Biosynthesis of proteoglycans by isolated rabbit glomeruli

Isolated rabbit glomeruli were incubated in vitro with 35SO4 in order to analyze the proteoglycans synthesized. Proteoglycans extracted with 4 M guanidine HCl from whole isolated glomeruli and from purified glomerular basement membrane (GBM) were analyzed by gel filtration chromatography. Two types of sulfated proteoglycans were found to be synthesized by rabbit glomeruli and these contained either heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycan chains. These glycosaminoglycans were characterized by their sensitivity to selective degradation by nitrous acid or chondroitinase ABC, respectively. The major proteoglycan extracted from the whole glomeruli was a chondroitin/dermatan sulfate species (75%), while purified GBM contained mostly heparan sulfate (70%). The glycosaminoglycan chains were estimated to be about 12,000 molecular weight which is consistent with previous estimates for similar molecules extracted from the rat GBM.     

Heterogeneity of tachykinin receptors in the rabbit lung

The tachykinins, substance P (SP) and neurokinin A (NKA), are agonists for the NK₁ and NK₂ receptors, respectively. Tachykinins have various respiratory effects, including bronchoconstriction. This study characterizes tachykinin binding sites in the rabbit lung. We hypothesize that (2-[¹²⁵I]iodohistidyl¹)Neurokinin A ([¹²⁵I]NKA) interacts with NK₁ and NK₂ binding sites in the rabbit lung. The K_d determined from saturation isotherms was 0.69 X/÷1.14 nM (geometic mean X/÷ SEM) and the B_max was 4.15±0.22 femtomole/mg protein (arithmetic mean±SEM). Competitive inhibition studies with NKA, SP and various selective tachykinin agonists showed the rank order of potency: [β-Ala⁸]-Neurokinin A 4–10=SP ≫ NKA ≫ [Sar⁹,Met(O₂)¹¹]-Substance P. [β-Ala⁸]-Neurokinin A 4–10, a selective NK₂ agonist, and SP inhibition of [¹²⁵I]NKA binding were best described using a two-site model. Competitive inhibition studies using the selective nonpeptide NK₂ antagonist (SR 48968) and the selective nonpeptide NK₁ antagonist (CP 96,345) revealed K_i's of 5.5 nM and 8.1 nM, respectively. Our data therefore suggest that [¹²⁵I]NKA binds to both the NK₁ and NK₂ receptors in the lung. Special issue dedicated to Dr. Kinya Kuriyama.     

Benzodiazepine receptor: Heterogeneity in rabbit brain

Binding characteristics of benzodiazepine receptors were studied with synaptosomal and microsomal membranes from rabbit brain $\text{in}$$\text{vitro}$ utilizing [methyl-³H]diazepam. In synaptosomal membranes, both high and low affinity binding sites were identified with the dissociation constants (K_d) of 4.92 nM and 83.8 nM, respectively. However, only the high affinity site was identified with K_d of 3.96 nM with microsomal membranes. Benzodiazepine binding sites appear to include at least two subpopulations of receptors, one with high affinity and another with low affinity binding site.     

Heterogeneity of acid beta-galactosidase from rabbit kidney

1. Rabbit kidney acid beta-galactosidase can be resolved into three peaks (named A3, A2 and A1) by gel-filtration chromatography. Their estimated molecular weights were: more than 250,000, 150,000 and 17,000 respectively. 2. The purified acid form appeared as a single band of protein (Mr = 28,000) on electrophoresis in the presence of sodium dodecyl sulphate, suggesting that forms A3 and A2 are multimeric forms of beta-galactosidase A1. 3. Treatment with neuraminidase from Clostridium perfringens converts form A3 into a more basic form. This phenomenon occurs also when this form is stored for a week at 4 degrees C and parallels its disaggregation. 4. The data suggest that the sialic acids present in the multimeric forms are involved in the aggregation of the acidic form of beta-galactosidase.     

Heterogeneity of rabbit aortic endothelial cells in primary culture

Factor VIII-related antigen (F8-RAg) and angiotensin-converting enzyme (ACE) are accepted diagnostic markers of endothelial cells in culture. However, when we isolated cells from rabbit thoracic aorta (after collagenase treatment and gentle scraping of the intima) and examined them with immunoperoxidase techniques, we observed two cell types which stained specifically for either F8-RAg or ACE, but not both. Each cell type was morphologically distinguishable in primary culture. F8-RAg-positive cells were recognizable in distinct patches as more elongated, tightly apposed, and firmly adherent cells; they exhibited only faint or no staining for ACE and no accumulation of a fluorescent, acetylated low-density lipoprotein probe (DiI-Ac-LDL), another endothelial cell marker. In contrast, ACE-positive cells were more rounded, less closely apposed, and grew as strict monolayers that exhibited a characteristic cobblestone appearance at confluence; ACE-positive cells were F8-RAg negative, but demonstrated intense labeling with DiI-Ac-LDL. Subcultures of ACE-positive cells were also stained by anti-rabbit thrombomodulin.     

Heterogeneity and ontogenesis of progestin receptors in rabbit lung

In order to evaluate the possible role of progesterone in fetal lung development, the presence of specific pulmonary progestin receptors and their ontogenesis were investigated in the rabbit fetus. Scatchard analysis of binding in lung cytosol from 29-day fetuses over a wide range of [³H]-R5020 concentrations indicates the presence of at least two binding sites. One of these sites, type I, is of very high affinity (K_D = 0.12 nM) and low capacity (26fmol per mg protein). The second binding site, type II, is of lower affinity (K_D = 36 nM) and higher capacity (240 fmol per mg protein). These two binding sites can be distinguished by sucrose density gradient centrifugation, the type I component sedimenting at 7.1 S and the type II component sedimenting at 4.5 S. Similar type I and type II sites are present in adult lung cytosol except that the type II binding component in adult lung sediments at 2.8 S rather than 4.5 S. Progesterone and R5020 compete well with [³H]-R5020 for binding to both sites while dexamethasone and cortisol do not compete. Thus the type I and type II binding sites appear to represent specific progestin receptors distinct from transcortin or the glucocorticoid receptor. The concentration of the type I sites increases significantly between the 20th and 29th day of gestation, with a further increase being observed in adult animals. The type II site is not measurable until 26 days of gestation and attains adult levels by day 29. Among a large number of fetal tissues examined, the lung contained the highest concentration of type I progestin receptor sites.Although cortisol and dexamethasone, even at very high concentrations, do not compete with [³H]-R5020 for binding to lung cytosol, the binding of [³H]-dexamethasone is inhibited significantly by nonlabeled progesterone or R5020 and this inhibition appears to be due to dissociation of [³H]-dexamethasone-receptor complexes. These results indicate that, in addition to type I and type II progestin receptor sites, fetal lung cytosol contains a third binding site, type III, which appears to be different from the glucocorticoid receptor site. Occupation of the type III site by progestins interferes with the binding of glucocorticoids to glucocorticoid receptors perhaps by increasing the rate of dissociation of glucoeortieoid-receptor complexes.     

Heterogeneity of Es-1 esterases in the rabbit (Oryctolagus cuniculus)

Analysis of the Es-1 system in the rabbit with polyacrylamide gel electrophoresis (PAGE) revealed a high degree of individual variation. In the liver the number of esterase bands found in the Es-1 region of the gels ranged from 2 to 16. The results indicate that one locus with three alleles is responsible for all of the esterase bands in the Es-1 region. The most plausible explanation for the observed heterogeneity is that each of the alleles codes for a protein (MW 65,000±2000) that is changed by posttranslational modifications, thus giving rise to two to five monomeric enzymes with esterase activity. Polymerization of these monomers then results in 1–11 dimers. Based on similarities with mouse Es-9, chromosomal homology between rabbit Es-1 and mouse Es-9 is proposed.     

Distribution of parathyroid hormone-sensitive adenylate cyclase in isolated rabbit renal cortex microvessels and glomeruli

The effect of the synthetic amino-terminal fragment of bovine parathyroid hormone, bPTH-(1-34), on the adenylate cyclase of microvessels and glomeruli isolated from rabbit kidney cortex was studied in the presence and absence of guanosine triphosphate (GTP). bPTH-(1-34) stimulated the vascular and glomerular adenylate cyclase in a dose-dependent manner with apparent ED50 values of 11.5 nM and 64 nM respectively, in the absence of GTP. 10(-4)M GTP greatly amplified the vascular response to bPTH-(1-34) while, in the glomeruli, both GTP and bPTH-(1-34) had only additive effects. In the presence of GTP, vascular and glomerular apparent ED50 were 190 nM and 64 nM respectively. [Nle8, Nle18, Tyr34] -bPTH-(3-34) amide, described as a PTH antagonist, inhibited the action of bPTH-(1-34) in the microvessels and to a lesser extent in the glomeruli. PTH is therefore a potent stimulator of adenylate cyclase in rabbit renal microvessels and glomeruli, and may play a role in the regulation of renal blood flow and glomerulo-tubular feedback control.     

Heterogeneity of intermediate filament proteins from rabbit spinal cord

Large amounts of a neurofilament-enriched fraction may be prepared from spinal cord homogenates by a simple, three-step procedure. This involves flotation of filament-containing axon fragments, extraction with Triton X-100, and washing by sedimentation through a sucrose density gradient. The material obtained by this procedure includes both large mats of individual 10-nm filaments and tightly packed bundles of filaments. SDS-gel electrophoresis of these fractions indicates that the fractions are formed of four polypeptides: the three which are generally considered to form neurofilaments (P200, P150, and P68) and another, with a molecular weight of about 50,000 daltons (P50), which is thought to be derived from fibrous astrocytes. Analysis of these filament fractions on two-dimensional gels indicates heterogeneity among each of the different molecular weight classes. The largest polypeptide of neurofilaments, P200, focuses at several spots in the pH gradient. P68 and P150 are more acidic: each appears as a pair of overlapping spots. P50 resolves into a complex of spots of about the same molecular weight but with different isoelectric points. Heterogeneity is not unique to these filament polypeptides but appears to be a characteristic of all fibrous proteins of the nervous system.     

兔左室壁三层心肌细胞的分离及动作电位、钙和钾电流分布的异质性

探讨兔左室壁三层心肌单个细胞的分离方法以及电生理特征,实验以胶原酶按二步消化法分离兔心肌细胞,其中用剃须刀分离左室游离壁内,中,外三层心肌,采用全细胞膜片钳记录AP和离子电流,结果显示：（1）中层细胞上的动作电位时程明显长于内膜下心肌和外膜下心肌,且存在显著的1相切迹和2相驼峰;（2）中层细胞的Ica,L和Lto较内,外膜下的大,IK,s相反,可见三层心肌细胞上多种电流存在显著差异。     

Heterogeneity of NK-2 tachykinin receptors in hamster and rabbit smooth muscles.

The possible existence of NK-2 receptor subtypes in peripheral smooth muscle preparations from rabbit and hamster was investigated by studying the effect of neurokinin A, the selective NK-2 receptor agonist [beta Ala8] neurokinin A (4-10), the selective NK-2 tachykinin receptor antagonists, MEN 10,376, L 659,877 and R 396, and the pseudopeptide derivative of neurokinin A (4-10), MDL 28,564. All experiments were performed in the presence of peptidase inhibitors (captopril, bestatin and thiorphan, 1 microM each). Both neurokinin A and [beta Ala8] neurokinin A (4-10) produced concentration-dependent contractions of the rabbit isolated bronchus and hamster isolated stomach and colon, as well as enhancement of the nerve-mediated twitches of rabbit isolated vas deferens (pars prostatica). MEN 10,376, L 659,877 and R 396 antagonized the effect of the NK-2 receptor selective agonist in all four tissues under study, although marked differences in antagonist potency were evident for the three antagonists. Thus MEN 10,376 was distinctly more potent (about 100 times) in rabbit than in hamster preparations while L 659,877 and R 396 were more potent in hamster than rabbit preparations. MDL 28,564 showed a distinct agonist character in rabbit preparations while it was virtually inactive in hamster preparations, where it antagonized the effect of the NK-2 receptor selective agonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid preparation of pure glomeruli and tubular fragments from rabbit kidney : Distribution of alkaline phosphatase

A simple rapid method for separating glomeruli and tubular fragments is described. The alkaline phosphatase activity of the glomeruli based on the tissue proteins, or on the DNA, was about 4%, or 1%, respectively of that of the tubular fraction. Alkaline phosphatase can be used as a chemical marker of the purity of glomerular fractions in studies on the structure, antigenicity, and biochemical function of glomeruli.     

Aggregation of f-actin in olfactory glomeruli: a common feature of glomeruli across phyla

Using fluorophore-conjugated phalloidin, we show that filamentous (F)-actin is strongly aggregated in olfactory glomeruli within primary olfactory centers of vertebrates and insects. Our comparative study demonstrates that aggregation of F-actin is a common feature of glomeruli across phyla, and is independent of glomerular architecture and/or the presence or absence of cellular borders around glomeruli formed by neurons or glial cells. The distribution of F-actin in axonal and dendritic compartments within glomeruli, however, appears different between vertebrates and insects. The potential role of the actin-based cytoskeleton in synaptic and structural plasticity within glomeruli is discussed.     

Prostaglandin synthesis in isolated glomeruli

Prostaglandins are thought to play an important role in the local regulation of glomerular blood flow and in the release of renin from the juxtaglomerular apparatus. We therefore examined prostaglandin synthesis by isolated rat glomeruli. Isolated glomeruli were either prelabeled with [14_C] arachidonic acid or were incubated with [14_C] arachidonic acid for the entire experimental incubation in Krebs buffer. Prostaglandin synthesis was determined by thin layer radiochromatography of acid extracts of the supernatant solutions. Indomethacin inhibitable synthesis of small amounts of 6-keto-PGF_1α, the metabolite of prostacyclin(PGI₂,) and larger amounts of PGF_2α, and PGE₂, and possibly thromboxane B₂ (TXB₂) by isolated glomeruli could be demonstrated with either prelabeling or direct incubation. These findings support the hypothesis that prostaglandins are produced within the glomerulus where they may affect local glomerular blood flow and function.     

Heterogeneity of rabbit muscle creatine kinase and limited proteolysis by proteinase K.

By using sodium dodecyl sulphage/polyacrylamide-gel electrophoresis it was shown that rabbit muscle creatine kinase, both in a homogenate and purified, appears to be composed of a mixture of two peptides (mol.wts. 42100 and 40300) differing in length by about 15 amino acids. It is found that low concentrations of proteinase K from the fungus Tritirachium album can cleave about 38 amino acids from each chain of creatine kinase, leaving two large fragments (mol.wts 37700 and 35500). Scission of the whole enzyme was found to be concomitant with complete loss of enzyme activity. MgADP in the presence of absence of creatine slowed the rate of proteolysis by about 50%, but the transition-state analogue complex creatine-NO3--MgADP appeared to protect completely. The time course for the proteolytic inactivation in the presence of this complex, but not in its absence, was biphasic.     

Development of the cerebellar glomeruli in the ontogeny of hens

By means of light and electron microscopy development of the cerebellar glomeruli has been studied in the hen ontogenesis. Two successive stages have been revealed: protoglomerular (the 16th-18th days of incubation) and glomerular (the 19th day of incubation--the end of the 3d week after hatching). Specific peculiarities of their structural organization are presented. A suggestion is made on an inductive effect of the afferent mossy fibers on processes of morphological formation of the cerebellar glomeruli.