共查询到20条相似文献,搜索用时 15 毫秒
1.
M Jaziri D Migliore-Samour M R Casabianca-Pignède K Keddad J L Morgat P Jollès 《Biochimica et biophysica acta》1992,1160(3):251-261
Two immunostimulating peptides were isolated from human milk proteins by enzymatic digestion, the tripeptide GLF and the hexapeptide VEPIPY. These peptides increased the phagocytosis of human and murine macrophages and protected mice against Klebsiella pneumoniae infection. The present study showed that this activity may be correlated to the presence of specific binding sites on human blood phagocytic cells. The receptor molecules implicated were different for the two peptides. [3H]GLF specifically bound to PMNL and monocytes, whereas [3H]VEPIPY only bound to monocytes. The leukemic promyelocytic cell line HL-60 differentiated into granulocytes or into macrophages (depending on inducer used) coroborated these results. Specific binding of [3H]GLF on plasma membrane preparations of human PMNL (20 degrees C) was saturable and Scatchard analysis indicated two classes of binding sites: high-affinity sites of Kd 2.3 +/- 1.0 nM and Bm 60 +/- 9 fmol/mg protein and low-affinity sites of Kd 26.0 +/- 3.5 nM and Bm 208 +/- 45 fmol/mg protein. [3H]GLF binding was inhibited in a concentration-dependent manner by various analogous peptides, such as LLF, GLY, LLY and RGDGLF, but not by RGD, RGDS, VEPIPY and the chemotactic peptide f-Met-Leu-Phe (f-MLF). Only at high concentrations the direct analog MLF competed with labeled GLF. An important inhibitory effect was also observed with C1q component of the complement whereas C3 and BSA were uneffective. Specific binding of [3H]VEPIPY on monocyte membranes (20 degrees C) was saturable and Scatchard analysis was consistent with one class of binding sites of Kd 3.7 +/- 0.3 nM and Bm 150 +/- 6 fmol/mg protein. 相似文献
2.
Effect of diabetes and Sorbinil treatment on phospholipid metabolism in rat glomeruli 总被引:1,自引:0,他引:1
To explore the hypothesis that changes in membrane phospholipids accompany tissue myo-inositol depletion and reduced (Na+ + K+)-ATPase activity in diabetes, we examined phospholipid concentrations in glomeruli isolated from control and streptozotocin-diabetic rats and the effect of diabetes on myo-[3H]inositol incorporation in vitro into glomerular phosphatidylinositol. Since the aldose reductase inhibitor, Sorbinil, prevents the fall in myo-inositol and the decrease in (Na+ + K+)-ATPase activity associated with diabetes, phospholipid and phosphatidylinositol content were also examined in glomeruli isolated from Sorbinil-treated diabetic rats. Total phospholipids (microgram phosphorus/mg dry weight) did not differ in the three groups of animals. The concentration of phosphatidylcholine was elevated in preparations from diabetic rats, both untreated and Sorbinil-treated. Phosphatidylethanolamine was reduced in glomeruli from Sorbinil-treated rats. Neither acute experimental diabetes nor Sorbinil treatment produced detectable changes in the glomerular concentration of phosphatidylinositol. In vitro incubations with glomeruli isolated from control and diabetic animals resulted in increased levels of incorporation of myo-[3H]inositol into phospholipids of diabetic glomeruli. The specific activity of [3H]phosphatidylinositol in glomeruli from diabetic rats was significantly greater than that in control samples. The findings do not support the postulate invoking correspondent changes in myo-inositol and phosphatidylinositol contents as contributory to diminished glomerular (Na+ + K+)-ATPase activity in diabetes, but are compatible with depletion of glomerular intracellular myo-inositol in diabetes. 相似文献
3.
Angiotensin II (AII) binding sites were localized and quantified in kidney and adrenal of the frog Rana temporaria by quantitative in vitro autoradiography. AII binding was present in kidney glomeruli and in interrenal tissue of the outer zone of the adrenal gland. Saturation experiments showed that [125I]-[Val5]AII binds to a single class of binding sites with a dissociation constant (Kd) of 548 +/- 125 pM in glomeruli and 593 +/- 185 pM in interrenal tissue (n = 8). The corresponding maximal binding capacities (Bmax) were 2.48 +/- 0.71 and 3.05 +/- 1.02 fmol/mm2, respectively. AII binding was displaced by unlabeled angiotensin analogues in the rank order: [Sar1]AII greater than human AII greater than [125I]-[Val5]AII = [Val5]AII = human AIII much greater than human AI. The AII binding sites in glomeruli and interrenal tissue suggest an influence of AII on glomerular filtration rate and adrenal steroid secretion to take part in osmomineral regulation of the frog. 相似文献
4.
M C Sekar M Yang E Meezan D J Pillion 《Biochemical and biophysical research communications》1990,166(1):373-379
We have investigated the effect of angiotensin II, bradykinin, insulin and insulin-like growth factor I on phosphoinositide turnover in intact rat glomeruli and tubules. Angiotensin II produced a dose-dependent increase in inositol monophosphate formation with an IC50 of 10(-7)M, when added to isolated rat glomeruli. Angiotensin II-stimulated inositol phosphates formation was inhibited by the angiotensin receptor antagonist [Sar-Leu8]angiotensin II, indicating that the above response was mediated through activation of an angiotensin receptor in intact glomeruli. Besides angiotensin, in intact glomeruli, only bradykinin stimulated a phosphoinositide response, while in intact proximal tubules, none of the agonists tested produced an activation of the inositol phosphate formation. Angiotensin II- and bradykinin-stimulated inositol phosphate accumulation in intact glomeruli was inhibited by phorbol myristate acetate, an activator of protein kinase C. 相似文献
5.
Saeki T Matoba K Furukawa H Kirifuji K Kanamoto R Iwami K 《Journal of biochemistry》1999,125(4):846-851
Mouse ileal sodium dependent bile acid transporter (ISBT) was characterized using isolated enterocytes. Only enterocytes from the most distal portion showed Na+-dependent [3H]taurocholate uptake. Northern blot analysis using a probe against mouse ISBT revealed the expression of mouse ISBT mRNA to be restricted to the distal ileum. The Km and Vmax for Na+-dependent [3H]taurocholate transport into isolated ileocytes were calculated as 27 microM and 360 pmol/mg protein/min, respectively. Uptake of [3H]taurocholate was inhibited by N-ethylmaleimide. We have cloned ISBT cDNA from mouse ileum. The cDNA included the entire open reading frame coding 348 amino acid protein with seven hydrophobic segments and two N-glycosylation sites. COS-7 cells transfected with the expression vector containing this cDNA expressed Na+-dependent [3H]taurocholate uptake activity with a Km of 34 microM. 相似文献
6.
The present study investigated whether specific [3H]oxytocin binding sites previously demonstrated in estrogen-dominated rabbit uterus have properties expected of physiologic receptors coupled to uterine contraction. Microsomal membranes from estrogen-dominated rabbit uterus were found to contain high-affinity specific oxytocin binding sites with Kd = 2-3 nM. These sites were predominantly myometrial in locus. Specific oxytocin binding exhibited a pH optimum between 7.5 and 8.0. Mg2+ or Mn2+ was necessary for maximal specific [3H]oxytocin binding; in contrast, Ca2+ at submillimolar concentrations inhibited specific binding. Oxytocin binding sites were not detectable in microsomal membranes isolated from progesterone-dominated rabbit uterus. Relative binding and uterotonic activities of 10 synthetic neurohypophyseal hormone analogues were determined in estrogen-dominated rabbit uterus. A qualitative correlation was observed between binding and uterotonic responses. Angiotensin II and insulin did not compete with [3H]oxytocin for uterine binding sites. It is concluded that the specific high affinity [3H]oxytocin binding sites demonstrated in estrogen-dominated rabbit uterus have the selectivity for neurohypophyseal hormone analogues expected for physiologic receptors coupled to uterine contraction. 相似文献
7.
Pulmonary angtiotensin-converting enzyme (ACE) is located on the luminal surface of pulmonary microvasculature. Multiple indicator-dilution techniques have been used to measure pulmonary ACE activity in vivo and in isolated lungs. These studies suggest that ACE activity is depressed in several forms of acute lung injury. Depression of ACE activity may reflect impaired substrate delivery to enzyme sites because of flow-related reduction of perfused surface area. To assess the role of altered microvascular flow and surface area in the measurement of ACE activity, we utilized similar techniques to estimate the apparent Km and Vmax of pulmonary ACE in isolated, Krebs-perfused rabbit lungs. Km is an estimate of the affinity of a synthetic ACE substrate, [3H]benzoyl-phenyl-alanyl-alanyl-proline ([3H]BPAP), for ACE and should not be influenced by the rate of substrate delivery to luminal enzyme sites. Conversely, Vmax is an index of the number of ACE sites and should be influenced by perfusion changes that alter the number of perfused sites (recruitment or derecruitment). When isolated lungs were subjected to physiological maneuvers designed to increase or decrease perfused surface area, apparent Vmax increased or decreased respectively. Apparent Km was not altered by these maneuvers. Km and Vmax were independent of changes in perfusion rate when surface area was held constant. Thus these parameters should be useful in evaluating perfusion changes in normal and injured lungs. 相似文献
8.
S Ochi Y Fujiwara Y Orita Y Tanaka S Shin T Takama A Wada N Ueda T Kamada 《Biochimica et biophysica acta》1987,927(1):100-105
To clarify the signal transduction mechanism of angiotensin II in renal glomeruli, we studied the effect of the hormone on phospholipid metabolism using isolated rat glomeruli. Stimulation of the glomeruli pulse-chase labeled with [3H]glycerol by angiotensin II caused a rapid (within 15 s) breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) with a concurrent production of 1,2-diacylglycerol. This effect of angiotensin II was in a dose-dependent manner within the range from 10(-12) M to 10(-6) M, and was inhibited by saralasin. Angiotensin II also decreased the 3H radioactivity of PIP slightly only at 15 s and increased that of phosphatidic acid after 15 s, with no significant effect upon the labelings of phosphatidylinositol (PI), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) within 1 min. The change in phospholipid metabolism by angiotensin II was similar when the glomeruli were labeled with [32P]orthophosphate: the decrease in the labeling of PIP2 and the increase in the labeling of phosphatidic acid after 15 s. In addition, 32P labeling of PI increased after 2 min. These results suggest that angiotensin II, after binding to glomerular receptors, induces initial PIP2 hydrolysis to diacylglycerol and subsequent resynthesis of PIP2 through phosphoinositide turnover. 相似文献
9.
Thromboxane (TX) A2 effects in the kidneys include contraction of glomerular mesangial cells and intrarenal vascular tissue. A kidney cDNA encoding a TX receptor expressed in rat renal glomeruli and rat renal arterial smooth muscle cells has been reported. However, TXA2 receptors in human kidneys have not been documented. The purpose of this study was to identify and characterize TXA2 receptors in glomeruli and intrarenal arteries isolated from human kidneys. Normal kidneys, not used for transplant because of technical reasons, were kept at -70 degrees C and used for research purposes. The glomeruli and intrarenal arteries were isolated from renal cortical tissue by a mechanical sieving technique. The equilibrium dissociation constant and receptor number were determined by nonlinear analysis of binding inhibition data. The data were generated in radioreceptor assays using [125I]-BOP, a stable analog of TXA2. The dissociation constants (mean +/- SEM) for binding of I-BOP to human glomeruli and intrarenal arterial membranes were 6.6 +/- 1.1 nM (n = 7) and 20 +/- 6 nM (n = 7), respectively (p < 0.05). The receptor number was 311 +/- 91 fmol/mg protein (n = 7) in glomeruli and 74 +/- 16 fmol/mg protein (n = 7) in intrarenal arterial membranes (p < 0.04). The order of specificity of TXA2 analogs for [125I]-BOP binding sites was similar in glomeruli and in arterial membranes and was I-BOP > or = U46619 > or = pinane TXA2 > or = carbocyclic TXA2 > or = PGH2. These findings provide direct evidence for the presence of specific, high-affinity [125I]-BOP binding sites in human renal glomeruli and extraglomerular vascular tissue. These data also indicate that the human binding sites have higher affinity for the TXA2 agonist I-BOP than for PGH2. 相似文献
10.
We characterized thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors and histamine H1 receptors in Guinea-pig cultured tracheal smooth-muscle cells (TSMC). [3H]SQ 29,548 (a TXA2 antagonist)-binding sites were saturable and a high affinity with a dissociation constant of 6.2 +/- 0.60 nM (mean +/- S.E.) and a receptor density of 46 +/- 4.6 fmol/10(6) cells. [3H]SQ 29548 binding was completely inhibited by TXA2 mimetics or antagonists. Intracellular calcium concentration ([Ca2+]i) in TSMC was increased with U46619 stimulation and the increase was attenuated by TXA2 antagonists, the potencies of which correlated with those inhibiting the activities of the [3H]SQ 29548 binding. [3H]Mepyramine (a H1 antagonist)-binding sites were also present in TSMC. [3H]Mepyramine had a single class of low-affinity-binding sites with a dissociation constant of 2.6 +/- 0.081 microM and a receptor density of 10.6 +/- 0.11 nmol/mg protein. [3H]Mepyramine binding in TSMC membrane was inhibited by H1 antagonists, but not by H2 antagonists. The inhibition constants of mepyramine in TSMC were 910-times lower than those in tracheal membranes. In contrast, the histamine-induced increase in [Ca2+]i in TSMC was inhibited in the presence of low concentrations of H1 antagonists. All these observations provide evidence that TXA2/PGH2 receptors, mepyramine-binding sites and/or H1 receptors are expressed in cultured TSMC. 相似文献
11.
Angiotensin II (Ang II) binding sites were characterized in primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. Binding of [3H]Ang II to BMECs was time dependent and saturable. Scatchard plot analysis of dose-dependent [3H]Ang II binding revealed a single population of binding sites (Kd = 3.1 nM, Bmax = 52 fmoles/mg protein). Sarathrin, an Ang II antagonist, and saralsin, a partial agonist, inhibited [3H]Ang II binding to BMEC monolayers, whereas two unrelated peptides, bradykinin and arginine-vasopressin, had no effect on the specific binding of [3H]Ang II. At 37 degrees C, [3H]Ang II was internalized in BMECs and this uptake appeared to be saturable. Nanomolar concentrations of Ang II and saralasin stimulated [3H]thymidine uptake in serum-free starved BMEC monolayers, corresponding to an increase in DNA synthesis. On the other hand, sarathrin had no effect on [3H]thymidine uptake. The affinity of the single population of Ang II of binding sites was consistent with the concentration range of Ang II actions demonstrated in several cell types including BMECs. The Ang II-mediated actions on DNA synthesis suggest that this peptide-hormone may possess growth regulating properties in BMECs through either surface or internal site interactions. Collective findings support the complex nature of Ang II in regulating vascular and nonvascular cell growth and permeability characteristics. 相似文献
12.
Identification and functional importance of tyrosine sulfate residues within recombinant factor VIII. 总被引:3,自引:0,他引:3
Sulfated tyrosine residues within recombinant human factor VIII were identified by [35S]sulfate biosynthetic labeling of Chinese hamster ovary cells which express human recombinant factor VIII. Alkaline hydrolysis of purified [35S]sulfate-labeled factor VIII showed that greater than 95% of the [35S]sulfate was incorporated into tyrosine. [3H]Tyrosine and [35S]sulfate double labeling was used to quantify the presence of 6 mol of tyrosine sulfate per mole of factor VIII. Amino acid sequence analysis of thrombin and tryptic peptides isolated from [35S]sulfate-labeled factor VIII demonstrated tyrosine sulfate at residue 346 in the factor VIII heavy chain and at residues 1664 and 1680 in the factor VIII light chain. In addition, the carboxyl-terminal half of the A2 domain contained three tyrosine sulfate residues, likely at positions 718, 719, and 723. Interestingly, all sites of tyrosine sulfation border thrombin cleavage sites. The functional importance of tyrosine sulfation was examined by treatment of cells expressing factor VIII with sodium chlorate, a potent inhibitor of tyrosine sulfation. Increasing concentrations of sodium chlorate inhibited sulfate incorporation into factor VIII without affecting its synthesis and/or secretion. However, factor VIII secreted in the presence of sodium chlorate exhibited a 5-fold reduction in procoagulant activity, although the protein was susceptible to thrombin cleavage. These results suggest that tyrosine sulfation is required for full factor VIII activity and may affect the interaction of factor VIII with other components of the coagulation cascade. 相似文献
13.
The binding of [3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [3H]forskolin bound to protein from free [3H]forskolin by rapid filtration. The Kd for [3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (GS). 相似文献
14.
A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex 总被引:18,自引:0,他引:18
E Sigel F A Stephenson C Mamalaki E A Barnard 《The Journal of biological chemistry》1983,258(11):6965-6971
The gamma-aminobutyric acid/benzodiazepine receptor from bovine cerebral cortex was solubilized with sodium deoxycholate and purified by affinity chromatography on benzodiazepine-agarose and ion exchange chromatography. The benzodiazepine binding protein was enriched 1800-fold. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of two major bands of Mr = 57,000 and 53,000. [3H]Flunitrazepam, after UV irradiation, was incorporated irreversibly into both bands of the isolated protein. A high affinity binding site for gamma-aminobutyric acid was co-purified with the benzodiazepine binding site and the two sites were shown to reside on the same physical structure. The dissociation constants were 10 +/- 4 nM for [3H] flunitrazepam and 12 +/- 3 nM for the gamma-aminobutyric acid agonist [3H]muscimol. The maximum specific activity for [3H] muscimol binding was 4.3 nmol/mg of protein. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was between 3 and 4. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 7.3 +/- 0.5 nm and a sedimentation coefficient of 11.1 +/- 0.3 S, respectively. The purified complex had a pharmacological profile that corresponds to the receptor specificity found in membranes and crude soluble extracts. 相似文献
15.
Characterization of alpha 2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine 总被引:3,自引:0,他引:3
A Mukherjee 《Biochimica et biophysica acta》1981,676(2):148-154
[3H]Yohimbine, a potent alpha 2-adrenergic antagonist, was used to label the alpha-adrenergic receptors in membranes isolated from human platelets. Binding of [3H]yohimbine to platelet membranes appears to have all the characteristics of binding to alpha-adrenergic receptors. Binding reached a steady state in 2-3 min at 37 degrees C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10(-5) M; t1/2 = 2.37 min). [3H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [3H]Yohimbine binding was stereo-specifically inhibited by epinephrine: the (-) isomer was 11-times more potent that the (+) isomer. Catecholamine agonists competed for the occupancy of the [3H]yohimbine-binding sites with an order of potency: clonidine greater than (-)-epinephrine greater than (-)-norepinephrine much greater than (-)-isoproterenol. The potent alpha-adrenergic antagonist, phentolamine, competed for the sites whereas the beta-antagonist, (+/-)-propranolol, was very weak inhibitor. 0.1 mM GTP reduced the binding affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonin competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [3H]yohimbine binding. These results suggest that [3H]yohimbine binding to hunan platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label alpha 2-adrenergic receptors. 相似文献
16.
A comparison of methods measuring aromatase activity in human placenta and rat ovary 总被引:1,自引:0,他引:1
The single step chromatographic product isolation method using [4-14C]-androstenedione and the tritiated water method using either [1 beta, 2 beta-3H]- or [1 beta-3H]-androstenedione have been used to determine a suitable method to measure the aromatase activity in rat ovarian 1000 g supernatant and human placental microsomes. The single step product isolation method using [4-14C]A4 reveals the presence of four distinct [4-14C]-labelled products in the rat ovary of which only the synthesis of estradiol is markedly inhibited by CGS 16949A, a well established aromatase inhibitor. In the human placenta, the formation of both [4-14C]-estrone and [4-14C]-estradiol is strongly inhibited by CGS 6949A. Therefore, in the rat ovary spurious results are obtained if accumulative radiolabelled product formation is measured without characterisation of the products. The Vmax in the rat ovary using [1 beta, 2 beta-3H]-A4 as a substrate is 13.7 pmol/h/mg compared to 2.9 pmol/h/mg when [1 beta-3H]-A4 is used. In the human placenta, the Vmax is similar using either [1 beta, 2 betat-3H]-A4 or [1 beta-3H]-A4 (1.21 and 1.27 nmol/h/mg, respectively). Consistent results are obtained for the human placenta using either the single step chromatographic product isolation method or the tritiated water method. However, in the rat ovary the more suitable method of the two used to measure the aromatase activity is the tritiated water method employing [1 beta-3H]-A4 as a substrate. Aromatase activity in the rat ovary during estrus cycle was measured using the tritiated water method employing [1 beta-3H]-A4 as a substrate. A peak of aromatase activity at proestrus was seen which returned rapidly to its basal level at estrus. Plasma estradiol concentrations were in parallel with the aromatase activity. 相似文献
17.
At different concentrations, prostaglandin E2 (PGE2) can either stimulate or inhibit cAMP formation in freshly isolated rabbit cortical collecting tubule (RCCT) cells, but in cultured RCCT cells PGE2 can only stimulate cAMP synthesis (Sonnenburg, W. K., and Smith W. L. (1989) J. Biol. Chem. 263, 6155-6160). Here, we report characteristics of [3H]PGE2 binding to membrane receptor preparations from both freshly isolated and cultured RCCT cells. [3H]PGE2 binding to membranes from freshly isolated RCCT cells was saturable and partially reversible. Equilibrium binding analyses indicated that in the absence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) there is a single class of PGE2 binding sites (KD = 4.2 +/- 0.4 nM; Bmax = 583 +/- 28 fmol/mg); in the presence of 100 microM GTP gamma S, there is also only one class of binding sites but with a somewhat lower KD = 1.2 +/- 0.5 nM (Bmax = 370 +/- 40 fmol/mg). This stimulatory effect of GTP gamma S was blocked by pretreatment of the freshly isolated RCCT cells with pertussis toxin. The relative affinities of prostanoids for the [3H]PGE2-binding site were determined to be 17,18,19,20-tetranor-16-phenoxy-PGE2-methylsulfonylamide (sulprostone) approximately PGE2 approximately PGE1 approximately 16,16-dimethyl-PGE2 greater than carbacyclin approximately PGF2 alpha greater than PGD2. This is the order of potency with which prostaglandins inhibit arginine vasopressin-induced cAMP formation in fresh RCCT cells. Interestingly, [3H]PGE2 binding to membranes from cultured cells, which, unlike fresh cells, fail to show an inhibitory response to PGE2, was only 10-20% of that observed with membranes from fresh cells; moreover, binding of [3H]PGE2 to membranes from cultured cells was neither stimulated by GTP gamma S nor inhibited by sulprostone. The prostanoid binding specificities and the unusual pertussis toxin-sensitive, stimulatory effect of GTP gamma S on binding of [3H]PGE2 to membranes from freshly isolated RCCT cells are characteristics shared by a Gi-linked PGE receptor from renal medulla (Watanabe, T., Umegaki, K., and Smith, W. L. (1986) J. Biol. Chem. 261, 14340-14349). Our results suggest that the [3H]PGE2 binding site of freshly isolated RCCT cells is the PGE receptor which is coupled to a Gi to attenuate arginine vasopressin-induced cAMP synthesis in the renal collecting tubule. 相似文献
18.
Enalapril improves albuminuria by preventing glomerular loss of heparan sulfate in diabetic rats 总被引:5,自引:0,他引:5
A S Reddi R Ramamurthi M Miller S Dhuper N Lasker 《Biochemical medicine and metabolic biology》1991,45(1):119-131
Angiotensin converting enzyme (ACE) inhibitors, particularly enalapril and captopril, have been shown to decrease proteinuria in diabetic animals and human subjects. Since heparan sulfate proteoglycan confers a negative charge on the glomerular basement membrane, and either decreased synthesis or loss of this charge causes albuminuria in diabetic animals, we examined the possibility that enalapril prevents albuminuria through glomerular preservation of heparan sulfate in long-term diabetic rats. A total of 22 male Wistar rats were used in the study. Diabetes was induced in 15 rats by a single intraperitoneal injection of streptozotocin (60 mg/kg). The remaining 7 rats received buffer. One week following induction of diabetes, 8 diabetic rats were allowed to drink tap water containing enalapril at a concentration of 50 mg/liter; the remaining 7 diabetic and 7 nondiabetic rats were given only tap water. The drug treatment was continued for 20 weeks. Systolic blood pressure and 24-hr urinary excretion of albumin were measured at 2, 8, 16, and 20 weeks. At the end of 20 weeks, all rats were killed, kidneys were removed, and glomeruli were isolated by differential sieving technique. Total glycosaminoglycan and heparan sulfate synthesis was determined by incubating glomeruli in the presence of [35S]sulfate. Characterization of heparan sulfate was performed by ion-exchange chromatography. Systolic blood pressures were significantly lower in enalapril-treated diabetic rats compared to untreated diabetic rats. Diabetic glomeruli synthesized less heparan sulfate than glomeruli from nondiabetic rats. Also, glomerular heparan sulfate content of diabetics was significantly lower than that of nondiabetics. Further characterization of heparan sulfate showed that the fraction eluted with 1 M NaCl was significantly lower and the fraction eluted with 1.25 M NaCl significantly higher in diabetic than in normal rats. Enalapril treatment normalized not only glomerular synthesis and content but also various fractions of heparan sulfate in diabetic rats. Diabetic rats excreted increased quantities of heparan sulfate and albumin than nondiabetic rats. Enalapril therapy prevented both these increases in diabetic rats. These data suggest that enalapril treatment improves albuminuria through preservation of glomerular heparan sulfate and prevention of its urinary loss in diabetic rats. 相似文献
19.
J. E. Steele T. N. Robinson A. J. Cross D. M. Bowen A. R. Green 《Journal of neurochemistry》1991,56(4):1248-1254
The binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate ([3H]MK-801) and N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine ([3H]TCP) to the N-methyl-D-aspartate (NMDA) receptor complex of human brain has been investigated. Significant differences were noted between the binding of the two ligands in the same tissue samples. Binding of both ligands was stimulated by addition of glutamic acid or glycine. However, addition of both compounds resulted in an additional effect with [3H]MK-801 but not [3H]TCP binding. Saturation analysis revealed approximately twice as many high-affinity sites for [3H]MK-801 (Bmax, 1,500 +/- 300 fmol/mg of protein) than for [3H]TCP (Bmax, 660 +/- 170 fmol/mg of protein). In addition, a low-affinity site was detected for [3H]MK-801 binding but not [3H]TCP binding. The pharmacology of the high-affinity [3H]MK-801 and [3H]TCP binding sites was similar with rank order of potency of inhibitors being MK801 greater than TCP greater than phencyclidine greater than N-allylnormetazocine (SKF 10047). 2-Amino-5-phosphonopentanoate inhibited binding of both ligands with comparable potency whereas both 7-chlorokynurenic acid and ZnCl2 were more potent inhibitors of [3H]MK-801 than of [3H]TCP binding. All compounds examined exhibited Hill coefficients of significantly less than unity. Saturation analysis performed in the striatum revealed that the number of binding sites was the same for both [3H]MK-801 (Bmax, 1,403 +/- 394 fmol/mg) and [3H]TCP (Bmax, 1,292 +/- 305 fmol/mg). Addition of glutamate or glycine stimulated striatal binding but there was no further increase on addition of both together.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
Isolated liver granulomas of murine Schistosoma mansoni contain components of the angiotensin system 总被引:2,自引:0,他引:2
Angiotensin I (AI) and angiotensin II/III (AII/III) were detected by radioimmunoassay in homogenates of isolated liver granulomas from mice infected for 8 wk with Schistosoma mansoni. Angiotensin I converting enzyme (ACE) activity, which could be completely inhibited by captopril, a specific ACE inhibitor, was also present as determined by radioassay. Spontaneous angiotensin I-generating activity was detected in homogenates that received supplemental angiotensinogen (protein renin substrate). This activity was partly inhibited by pepstatin, an acid protease inhibitor, indicating the presence of angiotensinogenase(s). Trypsinization of homogenates resulted in some AI generation, which suggests that homogenates had AI precursor. Treatment of infected mice with MK421, another specific ACE inhibitor, decreased granuloma ACE activity and AII content and size. AII, and to a lesser extent AIII, inhibited mouse peritoneal macrophage migration in an in vitro assay. These data support the contention that components of the angiotensin system are in the granuloma and may serve a function in regulation of the inflammation. 相似文献