediated predominantly through tachykinin NK2 receptors. In the present study, the NK2 receptor radioligand [125I]-NKA has been used to characterize binding sites in this tissue, using tachykinin agonists and antagonists. 125INKA labelled a single, high affinity binding site. Specific binding (95% of total binding) of [125I]-NKA was saturable (K(D) 0.47+/-0.05 nM), of high capacity (Bmax 2.1+/-0.1 fmol mg(-1) wet weight tissue) and reversible (kinetically derived K(D) 0.36+/-0.07 nM). The rank order of agonists competing for the [125I]-NKA binding site was neuropeptide gamma (NPgamma) > or = NKA > or = [Lys5, MeLeu9,Nle10]NKA (4-10) (NK2 agonist) >> substance P (SP) > neurokinin B (NKB) > or = [Pro9]SP (NK1 agonist) >> senktide (NK3 agonist), indicating binding to an NK2 site. The nonpeptide selective NK2 antagonist SR48968 showed higher affinity for the [125I]-NKA site than selective peptide NK2 antagonists. The rank order of potency for NK2 antagonists was SR48968 > or = MEN11420 > GR94800 > or = MEN10627 > MEN10376 > or = R396. The NK1 antagonist SR140333 was a weak competitor. The competition curve for SP could be resolved into two sites. When experiments were repeated in the presence of SR140333 (0.1 microM), the curve for SP became monophasic and showed a significant shift to the right, whereas curves to NKA and NKB were unaffected. In conclusion, binding of the radioligand [125I]-NKA to membranes from circular muscle is predominantly to the NK2 receptor. There may be a small component of binding to the NK1 receptor. The NK2 receptor mediates circular muscle contraction, whereas the role of the NK1 receptor in circular muscle is unclear.

Relevance of aromatic residues in transmembrane segments V to VII for binding of peptide and nonpeptide antagonists to the human tachykinin NK(2) receptor.

We used membranes from Chinese hamster ovary cells stably transfected with the human tachykinin NK(2) receptor, either wild-type or mutated, at four aromatic residues (His(198), Tyr(266), Phe(270), Tyr(289)) located in transmembrane segments V to VII, to assess the role of these residues in the binding of natural tachykinins and peptide and nonpeptide antagonists. Three radioligands, the agonist [(125)I]neurokinin A (NKA), the peptide antagonist [(3)H]MEN 11420, and the nonpeptide antagonist [(3)H]SR 48968 bound to the wild-type receptor with high affinity (K(d) = 2.4 nM, 0.3 nM, and 4.0 nM, respectively). Four of the six mutant receptors tested retained high affinity for at least one of the radioligands. H(198)A mutation abrogated the binding of NKA but not that of MEN 11420 or SR 48968 (K(d) = 4.8 and 11.5 nM, respectively); Y(266)F mutation abrogated the binding of MEN 11420 but not that of NKA or SR 48968 (K(d) = 2.8 nM and 1.2 nM, respectively); F(270)A mutation abrogated the binding of both NKA and MEN 11420 but not that of SR 48968 (K(d) = 1.6 nM); Y(289)F mutation abrogated the binding of SR 48968 but not that of NKA and MEN 11420 (K(d) = 2.0 and 2.9 nM, respectively). Y(266)A and Y(289)A mutations abrogated the binding of all radioligands. Among the unlabeled antagonists, the affinity of the nonpeptide GR 159897, at variance with SR 48968, resulted heavily compromised by H(198)A and Y(266)F mutations; the peptide antagonists R396 and MEN 10376 essentially followed the binding profile of NKA, but R396 showed markedly increased affinity for the Y(289)F mutant receptor. Taken together, these results indicate that different, partially overlapping sets of sites may be involved in the binding of agonists and diverse antagonists to the human tachykinin NK(2) receptor.

Molecular characterization of two novel transporters from human and mouse kidney and from LLC-PK1 cells reveals a novel conserved family that is homologous to bacterial and Aspergillus nucleobase transporters.

Nucleobase transport is important for the metabolism of nucleic acids and antiviral and antineoplastic drugs. This transport has been functionally described in several mammalian cells but has not been well characterized molecularly. We report the cloning of two novel transporters. YSPL2 encodes a 650-residue protein and has an ubiquitous 8 kb transcript. The human and pig homologs are 95% similar. YSPL3 encodes a 598-residue protein with a 3 kb transcript that is expressed only in kidney and liver. Human YSPL2 and YSPL3 are 60% similar at the amino acid level and both show 31% similarity to the first nucleobase permease gene described in vertebrates, YSPL1. These proteins appear to be members of a new family of possible nucleobase transporters with significant sequence similarities with bacterial and Aspergillus nucleobase transporters. Further functional studies will be needed to unveil the role of these transporters in nucleic acid metabolism in normal and in disease states.

Purification, characterization, and spasmogenic activity of neurotensin from the toad Bufo marinus.

Neurotensin (NT) was isolated from an extract of the intestine of the cane toad, Bufo marinus and its primary structure established as: pGlu-Ala-Ile-Val-Ser-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu. This amino acid sequence shows five substitutions (Leu2 --> Ala, Tyr3 --> Ile, Glu4 --> Val, Asn5 --> Ser, and Pro7 --> Ala) compared with bovine NT. Synthetic Bufo NT (pD2 = 8.05 +/- 0.28) was equipotent and equally effective as bovine NT (pD2 = 8.24 +/- 0.38) in producing spasmogenic contraction of isolated segments of toad small intestine. However, the maximum response produced by Bufo NT was only 35 +/- 2% of that produced by substance P. The potencies, but not the maximum responses, to Bufo and bovine NT were significantly (p < 0.05) attenuated by pre-treatment with atropine but neither parameter was significantly diminished by tetrodotoxin and indomethacin. The data suggest that the action of NT involves interaction with receptors on toad intestinal smooth muscle that recognize the C-terminal region of NT (residues 8-13) that has been fully conserved during evolution of tetrapods. Contractile activity is mediated, at least in part, by release of acetylcholine.

Tachykinin receptors in the small intestine of the cane toad (Bufo marinus): a radioligand binding and functional study.

In this study, we have used radioligand binding and functional techniques to investigate tachykinin receptors in the small intestine of the cane toad Bufo marinus. The radioligand [125I]Bolton-Hunter [Sar9,Met(O2)11]substance P (selective at mammalian NK-1 receptors) showed no specific binding. Specific binding of [125I]Bolton-Hunter substance P ([125I]BHSP) was saturable, of high affinity (Kd 0.3 nM) and was inhibited by SP (IC50 0.64 nM) > ranakinin approximately neurokinin A (NKA) > or = SP(5-11) > or = neuropeptide gamma > or = scyliorhinin II > scyliorhinin I > or = [Sar9]-SP > or = neurokinin B approximately physalaemin approximately carassin >> SP(7-11) approximately eledoisin > or = SP(4-11) approximately SP(6-11). Binding was also inhibited by Gpp[NH]p > or = GTPgammaS > App[NH]p, indicating a G-protein coupled receptor. The order of potency of tachykinins and analogues in contracting the isolated lower small intestine was carassin (EC50 1.4 nM) > eledoisin approximately SP > or = physalaemin > or = ranakinin > SP(6-11) > scyliorhinin II > or = neuropeptide gamma > neurokinin B approximately NKA approximately scyliorhinin I > or = SP(4-11) > or = SP(5-11) > [Sar9]SP > SP(7-11). In both studies, the selective mammalian NK-1, NK-2 and NK-3 receptor agonists [Sar9,Met(O2)11]SP, [Lys5,Me-Leu9,Nle10]NKA(4-10) and senktide were weak or ineffective. There was a strong positive correlation between the pD2 and pIC50 values for mammalian tachykinins and analogues (r = 0.907), but not for the non-mammalian tachykinins, which were all full agonists but variable binding competitors. [Sar9,Met(O2)11]-SP(pD2 5.7) was approximately 25-fold less potent as an agonist than [Sar9]SP, which was itself 25-fold weaker than SP. Responses to SP were significantly reduced (n = 8, P<0.001) by the antagonist [D-Arg1,D-Trp7,9,Leu11]-SP (spantide; 1 microM). Highly selective NK-1 receptor antagonists including CP 99994 and GR 82334 (both 1 microM) were ineffective in both functional and binding studies. Tetrodotoxin (1 microM) did not inhibit contractile responses to SP, NKA and senktide. In summary, this study has shown the presence of one or more tachykinin receptor in the toad intestine. The binding site recognised by [125I]BHSP prefers SP and ranakinin. This toad "NK-1-like receptor" differs from the mammalian NK-1 receptor in having a low affinity for all mammalian NK-1 selective ligands, including antagonists. For some non-mammalian peptides, their high potency as contractile agonists relative to their poor binding affinity suggests the existence of other tachykinin receptors in the toad small intestine.

Bufokinin: a substance P-related peptide from the gut of the toad, Bufo marinus with high binding affinity but low selectivity for mammalian tachykinin receptors.

A tachykinin peptide, termed bufokinin, was isolated in pure form from an extract of the intestine of the toad, Bufo marinus, and its primary structure was established as: Lys-Pro-Arg-Pro-Asp-Gln-Phe-Tyr-Gly-Leu-Met.NH2. This sequence was confirmed by chemical synthesis and shows four amino acid substitutions (Arg1 --> Lys,Lys3 --> Arg,Gln5 --> Asp and Phe8 --> Tyr) compared with substance P. Binding parameters for synthetic bufokinin and mammalian tachykinins were compared using receptor-selective radioligands and crude membranes from rat tissues enriched in the NK-1 (submandibular gland) , NK-2 (stomach fundus) and NK-3 (brain) receptors. In terms of inhibiting the binding of the selective radioligands, bufokinin (Kd = 0.3 nM) was 1.8-fold more potent than substance P at the rat NK-1 site, but it was only 2-fold less potent (Kd = 2.8 nM) than neurokinin A at the NK-2 site and only 2-fold less potent (Kd = 48 nM) than neurokinin B at the NK-3 site. Thus, bufokinin shows relatively high affinity but lack of selectivity for all three tachykinin binding sites in rat tissues.