- Email: [email protected]
RPR 102359 upregulates LDL-receptors in a cultured human hepatocyte cell line by a sterol-independent mechanism
Tuesday 11 October 1994: Poster Abstracts Receptors
113
VIC JT, Kroon PA, Dept. of Biochem., Univ. of Queensland., Brisbane, Queensland 4072, Australia
icina i Hospital de Sant Joan, C/ Sant Llorenc 21. 43201 Reus, Spain
Numerous mutant alleles have been reported for the human LDL receptor (LDLR). The FH Cape Town-l allele produces an LDLR which lacks two contiguous amino acids, Asp-26 and Gly-27,
Many mutations of the LDL receptor gene have been identified in diverse populations. The ‘hot spot’ for these mutations appears to be in exon 4. The objective of the present study was to apply the SSCP technique in a rapid screening of our patients as a preliminary to follow-up studies and early diagnosis in family members. Selection criteria were plasma cholesterol >8 mmol/l, plasma triglycerides ~2.8 mmol/I, and at least one first-degree relative with a similar profile or with xanthomas. An exclusion criterion was any indication of a mis-diagnosis of the apo B 3500 mutation as FH using the amplification and specific enzymatic cleavage with MspI. As a preliminary screen, PCR was used to amplify exons 4 and 6 using published sequences of oligonucleotides followed by SSCP. Contrary to expectation, none of our patients showed any mutation in exon 4 while, conversely, four of the patients appeared to have the same mutation in exon 6 as evidenced by the identical banding patterns on SSCP. It seems unlikely that all of the latter four patients have exactly the same mutation; it is possible that nearly adjacent basepair mutations could give rise to very similar SSCP banding patterns. Conversely, if real, the finding of a higher frequency of mutations in this exon relative to exon 4 would indicate that a sub-set of our Catalan FH population is more genetically defined. We are in the process of direct sequencing of this PCR product to confirm the precise point of mutation in these patients.
from the amino-terminal cysteine-rich repeat (LBl) of the ligandbinding domain. Transport of these receptors to the cell surface is retarded and inefficient. To investigate the effects of this deletion on the folding of LBl. we exoressed LB1 and LBlA26-27 as thrombin-cleavable GST’ fusion’proteins in E. coli. Reverse-phase HPLC analysis
showed that the thrombin-released peptides existed in multiple forms. For LBl, these forms converged to a single molecular species following disultide exchange in the presence of oxidized and reduced glutathione. This form contained three disulfide bonds, and was recognized by a conformation-specific monoclonal antibody (IgGC7) for the LDLR, suggesting that it had the same conformation as LB1 in the native LDLR. In contrast, when LB 1A26-27 was incubated under the same conditions, a number of closely-eluting forms remained, none of which were reco nized by IgGC7. These data indicate that the deletion in LB1A%27 , which shortens the distance between the 4th and 5th cysteine residues, prevents this peptide from folding into a single form, but instead leads to multiple forms which are immunologically distinct from native LB 1. The results support a model for the FH Cape Town-l LDLR, in which the two-amino acid deletion causes the ligand binding repeat to m&fold, leading to retarded, inefficient transport through the endoplasmic reticulum.
1 @I m,
Expression of the first and fourth repeats of the LDL receptor ligand-binding domain: evidence for sequence-dependent requirements for folding Djordjevic JT, Kroon PA, Dept. of Biochem., univ. of
+ Recognition of lactoferrin and aminopeptidase Mmodified lactoferrin by the liver: involvement of proteoglycans
and the remnant receptor
Ziere GJ, Kruijt JK, Bijsterbosch MK, van Berkel TJC, Div. of Biophannaceutics, LACDR, Sylvius Lab., PO Box 9503, 2300 RA Leiden, The Netherlands
Queensland, Brisbane, Queensland 4072, Australia
The ligand-binding (LB) domain of the LDL receptor consists of seven imperfect repeats of approximately 40 residues each. Each LB repeat contains six highly conserved cysteine residues which are believed to form three i&a-repeat disulfide bonds. In view of their conserved nature it has been hypothesized that LB repeats share a similar overall structure. Here we investigate whether the sequence of LB repeats is sufficient to direct their folding into unique disulfide-bonded structures. We expressed the first (LBl) and fourth (LB4) LB repeats as thrombin-cleavable GST fusion proteins. Following release by thrombin cleavage, reverse-phase HPLC analysis showed that LB1 existed in a number of distinct forms. These were converted to a single molecular species following incubation at 4°C in a refolding buffer (50 mmol/l Tris-HCI, pH 8.0, 150 mmolll NaCI, 2.5 mmol/l CaCl2) which contained 3 mmol/l reduced glutathione and 0.3 mmoY1oxidized glutathione to facilitate thiol-disulfide exchange. The refolded form of LB1 contained three disultide bonds and was recognized by the conformationspecific monoclonal antibody IgG-C7. Under the same refolding conditions, LB4 eluted as four overlapping peaks. Attempts to refold LB4 under different conditions failed to produce a unique form. We conclude that the amino acid sequence of individual LB repeats is not always sufficient to specify a unique disulfidebonded structure. Correct folding of LB4 may require the presence of one or more of the N-terminal three LB repeats.
Lactoferrin inhibits the hepatic uptake of lipoprotein remnants, and we showed earlier that arginine residues of lactoferrin are involved. In this study, lactoferrin was treated with aminopeptidase-M (APM), which resulted in removal of 14 N-terminal amino acids, including 4 clustered arginines at positions 2-5 (APM-lactoferrin). After i.v. injection into rats, ‘251-labeled APM-lactoferrin was cleared within 10 min by the liver parenchymal cells (74.7% of the dose). Binding of APM-lactoferrin to isolated parenchymal liver cells was saturable, with a Kd of 186 nM (750 000 sites/cell). This is in striking contrast to the binding of lactofenin (& 1OpM; 20 X lo6 sites/cell). Prior injection of rats with 20 mg of APM-lactoferrin/kg body weight reduced the liver association of /3-VLDL by 501, whereas lactoferrin had no effect at this dose. With isolated parenchymal liver cells, APM-lactoferrin was a more effective competitor for /?VLDL binding than native lactoferrin (50% inhibition at 0.5 mg/ml vs. 8.0 mg/ml). Removal of chondroitin sulfate proteoglycans from the surface of parenchymal cells lowered the Kd and the amount of binding sites for lactoferrin 10 and 7 times, respectively. The binding of APM-lactoferrin and B-VLDL were not affected. We conclude that the 4-arginine cluster of lactoferrin at position 2-5 may be involved in the association of lactofenin with chondroitin sulfate proteoglycans on the parcnchymal liver cell, but does not inhibit lipoprotein remnant uptake. The Arg/Lys sequence at position 25-30, which resembles the binding site of apo E, may mediate the high affinity binding of lactofenin and block the binding of j%VLDL to the remnant receptor efficiently.
Unexpected
result
from
using
SSCP
to screen
for
El
LDL receptor mutations Alonso-Villaverde C, Margalef J, VallvC J-C, Ribalta J, Masana L, Turner PR, Unitat de Recerca de Lipids, Facultat de Med-
El
RPR 102359 upregulates LDL receptors in a cultured human hepatocyte cell line by a sterol-independent mechanism
Atherosclerosis X, Montreal, October I994
114
Tuesday I1 October 1994: Poster Abstracts Receptors
Brown T,!, White A, Lloyd J, Wong M, Vicker N, Lackey P, Rhone Poulenc Rarer, Dane&am Research Centre, Rainham Road South, Dagenham, Essex RMIO 7XS, UK
Upregulation of hepatic LDL receptors can be achieved by inhibition of cholesterol and oxysterol biosynthesis or by mechanisms independent of the cellular sterol pool. RPR 102359 (N-(5-3’cyclohexylpropanamido-2-methylphenyl)-4-hydroxy-~nz~ide) is a member of a novel series of upregulators of compounds which upregulate LDL receptor expression in cultured Hep G2 cells. The purpose of this study was to determine whether RPR 102359 was able to upregulate LDL receptors via a sterol-independent mechanism. LDL receptor expression was measured in Hep G2 cells cultured in DME medium using a mouse anti-LDL receptor monoclonal antibody specific for the human LDL receptor and an alkaline phosphatase-conjugated anti-mouse antibody with attaphos as substrate. RPR 102359 (l-10pM) increased in a concentrationdependent manner the expression of LDL receptor, with an EC50 of 1.7 pM and a maximum upregulation of 290%. In the presence of human LDL (5-lOO,uLg/ml) or 25-hydroxycholesterol (0.35 PM), which downregulate receptor expression, RPR 102359 still significantly increased receptor expression. This profile of activity distinguished RPR 102359 from the HMG-CoA reductase inhibitor mevinolin, which was inactive in the presence of LDL and 25hydroxycholesterol, and also from ketoconazole whose activity can be abolished by 25-hydroxycholesterol. Furthermore, in the presence of maximally upregulating concentrations of mevinolin (O.l-3pM) RPR 102359 was able to produce an additional increase in LDL receptor expression. These data suggest that RPR 102359 stimulates LDL receptor expression by a sterol-independent mechanism. 1 n,
Analysis of the lipoprotein binding site of rat liver membrane Adam L, Falstrault L, Charest M-C, Dept. des Sci-
ences Biol., Univ. du Quebec a Montreal, Montreal, Canada
Intermediate density lipoproteins (IDL) were shown to bind to high- and low-affinity binding sites on rat liver membranes. The low-affinity sites were named the ‘lipoprotein binding site’ (LBS) since they bind ah classes of lipoproteins (Brissette et al J Biol Chem 1986; 261: 11631-11638). This study was undertaken to further characterize the interaction of 1251-labeled IDL with the LBS of rat liver membranes and to determine the chemical nature of the LBS. We found that the binding of IDL to the LBS is insensitive to EDTA and is sensitive to heparin. Membranes were pre-treated with various enzymes that have an effect on the membrane constituents and the activity of the LBS on these treated membranes was determined. The LBS was insensitive to heparinase I, chondroitinase ABC and phospholipase C while being partially sensitive to phospholipase A2 and sensitive to proteases and heat. Rat liver membrane proteins were solubilized with Triton X-100, reconstituted in liposomes and analyzed for their ability to bind lipoproteins. 12?-labeled IDL was shown to bind to high- and low-affinity sites that are similar in affinity and specificity to those in intact rat liver membranes, which indicates that a LBS activity is detectable on these liposomes. We found that the binding capacity of liposomes containing either no protein or proteins solubilized from E. coli membranes is 5 times weaker. Thus, a specific protein is responsible for the LBS activity of the liposomes containing solubilized rat liver membranes. Taken together, our results indicate that LBS activity is in part mediated by a protein. m
Mato’s fluorescent granular perithelial cells express macrophage scavenger receptors, mediate scavenger
function and cause the narrowing of microarteries in brain cortex ltakura, Matsumoto A, Honda M, Kodama T, Sakamoto A, Aikawa E, Ogawa T, Gokawara S, Mitsuhashi U, Mato M, National Inst. of Health and Nutrition, l-23-1, Toyama, Shinjuku-ku, Tokyo, University of Tokyo and Jichi Med. Sch., Japan
Mato’s fluorescent granular perithelial cells (Mato’s FGP cells) are perivasculsr cells with fluorescent granules distributed along the cerebral small vessels in mammalian brains. In the bovine cerebral cortex, cells which express macrophage scavenger receptors are identified as FGP cells. Developmental analysis of rat FGP cells revealed that: (1) they express macrophage-related antigens, which are detected as early as l-3 weeks after birth, (2) they contain substances from the blood, including low density LDL, and from neural tissue, including neurotransmitters and degradation products, (3) the lipid contents and lipase activities of inclusion bodies increase during aging, (4) the enlargement of inclusion bodies results in the formation of foam cells containing honeycomb structures, and (5) swollen FGP cells apply pressure to adjacent vessels, which causes narrowing of the lumen of small vessels. These results indicate that Mato’s FGP cells belong to the macrophage lineage, mediate scavenger function, and may play an important role in microvessel injury in the brain cortex. Nitric oxide production and oxy_LDL metabolism in 1 rat peritoneal macrophages Dulak J, Wybrat?ska I, Baczydska E, Pawelec M, DembiiiskaKieC A, Dept. of Clin. Biochem., Coil. Med., Jagiellonian Univ., 31501 Krakow, Kopemika ISa, Poland
The role of LDL in the development of atherosclerosis is well known and recent evidence suggests that oxidation of LDL and their accumulation by macrophages in the arterial wall is a key event in this process. The aim of this study was to investigate the influence of NO donors 3-morpholinosyndnonimine (SIN-l) and sodium nitroprusside (NaNP) as well as an NO synthesis inhibitor, NG-monomethyl+arginine (LNMMA), on metabolism of oxLDL by rat peritoneal macrophages. The cells were first incubated without (control) and with SIN1 (30-300pM) or with NaNP (30-3OOpM). These pre-incubations were also performed in the presence or absence of LNMMA. Afterwards, the macrophages were incubated with lOO~g/ml of 1251-labe1edox-LDL with or without NO donors and inhibitor. The TCA- and AgNOs-soluble degradation products of 12sI-labeled ox-LDL were measured in a gamma counter and NO generation by the cells was studied in culture media by Griess’ method. We found a tendency of ox-LDL, but not n-LDL, to suppress the lipopolysacchatide-stimulated biosynthesis of NO by macrophages. SIN-l at high concentration (3OOpM) and NaNP (30-3OOpM) stimulated the accumulation and degradation of oxLDL by macrophages. Prior treatment of macrophages with LNMMA had a similar stimulatory effect. However, in the presence of L-NMMA the stimulatory action of SIN-l was transformed to inhibition of the accumulation and degradation of ox-LDL, while NaNP lost its stimulatory action entirely. These observations suggest that endogenous NO in macrophages inhibits the accumulation of ox-LDL, but NO at high concentrations promotes lipid accumulation in macrophages. We further suppose that NO at low physiological concentration keeps scavenger receptors of macrophages downregulated, so that endogenous NO may show anti-atherogenic properties. Modeling and creation of the LDL receptor based on 1 the apo B-binding peptide ,&h&ov AL, Markin SS, Inst. of Biomed. Chem.. Russia, Acad. of Med. Sci., Pogodinskaya str. 10, Moscow, Russia
Atherosclerosis X, Montreal, October 1994
113
VIC JT, Kroon PA, Dept. of Biochem., Univ. of Queensland., Brisbane, Queensland 4072, Australia
icina i Hospital de Sant Joan, C/ Sant Llorenc 21. 43201 Reus, Spain
Numerous mutant alleles have been reported for the human LDL receptor (LDLR). The FH Cape Town-l allele produces an LDLR which lacks two contiguous amino acids, Asp-26 and Gly-27,
Many mutations of the LDL receptor gene have been identified in diverse populations. The ‘hot spot’ for these mutations appears to be in exon 4. The objective of the present study was to apply the SSCP technique in a rapid screening of our patients as a preliminary to follow-up studies and early diagnosis in family members. Selection criteria were plasma cholesterol >8 mmol/l, plasma triglycerides ~2.8 mmol/I, and at least one first-degree relative with a similar profile or with xanthomas. An exclusion criterion was any indication of a mis-diagnosis of the apo B 3500 mutation as FH using the amplification and specific enzymatic cleavage with MspI. As a preliminary screen, PCR was used to amplify exons 4 and 6 using published sequences of oligonucleotides followed by SSCP. Contrary to expectation, none of our patients showed any mutation in exon 4 while, conversely, four of the patients appeared to have the same mutation in exon 6 as evidenced by the identical banding patterns on SSCP. It seems unlikely that all of the latter four patients have exactly the same mutation; it is possible that nearly adjacent basepair mutations could give rise to very similar SSCP banding patterns. Conversely, if real, the finding of a higher frequency of mutations in this exon relative to exon 4 would indicate that a sub-set of our Catalan FH population is more genetically defined. We are in the process of direct sequencing of this PCR product to confirm the precise point of mutation in these patients.
from the amino-terminal cysteine-rich repeat (LBl) of the ligandbinding domain. Transport of these receptors to the cell surface is retarded and inefficient. To investigate the effects of this deletion on the folding of LBl. we exoressed LB1 and LBlA26-27 as thrombin-cleavable GST’ fusion’proteins in E. coli. Reverse-phase HPLC analysis
showed that the thrombin-released peptides existed in multiple forms. For LBl, these forms converged to a single molecular species following disultide exchange in the presence of oxidized and reduced glutathione. This form contained three disulfide bonds, and was recognized by a conformation-specific monoclonal antibody (IgGC7) for the LDLR, suggesting that it had the same conformation as LB1 in the native LDLR. In contrast, when LB 1A26-27 was incubated under the same conditions, a number of closely-eluting forms remained, none of which were reco nized by IgGC7. These data indicate that the deletion in LB1A%27 , which shortens the distance between the 4th and 5th cysteine residues, prevents this peptide from folding into a single form, but instead leads to multiple forms which are immunologically distinct from native LB 1. The results support a model for the FH Cape Town-l LDLR, in which the two-amino acid deletion causes the ligand binding repeat to m&fold, leading to retarded, inefficient transport through the endoplasmic reticulum.
1 @I m,
Expression of the first and fourth repeats of the LDL receptor ligand-binding domain: evidence for sequence-dependent requirements for folding Djordjevic JT, Kroon PA, Dept. of Biochem., univ. of
+ Recognition of lactoferrin and aminopeptidase Mmodified lactoferrin by the liver: involvement of proteoglycans
and the remnant receptor
Ziere GJ, Kruijt JK, Bijsterbosch MK, van Berkel TJC, Div. of Biophannaceutics, LACDR, Sylvius Lab., PO Box 9503, 2300 RA Leiden, The Netherlands
Queensland, Brisbane, Queensland 4072, Australia
The ligand-binding (LB) domain of the LDL receptor consists of seven imperfect repeats of approximately 40 residues each. Each LB repeat contains six highly conserved cysteine residues which are believed to form three i&a-repeat disulfide bonds. In view of their conserved nature it has been hypothesized that LB repeats share a similar overall structure. Here we investigate whether the sequence of LB repeats is sufficient to direct their folding into unique disulfide-bonded structures. We expressed the first (LBl) and fourth (LB4) LB repeats as thrombin-cleavable GST fusion proteins. Following release by thrombin cleavage, reverse-phase HPLC analysis showed that LB1 existed in a number of distinct forms. These were converted to a single molecular species following incubation at 4°C in a refolding buffer (50 mmol/l Tris-HCI, pH 8.0, 150 mmolll NaCI, 2.5 mmol/l CaCl2) which contained 3 mmol/l reduced glutathione and 0.3 mmoY1oxidized glutathione to facilitate thiol-disulfide exchange. The refolded form of LB1 contained three disultide bonds and was recognized by the conformationspecific monoclonal antibody IgG-C7. Under the same refolding conditions, LB4 eluted as four overlapping peaks. Attempts to refold LB4 under different conditions failed to produce a unique form. We conclude that the amino acid sequence of individual LB repeats is not always sufficient to specify a unique disulfidebonded structure. Correct folding of LB4 may require the presence of one or more of the N-terminal three LB repeats.
Lactoferrin inhibits the hepatic uptake of lipoprotein remnants, and we showed earlier that arginine residues of lactoferrin are involved. In this study, lactoferrin was treated with aminopeptidase-M (APM), which resulted in removal of 14 N-terminal amino acids, including 4 clustered arginines at positions 2-5 (APM-lactoferrin). After i.v. injection into rats, ‘251-labeled APM-lactoferrin was cleared within 10 min by the liver parenchymal cells (74.7% of the dose). Binding of APM-lactoferrin to isolated parenchymal liver cells was saturable, with a Kd of 186 nM (750 000 sites/cell). This is in striking contrast to the binding of lactofenin (& 1OpM; 20 X lo6 sites/cell). Prior injection of rats with 20 mg of APM-lactoferrin/kg body weight reduced the liver association of /3-VLDL by 501, whereas lactoferrin had no effect at this dose. With isolated parenchymal liver cells, APM-lactoferrin was a more effective competitor for /?VLDL binding than native lactoferrin (50% inhibition at 0.5 mg/ml vs. 8.0 mg/ml). Removal of chondroitin sulfate proteoglycans from the surface of parenchymal cells lowered the Kd and the amount of binding sites for lactoferrin 10 and 7 times, respectively. The binding of APM-lactoferrin and B-VLDL were not affected. We conclude that the 4-arginine cluster of lactoferrin at position 2-5 may be involved in the association of lactofenin with chondroitin sulfate proteoglycans on the parcnchymal liver cell, but does not inhibit lipoprotein remnant uptake. The Arg/Lys sequence at position 25-30, which resembles the binding site of apo E, may mediate the high affinity binding of lactofenin and block the binding of j%VLDL to the remnant receptor efficiently.
Unexpected
result
from
using
SSCP
to screen
for
El
LDL receptor mutations Alonso-Villaverde C, Margalef J, VallvC J-C, Ribalta J, Masana L, Turner PR, Unitat de Recerca de Lipids, Facultat de Med-
El
RPR 102359 upregulates LDL receptors in a cultured human hepatocyte cell line by a sterol-independent mechanism
Atherosclerosis X, Montreal, October I994
114
Tuesday I1 October 1994: Poster Abstracts Receptors
Brown T,!, White A, Lloyd J, Wong M, Vicker N, Lackey P, Rhone Poulenc Rarer, Dane&am Research Centre, Rainham Road South, Dagenham, Essex RMIO 7XS, UK
Upregulation of hepatic LDL receptors can be achieved by inhibition of cholesterol and oxysterol biosynthesis or by mechanisms independent of the cellular sterol pool. RPR 102359 (N-(5-3’cyclohexylpropanamido-2-methylphenyl)-4-hydroxy-~nz~ide) is a member of a novel series of upregulators of compounds which upregulate LDL receptor expression in cultured Hep G2 cells. The purpose of this study was to determine whether RPR 102359 was able to upregulate LDL receptors via a sterol-independent mechanism. LDL receptor expression was measured in Hep G2 cells cultured in DME medium using a mouse anti-LDL receptor monoclonal antibody specific for the human LDL receptor and an alkaline phosphatase-conjugated anti-mouse antibody with attaphos as substrate. RPR 102359 (l-10pM) increased in a concentrationdependent manner the expression of LDL receptor, with an EC50 of 1.7 pM and a maximum upregulation of 290%. In the presence of human LDL (5-lOO,uLg/ml) or 25-hydroxycholesterol (0.35 PM), which downregulate receptor expression, RPR 102359 still significantly increased receptor expression. This profile of activity distinguished RPR 102359 from the HMG-CoA reductase inhibitor mevinolin, which was inactive in the presence of LDL and 25hydroxycholesterol, and also from ketoconazole whose activity can be abolished by 25-hydroxycholesterol. Furthermore, in the presence of maximally upregulating concentrations of mevinolin (O.l-3pM) RPR 102359 was able to produce an additional increase in LDL receptor expression. These data suggest that RPR 102359 stimulates LDL receptor expression by a sterol-independent mechanism. 1 n,
Analysis of the lipoprotein binding site of rat liver membrane Adam L, Falstrault L, Charest M-C, Dept. des Sci-
ences Biol., Univ. du Quebec a Montreal, Montreal, Canada
Intermediate density lipoproteins (IDL) were shown to bind to high- and low-affinity binding sites on rat liver membranes. The low-affinity sites were named the ‘lipoprotein binding site’ (LBS) since they bind ah classes of lipoproteins (Brissette et al J Biol Chem 1986; 261: 11631-11638). This study was undertaken to further characterize the interaction of 1251-labeled IDL with the LBS of rat liver membranes and to determine the chemical nature of the LBS. We found that the binding of IDL to the LBS is insensitive to EDTA and is sensitive to heparin. Membranes were pre-treated with various enzymes that have an effect on the membrane constituents and the activity of the LBS on these treated membranes was determined. The LBS was insensitive to heparinase I, chondroitinase ABC and phospholipase C while being partially sensitive to phospholipase A2 and sensitive to proteases and heat. Rat liver membrane proteins were solubilized with Triton X-100, reconstituted in liposomes and analyzed for their ability to bind lipoproteins. 12?-labeled IDL was shown to bind to high- and low-affinity sites that are similar in affinity and specificity to those in intact rat liver membranes, which indicates that a LBS activity is detectable on these liposomes. We found that the binding capacity of liposomes containing either no protein or proteins solubilized from E. coli membranes is 5 times weaker. Thus, a specific protein is responsible for the LBS activity of the liposomes containing solubilized rat liver membranes. Taken together, our results indicate that LBS activity is in part mediated by a protein. m
Mato’s fluorescent granular perithelial cells express macrophage scavenger receptors, mediate scavenger
function and cause the narrowing of microarteries in brain cortex ltakura, Matsumoto A, Honda M, Kodama T, Sakamoto A, Aikawa E, Ogawa T, Gokawara S, Mitsuhashi U, Mato M, National Inst. of Health and Nutrition, l-23-1, Toyama, Shinjuku-ku, Tokyo, University of Tokyo and Jichi Med. Sch., Japan
Mato’s fluorescent granular perithelial cells (Mato’s FGP cells) are perivasculsr cells with fluorescent granules distributed along the cerebral small vessels in mammalian brains. In the bovine cerebral cortex, cells which express macrophage scavenger receptors are identified as FGP cells. Developmental analysis of rat FGP cells revealed that: (1) they express macrophage-related antigens, which are detected as early as l-3 weeks after birth, (2) they contain substances from the blood, including low density LDL, and from neural tissue, including neurotransmitters and degradation products, (3) the lipid contents and lipase activities of inclusion bodies increase during aging, (4) the enlargement of inclusion bodies results in the formation of foam cells containing honeycomb structures, and (5) swollen FGP cells apply pressure to adjacent vessels, which causes narrowing of the lumen of small vessels. These results indicate that Mato’s FGP cells belong to the macrophage lineage, mediate scavenger function, and may play an important role in microvessel injury in the brain cortex. Nitric oxide production and oxy_LDL metabolism in 1 rat peritoneal macrophages Dulak J, Wybrat?ska I, Baczydska E, Pawelec M, DembiiiskaKieC A, Dept. of Clin. Biochem., Coil. Med., Jagiellonian Univ., 31501 Krakow, Kopemika ISa, Poland
The role of LDL in the development of atherosclerosis is well known and recent evidence suggests that oxidation of LDL and their accumulation by macrophages in the arterial wall is a key event in this process. The aim of this study was to investigate the influence of NO donors 3-morpholinosyndnonimine (SIN-l) and sodium nitroprusside (NaNP) as well as an NO synthesis inhibitor, NG-monomethyl+arginine (LNMMA), on metabolism of oxLDL by rat peritoneal macrophages. The cells were first incubated without (control) and with SIN1 (30-300pM) or with NaNP (30-3OOpM). These pre-incubations were also performed in the presence or absence of LNMMA. Afterwards, the macrophages were incubated with lOO~g/ml of 1251-labe1edox-LDL with or without NO donors and inhibitor. The TCA- and AgNOs-soluble degradation products of 12sI-labeled ox-LDL were measured in a gamma counter and NO generation by the cells was studied in culture media by Griess’ method. We found a tendency of ox-LDL, but not n-LDL, to suppress the lipopolysacchatide-stimulated biosynthesis of NO by macrophages. SIN-l at high concentration (3OOpM) and NaNP (30-3OOpM) stimulated the accumulation and degradation of oxLDL by macrophages. Prior treatment of macrophages with LNMMA had a similar stimulatory effect. However, in the presence of L-NMMA the stimulatory action of SIN-l was transformed to inhibition of the accumulation and degradation of ox-LDL, while NaNP lost its stimulatory action entirely. These observations suggest that endogenous NO in macrophages inhibits the accumulation of ox-LDL, but NO at high concentrations promotes lipid accumulation in macrophages. We further suppose that NO at low physiological concentration keeps scavenger receptors of macrophages downregulated, so that endogenous NO may show anti-atherogenic properties. Modeling and creation of the LDL receptor based on 1 the apo B-binding peptide ,&h&ov AL, Markin SS, Inst. of Biomed. Chem.. Russia, Acad. of Med. Sci., Pogodinskaya str. 10, Moscow, Russia
Atherosclerosis X, Montreal, October 1994