Effects of physiological concentrations of parathyroid hormone on acid phosphatase activity in cultured rat bone cells

The mechanism by which parathyroid hormone (PTH) induces osteoclastic bone resorption is still incompletely understood. Recent evidence suggests that the hormone exerts its effects indirectly, via the osteoblasts. Bone cells isolated from fetal rat calvaria by enzymatic digestion were used. Two heterogeneous cell populations were isolated by equilibrium density centrifugation on Percoll gradients and maintained by differential culture conditions. These two populations, which are morphologically distinguishable from one another by light and electron microscopy, have been characterized previously both biochemically and with regard to their hormonal (PTH and calcitonin) responses. We have called them type C cells (containing cells with some of the properties of osteoclasts) and type B cells (containing osteoblast-like cells, as well as fibroblasts, chondrocytes and other stromal cells). In the present study, we have further characterized the functional relationship between the two cell populations, with particular regard to the hormonal responses of type C cultures. Acid phosphatase, measured cytochemically in individual cells, was used as a marker for C cell responses. C cells had significantly higher levels of acid phosphatase activity than either B cells or spleen macrophages. Calcitonin (0-10 pg/ml) decreased C cell acid phosphatase activity but was without effect on B cells or spleen macrophages. Co-culture of C cells with B cells produced increased enzyme activity only in the former; this effect could be mimicked if fibroblasts replaced B cells and cell contact was essential for this response. PTH (0-10 pg/ml) raised enzyme activity further in C cells only when they were cultured with B cells. When C cells were cultured so that they shared medium, but were not in contact, with B cells, PTH (2 pg/ml) still increased enzyme activity in the former. Fibroblasts were ineffective in this system. Spleen macrophages were also unresponsive to PTH when substituted for C cells. Calcitonin (10 pg/ml) blocked the effects of PTH on C cells. These results indicate that macrophages are probably not a significant proportion of the C cell population, and that PTH may produce increased acid phosphatase activity in C cells via a humoral factor produced by cells present in B cell cultures.     

Effects of alpha-adrenergic stimulation and blockade of plasma parathyroid hormone concentrations in cows.

Experiments were designed to investigate responses of immunoreactive parathyroid hormone (PTH) during alpha-adrenergic stimulation and blockade in cows. Alpha-adrenergic agonists (methoxamine, phenylephrine and noradrenaline, the beta-adrenergic action of which was blocked by propranolol) did not change PTH and free fatty acid levels, whereas they characteristically increased the blood pressure and decreased the heart rate. In contrast, alpha-adrenergic blockade by phentolamine progressively increased PTH levels. The elevated PTH concentrations, associated with increased plasma noradrenaline and free fatty acid levels, rising heart rate and decreasing blood pressure, indicated that all these changes can be related to a beta-adrenergic stimulatory mechanism. Beta-adrenergic stimulation was presumably responsible for the initial elevation of PTH concentrations, whereas, during the later phase of the phentolamine infusions, a concomitant hypocalcaemia probably also produced a stimulatory effect.     

Influence of temperature and photoperiod on plasma melatonin in the mudpuppy, Necturus maculosus.

A melatonin (MEL) radioimmunoassay employed previously only in mammals was used to estimate plasma MEL in a salamander, Necturus maculosus. Validation procedures included thin-layer chromatography of plasma extracts, parallel inhibition curves of authentic MEL and serially diluted plasma and plasma extracts and quantitative recovery of authentic MEL added to pooled Necturus plasma. A diel cycle of plasma MEL was demonstrated in mudpuppies acclimatized for a minimum of 3 weeks under a 12L:12D photoperiod and 15 +/- 1 degrees and sampled in late March. The MEL cycle persisted under a reversed photoperiod (lights on, 1800 hr), but the amplitude of the MEL peak was diminished, and the peak was more than 180 degrees out of phase with the corresponding peak under a normal photoperiod. In animals acclimated to 5 degrees in mid-June under a 12L:12D photoperiod, the diel cycle of plasma MEL continued, but both midphotophase and midscotophase concentrations were reduced compared with 15 degrees controls. The diel cycle was also present in animals acclimated to 25 degrees, but the decrease in MEL concentrations was less marked than that in 5 degrees animals. Photoperiod is apparently the primary cue for cycles in plasma MEL in Necturus, but the cycle can be influenced by temperature.     

Immunocytochemical localization of prolactin in pars distalis of the salamander Necturus maculosus.

The prolactin cells in the anterior pituitary of Necutrus maculosus have been localized with anti-ovine prolactin using the peroxidase-labeled antibody technique. Two histological stains, carmoisine-L, orange G and hematolylin and eosin, were used to corroborate the immunocytochemical results. In low magnification micrographs, the peroxidase positive area corresponds to comparable acidophilic and carmoisine-L staining areas of other sections from the same part of the gland.Carmoisine-L stains the heavily granulated prolactin cells bright orange. These cells correspond in morphology and arrangement to those containing the dark brown precipitate which indicates the site where antigen bound peroxidase has reacted with its substrate. When tissue was stained with nonimmune serum or with immune serum adsorbed with prolactin instead of the first antibody, the results were negative.     

Effects of aspirin on pathways of ion permeation in Necturus antrum: role of nutrient HCO3.

Intracellular microelectrodes were used to evaluate electrical properties of the cell membranes in Necturus antral mucosa during exposure to luminal acid alone (pH 4) or to 5 mmol/L aspirin [acetylsalicylic acid (ASA)] in the presence of luminal acid. When nutrient solutions were buffered by HCO3- (pH 7.3), ASA moderately depolarized and increased the resistances of both cell membranes. When nutrient solutions were buffered by HEPES (pH 7.3), ASA induced even greater depolarizations of the cell membranes. In addition, resistance of the apical membrane did not increase and resistance of the basolateral membrane decreased. The changes in basolateral membrane resistance were observed when tissues were exposed to 5 mmol/L salicylate but not during exposure to luminal acid alone or to acidified luminal solutions containing 5 mmol/L acetate, a small and permeable organic acid. Electron microscopy confirmed that these initial electrophysiological changes precede alterations in cell morphology. The findings suggest that nutrient HCO3- attenuates changes in membrane potentials caused by ASA. Loss of nutrient HCO3- seems to accelerate alterations in basolateral membrane resistance caused by ASA and its salicylate moiety.     

Increasing parathyroid hormone concentrations in untreated primary hyperparathyroidism.

Twenty-four patients with mild to moderate primary hyperparathyroidism were followed for an average of 2.45 years with serial determinations of serum ionized calcium and intact parathyroid hormone (PTH). For the entire group serum ionized calcium remained stable, whereas serum PTH increased significantly. Eleven patients (group 1) demonstrated a significant increase in PTH with time. The remaining 13 patients formed group 2. Comparison of the changes (%) in each subgroup showed a small but significant increase in serum ionized calcium of 2.6% with time in group 1, while serum PTH increased by 78%. In group 2 serum ionized calcium remained stable whereas PTH increased modestly by 22%. Serum concentrations of creatinine were stable throughout the follow-up period in both groups. Despite the greater precision of serum ionized calcium, measurements of intact PTH are evidently more sensitive than measurements of serum ionized calcium for the detection of progression in primary hyperparathyroidism.     

Effects of parathyroid hormone fragments on the growth of murine mandibular condylar cartilage in vitro

The present study showed that the fragment hPTH (1-34) is mitogenic in organ cultures of neonatal mandibular condylar cartilage, and even more so in late fetal condyles. Three fragments of hPTH were used to clarify which part of the molecule possesses a mitogenic effect alike that of the native hormone: hPTH (1-34), (28-48) and (53-84). [3H]thymidine incorporation into trichloracetic acid insoluble material and quantitative autoradiography were employed in a serum-free medium to assess the effects of these fragments while light and electron microscopy studies served for morphological evaluations. It became evident that the fragment hPTH (1-34) enhanced the incorporation of [3H]thymidine, a fact that could be noted only in serum-free medium. The putative target cells for the effects of hPTH (1-34) were the chondroporogenitor cells which also appeared to have experienced a blockage in their differentiation into chondroblasts. Ultrastructurally, the latter cells responded in the formation of adherent profiles of plasma membranes, whereas the differentiated zone of the cartilage reduced its size. Using serum-free medium, hPTH (1-34) also brought about an inhibition in alkaline phosphatase activity, a fact that was not encountered in medium containing serum. By contrast, hPTH (28-48) had no mitogenic effect, although treated specimens revealed morphological changes in the chondroprogenitor cell zone along with an enhancement of cartilage cells hypertrophy. No significant effects on either mitogenecity or morphology could be noted in hPTH (53-84)-treated cultures.     

Effects of parathyroid hormone and cortisol on prostaglandin production by neonatal rat calvaria in vitro

We have examined the effects of parathyroid hormone (PTH) and cortisol on the production of prostaglandins, particularly PGE2, by neonatal rat calvaria cultured in a chemically-defined medium. Although there was considerable variability, calvaria produced large amounts of PGE2 in control cultures, reaching medium concentrations of 40 to 200 nM. PGE2 release was partially inhibited by cortisol at 10 nM and markedly inhibited at 100 nM. Bovine 1-34 synthetic PTH produced an increase in PGE2 concentration which was most striking in the presence of a low concentration of cortisol (10 nM). The medium also contained large amounts of 6-keto PGF1 alpha, the metabolite of prostacyclin, which showed similar changes in response to PTH and cortisol. Thromboxane B2 concentrations were low and unaffected by these hormones. 1,25-dihydroxyvitamin D did not increase medium PGE2 concentration. Since PGE2 is a potent stimulator of bone resorption and formation, some of the effects of PTH as well as cortisol may be mediated by their ability to alter PGE2 production in skeletal tissue.     

Plasma steroid profiles in male and female mudpuppies, Necturus maculosus (Rafinesque).

Plasma steroid profiles of healthy adult male and female mudpuppies, Necturus maculosus Rafinesque, were measured in animals sampled from November–June. Steroids in ether extracted plasma were analyzed directly by radioimmunoassay (RIA) for progesterone (P), total androgens, and total estrogens, or after Celite-ethylene glycol column chromatography for P, dihydrotestosterone (DHT), testosterone (T), estrone (E₁), and estradiol-17β (E₂). Steroid levels in both sexes were high compared to other vertebrates (10- to 100-fold greater), and highly variable at all times of the year. Comparison of the steroid profiles of males and females showed no qualitative differences; howeve, titers of all steroids (in ng/ml) were higher in males. Progesterone (P) ranged from 2.0–64.0, dihydrotestosterone (DHT) from 2.0–133, testosterone (T) from 4.0–202, estrone (E₁) from N.D. to 1.87, and estradiol (E₂) from 2.0–9.85 in adult males. In adult female Necturus, P ranged from N.D. to 15.5, DHT from 2.9–11.4, T from 1.5–8.3, and E₂ from −0.4–6.35. E₁ was not detectable. No clear seasonal variations in steroid titers or gonadal structure were observed although holding conditions at 5–8° did not approximate those in nature.     

Effects of the calcium channel activator BAY-K-8644 on in vitro secretion of calcitonin and parathyroid hormone

The recently discovered calcium (Ca) channel activator BAY-K-8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate], an analog of the calcium channel blockers nifedipine and nitrendipine, was tested to determine its potential for altering hormone secretion in an in vitro system designed to study concurrent secretion of calcitonin (CT) and PTH. Addition of BAY-K-8644 (10(-4)-10(-5) M) to medium (1 mM Ca) bathing baby rat thyroparathyroids enhanced secretion of CT at least 2- to 4-fold and suppressed PTH release by as much as 75-85%. Addition of BAY-K-8644 alone to medium containing high (2.5 mM) Ca did not further enhance the already high rate of CT release, nor did it cause any further suppression of PTH secretion. BAY-K-8644 did not stimulate CT release or suppress PTH release in the absence of medium Ca. Addition of the Ca channel blocker nitrendipine (10(-5) M) inhibited CT release at either 1 or 2.5 mM Ca, and at 1 mM Ca, nitrendipine negated the simulatory effect of 10(-5) M BAY-K-8644 on CT release. However, at 2.5 mM Ca, 10(-5) M BAY-K-8644 reversed the marked inhibitory effect of 10(-5) M nitrendipine on CT release. At 1 mM Ca, PTH secretion was inhibited equally well by BAY-K-8644 and nitrendipine, and both agents together caused a further suppression of PTH release. The results indicate that Ca entry into the thyroid C-cell and parathyroid chief cell may occur via classical voltage-sensitive Ca channels and that the newly described Ca channel activator BAY-K-8644 should prove useful as a probe for studying hormone secretion in Ca-dependent secretory systems.     

Effects of estradiol and progesterone on parathyroid hormone secretion from human parathyroid tissue

Estrogens have been used to treat the hypercalcemia in primary hyperparathyroidism. Estrogens and progesterone both stimulate PTH secretion from bovine parathyroid tissue. The effects of these agents on PTH secretion from human parathyroid tissue are not known. In this study, we evaluated the effects of 17 beta-estradiol and progesterone on PTH secretion from abnormal parathyroid tissue from seven patients (four adenomas, three hyperplasia). Both estradiol (10(-9)-10(-6) mol/L) and progesterone (10(-7) and 10(-6) mol/L) significantly stimulated PTH secretion in a time- and dose-dependent manner during a 3-h period. These results indicate the need for a careful evaluation of serum PTH levels and other parameters of parathyroid function in estrogen- and/or progesterone-treated patients with primary hyperparathyroidism.     

Identification of an androgen receptor in the zonal testis of the salamander (Necturus maculosus).

The testis of the salamander, Necturus maculosus, is advantageous for studying biochemical changes during spermatogenesis because germ cells and associated Sertoli and Leydig cells are topographically separated by stage of development. Using extracts of staged tissue samples and [3H]testosterone (T) in a standard binding assay, followed by Sephadex LH-20 or DNA-cellulose chromatography to separate free and bound steroid, we have identified a T-binding protein having physicochemical characteristics of a classical androgen receptor (AR): high affinity (Kd = 10(-9) M), limited capacity (Bmax) = 10(-10) M or 350 fmol/g tissue) and androgen specificity (T = 5 alpha - dihydrotestosterone greater than progesterone = corticosterone greater than estradiol). AR was present in nuclear extracts, where greater than 80% of binding sites were occupied by endogenous ligand, but was not detectable in cytosol. On linear sucrose gradients, nuclear AR sedimented at 3-4 S in both low and high ionic-strength buffers and, by this and other criteria, was distinguishable from the nonreceptor androgen binding protein (ABP) of the same species. The diffuse distribution of AR in germinal and nongerminal (glandular) tissues at all developmental stages is consistent with a dual localization in Sertoli cells and Leydig cells, as previously reported in mammals, and further suggests a regulatory role of androgen throughout spermatogenesis.     

The effect of 1,25-dihydroxycholecalciferol on the parathyroid hormone secretion of porcine parathyroid glands and human parathyroid adenomas in vitro

The effect of 1,25-dihydroxycholecalciferol (1,25-(OH)2-D3) on parathyroid hormone secretion by porcine parathyroid glands and human parathyroid adenoma tissue was investigated by in vitro incubation. The addition of 100 nmoles 1,25-(OH)2-D3 to the medium inhibited significantly the release of immunoreactive parathyroid hormone by 63--65%. This suppression was reversible when 1,25-(OH)2-D3 was removed again. The inhibition of parathyroid hormone release observed in human parathyroid adenoma tissue was similar to that in normal porcine parathyroid glands. This indicates that adenoma tissue is sensitive to regulatory influences. As well as calcium, 1,25-(OH)2-D3 may act as another feedback inhibitor of parathyroid hormone secretion.     

Effects of transdermal estrogen replacement on parathyroid hormone secretion

The effect of transdermal 17 beta-estradiol replacement on ionized calcium and PTH levels was examined in 15 postmenopausal women. After baseline studies in the fasting state, the effect of a calcium infusion on PTH levels was studied. Estrogen replacement resulted in a fall in fasting resting ionized calcium and a rise in PTH levels. After calcium infusion there was no change in the shape of the relationship between plasma calcium and PTH. The level of nonsuppressible PTH secretion was not altered. Transdermal estrogen did not alter basal vitamin D-binding protein levels, 25-hydroxyvitamin D levels, or calcitriol levels. We conclude that the effect of transdermal estrogen replacement on PTH secretion is completely explained by the lowering of ionized calcium, causing a rise in PTH secretion. Thus, with this route of estrogen replacement, there is no necessity to postulate a direct effect of 17 beta-estradiol on the parathyroid gland.