Quantitative morphological assessment of parathyroid activity in response to variations in calcium concentration in vitro

Isolated parathyroid glands from gerbils incubated at low (0.5 mM), normal (1.25 mM) or high (3 mM) calcium concentration for 1 or 6 h were studied with regard to the following stereological parameters: the volume densities of the nucleus, mitochondria, endoplasmic reticulum, Golgi zone, lipid bodies, lysosomes, secretory granules and storage granules, and the surface densities of the cell membrane and endoplasmic reticulum. In glands incubated for 1 h no significant differences were seen between the experimental groups in any of the parameters investigated, however, the volume density of the Golgi zone and the surface density of the cell membrane were significantly increased in glands incubated for 6 h at low calcium concentration, compared with those incubated at high calcium concentration. The enlargement of the Golgi zone in stimulated glands was also evident when the two glands from the same animal incubated at different calcium concentrations were compared. The data suggest that the early response of parathyroid glands to altered concentration of extracellular calcium includes functional activities associated with the Golgi zone.     

Abnormal calcium distribution in human parathyroid adenomas as possible cause of primary hyperparathyroidism

Current knowledge suggests that normal parathyroid glands and parathyroid adenomas have different sensitivities to environmental calcium. In search for morphological equivalents, 5 normal human and 10 porcine parathyroid glands, as well as 10 human parathyroid adenomas were investigated with regard to intracellular and extracellular calcium distribution. The glands were incubated for 2, 4, 6 and 20 h in tissue cultures using HAM's F10 medium with various calcium concentrations. For visualization of the calcium distribution in the tissue the method of pyroantimonate precipitation was applied. Specificity of the reaction was controlled by X-ray microanalysis. Shifts of the calcium pyroantimonate precipitates were quantitated by morphometry using an area-counting system. The results demonstrate that in normal parathyroid glands calcium precipitates are distributed randomly. Incubation of normal glands in medium with low calcium concentration (0.6 mM) provoked reduced amounts of intracellular and extracellular calcium complexes. When the incubations were performed in medium with high calcium content (2.6 mM), calcium accumulated inside parathyroid and stroma cells. In contrast to normal parathyroid glands, parathyroid adenomas fixed immediately after surgery showed an atypical calcium distribution with low amounts of intracellular and high amounts of extracellular calcium grains. The data suggest that in normal parathyroid glands the intracellular calcium concentration follows the extracellular environmental calcium concentration. Thus, calcium modulates parathyroid hormone (PTH) secretion via intracellular regulatory mechanisms. In parathyroid adenomas the calcium transport via the tumor cell membrane appears to be disturbed resulting in lowered intracellular calcium levels. This is remarkable since the environmental calcium concentration is elevated due to the hypercalcemia of primary hyperparathyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence measurements of cytosolic free Na concentration, influx and efflux in gastric cells.

Regulation of cytosolic free Na (Nai) was measured in isolated rabbit gastric glands with the use of a recently developed fluorescent indicator for sodium, SBFI. Intracellular loading of the indicator was achieved by incubation with an acetoxymethyl ester of the dye. Digital imaging of fluorescence was used to monitor Nai in both acid-secreting parietal cells and enzyme-secreting chief cells within intact glands. In situ calibration of Nai with ionophores indicated that SBFI fluorescence (345/385 nm excitation ratio) could resolve 2 mM changes in Nai and was relatively insensitive to changes in K or pH. Measurements on intact glands showed that basal Nai was 8.5 +/- 2.2 mM in parietal cells and 9.2 +/- 3 mM in chief cells. Estimates of Na influx and efflux were made by measuring rates of Nai change after inactivation or reactivation of the Na/K ATPase in a rapid perfusion system. Na/K ATPase inhibition resulting from the removal of extracellular K (Ko) caused Nai to increase at 3.2 +/- 1.5 mM/min and 3.5 +/- 2.7 mM/min in parietal and chief cells, respectively. Na buffering was found to be negligible. Addition of 5 mM Ko and removal of extracellular Na (Nao) caused Nai to decrease rapidly toward 0 mM Na. By subtracting passive Na efflux under these conditions (the rate at which Nai decreased in Na-free solution containing ouabain), an activation curve (dNai/Nai) for the Na/K ATPase was calculated. The pump demonstrated the greatest sensitivity between 5 and 20 mM Nai. At 37 degrees C the pump rate was less than 3 mM/min at 5 mM Nai and 26 mM/min at 25 mM Nai, indicating that the pump has a great ability to respond to changes in Nai in this range. Carbachol, which stimulates secretion from both cell types, was found to stimulate Na influx in both cell types, but did not have detectable effects on Na efflux. dbcAMP+IBMX, potent stimulants of acid secretion, had no effect on Na metabolism.     

Ultrastructural alterations in mammalian parathyroid glands induced by fixation

The influence of fixation methods, buffers and ions on the ultrastructure of parathyroid cells was studied in dogs, cats, rats and mice. Parathyroids fixed by immersion showed 3 chief cell variants referred to as cells in active, intermediate and resting stages, multinucleated syncytial cells, atrophic cells and, only in 1 feline parathyroid, a few oxyphil cells. Parathyroid glands fixed by perfusion, however, consisted only of 1 cell type. Satisfactory preservation was achieved by perfusion with 2.5% glutaraldehyde in 0.1 M Na cacodylate containing 0.25 mM CaCl2 and 0.5 mM MgCl2, and postfixation with 1% OsO4 in 0.1 M s-collidine containing 0.5 mM CaCl2 and 1.0 mM MgCl2. Good preservation was also obtained using Na phosphate during prefixation and postfixation. Other combinations of buffers led to shrinkage, dilation of rough endoplasmic reticulum cisternae, disruption of membranes or loss of matrix and secretory granules. The results demonstrate that the variants of parathyroid chief cells, multinucleated syncytial cells and atrophic cells arise during fixation.     

Parvalbumin is expressed in normal and pathological human parathyroid glands.

The parathyroid glands are of major importance in calcium homeostasis. Small changes in the plasma calcium (Ca2+) concentration induce rapid changes in parathyroid hormone (PTH) secretion to maintain the extracellular Ca2+ levels within the physiological range. Extracellular Ca2+ concentration is continuously measured by a G-protein-coupled Ca2+-sensing receptor, which influences the expression and secretion of PTH. The mechanism of signal transduction from receptor sensing to PTH secretion is not well understood, but changes in PTH secretion are tightly linked to changes in the cytosolic Ca2+ concentration. Using immunohistochemistry and Western blot analysis, we detected the EF Ca2+ binding protein parvalbumin (PV) in normal and in hyperplastic and adenomatous human parathyroid glands. The strongest PV signal was present in chief cells and water clear cells, whereas in oxyphilic cells only a weak signal was observed. Immunohistochemistry and in situ hybridization of the PTH indicated a co-localization of PV and PTH in the same cell types. Because changes in the cytosolic Ca2+ concentration are believed to influence the process of PTH secretion, a possible role of PV as a modulator of this Ca2+ signaling is envisaged.     

Occurrence of oxyphil cells in suppressed parathyroid glands

Parathyroid glands from Mongolian gerbils cultured for 7 days at a high calcium concentration and examined by electron microscopy, were found to contain suppressed chief cells containing numerous mitochondria, and some oxyphil cells containing an abundance of medium-sized or large mitochondria with calcium-containing precipitates. It is suggested that parathyroid chief cells may be transformed into oxyphil cells, under certain conditions, and that the associated increase in the number and size of the mitochondria may be related to an intracellular accumulation of calcium.     

Fine structure of the parathyroid glands in baboons, Papio hamadryas in response to experimental hypercorticoidism

Female baboons maintained under laboratory conditions were subjected to a series of 10 weekly injections (4 mg/kg body weight) of the synthetic glucocorticoid, triamcinolone hexacetonide. In response to the treatment, serum immunoreactive parathyroid hormone (PTH) levels were raised, though blood calcium levels remained within normal physiological limits. Light and electron-microscopic studies were made on the parathyroid glands at the end of the experimental period. The baboon parathyroid glands were composed of 'light' and 'dark' forms of the chief cells in varying ratios from gland to gland even within a single animal. Glucocorticoid-induced parathyroid hyperactivity as measured by circulating PTH levels was not accompanied by cellular hypertrophy, though there was an increase in the relative number of 'light' cells. At the ultrastructural level, after treatment, many of the 'light' cells were found to contain more free ribosomes, larger profiles of granular endoplasmic reticulum and had better developed mitochondria. Interdigitations between adjacent chief cells were more complex in treated glands. Apart from these features, chief cells of treated glands were basically similar to those of untreated controls. Our study showed that functional parathyroid hyperactivity in baboons is not necessarily accompanied by significant ultrastructural changes in chief cells.     

Activation of calcium-sensing receptor accelerates apoptosis in hyperplastic parathyroid cells

Calcimimetic compounds inhibit not only parathyroid hormone (PTH) synthesis and secretion, but also parathyroid cell proliferation. The aim of this investigation is to examine the effect of the calcimimetic compound NPS R-568 (R-568) on parathyroid cell death in uremic rats. Hyperplastic parathyroid glands were obtained from uremic rats (subtotal nephrectomy and high-phosphorus diet), and incubated in the media only or the media which contained high concentration of R-568 (10(-4)M), or 10% cyclodextrin, for 6h. R-568 treatment significantly suppressed medium PTH concentration compared with that of the other two groups. R-568 treatment not only increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay-positive cells, but also induced the morphologic changes of cell death determined by light or electron microscopy. These results suggest that CaR activation by R-568 accelerates parathyroid cell death, probably through an apoptotic mechanism in uremic rats in vitro.     

Modeling repetitive firing and bursting in a small unmyelinated nerve fiber.

The Hodgkin-Huxley equations, originally developed to describe the electrical events in the squid giant axon, have been modified to simulate the ionic and electrical events in a small unmyelinated nerve fiber. The modified equations incorporate an electrogenic sodium-potassium pump, finite intra-axonal volume, a periaxonal space, a calcium current, and calcium-dependent potassium conductance (GKCa). The model shows that adaptation can occur in two ways: increased Na-K pump activity because of periaxonal K accumulation or intra-axonal Na accumulation; or from an increase in (GKCa) caused by calcium accumulating within the axon. Bursting is an extension of adaptation and occurs when the sensitivity of the Na-K pump or (GKCa) to changes in ionic concentration is increased.     

Increases in Na/K pump numbers in isolated human lymphocytes exposed to lithium in vitro. Reversal by myo-inositol and by inhibitors of protein kinase C and the Na/H antiport

Lithium (1-8 mM) caused a dose-dependent increase in the number of [3H]ouabain binding sites and in sodium/potassium (Na/K) pump activity in normal lymphocytes after incubation for 72 h. The increase in Na/K pump activity was due to an increase in the Vmax of the pump, with no change in the apparent affinity (Km) for potassium (rubidium). There was no change in the turnover number of the pump and the intracellular sodium concentration fell. The increase in [3H]ouabain binding sites was prevented by the addition of myo-inositol (10 mM), by inhibition of the protein kinase C with staurosporine (100 nM) and by inhibition of the Na/H antiport with dimethylamiloride (50 microM). These results suggest that the increase in Na/K pump activity caused by lithium is due to an increase in pump numbers and not due to increased activity of individual pumps or to an alteration in the affinity of the pumps for potassium. The increase in Na/K pump numbers and activity in lymphocytes exposed to lithium for 72 h may be related to altered Na/H antiport activity secondary to inhibition of phosphoinositol breakdown by lithium.     

Caclium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. Requirement for potassium ions.

The effects of monovalent cations on calcium uptake by fragmented sarcoplasmic reticulum have been clarified. Homogenization of muscle tissue in salt-containing solutions leads to contamination of this subcellular fraction with actomyosin and mitochondrial membranes. When, in addition, inorganic cations are contributed by the microsomal suspension and in association with nucleotide triphosphate substrates there is an apparent inhibition of the calcium transport system by potassium and other cations. However, when purified preparations were obtained after homogenization in sucrose medium followed by centrifugation on a sucrose density gradient in a zonal rotor, calcium uptake and the associated adenosine triphosphatase activity were considerably activated by potassium and other univalent cations. When plotted against the log of the free calcium concentration there was only a slight increase in calcium uptake and ATPase activity in the absence of potassium ions but sigmoid-shaped curves were obtained in 100 mM K+ with half-maximal stimulation occurring at 2 muM Ca2+ for both calcium uptake and ATPase activity. The augmentation in calcium uptake was not due to an ionic strength effect as Tris cation at pH 6.6 was shown to be inactive in this respect. Other monovalent cations were effective in the order K+ greater than Na+ greater than NH4+=Rb+=Cs+ greater than Li+ with half-maximal stimulation in 11 mM K+, 16 mM Na+, 25 mM NH4+, Rb+, and Cs+ and in 50 mM Li+. There was nos synergistic action between K+ AND Na+ ions and both calcium uptak and associated ATPase were insensitive to ouabain. Thallous ions stimulate many K+-requiring enzymes and at one-tenth the concentration were nearly as effective as K+ ions in promoting calcium uptake. The ratio of Ca2+ ions transported to P1 released remained unchanged at 2 after addition of K+ ions indicating an effect on the rate of calcium uptake rather than an increased efficiency of uptake. In support of this it was found that during the stimulation of calcium uptake by Na+ ions there was a reduction in the steady state concentration of phosphorylated intermediate formed from [gamma-32P]ATP. It is considered that there is a physiological requirement for potassium ions in the relaxation process.     

Structural changes in parathyroid glands exposed to vitamin D metabolites in vitro

Isolated parathyroid glands from normal adult Mongolian gerbils were incubated for 10 minutes to 16 1/2 hours at high, medium or low concentrations of Ca2+, with or without added 1,25-dihydroxycholecalciferol (1.25-DHCC) or 25-hydroxycholecalciferol (25-HCC), after which they were studied electron microscopically. 1,25-DHCC appeared to potentiate the inhibitory action of high calcium on parathyroid activity, whereas 25-HCC had no structurally detectable effect.     

Light- and electron microscopic observations on parathyroid glands in different age groups of rats

The parathyroid hormone (PTH) acts on bones, intestines, and kidneys to maintain the calcium homeostasis which, in turn, is a main factor in controling the parathyroid (PT) gland activity. In all mammals studied, the chief cells of PT glands changed their size, shape, and cytoplasmic structure due to different functional states which vary the serum calcium levels. The chief cells of the rat PT glands were classified as dark and light. The dark cells may constitute an active form, characterized mainly by the abundant free ribosomes, conspicuous rough endoplasmic reticulum, and GOLGI complexes, greater number of secretory granules (SG) and increased tortuosity of the plasma membranes as compared to the light ones which were considered as a less active type of cells. Due to different calcium requirements in newborn and young rats for the ossification of growing skeleton and in adult and senile rats with consolidate mature bones, the PT glands studied with electron microscope showed various cytological features. The parenchyma of newborn and young PT glands was composed by dark chief cells. The light chief cells were more frequent in adult and senile animals as a less active type of cell. Mature SG were only occasionally observed in dark cells of newborn, young and adult PT glands. They may constitute a reserve supply of PTH but probably not the main way of secretion, according to their little number. Another pool of PTH probably answers the needs for the small basal variations in the steady-state secretion and may be represented by the vesicles observed in the chief cells cytoplasm.     

Effect of high potassium concentrations on juvenile hormone release in vitro from corpora allata of Locusta migratoria

ABSTRACT. Incubation of corpora allata (CA) from adult females of Locusta migratoria (L.) in vitro in medium with a potassium concentration of 50 mM results in an elevation of the rate of juvenile hormone (JH) release. This elevation is however delayed, becoming apparent after the glands have been returned to low concentration of potassium but it is also prolonged, lasting up to 270 min. The elevation is initially modest but becomes more marked with time. The response of glands to high concentration of potassium is heterogeneous and appears to some extent to be a function of the initial rate of JH release of the glands. Glands starting with low rates of JH release are stimulated strongly by potassium, those with high rates of JH release much less so. Co-incubation of glands with high concentration of potassium and with calcium channel blockers (both organic and ionic) prevents the elevation of release rates otherwise consequent on high concentrations of potassium.     

Anomalous potassium channel-gating rates as functions of calcium and potassium ion concentrations.

With near normal monovalent ionic concentrations, the rate of increase of the potassium conductance after a depolarizing voltage-clamp step is slowed when the external calcium concentration is increased. This trend in the rise-time with changes in calcium is reversed when the axointernal potassium concentration is reduced (150 mM) and the periaxonal concentration is increased (50 mM); that is, the rise-time decrease with increasing calcium concentration. Furthermore, the degree of sigmoidality of the K-conductance time-course always increase when the rise-times increase for a given test potential. In the case of calcium surface-charge screening, these effects may be caused by a shifted distribution of K-ions within the channels following the altered ion gradient, and by a consequent shift in the reciprocal electrostatic interactions between the ionic charges and channel-gate charges.     

Experimental study of follicle formation in suppressed parathyroid glands of Mongolian gerbils.

Parathyroid follicle formation was studied in Mongolian gerbils subjected to different concentrations of calcium in vivo and in vitro, using light and electron microscopic methods, including the potassium pyroantimonate technique and x-ray microanalysis for identification of cations. Follicles were frequent at high calcium concentration, but sparse at intermediate and low levels of calcium. Two main types of follicle were differentiated: "degenerative follicles" containing cellular debris and lined by smooth-surfaced epithelium which occasionally showed degenerative changes; and "secretory follicles" characterized by amorphous and granular contents, and an epithelium possessing microvilli and cytoplasmic projections. Amorphous masses were also seen in dilated intercellular spaces and in dilated cisterns of rough endoplasmic reticulum in the follicle epithelium. Calcium-containing precipitates were found in degenerating chief cells, and between degenerating cells and follicles. Parathyroid follicles are believed to be formed by degeneration of suppressed chief cells (degenerative follicles), and by secretion of hormonal and/or other substances into dilated intercellular spaces which progressively increase in size to form follicular cavities (secretory follicles), thereby possibly reducing the level of metabolically active parathyroid hormone. Functional suppression is believed to underlie the development of parathyroid follicles.     

Direct inhibitory effect of amino-terminal parathyroid hormone fragment [PTH(1-34)] on PTH secretion from bovine parathyroid primary cultured cells in vitro

We have examined the possibility of direct inhibitory effect of PTH(1-34) on PTH secretion in bovine parathyroid cells. As low as 10(-12) M PTH(1-34) completely inhibited low calcium (0.5 mM Ca2+)-stimulated PTH secretion by these cells. In the presence of 1.25 mM Ca2+, 10(-12) M PTH(1-34) inhibited PTH secretion by about 14.3% of the basal value, while 10(-11) M or higher concentration of PTH(1-34) showed potent inhibitory effects equivalent to the inhibitory action of high calcium concentration (2.5 mM Ca2+) on PTH secretion. At 2.5 mM Ca2+, as much as 10(-9) M PTH(1-34) failed to inhibit PTH secretion further. These results suggest that PTH(1-34) might directly, not via calcium concentration, inhibit PTH secretion by parathyroid cells and that a cooperative mechanism could exist between calcium and PTH(1-34) to inhibit PTH secretion.     

Proton-activated rubidium transport catalyzed by the sodium pump

Although the sodium pump normally exchanges three sodium for two potassium ions, experiments with inside-out red cell membrane vesicles show that the stoichiometry is reduced when the cytoplasmic sodium concentration is decreased to less than 1 mM. The present study was designed to gain insight into the question whether other monovalent cations, particularly protons, can act as sodium congeners in effecting pump-mediated potassium transport (ATP-dependent rubidium efflux from inside-out vesicles). The results show that at low cytoplasmic sodium concentration, an increase in proton concentration effects a further reduction in sodium:rubidium stoichiometry, to a value less than the minimal expected (1Na+:3Rb+). Furthermore, when vesicles containing 86RbCl are incubated in nominally sodium-free medium. ATP-dependent net rubidium efflux (normal influx) occurs when the pH is reduced from approximately 7.0 to 6.2 or less. This efflux is inhibited by strophanthidin and vanadate. These experiments support the notion that the sodium pump can operate as an ATP-dependent proton-activated rubidium (potassium) pump without obligatory countertransport of sodium ions.     

Electron microscopic study of the parathyroid gland of the calcium-treated hamster subjected to hypergravity environment

The ultrastructure of the parathyroid glands of calcium-treated golden hamsters subjected to 5 gravity environment was studied. In the calcium-treated animals exposed to hypergravity environment, the Golgi complexes and cisternae of the granular endoplasmic reticulum were significantly decreased compared with those of the animals exposed to hypergravity environment only and appeared to increase compared with those of the calcium-treated animals, but were almost similar to those of the control animals. In addition, many chief cells contained some prosecretory granules in the Golgi areas, some secretory granules situated close to the plasma membrane and many lysosomes. The morphology of the parathyroid glands in the calcium-treated animals exposed to hypergravity environment resembled that of the control animals. These results suggest that the parathyroid glands suppressed by treatment of calcium and stimulated in response to hypergravity environment may indicate the secretory activity of the parathyroid glands of the control animals.     

Characterization and Distribution of Transferrin Receptors in the Rat Brain

The mechanism of calcium transport across the plasma membrane of chromaffin cells was studied using plasma membrane vesicles prepared from cells of adrenal medulla. Purification of the plasma membrane was about 30-fold, based on the alpha-bungarotoxin binding activity. The isolated membrane vesicles have both Na+/Ca2+ exchange and calcium pump activities. The Na+/Ca2+ exchange activity increased with the free calcium concentration and was not saturated at 1 mM, the highest concentration tried. The K1/2 of the calcium pump for calcium is 0.06 microM. Part of the Na+/Ca2+ exchange activity was inhibited by preincubation of the membrane vesicles with veratridine and the effect of veratridine was reversed by tetrodotoxin. The calcium taken up by the calcium pump was released by 0.005% saponin, but was not affected by oxalate. The calcium taken up by the calcium pump was released by exchanging with the external sodium, which suggests that the two calcium transport systems are located on the same population of membrane vesicles. The above evidence indicates that both calcium transport activities are located on the plasma membrane and not on contaminating organelle membranes. The significance of the two calcium transport systems in regulation of cytosolic calcium concentration of chromaffin cells is discussed.