Histamine receptors of the microvascular endothelium revealed in situ with a histamine-ferritin conjugate: characteristic high-affinity binding sites in venules.

Histamine covalently bound to glutaraldehyde-activated ferritin was prepared as either monomers or as small aggregates of approximately 0.05 to 0.15 micrometer Diam, suitable for electron microscopic detection of histamine cellular binding sites. The histamine-ferritin conjugates (MF) maintain the histamine capability to induce the opening of endothelial junctions in venules. To investigate the distribution of histamine receptors in the vascular endothelium, monomers or aggregates of MF were perfused in situ (mice), and various vascular beds, particularly that of the diaphragm, were fixed and processed for electron microscopy. The conjugate was preferentially bound on restricted areas of luminal endothelial cell plasmalemma especially in regions rich in filaments, and near the junctions between endothelial cells. The density of histamine binding sites was characteristically high in venules; it occurred to a much lesser extent in arterioles, veins, and muscular arteries whereas capillaries and aorta showed the lowest values. A similar distribution was obtained after perfusion of H1 or H2 receptor agonists coupled to ferritin (2-pyridylethylamine-ferritin [PF], or 4-methylhistamine-ferritin [MF], respectively). The binding specificity was assessed through control experiments with either native or activated ferritin or by competition with histamine. The findings suggest that histamine receptors are largely represented in the cell membrane of the vascular endothelium, particularly in venules. Experiments using specific H1 and H2 receptor agonists (PF and MF) and antagonists (mepyramine and cimetidine) indicate that the venular endothelium contains mainly H2 receptors.

Albumin-binding proteins of endothelial cells: immunocytochemical detection of the 18 kDa peptide.

Extracts of isolated microvascular endothelial cells (MEC) and cultured bovine aortic endothelial cells (BAEC) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransfer and incubation with albumin either radioiodinated or adsorbed to 5-nm gold particles. Both ligands reacted exclusively with two peptides of 18 and 31 kDa. To the 18 kDa peptide (excised from preparative SDS-PAGE), an antibody was raised in rabbits and purified by affinity on 18 kDa obtained from two-dimensional gel electrophoresis and immobilized on nitrocellulose paper. The specificity of the anti-18 kDa was assessed by immunoblotting and immunoprecipitation of endothelial cell extracts. To check whether the 18 kDa peptide is exposed on the endothelial cell surface and/or its components (uncoated pits, open plasmalemmal vesicles), the apical membrane of BAEC was radioiodinated, the solubilized proteins incubated with the anti-18 kDa, and the immune complexes formed were precipitated with protein A-Sepharose CL-4B. The ensuing SDS-PAGE and autoradiography revealed that from all radioiodinatable surface proteins, the 18 kDa was the only polypeptide immunoprecipitated by the anti-18 kDa antibody. To localize the 18 kDa peptide, we applied indirect immunofluorescence technique on cultured MEC and BAEC and immunoelectron microscopy (EM) on ultrathin cryosections of mouse heart. Nonpermeabilized whole MEC and BAEC incubated with anti-18 kDa followed by rhodamine-conjugated second antibody showed a relatively intense surface fluorescence often appearing as small dots. At the EM level, heart ultrathin cryosections exposed anti-18 kDa followed by gold-conjugated second antibody revealed that 18 kDa was primarily associated with the membrane of plasmalemmal vesicles of capillary endothelia.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumin-binding proteins function in the receptor-mediated binding and transcytosis of albumin across cultured endothelial cells.

The functional significance of the endothelial albumin-binding proteins (ABP) was tested on cultured bovine aortic endothelial cells (BAEC) incubated with radiolabeled albumin ([125I]Alb) alone or carrying fatty acids (oleic acid (OA) or arachidonic acid (AA)) or triiodothyronine. The [125I]Alb binding was estimated on BAEC grown on 96-well plates, and the transport was evaluated on BAEC cultured in a dual chamber system. The probe interaction with the monolayer was monitored as a function of concentration and temperature in the presence or absence of either unlabeled Alb or an anti-albumin anti-idiotypic antibody (Ab2), which was previously demonstrated to specifically recognize the ABP of endothelial cell surface. Cultured BAEC bound specifically and with high affinity [125I]Alb. The binding of fluorescein isothiocyanate (FITC)-Alb to endothelial cells was inhibited by Ab2 in immunofluorescence studies. As compared to albumin, the binding of albumin carrying either OA or AA was higher and was diminished by Ab2. Transport of [125I]Alb across BAEC grown on gelatin-coated filters increased with time, and after 60 min, approximately 30% of [125I]Alb was transported from the upper to the lower compartment; unlabeled Alb or Ab2 reduced this process by approximately 75%. Colchicine decreased transcytosis of [125I]Alb by approximately 80%, whereas chloroquine by approximately 27%. Transendothelial transport of [125I]Alb carrying fatty acids was 40% and 20% higher for OA and AA, respectively, as compared to that of defatted albumin. The results suggest the coexistence of a receptor-mediated and a receptor-independent transcytosis of albumin across cultured endothelial cells; ABP of the endothelial cell surface appear to be involved in the specific binding and transport of albumin.

Expression of transferrin receptors in endothelial cells transfected by electroporation.

Transferrin is the primary iron-binding protein in the plasma. Transferrin receptors (TfR) were detected in brain and liver endothelial cells (EC); however, little information exists about their intracellular routes. To detect the EC structures involved in TfR biosynthetic and endocytotic pathways, cultured aortic EC were transfected with the plasmid pSR alpha containing a construct encoding the human TfR, to which horseradish peroxidase (HRP) was anchored as reporter molecule. Since EC are difficult to be transfected, we tried different techniques, and two forms of the plasmid (circular and linearized), of which the electroporation method and the linearized plasmid were the most efficient in producing stable cell lines. Transfected cells were selected with geneticin, and the expression of TfR-HRP tested by cytochemistry. The stable transformants preserved the general characteristics of EC. At the ultrastructural level, TfR-HRP was associated with the nuclear envelope, rough endoplasmic reticulum, Golgi complex and adjacent secretory vesicles, cytoplasmic vesicles of various sizes (50-130 nm diameter), endosomes, plasma membrane, plasmalemmal pits, and a fraction of plasmalemmal vesicles. The intensity of the reaction product varied, suggesting a different concentration of TfR, in specific organelles. For example, (i) a gradient of HRP-reaction product was found within the Golgi cisternae, (ii) the plasmalemmal pits were more intensely stained than the adjacent plasma membrane, and (iii) the vesicle membrane was decorated stronger than the endosomal membrane (to which it fuses). A striking feature was the coexistence within the same EC of two vesicle populations (or subtypes): some containing TfR-HRP, whereas others lack the receptor. Quantitative data indicated a stronger expression of TfR in confluent cells (approximately 8-fold higher) than in EC at 2 days after plating; a significant decrease (approximately 9-fold) of TfR was found in postconfluent transfectants. Together, the data demonstrate that (i) after electroporation of EC, the stable lines maintain the characteristics of native cells; (ii) the newly synthesized TfR is located in variable concentration within the organelles involved in endocytosis and exocytosis, and (iii) the expression of TfR-HRP is particularly high in confluent cells.

A method for selective radiolabeling of lung endothelium plasmalemmal vesicles, in situ.

Albumin-gold complex (Alb-Au) was previously shown to bind selectively to plasmalemmal vesicles of capillary endothelium. Based on these findings, as well as on the ability of lactoperoxidase (LPO) to mediate the radioiodination of proteins, we have prepared a complex of gold particles bearing both albumin and anionized lactoperoxidase (Alb-Au-aLPO). The complex had a pI of 5.8, largely preserved aLPO enzymatic activity (approximately 74%), and was able to catalyze protein radioiodination. Upon washing out the blood, the complex was perfused in the mouse lung, the excess tracer removed, and a Na125I/H2O2 solution was introduced in the vasculature. After extensive washing, lung fragments were processed for either electron microscopy (EM), or to prepare a membrane-enriched fraction. In control experiments, lungs were perfused with native LPO (pI 9.3), or with a LPO-Affi Gel conjugate and further radioiodinated as described for Alb-Au-aLPO. By EM, it was found that both in tissue and in the isolated membrane fraction, only Alb-Au-aLPO labeled markedly and preferentially some uncoated pits and most plasmalemmal vesicles. Analysis by SDS-PAGE and autoradiography of a membrane-enriched fraction prepared from lungs perfused with Alb-Au-aLPO had some major identified 125I-labeled polypeptides of apparent molecular masses of 16, 18, 31, 36, 55, and 77 kDa. A different subset of polypeptides was labeled in lungs perfused with LPO, whereas after administration of LPO-Affi Gel the major radiolabeled polypeptides had a molecular mass of 33, 55 kDa and several peptides in the range of 77 to 160 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigation of IgG receptors expressed on the surface of human placental endothelial cells.

Fetal passive immunity is acquired by transfer of maternal IgG through the placental syncytiotrophoblast and endothelium; few and contradictory data exist for IgG transcytosis in human placental endothelial cells (HPEC). In this study, we tested the binding and internalization of IgG by cultured HPEC and the expression of FcgammaRs. Biochemical analysis and microscopy revealed that the binding of IgG occurred through the Fc portion of the molecule and was greater on the basolateral than on the apical cell surface. IgG binding and internalization were saturable and the data calculated from Scatchard plot for IgG surface binding indicated a single interaction with an apparent K(d)of 2x10(-7)M. During 3 h of chase, approximately 10 per cent of IgG was released in an intact form in the medium. By electron microscopy, IgG was detected on HPEC surface, internalized in endothelial caveolae and within endosomal compartments. RT-PCR, blotting and microscopy failed to detect the presence of FcgammaRI-III in HPEC. However, the specific radioiodination and affinity chromatography revealed the presence of a 55 kDa-IgG binding polypeptide on cell surface. These findings indicate that HPEC (i) take up and internalize IgG via a receptor mediated process; (ii) bind IgG prevalently on the basolateral surface via the Fc fragment of the molecule and (iii) exhibit a novel FcgammaR of 55 kDa on the cell membrane.

Establishment of a pure vascular endothelial cell line from human placenta.

UNLABELLED: Endothelial cells (EC) from various sectors of the circulatory system have distinct characteristics, some of which have only been identified in cultures upon their isolation from specific organs or tissues. Cultured vascular EC, derived from the human placenta (HPEC), may be helpful for studying their specific function in the fetoplacental unit, such as in the control of maternofetal traffic. In this paper we report an improved method for isolation, purification and culture of HPEC, that implies an enzymatic perfusion of the term placenta, followed by separation of resulting cells on a Percoll density gradient. The inoculated starting suspension was purified by a two-step selection procedure, based on differential trypsinization, leading to a pure population of about 8x10(7)cells/placenta, with 2.7-3.4 population doublings. The average population doubling time during eight passages was 60-65 h and the life span of HPEC was approximately 45-50 population doublings. The cell morphology at optical and electron microscopical level revealed a good differentiation of HPEC, which were endowed with numerous plasmalemmal vesicles (caveolae) and Weibel-Palade bodies. The transendothelial electrical resistance of the HPEC monolayer varied between 22 and 52 Ohm/cm(2). The cultures were mycoplasma free, as revealed by fluorescence microscopy using DNA dyes and the polymerase chain reaction (PCR). The negative immunofluorescent reaction for keratin confirmed that the HPEC were not contaminated with either type of placenta cells, as syncytiotrophoblast. Cultured HPEC demonstrated a strong reaction for von Willebrand factor antigen (by fluorescence microscopy), took up AcLDL-DiI and expressed active angiotensin converting enzyme. These characteristics substantiate the endothelial nature of cultured cells. The interactions with different lectins (BS-I, SBA, RCA, UEA and WGA) assessed by fluorescence microscopy and blotting reveal a strong reaction of HPEC with UEA and a negligible reaction with BS-I lectin. WGA lectin displayed a marked fluorescence staining in subconfluent HPEC, and at the level of intracellular clefts in post-confluent cultures. IN CONCLUSION: (i) we have obtained a pure line of cultured EC originating from the human placental venous side of the circulatory tree; (ii) the cells have the general characteristics and markers ascribed to EC; (iii) as opposed to large human placental vessels, HPEC do not react to BS-I lectin and, unlike human umbilical vein EC, have a much higher proliferation rate and a long lifespan; (iv) HPEC expressed a characteristic glycosylated coat particularly rich in alpha- L -fucose and beta-GlcNAc containing glycocompounds.

Identification of albumin-binding proteins of thymocyte plasmalemma.

In the present work we examined whether the interaction between albumin molecules and thymocytes involves albumin-binding proteins (ABP). Two plasmalemma-rich fractions obtained by differential centrifugation from rat thymus lymphocytes were characterized biochemically and morphologically. These fractions were examined by ligand-blotting and ligand affinity chromatography techniques. Plasmalemma proteins separated by SDS-PAGE were electrotransferred onto nitrocellulose membranes and incubated with 125I-albumin, in the presence or absence of excess native albumin. The autoradiogram revealed specific binding to two sets of polypeptides of 16-18 and 29-31 kDa, which could be blocked by native albumin. To elucidate whether albumin-binding proteins are exposed on the cell surface, intact lymphocytes were surface radioiodinated and membrane fractions prepared from them were subjected to affinity chromatography on albumin-agarose beads. The protein thus purified had, like ABP, M(r) of 16 and 31. These data indicate that ABP (i) are components of thymocyte plasma membrane, (ii) have apparent molecular mass of 16-18 and 29-31 kDa, and (iii) are exposed on the outer membrane surface.

Immunocytochemical localization of glyceraldehyde-3-phosphate dehydrogenase in cultured endothelial and smooth muscle cells.

The presence and distribution of glyceraldehyde-3-phosphate dehydrogenase (GPDH) in cultured bovine aortic endothelial cell (EC) and smooth muscle cell (SMC) were studied. For this purpose, we purified GPDH from human and bovine red blood cell (RBC) membranes and used it as antigen; anti-GPDH serum and affinity purified IgG were prepared. GPDH has been identified in the whole extracts of EC and SMC as a polypeptide having the same electrophoretic mobility as RBC protein. In addition, GPDH digested with V8 protease and analyzed by one dimensional peptide mapping presented the same pattern for the three cell types examined. Anti-RBC-GPDH cross-reacted with the polypeptide from EC and SMC. The intracellular localization of GPDH in EC and SMC was investigated by indirect immunofluorescence microscopy using affinity purified anti-GPDH. We found that the antigen exhibits a diffuse cytoplasmic distribution in both cell types; in addition, EC contained the antigen in the nucleus. The nuclear GPDH-like protein in EC has a characteristic pattern, suggesting yet unknown implications of GPDH in the cell metabolism.

Immunological detection of an analogue of the erythroid protein 4.1 in endothelial cells.

Endothelial cells (EC) of arterial and venous origin were investigated by indirect immunofluorescence and immunoautoradiography for the presence of red cell membrane 4.1-like protein. By immunofluorescence, EC exhibited a relatively uniform fluorescent staining sometimes of a reticular pattern, distributed over the entire cell. All controls were negative. Immunoblot analysis of EC revealed a cross reactive band of a molecular weight comparable to that of the erythrocyte band 4.1. These findings indicate that endothelial cells of arterial and venous origin express a polypeptide immunologically related to the erythrocyte protein 4.1, which may play an important role in membrane-cytoskeleton interactions.

Further evidence for the distribution and nature of histamine receptors on microvascular endothelium.

The localization of histamine receptors and their classes on the microvascular endothelium was assessed by using the electron-opaque conjugate histamine-ferritin (HF). In order to further check the specific binding of this conjugate, two compounds chemically similar to histamine but biologically inactive, tele-methylhistamine (t-MH) and 4-pyridylethylamine (PEA), and a specific H2 histamine receptor antagonist, SK&F 93479, were used. Glutaraldehyde-activated ferritin was covalently coupled with either histamine, as a biologically active HF conjugate, or with tele-methylhistamine, as a biologically inactive conjugate tele-methylhistamine-ferritin (t-MHF). The purity of the conjugates was determined by thin-layer chromatography. The HF conjugate (25 mg protein/100 g body weight) was perfused into RAP mice and bipolar microvascular fields of the diaphragm were fixed and processed for electron microscopy. The HF conjugate decorated restricted domains of the luminal aspect of the microvascular endothelium. The specific density of HF binding, recorded as the average number of binding sites per square micrometer of the luminal endothelial surface, was relatively high in venules (18.46 +/- 5.73) and lower in arterioles (12.89 +/- 6.13) and capillaries (9.51 +/- 4.20). The binding specificity of the HF conjugate was assessed through six groups of experiments: perfusion before HF conjugate with either histamine, tele-methylhistamine or 4-pyridylethylamine, perfusion with t-MHF conjugate only, or administration before t-MHF conjugate of either histamine or tele-methylhistamine. The statistical significance of the data was analyzed by using the "F" distribution test and the "Wilcoxon-Mann-Whitney rank-sum test". Both tele-methylhistamine and 4-pyridylethylamine as well as histamine, showed a higher competition on arterioles and venules than on capillaries. Neither tele-methylhistamine nor histamine inhibited the binding of t-MHF conjugate on the endothelium. Experiments using SK&F 93479 as a very potent H2 histamine receptor antagonist perfused before HF administration, suggested the existence of H2 histamine receptors at a higher concentration in venules (47.03%) and arterioles (38.80%) than in capillaries (14.16%). These findings demonstrate that HF conjugate binds specifically to the histamine receptors of microvascular endothelium, which are characteristically frequent and predominantly of H2 type in venules.

Evidence for thyroxine transport by the lung and heart capillary endothelium.

The uptake and transport of carrier-bound thyroxine by the endothelium were investigated by perfusing through the heart and lung of young rats radiolabeled thyroxine bound to prealbumin ([125I]T4Pa) or serum ([125I]T4S). In addition these complexes were tagged to 5-nm gold particles to obtain quantitative (radioassay) and qualitative (autoradiography) data from the same experiment. The complexes (prewarmed at 37 degrees) were perfused in situ at various concentrations (1 to 50 muCi/ml) for time intervals ranging from 5 to 30 min. After thorough washing of the unbound probe, tissue fragments were either measured for total uptake in a gamma counter or processed for electron microscopy autoradiography. The results showed that both the lung and heart take up [125I]T4 complexes by a process that is saturable at low hormone concentration; uptake is completed by free T4 and Pa. In specimens perfused with double-labeled complexes (iodinated and tagged to gold) autoradiography revealed that silver grains and gold particles colocalize predominantly on endothelial plasmalemmal vesicles. The probe appeared first in vesicles open to the capillary lumen (5 min) and further on in vesicles apparently free within the cytoplasm or open to the abluminal front. At 30 min, only silver grains seem to be present in the pericapillary space, on the alveolar epithelial cells, as well as on the nucleus and mitochondria of heart myocytes. The findings suggest that (1) T4Pa uptake by the endothelial cell (EC) is a specific process (possibly via specific binding sites); (2) T4Pa is taken up and transported across capillary EC by plasmalemmal vesicles; (3) in the pericapillary space T4 seems to dissociate from its carrier.

Histamine receptor on mast cells.

Mast cells isolated from the peritoneal fluid of Wistar rats were purified by centrifugation on Percoll gradient with a yield of 2x10(6) cells/ml. Cell morphology was well preserved as shown by light and electron microscopy. The mast cells capacity to bind histamine was assayed using either [3H]-histamine or histamine-ferritin conjugate as electron-opaque probe for electron microscopic examination. The [3H]-histamine binding performed at 4 degrees C in Ca2+-free phosphate-buffered saline pH 7.3 completed within 30 min was found to be specific, with an IC50 value of 0.72 +/- 0.23 nM. The data analyses by Scatchard and Hill's representations showed a KD of 0.60 +/- 0.24 nM and Bmax of 4.9 +/- 1.2 pM/10(6) cells suggesting that on mast cells the histamine receptors are restricted to the plasma membrane. According to Hill's analysis neither positive nor negative cooperativity (n = 1.06) appeared to be involved in the specific histamine-receptor binding. Competition experiments with 4-methylhistamine and SK&F 93479, revealed that mast cells express H2-histamine receptors. At electron microscopic level, the histamine-ferritin conjugate interstitially injected in the hamster cheek pouch was localized on the mast cell membrane.

Albumin binding proteins of endothelial cells identified by anti-idiotypic antibodies.

Studies were conducted to identify and localize albumin binding proteins (ABPs) in endothelial cells using rabbits polyclonal anti-idiotypic antibodies (Ab2) raised against the affinity-purified anti-bovine serum albumin IgG. Ab2 were purified by fast performance liquid chromatography. The anti-idiotypic nature of the IgG was assessed by (i) the capacity to inhibit albumin binding to its specific antibody in a dose-dependent manner, (ii) the lack of interaction with albumin, and (iii) the interaction with anti-albumin antibodies of diverse origins. The latter characteristic indicates that although polyclonal, the purified anti-idiotypic antibodies contain some of the Ab2 beta type. The binding of Ab2 to cultured bovine aortic endothelial cell surfaces was saturable and specific as demonstrated by radioimmunoassay (RIA) and immunofluorescence studies respectively. A competitive RIA was used to test whether Ab2 competed for albumin binding to bovine aortic endothelial cells (BAECs) (presumably to ABPs). It was found that Ab2 inhibited binding of [125I]albumin to BAECs in a dose-dependent fashion. Immunoblot analysis of extracts of BAECs, microvascular endothelial cells, and lung showed that both Ab2 and albumin bind specifically to two polypeptides with an apparent molecular mass of 18 and 31 kDa. In addition, upon radioiodination of BAECs apical membrane proteins, Ab2 bound specifically and immunoprecipitated restrictively two radiolabeled cell surface proteins of 18 and 31 kDa. The results provide direct evidence for the presence of the 18 and 31 kDa peptides (ABPs) on the endothelial cell membrane and/or associated structures, i.e. open plasmalemmal vesicles and uncoated pits.

Endothelial cells express a spectrin-like cytoskeletal protein.

Vascular endothelium was investigated by indirect immunofluorescence and immunoautoradiography for the possible presence of spectrin-like molecules. Antibodies were raised against electrophoretically purified rat, rabbit, and bovine red blood cell spectrin and against rabbit brain fodrin. Antibody specificity was assessed by immunoblotting and double-diffusion technique. Homogenates of endothelial cells freshly isolated from heart microvasculature or aorta, as well as cultured aortic endothelial cells, were analyzed by gel electrophoresis. Immunoautoradiograms of gels incubated with spectrin specific antibody, followed by radio-labeled protein A, revealed two bands of electrophoretic mobility similar to that of the alpha- and beta-subunits of spectrin. Indirect immunofluorescence of endothelial cells, both in situ and in vitro, showed the existence of a protein which cross-reacted with the antibodies against spectrin and fodrin. Controls, in which endothelial cells were exposed to spectrin antibody absorbed with pure spectrin or preimmune serum, were negative. These findings indicate that endothelial cells express a protein antigenically related to the spectrin family; both spectrin- and fodrin-like molecules, in various proportions, may coexist. In the endothelial cell, these proteins may play an important role in modulation of the cytoskeleton in response to various stimuli, and in maintaining the biochemically differentiated microdomains of plasmalemma.

Ultrastructural evidence of differential solubility in Triton X-100 of endothelial vesicles and plasma membrane.

Endothelial plasmalemmal vesicles (EV) are distinct membrane-bound structures characteristic for all vascular endothelia, being involved in transcytosis of plasma macromolecules. EV are considered to be similar to the caveolae (characterized by a specific peptide called caveolin) found in other cell types. Caveolin-rich membrane domains were recently isolated from whole lung and chicken gizzard as a Triton X-100 (TX)-insoluble membrane fraction. However, ultrastructural data on the localization of these domains within cells have not yet been reported. We have examined whether EV are TX-insoluble structures. Cultured bovine aortic endothelial cells (BAEC) briefly fixed in paraformaldehyde (10 min, 37 degrees C) were exposed to 0.1% TX for 5 min at 22 degrees C and further subjected to standard electron microscopy procedure. The results showed an extensive solubilization of endothelial plasmalemma as well as other intracellular membranes. Individual or clusters of EV were not affected by TX extraction, retaining their trilaminar unit membrane appearance and dimensions. Moreover, a crude membrane fraction prepared from unfixed BAEC was also extracted with 1% TX for 20 min at 4 degrees C and the insoluble material was examined by electron microscopy. In this fraction clusters of about 10 membranous vesicles were found. These data suggest that EV and plasma membrane have a different lipid composition; the low TX solubility is a characteristic common to caveolin-rich domains (caveolae) of other cells types and EV, whereas the ultrastructural complexity and intracellular localization of the latter are specific for endothelia.

Hyperthyroidism and pregnancy. I. Serum TBG level in normal pregnancy.

Methods for preparation and qualitative evaluation of the reagents necessary for direct radioimmunoassay of thyroxin-binding globulin (TBG) in serum are described. The reagents are: purified TBG necessary as antigen in preparing the labelled product and plotting the reference curve; 125I-TBG; anti-TBG rabbit serum. The technical stages characteristic of the RIA system are presented. TBG level determined in normal volunteers (about 80% males and 20% females, young subjects living in Bucharest area) was found to be 15.6 +/- 2.23 mg L-1 (M +/- SD) and the PBI, T4, and T3 values at the onset were normal. In another lot formed only from women TBG was 17.3 +/- 3.50. Assay of 136 pregnant women between the 2nd an 9th month of pregnancy showed values of 22.46 (2nd month) and 32.23 mg L-1(8th month). The shape of the increase curve during pregnancy shows two main stages. The importance of the serum TBG level as an additional criterion in the diagnosis of pregnancy (even in the first weeks of life of the foetus) and as an indicator of the placentar function is discussed.

Experimental studies concerning the mechanism of the thyroid-pineal interrelationship. I. The action of the pineal peptides on the thyroid gland.

Subcutaneous administration of a pineal polypeptide extract, "Crinofizin", to adult male Wistar rats having the thyroid function inhibited by prolonged administration of MTU, has important effects on both the whole organism (slightly correcting the weight increase rate, loweri g cholesterolemia) and the thyroid gland (reducing thyroid hyperplasia and playing a part in various stages of thyroid hormones metabolism). Thus under pineal treatment one can observe a clear tendency to return to the normal values of the thyroid parameters strongly modified by MTU, as RIC, intrathyroid proteic iodine, tissue thyroid hormones distribution, blood PBI and total T4. It appears that these effects of pineal peptides are induced directly on the thyroid and/or via the hypophyso-thyroid axis which they intercept and equilibrate (moderate).