An electron microscope autoradiographic study of the neuronal and extraneuronal localization of labeled amine in the heart of the bat after administration of tritiated norepinephrine.

The localization of labeled amine in the heart of the bat after administration of tritiated norepinephrine (NE) was studied by means of electron microscope autoradiography. Monoamine oxidase was inhibited so that the distribution of amine in both neuronal (Uptake(1)) and extraneuronal (Uptake(2)) sites could be analyzed. Labeling was nonrandom in both the atrial and ventricular myocardium. The highest relative specific activity was found in neural processes which showed morphological criteria of terminal adrenergic axons. Analysis of the distribution of label around the labeled axonal varicosities indicated that the radioactive amine was more concentrated peripherally than centrally in these structures. Label was also found over cardiocytes in both atrium and ventricle. The pattern of this labeling indicated that the radioactive amine was associated with myofilaments. In the ventricle, I bands were most heavily labeled, indicating a probable association of radioactive amine with thin filaments. Labeling was prevented by administration of phenoxybenzamine and decreased only in cardiocytes by normetanephrine. The nonrandom distribution of labeled amine within cardiocytes supports the view that Uptake(2) represents not only a second mechanism of inactivation of the sympathetic neurotransmitter, but may also be involved in the mediation of some of the action of NE on cardiac muscle.

Subcellular storage organelles for 5-hydroxytryptamine in parafollicular cells of drugs which deplete the amine.

A study was made of the effect of the administration of reserpine and parachlorophenylalanine, an inhibitor of 5-hydroxytryptamine (serotonin; 5-HT), on the capacity of thyroid parafollicular cells to synthesize and store 5-HT. The two drugs were given to nonhibernating bats in doses which produced an equivalent degree of depletion of 5-HT from the thyroid. Tritiated 5-hydroxytryptophan, the precursor of 5-HT, was then given intravenously to assess the ability of parafollicular cell granules to take up and retain newly synthesized 5-HT. Reserpine, but not parachlorophenylalanine, decreased the amount of labeled 5-HT found in the thyroid and prevented autoradiographic labeling of parafollicular cell granules. Quantitative ultrastructural and stereological analysis demonstrated that the granules in untreated animals appeared to be nearly spherical prolate ellipsoids, with a uniformly electron-opaque inner matrix. In animals given reserpine, the axial ratio of the ellipsoidal granules increased greatly and a faint internal striation parallel to the long axis of the granules became apparent. Similar changes were not induced by parachlorophenylalanine. No other morphological changes in the thyroid epithelium were detected after administration of reserpine. This study confirms the association of 5-HT with the mature small secretory granules of thyroid parafollicular cells.

Separation of dissociated thyroid follicular and parafollicular cells: association of serotonin binding protein with parafollicular cells.

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.

Thyrotropin induces the acidification of the secretory granules of parafollicular cells by increasing the chloride conductance of the granular membrane.

Secretory granules of sheep thyroid parafollicular cells contain serotonin, a serotonin-binding protein, and calcitonin. Parafollicular cells, isolated by affinity chromatography, were found to secrete serotonin when activated by thyrotropin (TSH) or elevated [Ca2+]e. TSH also induced a rise in [Ca2+]i. We studied the effect of these secretogogues on the pH difference (delta pH) across the membranes of the secretory granules of isolated parafollicular cells. The trapping of the weak bases, acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine (DAMP), within the granules was used to evaluate delta pH. In contrast to lysosomes, which served as an internal control, the secretory granules of resting parafollicular cells displayed a limited and variable ability to trap either acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine; however, when parafollicular cells were stimulated with TSH or elevated [Ca2+]e, the granules acidified. Weak base trapping was also used to evaluate the ATP-driven H+ translocation into isolated parafollicular granules. The isolated parafollicular granules did not acidify in response to addition of ATP unless their transmembrane potential was collapsed by the K+ ionophore, valinomycin. Secretory granules isolated from TSH-treated parafollicular cells had a high chloride conductance than did granules isolated similarly from untreated cells. Furthermore, ATP-driven H+ translocation into parafollicular granules isolated from TSH-stimulated parafollicular cells occurred even in the absence of valinomycin. These results demonstrate that secretogogues can regulate the internal pH of the serotonin-storing secretory granules of parafollicular cells by opening a chloride channel associated with the granule membrane. This is the first demonstration that the pH of secretory vesicles may be modified by altering the conductance of a counterion for the H+ translocating ATPase.

Relations between fatty acids and oestrogen binding properties of pure rat alpha 1-foetoprotein.

A delipidation procedure based on treatment with charcoal at pH 3 has been applied to highly purified rat alpha 1-foetoprotein preparations. The oestrogen binding properties of the delipidated proteins have been studied with an equilibrium dialysis technique, and compared with the properties of the untreated foetal protein, as well as those of preparations reconstituted from the defatted alpha 1-foetoprotein and the removed lipids. An important increase has been evidenced for the binding levels of oestrone, oestradiol-17 beta and diethylstilboestrol by the delipidated alpha 1-foetoprotein. A reversal of this effect has been obtained by incubating the delipidated protein either with the lipids extracted from the purified alpha 1-foetoprotein or with a potent competitor of the rat alpha 1-foetoprotein-oestrogen interaction, designated as 'L', previously demonstrated and isolated from whole rat sera, and tentatively characterized as a mixture of fatty acids. Scatchard analysis of the oestrone and oestradiol-17 beta binding parameters show that the enhanced fixation of the hormones after defatting is primarily due to a two-fold increase of the apparent number of binding sites/mol alpha 1-foetoprotein. The results are interpreted in terms of the probable, at least partial, identity between the lipids closely associated with the pure alpha 1-foetoprotein and the fatty acid mixture 'L' isolated from whole sera. The possible biological role of complex interplay between oestrophilic alpha 1-foetoproteins, phenolsteroids and fatty acids in the control of oestrogen levels during development is discussed briefly.

A simplified method for the preparation of rat thyroxine-binding prealbumin. Factors influencing its circulating level.

Rat thyroxine-binding prealbumin (TBPA) was isolated in three simple steps by means of a serum precipitation by a 5% phenol solution and two consecutive semi-preparative polyacrylamide gel electrophoreses. The overall yield was 15% and the TBPA preparation contained less than 1% impurities. In addition a monospecific antiserum was raised in the rabbit. In polyacrylamide gel, rat TBPA, as with its human counterpart, migrated anodally to albumin while in agarose gel, its electrophoretic mobility was similar to that of albumin. Serum TBPA measured in adult male Wistar rats did not exhibit a circadian rhythm. However, a significant 13% decrease was observed between 9 and 15 h, followed by the restoration of the initial value by 21 h. TBPA concentration was measured in 1-, 15- and 28-day-old male and female pups as well as in adult rats. The level of this protein increased from 1 to 28 days of age and did not display any sexual difference. Yet, while TBPA concentrations in adult males were similar to those recorded in the 28-day-old pups, for adult females, they returned to the levels measured in the 1-day-old pups.

Conformational changes in rodent and human alpha-fetoprotein: influence of fatty acids.

Binding, spectral and immunological studies were performed to demonstrate the conformational changes in rodent and human alpha-fetoprotein (AFP) induced by a free fatty acid environment. Scatchard analysis of estradiol (E2) binding to purified rat AFP indicated that unsaturated fatty acids changed the number of binding E2 sites and the apparent E2 equilibrium dissociation constant which varied non-linearly with docosahexaenoic acid concentration. UV spectral analysis of rodent and human AFPs showed that the absorbance minimum of AFP incubated with unsaturated fatty acid (L-AFP) was red-shifted, broadened and less pronounced than that of purified native AFP (N-AFP). Immunochemical studies with specific polyclonal antibodies to purified rodent and human AFPs (N-AFP antibodies) showed that these proteins lost immunoreactivity after incubation with unsaturated fatty acid. N-AFP antibodies recognized fewer epitopes on L-AFP than on N-AFP, whatever the species. Specific anti-rat L-AFP antibodies were used to demonstrate specific epitopes on rat L-AFP. Rat L-AFP antibodies did not recognize rat N-AFP. Saturated fatty acids were without effect on the binding, spectral and immunological properties of rodent and human AFPs. RIA or ELISA values for human AFP from fetal serum, hepatoma serum, and cord serum, were reduced 80, 50 and 5%, respectively, by unsaturated fatty acids. This decrease correlated with the relative percentage of polyunsaturated fatty acid in each biological fluid. Such results indicate that an unsaturated fatty acid environment induces conformational changes in AFP which may modulate the endocrine and immune functions of this protein.

In vivo transient rise in plasma free fatty acids alters the functional properties of alpha-fetoprotein.

Previous in vitro studies have shown that unsaturated fatty acids (UFA) induce conformational changes in rodent and human alpha-fetoprotein (AFP). To determine whether such changes in the binding and immunological properties of rat AFP also occur in vivo, plasma free fatty acid (FFA) concentrations were increased in young male rats (15, 21 and 28 days old) by acute i.v. injection of heparin (200 IU/kg). Plasma estrogens (estrone and estradiol) did not change after injection of heparin. There was a large increase in plasma FFA 10-20 min post-heparin injection, with a return to normal 60 min later. This transient rise in FFA plasma was associated with a 50% drop (P less than 0.001) in the binding of estradiol to rat AFP of 15-, 21- and 28-day-old rats by reducing the number of binding sites (P less than 0.001), leaving the affinity constant (Ka) unchanged. FFA extracts from post-heparin plasma induced similar changes in estradiol binding to purified rat AFP. The rise in plasma FFA induced a loss of AFP immunoreactivity, in 21- (P less than 0.001) and 28-day-old rats (P less than 0.001), but not in 15-day-old rats. This age-dependent response correlated with the FFA/AFP molar ratio (38 in 15-day-old rats, 388 in 21-day-old rats, and 5600 in 28-day-old rats). These results indicate that an in vivo rise in FFA induces rapid and reversible conformational changes in AFP which may modulate the endocrine and immune function of this oncofetal protein.

High-affinity binding of testosterone in serum from normal developing chick embryos and during the graft-versus-host reaction.

The sera of developing chicken embryos contain high-affinity, low-capacity protein binding sites for testosterone. The affinities remain constant throughout development, with mean values for the association constants of approx. 3.6 X 10(8) M-1 at 25 degrees C, whereas the concentration of sites varies markedly as a function of age: from approx. 2 nmol/g serum proteins in 11-day embryos, it rises to a peak of approx. 5-8 nmol at 14-16 days, then drops to approx. 2.6 nmol at 18 days and only 0.8-1 nmol in adults. Testosterone binding is inhibited by corticosterone, progesterone and dihydrotestosterone, and is little affected by estradiol. The testosterone and corticosterone binding properties of chicken sera show close similarities: parallel ontogenic patterns; constant ratios, throughout development, of the equilibrium binding parameters of the two steroids; mutual binding inhibition. The evidence strongly suggests that the two activities are associated, at least in part, with a common protein carrier(s). In growing embryos which undergo a graft-versus-host reaction, elicited by the graft of adult spleen tissue at 9 days of age, the testosterone and corticosterone binding activities are significantly decreased. This decrease is due to a fall in the number of sites, whereas association constants are not affected. This is the first high-affinity, saturable, testosterone-binding property to be described in an embryonic serum.

Thyroxine-binding globulin and thyroxine-binding prealbumin in hypothyroid and hyperthyroid developing rats.

We present evidence based on equilibrium and non-equilibrium binding studies, as well as on immunological techniques, that of the two rat specific thyroid-hormone-binding proteins, i.e., thyroxine-binding globulin (TBG) and thyroxine-binding prealbumin (TBPA), TBG but not TBPA is regulated by the thyroid hormones (TH). Hypothyroidism, induced from the day of birth by daily treatment with propylthiouracil (PTU-rats), leads to dramatic and sustained increases of the TH-binding abilities of the sera measured at equilibrium, whereas hyperthyroidism, induced by treatment with thyroxine (T4-rats), leads to the decrease of these abilities. Polyacrylamide gel electrophoresis and isoelectrofocalisation of radioiodinated T4-labelled sera, together with immunoassay of TBPA, demonstrate that both effects are due to TBG, the levels of which rise in PTU-rats and decline in T4-rats, while TBPA levels do not respond to either depletion or excess of the thyroid hormones. TBG rather than TBPA appears as the key thyroid-hormone-binding protein of the rat, inasmuch as it alone expresses a regulatory function of the thyroid hormones at protein synthesis level.

A senescence up-regulated protein: the rat thyroxine-binding globulin (TBG).

Thyroxine-binding globulin (TBG), the major carrier of thyroid hormones in human serum, was thought to be absent in most species, including rodents. We demonstrated recently that in fact the rat possesses a TBG gene, virtually non-expressed in young adults, but actively transcribed during post-natal development. We now find that the TBG gene is also increasingly re-expressed during senescence. Evidence is presented suggesting that physiologically decreased thyroid hormone levels, characteristic of neonates and of ageing rats, might constitute a common factor inducing up-regulation of TBG in both developmental and ageing processes. Rat TBG is to our knowledge the first biochemical 'positive' (i.e. increasing) marker of non-pathological senescence, expressed at both biosynthetic and bloodstream levels.

Phytoestrogens: new ligands for rat and human alpha-fetoprotein.

The binding of the lignans, enterolactone, enterodiol, nordihydroguaiaretic acid (NDGA), and the isoflavonic phytoestrogen equol, to human and rat alpha-fetoprotein (AFP) was studied. They had differential inhibitory effects (NDGA greater than equol greater than enterolactone greater than enterodiol) on the binding of estrone and estradiol to rat AFP and the binding of unsaturated fatty acid to both rat and human AFP. Inhibition was dose-dependent. The apparent dissociation constants (Kd) for phytoestrogens binding to AFP were: Kd NDGA = 5 +/- 1.2.10(-7) M, Kd equol = 6.7 +/- 0.8.10(-6) M, Kd enterolactone = 1.7 +/- 0.4.10(-5) M and Kd enterodiol = 2.2 +/- 0.6.10(-5) M. The Kd for estrone binding to rat AFP was increased by increasing concentrations of equol, but the number of esterone binding sites remained unchanged. This, plus the results of double-reciprocal plots, suggests that they compete for the same site(s). NDGA also competitively inhibited estrone binding at low NDGA concentrations (increased Kd), but high concentrations induced conformational changes in rat AFP, as both Kd and the number of binding sites (n) were altered. Both rat and human AFPs underwent changes in electrophoretic behaviour and loss of immunoreactivity with increasing NDGA, suggesting that NDGA binding induces conformational changes in the AFPs. However, equol did not alter the electrophoretic or immunological properties of either rat or human AFP, providing further evidence for qualitative differences in the effects of these diphenols. These findings indicate that phytoestrogens could play a role in AFP-dependent normal and pathological growth and development.

Rapid and long-term effects of 17 beta-estradiol on PIP2-phospholipase C-specific activity of MCF-7 cells.

Activity of the enzyme phosphatidylinositol 4,5-bisphosphate phospholipase C (PIP2-PLC) was demonstrated in MCF-7 human breast cancer cell homogenate. The addition of 10(-9) M 17 beta-estradiol to the culture medium elicited in the cells two types of responses depending on the period of exposure. Enzyme activity was rapidly activated at 15 s of incubation. After 5 min, PIP2-PLC activity was inhibited, and this effect continued at least until 24 h of exposure to the hormone. When 17 beta-estradiol was added in vitro to the total homogenate of untreated cells, enzyme activity was stimulated in a dose-dependent manner. These findings indicate that 17 beta-estradiol induces early and long-term modifications of the phosphoinositide signal pathway in intact MCF-7 cells as well as in vitro. The rapidity of the early effect suggests a non-genomic action of estradiol.

Unsaturated fatty acids synergistically enhance glucocorticoid-induced gene expression.

Regulation by unsaturated fatty acids of glucocorticoid-sensitive gene transcription was studied in HeLa cells transiently transfected with a mouse mammary tumour virus-luciferase reporter gene. Arachidonic acid and docosahexaenoic acid by themselves had no effect on basal levels of luciferase expression. However, they were able to enhance dexamethasone-induced transcription by 1.4-2.3 times (25-42 times the control levels) in a dose-dependent manner (ED50: 18 and 8 microM) for arachidonic and docosahexaenoic acid, respectively. The glucocorticoid antagonist RU486 effectively antagonized the dexamethasone response as well as the synergistic effect observed in the presence of arachidonic and docosahexaenoic acids, suggesting that the glucocorticoid receptor was an intermediate in the fatty acid synergism of the dexamethasone response. These studies show that fatty acids may be playing a role in modulating the intracellular steroid hormone signalling pathway to co-regulate a glucocorticoid-sensitive promoter.

Lipodystrophy defined by a clinical score in HIV-infected men on highly active antiretroviral therapy: correlation between dyslipidaemia and steroid hormone alterations.

BACKGROUND: A syndrome of lipodystrophy, associated with hypertriglyceridaemia, hypercholesterolaemia, hyperinsulinaemia and peripheral insulin resistance has been reported in protease inhibitor (PI)-treated HIV-infected patients. Because lipid metabolism, fat mass distribution and insulin resistance are partly regulated by steroid hormones, we questioned whether lipodystrophy is related to hormonal perturbations. OBJECTIVE: To evaluate serum lipid and steroid hormone concentrations in HIV-positive men on highly active antiretroviral therapy (HAART) in order to determine whether dyslipidaemia, peripheral loss of fatty tissue and central fat accumulation are related to steroid hormone modifications. DESIGN: A cross-sectional study. METHODS: Thirty-seven HIV-1-positive men on HAART, 23 of whom had symptoms of lipodystrophy, according to a subjective clinical score of lipodystrophy (SCSL), were tested. Serum concentrations of cholesterol, triglycerides and their subclasses, apolipoproteins and steroid hormones, including cortisol, dehydroepiandrosterone (DHEA), DHEA sulphate, androstenedione, testosterone and dihydrotestosterone were measured. RESULTS: Serum cholesterol, very low density lipoprotein (VLDL) cholesterol, triglycerides, VLDL triglycerides, high density lipoprotein (HDL) and low density lipoprotein (LDL) triglycerides, apolipoprotein B (ApoB) and atherogenic ratios of cholesterol:HDL cholesterol, LDL cholesterol:HDL cholesterol and ApoB:apolipoprotein A1 (ApoA1) were significantly increased in lipodystrophy-positive compared with lipodystrophy-negative men. The serum cortisol level was similar in lipodystrophy-positive versus lipodystrophy-negative men, but was elevated compared with controls. Serum DHEA was significantly lower in lipodystrophy-positive versus lipodystrophy-negative men and, consequently, the cortisol:DHEA ratio was increased in lipodystrophy-positive patients. A positive correlation was found between the cortisol:DHEA ratio and increased levels of atherogenic lipids. In addition, the SCSL was positively correlated with dyslipidaemia and the cortisol:DHEA ratio. CONCLUSION: This study demonstrates an association between the cortisol:DHEA ratio, lipid alterations and lipodystrophy. This syndrome might result from an imbalance between peripheral lipolysis and lipogenesis, both regulated by cortisol and DHEA.

Thyrotropin-induced thyroidal release of 5-hydroxytryptamine and accompanying ultrastructural changes in parafollicular cells.

The action of TSH on parafollicular cells of the bat thyroid was examined. Parafollicular cells were loaded with [3H]5-hydroxytryptamine ([3H]5-HT) by incubation with the precursor [3H]5-hydroxytryptophan in vitro. Subsequent exposure to TSH released [3H]5-HT from the glands, but not [3H]5-hydroxytryptophan. When thyroids were loaded with [3H]norepinephrine, TSH failed to release that amine. The [3H]5-HT-releasing effects of TSH were blocked by dinitrophenol and antimycin A and so were energy dependent. [3H]5-HT was not released by pentagastrin or calcitonin. Electron microscopic examination of thyroid glands exposed to TSH in vitro revealed degranulation of some parafollicular cells as well as the presence of abnormal appearing microgranules and large intracisternal (within rough endoplasmic reticulum) accumulations of secretory material. These results demonstrate for the first time direct effects of TSH on parafollicular cells. The results are consistent with the hypothesis that 5-HT, a normal constituent of bat parafollicular cells and an activator of follicular cells, may act as an intrathyroid local hormone.

Selective changes in binding and immunological properties of human corticosteroid binding globulin by free fatty acids.

The steroid hormones, progesterone (P4) and cortisol (F), have different biological activities but are both bound to human corticosteroid binding globulin (CBG) with similar affinity. This study examines the effect of physiological concentrations of FFA on the binding of these steroids to purified CBG and to the serum of pregnant women. It also analyzes the influence of the FFA environment on the immunological behavior of CBG. Unsaturated fatty acids (UFA) had a dose-dependent inhibitory effect (P less than 0.001) on steroid binding to CBG which was offset by saturated fatty acid-induced potentiation of binding (P less than 0.01) when both were present with CBG. UFAs inhibited P4 binding more than F binding. Comparable results were obtained with pregnant serum or with pure CBG. UFAs seemed able, depending on their concentration, to promote different molecular states of CBG, some with enhanced F binding and significantly reduced P4 binding, and others in which both P4 and F binding was markedly reduced. Scatchard analysis of steroid binding to purified CBG indicated that the UFAs influenced the association constant (Ka) and the number of binding sites (n) for F and P4 binding differently. Low concentrations (less than 16 microM) of arachidonic acid (C20:4) slightly potentiated F binding, with no change in Ka and a 1.6-fold increase in n; this concentration of C20:4 reduced n for P4 binding by 40% and did not affect Ka. Higher C20:4 concentrations (greater than 32 microM), reduced the Ka for F binding but did not apparently change n; for P4 binding, Ka was sharply reduced and n increased. The apparent equilibrium dissociation constant (Kd) for both F and P4 binding varied nonlinearly and differently with increasing C20:4 concentration. Immunoelectrophoresis and immunoautoradiography showed a reduction, or loss, of CBG immunoreactivity in the presence of UFA. The extent of these changes varied with the concentration and class of the UFA. These results indicate that FFA induce conformational changes in CBG which may modulate its activity and so influence the role of this protein in both the endocrine and immune systems.

Calcium-induced release of 5-hydroxytryptamine from thyroid lobes in vitro and accompanying ultrastructural changes in parafollicular and follicular cells.

Biogenic amines, including serotonin (5-HT), have been shown to activate follicular cells of the thyroid. 5-HT is stored in bat's parafollicular cells. Previous radioautographical evidence indicates that this 5-HT is present in calcitonin granules. The present study was done to determine if Ca++, the natural stimulus to calcitonin release, would also release parafollicular cell 5-HT and, if so, whether this release would be accompanied by activation of follicular cells. Parafollicular cells were filled with labeled 5-HT by incubation of thyroid lobes of bats or mice with 5-[3H]hydroxytryptophan, the precursor of [3H]5-HT. Thyroid lobes were incubated in vitro in Ca++-free medium containing a chelating agent and were then challenged with Ca++ (0-30 mM). Release of [3H]5-HT was defected beginning at 5 mM Ca++. [3H]5-HT release was roughly proportional to the Ca++ concentration. Calcium challenge also affected thyroid ultrastructure in bats. After 10-min exposure to 30 mM Ca++, there were an increased number and centripetal movement of follicular cell lysosomes, development of apical pseudopods, and formation of colloid droplets. Many lysosomes also developed a crystalline-like matrix. An unique membrane-enclosed, rod-shaped organelle appeared in a small number of follicular cells. Parafollicular cells exposed to high concentrations of Ca++ were often degranulated, although many appeared unchanged. These changes confirm that 5-HT is released from parafollicular cells by elevating the external Ca++ concentration, supporting the hypothesis that 5-HT and calcitonin share storage granules. 5-HT release may mediate the activation of follicular cells by Ca++.

alpha-Fetoprotein and transcortin behave as acute phase reactants in the maternal and fetal compartments of the inflammatory pregnant mouse.

Immunological and binding methods have been used to demonstrate that acute inflammation induced in the pregnant mouse by a single sc turpentine injection elicits plasma protein responses in the fetal as well as in the maternal compartment. The maternal response involves, along with the classical pattern of positive and negative acute phase reactants seen in the inflammatory nonpregnant animal, a highly specific approximately 2-fold increase of alpha-fetoprotein (AFP) concentrations. In addition, the high pregnancy-associated corticosteroid binding globulin (CBG) levels drop dramatically (2-3 times) in response to inflammation. The fetal response is characterized by small (10-25%) but statistically significant declines of AFP, CBG, and albumin concentrations, without any increase in levels of the positive classical acute phase reactants. The divergent responses of the estrophilic mouse AFP on the two sides of the placental barrier result in a 3- to 4-fold enrichment of the maternal serum vs. an approximately 20% impoverishment of the fetal serum in high affinity estrogen binding sites. The similar decrease in levels of CBG in mother and fetus leads to marked losses of high affinity corticosteroid sites for both. Neither the affinity constants for the estrogen-AFP interactions nor those for the corticosterone-CBG interactions are affected by inflammation. This is the first report of AFP as a positive marker of acute inflammation, of AFP as a pregnancy-specific inflammatory reactant in the mouse, and of a plasma protein response of the fetus in utero to an inflammatory stress undergone by the mother.